ANTIGEN-BINDING CONSTRUCTS

BACKGROUND

Antibodies are well known for use in therapeutic applications.

Antibodies are heteromultimeric glycoproteins comprising at least two heavy and two light chains. Aside from IgM, intact antibodies are usually heterotetrameric glycoproteins of approximately 150 Kda, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond while the number of disulfide linkages between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant regions. Each light chain has a variable domain (VL) and a constant region at its other end; the constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. The light chains of antibodies from most vertebrate species can be assigned to one of two types called Kappa and Lambda based on the amino acid sequence of the constant region. Depending on the amino acid sequence of the constant region of their heavy chains, human antibodies can be assigned to five different classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3 and IgG4; and IgA1 and IgA2. Species variants exist with mouse and rat having at least IgG2a, IgG2b. The variable domain of the antibody confers binding specificity upon the antibody with certain regions displaying particular variability called complementarity determining regions (CDRs). The more conserved portions of the variable region are called Framework regions (FR). The variable domains of intact heavy and light chains each comprise four FR connected by three CDRs. The CDRs in each chain are held together in close proximity by the FR regions and with the CDRs from the other chain contribute to the formation of the antigen binding site of antibodies. The constant regions are not directly involved in the binding of the antibody to the antigen but exhibit various effector functions such as participation in antibody dependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding to Fcγ receptor, half-life/clearance rate via neonatal Fc receptor (FcRn) and complement dependent cytotoxicity via the C1q component of the complement cascade.

The nature of the structure of an IgG antibody is such that there are two antigen-binding sites, both of which are specific for the same epitope. They are therefore, monospecific.

A bispecific antibody is an antibody having binding specificities for at least two different epitopes. Methods of making such antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin H chain-L chain pairs, where the two H chains have different binding specificities see Millstein et al, Nature 305 537-539 (1983), WO93/08829 and Traunecker et al EMBO, 10, 1991, 3655-3659. Because of the random assortment of H and L chains, a potential mixture of ten different antibody structures are produced of which only one has the desired binding specificity. An alternative approach involves fusing the variable domains with the desired binding specificities to heavy chain constant region comprising at least part of the hinge region, CH2 and CH3 regions. It is preferred to have the CH1 region containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding these fusions, and if desired the L chain are inserted into separate expression vectors and are then cotransfected into a suitable host organism. It is possible though to insert the coding sequences for two or all three chains into one expression vector. In one approach, a bispecific antibody is composed of a H chain with a first binding specificity in one arm and a H-L chain pair, providing a second binding specificity in the other arm, see WO94/04690. Also see Suresh et al Methods in Enzymology 121, 210, 1986. Other approaches include antibody molecules which comprise single domain binding sites which is set out in WO2007/095338.

SUMMARY OF INVENTION

The present invention relates to an antigen-binding construct comprising a protein scaffold which is linked to one or more epitope-binding domains wherein the antigen-binding construct has at least two antigen binding sites at least one of which is from an epitope binding domain and at least one of which is from a paired VH/VL domain.

The invention further relates to antigen-binding constructs comprising at least one homodimer comprising two or more structures of formula I:

wherein

X represents a constant antibody region comprising constant heavy domain 2 and constant heavy domain 3;

R¹, R⁴, R⁷and R⁸represent a domain independently selected from an epitope-binding domain;

R²represents a domain selected from the group consisting of constant heavy chain 1, and an epitope-binding domain;

R³represents a domain selected from the group consisting of a paired VH and an epitope-binding domain;

R⁵represents a domain selected from the group consisting of constant light chain, and an epitope-binding domain;

R⁶represents a domain selected from the group consisting of a paired VL and an epitope-binding domain;

n represents an integer independently selected from: 0, 1, 2, 3 and 4;

m represents an integer independently selected from: 0 and 1,

wherein the Constant Heavy chain 1 and the Constant Light chain domains are associated;

wherein at least one epitope binding domain is present;

and when R³represents a paired VH domain, R⁶represents a paired VL domain, so that the two domains are together capable of binding antigen.

The invention relates to IgG based structures which comprise monoclonal antibodies, or fragments linked to one or more domain antibodies, and to methods of making such constructs and uses thereof, particularly uses in therapy.

The invention also provides a polynucleotide sequence encoding a heavy chain of any of the antigen binding constructs described herein, and a polynucleotide encoding a light chain of any of the antigen binding constructs described herein. Such polynucleotides represent the coding sequence which corresponds to the equivalent polypeptide sequences, however it will be understood that such polynucleotide sequences could be cloned into an expression vector along with a start codon, an appropriate signal sequence and a stop codon.

The invention also provides a recombinant transformed or transfected host cell comprising one or more polynucleotides encoding a heavy chain and a light chain of any of the antigen binding constructs described herein.

The invention further provides a method for the production of any of the antigen binding constructs described herein which method comprises the step of culturing a host cell comprising a first and second vector, said first vector comprising a polynucleotide encoding a heavy chain of any of the antigen binding constructs described herein and said second vector comprising a polynucleotide encoding a light chain of any of the antigen binding constructs described herein, in a serum-free culture media.

The invention further provides a pharmaceutical composition comprising an antigen binding construct as described herein a pharmaceutically acceptable carrier.

The invention also provides a domain antibody comprising or consisting of the polypeptide sequence set out in SEQ ID NO: 2 or SEQ ID NO: 3. In one aspect the invention provides a protein which is expressed from the polynucleotide sequence set out in SEQ ID NO: 60 or SEQ ID NO: 61.

DEFINITIONS

The term ‘Protein Scaffold’ as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions. Such protein scaffolds may comprise antigen-binding sites in addition to the one or more constant regions, for example where the protein scaffold comprises a full IgG. Such protein scaffolds will be capable of being linked to other protein domains, for example protein domains which have antigen-binding sites, for example epitope-binding domains or ScFv domains.

A “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. A “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.

The phrase “immunoglobulin single variable domain” refers to an antibody variable domain (V_H, V_HH, V_L) that specifically binds an antigen or epitope independently of a different V region or domain. An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other, different variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., where the immunoglobulin single variable domain binds antigen independently of the additional variable domains). A “domain antibody” or “dAb” is the same as an “immunoglobulin single variable domain” which is capable of binding to an antigen as the term is used herein. An immunoglobulin single variable domain may be a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V_HHdAbs. Camelid V_HHare immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such V_HHdomains may be humanised according to standard techniques available in the art, and such domains are still considered to be “domain antibodies” according to the invention. As used herein “V_Hincludes camelid V_HHdomains. NARV are another type of immunoglobulin single variable domain which were identified in cartilaginous fish including the nurse shark. These domains are also known as Novel Antigen Receptor variable region (commonly abbreviated to V(NAR) or NARV). For further details see Mol. Immunol. 44, 656-665 (2006) and US20050043519A.

The term “Epitope-binding domain” refers to a domain that specifically binds an antigen or epitope independently of a different V region or domain, this may be a domain antibody (dAb), for example a human, camelid or shark immunoglobulin single variable domain or it may be a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human γ-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a ligand other than the natural ligand.

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptor expressed on mainly CD4+T-cells. Its extracellular domain has a variable domain-like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies. For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001)

Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid 13-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 and US20070224633

An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomisation of surface residues. For further details see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP1641818A1

Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007)

A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem. 274, 24066-24073 (1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two α-helices and a β-turn. They can be engineered to bind different target antigens by randomising residues in the first α-helix and a β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges—examples of microproteins include KalataB1 and conotoxin and knottins. The microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see WO2008098796.

Other epitope binding domains include proteins which have been used as a scaffold to engineer different target antigen binding properties include human γ-crystallin and human ubiquitin (affilins), kunitz type domains of human protease inhibitors, PDZ-domains of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Chapter 7—Non-Antibody Scaffolds from Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). Epitope binding domains of the present invention could be derived from any of these alternative protein domains.

As used herein, the terms “paired VH domain”, “paired VL domain”, and “paired VH/VL domains” refer to antibody variable domains which specifically bind antigen only when paired with their partner variable domain. There is always one VH and one VL in any pairing, and the term “paired VH domain” refers to the VH partner, the term “paired VL domain” refers to the VL partner, and the term “paired VH/VL domains” refers to the two domains together.

In one embodiment of the invention the antigen binding site binds to antigen with a Kd of at least 1 mM, for example a Kd of 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, to each antigen as measured by Biacore™, such as the Biacore™ method as described in method 4 or 5.

As used herein, the term “antigen binding site” refers to a site on a construct which is capable of specifically binding to antigen, this may be a single domain, for example an epitope-binding domain, or it may be paired VH/VL domains as can be found on a standard antibody. In some aspects of the invention single-chain Fv (ScFv) domains can provide antigen-binding sites.

The terms “mAb/dAb” and dAb/mAb” are used herein to refer to antigen-binding constructs of the present invention. The two terms can be used interchangeably, and are intended to have the same meaning as used herein.

The term “constant heavy chain 1” is used herein to refer to the CH1 domain of an immunoglobulin heavy chain.

The term “constant light chain” is used herein to refer to the constant domain of an immunoglobulin light chain.

DETAILED DESCRIPTION OF INVENTION

Such antigen-binding constructs comprise a protein scaffold, for example an Ig scaffold such as IgG, for example a monoclonal antibody, which is linked to one or more epitope-binding domains, for example a domain antibody, wherein the binding construct has at least two antigen binding sites, at least one of which is from an epitope binding domain, and to methods of producing and uses thereof, particularly uses in therapy.

Some examples of antigen-binding constructs according to the invention are set out in FIG. 1.

The antigen-binding constructs of the present invention are also referred to as mAbdAbs.

In one embodiment the protein scaffold of the antigen-binding construct of the present invention is an Ig scaffold, for example an IgG scaffold or IgA scaffold. The IgG scaffold may comprise all the domains of an antibody (i.e. CH1, CH2, CH3, VH, VL). The antigen-binding construct of the present invention may comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4 or IgG4PE.

The antigen-binding construct of the present invention has at least two antigen binding sites, for examples it has two binding sites, for example where the first binding site has specificity for a first epitope on an antigen and the second binding site has specificity for a second epitope on the same antigen. In a further embodiment there are 4 antigen binding sites, or 6 antigen binding sites, or 8 antigen binding sites, or 10 or more antigen-binding sites. In one embodiment the antigen binding construct has specificity for more than one antigen, for example two antigens, or for three antigens, or for four antigens.

In another aspect the invention relates to an antigen-binding construct comprising at least one homodimer comprising two or more structures of formula I:

- wherein
- X represents a constant antibody region comprising constant heavy domain 2 and constant heavy domain 3;
- R¹, R⁴, R⁷and R⁸represent a domain independently selected from an epitope-binding domain;
- R²represents a domain selected from the group consisting of constant heavy chain 1, and an epitope-binding domain;
- R³represents a domain selected from the group consisting of a paired VH and an epitope-binding domain;
- R⁵represents a domain selected from the group consisting of constant light chain, and an epitope-binding domain;
- R⁶represents a domain selected from the group consisting of a paired VL and an epitope-binding domain;
- n represents an integer independently selected from: 0, 1, 2, 3 and 4;
- m represents an integer independently selected from: 0 and 1,
- wherein the Constant Heavy chain 1 and the Constant Light chain domains are associated;
- wherein at least one epitope binding domain is present;
- and when R³represents a paired VH domain, R⁶represents a paired VL domain, so that the two domains are together capable of binding antigen.
- In one embodiment R⁶represents a paired VL and R³represents a paired VH.
- In a further embodiment either one or both of R⁷and R⁸represent an epitope binding domain.
- In yet a further embodiment either one or both of R¹and R⁴represent an epitope binding domain.
- In one embodiment R⁴is present.
- In one embodiment R¹, R⁷and R⁸represent an epitope binding domain.
- In one embodiment R¹R⁷and R⁸, and R⁴represent an epitope binding domain.
- In one embodiment (R¹)_n, (R²)_m, (R⁴)_mand (R⁵)_m=0, i.e. are not present, R³is a paired VH domain, R⁶is a paired VL domain, R⁸is a VH dAb, and R⁷is a VL dAb.
- In another embodiment (R¹)_n, (R²)_m, (R⁴)_mand (R⁵)_mare 0, i.e. are not present, R³is a paired VH domain, R⁶is a paired VL domain, R⁸is a VH dAb, and (R⁷)_m=0 i.e. not present.
- In another embodiment (R²)_m, and (R⁵)_mare 0, i.e. are not present, R¹is a dAb, R⁴is a dAb, R³is a paired VH domain, R⁶is a paired VL domain, (R⁸)_mand (R⁷)_m=0 i.e. not present.

In one embodiment of the present invention the epitope binding domain is a dAb.

It will be understood that any of the antigen-binding constructs described herein will be capable of neutralising one or more antigens.

The term “neutralises” and grammatical variations thereof as used throughout the present specification in relation to antigen binding constructs of the invention means that a biological activity of the target is reduced, either totally or partially, in the presence of the antigen binding constructs of the present invention in comparison to the activity of the target in the absence of such antigen binding constructs. Neutralisation may be due to but not limited to one or more of blocking ligand binding, preventing the ligand activating the receptor, down regulating the receptor or affecting effector functionality.

Levels of neutralisation can be measured in several ways, for example by use of any of the assays as set out in the examples and methods below, for example in an assay which measures inhibition of ligand binding to receptor which may be carried out for example as described in any one of Methods 12, 19 or 21 or Example 32. The neutralisation of VEGF, IL-4, IL-13 or HGF in these assays is measured by assessing the decreased binding between the ligand and its receptor in the presence of neutralising antigen binding construct.

Levels of neutralisation can also be measured, for example in a TF1 assay which may be carried out for example as described in Method 8, 9, 10, 20 or 21. The neutralisation of IL-13, IL-4 or both of these cytokines in this assay is measured by assessing the inhibition of TF1 cell proliferation in the presence of neutralising antigen binding construct. Alternatively neutralisation could be measured in an EGFR phosphorylation assay which may be carried out for example as described in Method 13. The neutralisation of EGFR in this assay is measured by assessing the inhibition of tyrosine kinase phosphorylation of the receptor in the presence of neutralising antigen binding construct. Or, neutralisation could be measured in an IL-8 secretion assay in MRC-5 cells which may be carried out for example as described in Method 14 or 15. The neutralisation of TNFα or IL-1R1 in this assay is measured by assessing the inhibition of IL-8 secretion in the presence of neutralising antigen binding construct.

Other methods of assessing neutralisation, for example, by assessing the decreased binding between the ligand and its receptor in the presence of neutralising antigen binding construct are known in the art, and include, for example, Biacore™ assays.

In an alternative aspect of the present invention there is provided antigen binding constructs which have at least substantially equivalent neutralising activity to the antibodies exemplified herein, for example antigen binding constructs which retain the neutralising activity of 586H-TVAAPS-210, or PascoH-G4S-474, or PascoH-474, PascoH-474 GS removed, PascoL-G4S-474 or PascoHL-G4S-474 in the TF1 cell proliferation assay, or inhibition of pSTAT6 signalling assay as set out in Examples 4 and 20 respectively, or for example antigen binding constructs which retain the neutralising activity of BPC1603, BPC1604, BPC1605, BPC1606 in the VEGFR binding assay or inhibition of IGF-1R receptor phosphorylation as set out in Examples 14.6 and 14.7.

The antigen binding constructs of the invention include those which have specificity for IL-13, for example which comprise an epitope-binding domain which is capable of binding to IL-13, or which comprise a paired VH/VL which binds to IL-13. The antigen binding construct may comprise an antibody which is capable of binding to IL-13. The antigen binding construct may comprise a dAb which is capable of binding to IL-13.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens selected from IL-13, IL-5, and IL-4, for example where it is capable of binding IL-13 and IL-4, or where it is capable of binding IL-13 and IL-5, or where it is capable of binding IL-5 and IL-4.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens selected from IL-13, IL-5, and IL-4, for example where it is capable of binding IL-13 and IL-4 simultaneously, or where it is capable of binding IL-13 and IL-5 simultaneously, or where it is capable of binding IL-5 and IL-4 simultaneously.

It will be understood that any of the antigen-binding constructs described herein may be capable of binding two or more antigens simultaneously, for example, as determined by stochiometry analysis by using a suitable assay such as that described in the Examples section, method 7.

Examples of antigen-binding constructs of the invention include IL-13 antibodies which have an epitope binding domain with a specificity for IL-4, for example an anti-IL-4 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO:16 to 39, SEQ ID NO:41 to 43, SEQ ID NO:87 to 90, SEQ ID NO:151, SEQ ID NO:152 or SEQ ID NO:155. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include IL-4 antibodies which have an epitope binding domain with a specificity for IL-13, for example an anti-IL-13 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO:48 to 53, SEQ ID NO:91 SEQ ID NO:92, SEQ ID NO:149, SEQ ID NO:150, or SEQ ID NO:157 to 160, and/or the light chain sequence set out in SEQ ID NO:54 to 59.

Antigen binding constructs of the present invention include IL-4 antibodies with an IL-13 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-13 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-13 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-13 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include IL-13 antibodies which have an epitope binding domain with a specificity for IL-5, for example an anti-IL-5 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-5 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-5 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-5 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-13 antibodies with an IL-5 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include IL-5 antibodies which have an epitope binding domain with a specificity for IL-13, for example an anti-IL-13 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the light chain sequence set out in SEQ ID NO: 72.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-13 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-13 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-13 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-13 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include IL-4 antibodies which have an epitope binding domain with a specificity for IL-5, for example an anti-IL-5 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-5 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-5 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-5 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-4 antibodies with an IL-5 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include IL-5 antibodies which have an epitope binding domain with a specificity for IL-4, for example an anti-IL-4 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 71.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

The invention also provides a trispecific binding construct which is capable of binding to IL-4, IL-13 and IL-5.

Examples of such antigen-binding constructs include IL-5 antibodies which have an epitope binding domain with a specificity for IL-4, for example an anti-IL-4 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain and an epitope binding domain with a specificity for IL-13, for example an anti-IL-13 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the heavy chain and an IL-13 epitope binding domain attached to the n-terminus of the light chain.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the light chain and an IL-13 epitope binding domain attached to the c-terminus of the light chain.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the heavy chain and an IL-13 epitope binding domain attached to the c-terminus of the light chain.

Antigen binding constructs of the present invention include IL-5 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the light chain and an IL-13 epitope binding domain attached to the c-terminus of the heavy chain.

Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

The antigen binding constructs of the invention include those which have specificity for IL-18, for example which comprises an epitope-binding domain which is capable of binding to IL-18, or which comprises a paired VH/VL which binds to IL-18.

The antigen binding construct may comprise an antibody which is capable of binding to IL-18. The antigen binding construct may comprise a dAb which is capable of binding to IL-18.

The invention also provides a trispecific binding construct which is capable of binding to IL-4, IL-13 and IL-18.

Examples of such antigen-binding constructs include IL-18 antibodies which have an epitope binding domain with a specificity for IL-4, for example an anti-IL-4 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain and an epitope binding domain with a specificity for IL-13, for example an anti-IL-13 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain.

Antigen binding constructs of the present invention include IL-18 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the heavy chain and an IL-13 epitope binding domain attached to the n-terminus of the light chain.

Antigen binding constructs of the present invention include IL-18 antibodies with an IL-4 epitope binding domain attached to the n-terminus of the light chain and an IL-13 epitope binding domain attached to the c-terminus of the light chain.

Antigen binding constructs of the present invention include IL-18 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the heavy chain and an IL-13 epitope binding domain attached to the c-terminus of the light chain.

Antigen binding constructs of the present invention include IL-18 antibodies with an IL-4 epitope binding domain attached to the c-terminus of the light chain and an IL-13 epitope binding domain attached to the c-terminus of the heavy chain.

The antigen binding constructs of the invention include those which have specificity for TNFα, for example which comprises an epitope-binding domain which is capable of binding to TNFα, or which comprises a paired VH/VL which binds to TNFα. The antigen binding construct may comprise an antibody which is capable of binding to TNFα. The antigen binding construct may comprise a dAb which is capable of binding to TNFα.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens selected from TNFα, EGFR and VEGF, for example where it is capable of binding TNFα and EGFR, or where it is capable of binding TNFα and VEGF, or where it is capable of binding EGFR and VEGF. Examples of such antigen-binding constructs include TNFα antibodies which have an epitope binding domain with a specificity for EGFR, for example an anti-EGFR dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example a mAbdAb having the heavy chain sequence set out in SEQ ID NO: 74, and/or the light chain sequence set out in SEQ ID NO: 79.

Antigen binding constructs of the present invention include TNFα antibodies with an EGFR epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include TNFα antibodies with an EGFR epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include TNFα antibodies with an EGFR epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include TNFα antibodies with an EGFR epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Antigen binding constructs of the present invention include EGFR antibodies with an TNFα epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include EGFR antibodies with an TNFα epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include EGFR antibodies with an TNFα epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include EGFR antibodies with an TNFα epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Examples of such antigen-binding constructs include TNFα antibodies which have an epitope binding domain with a specificity for VEGF, for example an anti-VEGF dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example a mAbdAb having the heavy chain sequence set out in SEQ ID NO: 75, 78 or 185.

The antigen-binding construct of the present invention may have specificity for more than one antigen, for example where it is capable of binding TNFα, and one or both antigens selected from IL-4 and IL-13, for example where it is capable of binding TNFα and IL-4, or where it is capable of binding TNFα and IL-13, or where it is capable of binding TNFα and IL-13 and IL-4. Examples of such antigen-binding constructs include IL-13 antibodies which have an epitope binding domain with a specificity for TNFα, for example an anti-TNFα adnectin, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example a mAbdAb having the heavy chain sequence set out in SEQ ID NO: 134 or 135. Other examples of such antigen-binding constructs include IL-4 antibodies which have an epitope binding domain with a specificity for TNFα, for example an anti-TNFα adnectin, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example a mAbdAb having the heavy chain sequence set out in SEQ ID NO: 146 or 147.

Antigen binding constructs of the present invention include TNFα antibodies with an VEGF epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include TNFα antibodies with an VEGF epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include TNFα antibodies with an VEGF epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include TNFα antibodies with an VEGF epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Antigen binding constructs of the present invention include VEGF antibodies with an TNFα epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include VEGF antibodies with an TNFα epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include VEGF antibodies with an TNFα epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include VEGF antibodies with an TNFα epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

The antigen binding constructs of the invention include those which have specificity for CD-20, for example which comprises an epitope-binding domain which is capable of binding to CD-20, or which comprises a paired VH/VL which binds to CD-20.

The antigen binding construct may comprise an antibody which is capable of binding to CD-20, for example it may comprise an antibody having the heavy and light chain sequences of SEQ ID NO: 120 and 117. The antigen binding construct may comprise a dAb which is capable of binding to CD-20. Examples of mAbdAbs with specificity for CD-20 are those having the heavy chain sequence set out in SEQ ID NO: 116, 118 or those having the light chain sequence set out in SEQ ID NO: 119 or 121. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

The antigen binding constructs of the invention include those which have specificity for IL1R1, for example which comprise an epitope-binding domain which is capable of binding to IL1R1, or which comprises a paired VH/VL which binds to IL1R1.

The antigen binding construct may comprise an antibody which is capable of binding to IL1R1. The antigen binding construct may comprise a dAb which is capable of binding to IL1R1.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding IL1R1 and a second antigen, for example where it is capable of binding IL1R1 and VEGF. Examples of such antigen-binding constructs include IL1R1 antibodies which have an epitope binding domain with a specificity for VEGF, for example an anti-VEGF dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example a mAbdAb having the light chain sequence set out in SEQ ID NO: 77.

Antigen binding constructs of the present invention include IL1R1 antibodies with an VEGF epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include IL1R1 antibodies with an VEGF epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include IL1R1 antibodies with an VEGF epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include IL1R1 antibodies with an VEGF epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

Antigen binding constructs of the present invention include VEGF antibodies with an IL1R1 epitope binding domain attached to the n-terminus of the heavy chain. Antigen binding constructs of the present invention include VEGF antibodies with an IL1R1 epitope binding domain attached to the n-terminus of the light chain. Antigen binding constructs of the present invention include VEGF antibodies with an IL1R1 epitope binding domain attached to the c-terminus of the heavy chain. Antigen binding constructs of the present invention include VEGF antibodies with an IL1R1 epitope binding domain attached to the c-terminus of the light chain. Such antigen-binding constructs may also have one or more further epitope binding domains with the same or different antigen-specificity attached to the c-terminus and/or the n-terminus of the heavy chain and/or the c-terminus and/or n-terminus of the light chain.

The antigen binding constructs of the invention include those which have specificity for EGFR, for example which comprises an epitope-binding domain which is capable of binding to EGFR, or which comprises a paired VH/VL which binds to EGFR. The antigen binding construct may comprise an antibody which is capable of binding to EGFR. The antigen binding construct may comprise a dAb which is capable of binding to EGFR. Some examples of such antigen binding construct will be capable of binding to an epitope on EGFR comprising SEQ ID NO:103, for example an antigen binding construct comprising one or more of the CDRs set out in SEQ ID NO: 97 to SEQ ID NO: 102 and SEQ ID NO: 104 to SEQ ID NO: 107.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens selected from EGFR, IGF-1R, VEGFR2 and VEGF, for example where it is capable of binding EGFR and IGF-1R, or where it is capable of binding EGFR and VEGF, or where it is capable of binding VEGF and IGF-1R, or where it is capable of binding EGFR and VEGFR2, or where it is capable of binding IGF-1R and VEGFR2, or where it is capable of binding VEGF and VEGFR2, or where it is capable of binding EGFR, IGF-1R and VEGFR2, or where it is capable of binding VEGF, IGF-1R and VEGFR2, or where it is capable of binding EGFR, VEGF and VEGFR2, or where it is capable of binding EGFR, VEGF and IGF1R. Examples of such antigen-binding constructs include EGFR antibodies which have an epitope binding domain with a specificity for VEGFR2, for example an anti-VEGFR2 adnectin, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 136, 140 or 144 and/or the light chain sequence set out in SEQ ID NO: 138, 142 or 145.

Examples of such antigen-binding constructs include EGFR antibodies which have an epitope binding domain with a specificity for VEGF, for example an anti-VEGF dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 165, 174, 176, 178, 184 or 186 and/or the light chain sequence set out in SEQ ID NO: 188 or 190.

Examples of such antigen-binding constructs include VEGF antibodies which have an epitope binding domain with a specificity for EGFR, for example an anti-EGFR dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 180. Such mAbdAbs may also comprise the light chain sequence set out in SEQ ID NO: 182.

Examples of such antigen-binding constructs include IGF-1R antibodies which have an epitope binding domain with a specificity for VEGF, for example an anti-VEGF lipocalin, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 123 or 125. Such mAbdAbs may also comprise the light chain sequence set out in SEQ ID NO:113 Examples of such antigen-binding constructs include IGF-1R antibodies which have an epitope binding domain with a specificity for VEGFR2, for example an anti-VEGFR2 adnectin, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain, for example the mAbdAb having the heavy chain sequence set out in SEQ ID NO: 124 or 133. Such mAbdAbs may also comprise the light chain sequence set out in SEQ ID NO: 113.

The antigen binding constructs of the invention include those which have specificity for IL-23, for example which comprises an epitope-binding domain which is capable of binding to IL-23, or which comprises a paired VH/VL which binds to IL-23. The antigen binding construct may comprise an antibody which is capable of binding to IL-23. The antigen binding construct may comprise a dAb which is capable of binding to IL-23.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens selected from TH17 type cytokines, for example. IL-17, IL-22, or IL-21, for example where it is capable of binding IL-23 and IL-17, or where it is capable of binding IL-23 and IL-21, or where it is capable of binding IL-23 and IL-22. Examples of such antigen-binding constructs include IL-23 antibodies which have an epitope binding domain with a specificity for IL-17, for example an anti-IL-17 dAb, attached to the c-terminus or the n-terminus of the heavy chain or the c-terminus or n-terminus of the light chain.

The antigen binding constructs of the invention include those which have specificity for PDGFRα, for example which comprises an epitope-binding domain which is capable of binding to PDGFRα, or which comprises a paired VH/VL which binds to PDGFRα. The antigen binding construct may comprise an antibody which is capable of binding to PDGFRα. The antigen binding construct may comprise a dAb which is capable of binding to PDGFRα.

The antigen binding constructs of the invention include those which have specificity for FGFR1, for example which comprises an epitope-binding domain which is capable of binding to FGFR1, or which comprises a paired VH/VL which binds to FGFR1. The antigen binding construct may comprise an antibody which is capable of binding to FGFR1. The antigen binding construct may comprise a dAb which is capable of binding to FGFR1.

The antigen binding constructs of the invention include those which have specificity for FGFR3, for example which comprises an epitope-binding domain which is capable of binding to FGFR3, or which comprises a paired VH/VL which binds to FGFR3. The antigen binding construct may comprise an antibody which is capable of binding to FGFR3. The antigen binding construct may comprise a dAb which is capable of binding to FGFR3.

The antigen binding constructs of the invention include those which have specificity for VEGFR2, for example which comprises an epitope-binding domain which is capable of binding to VEGFR2, or which comprises a paired VH/VL which binds to VEGFR2.

The antigen binding construct may comprise an antibody which is capable of binding to VEGFR2. The antigen binding construct may comprise a dAb which is capable of binding to VEGFR2.

The antigen binding constructs of the invention include those which have specificity for VEGFR3, for example which comprises an epitope-binding domain which is capable of binding to VEGFR3, or which comprises a paired VH/VL which binds to VEGFR3.

The antigen binding construct may comprise an antibody which is capable of binding to VEGFR3. The antigen binding construct may comprise a dAb which is capable of binding to VEGFR3.

The antigen binding constructs of the invention include those which have specificity for VE cadherin, for example which comprises an epitope-binding domain which is capable of binding to VE cadherin, or which comprises a paired VH/VL which binds to VE cadherin.

The antigen binding construct may comprise an antibody which is capable of binding to VE cadherin. The antigen binding construct may comprise a dAb which is capable of binding to VE cadherin.

The antigen binding constructs of the invention include those which have specificity for neuropilin, for example which comprises an epitope-binding domain which is capable of binding to neuropilin, or which comprises a paired VH/VL which binds to neuropilin.

The antigen binding construct may comprise an antibody which is capable of binding to neuropilin. The antigen binding construct may comprise a dAb which is capable of binding to neuropilin.

The antigen binding constructs of the invention include those which have specificity for Flt-3, for example which comprises an epitope-binding domain which is capable of binding to Flt-3, or which comprises a paired VH/VL which binds to Flt-3.

The antigen binding construct may comprise an antibody which is capable of binding to Flt-3. The antigen binding construct may comprise a dAb which is capable of binding to Flt-3.

The antigen binding constructs of the invention include those which have specificity for ron, for example which comprises an epitope-binding domain which is capable of binding ron, or which comprises a paired VH/VL which binds to ron.

The antigen binding construct may comprise an antibody which is capable of binding to ron. The antigen binding construct may comprise a dAb which is capable of binding to ron.

The antigen binding constructs of the invention include those which have specificity for Trp-1, for example which comprises an epitope-binding domain which is capable of binding Trp-1, or which comprises a paired VH/VL which binds to Trp-1. The antigen binding construct may comprise an antibody which is capable of binding to Trp-1. The antigen binding construct may comprise a dAb which is capable of binding to Trp-1.

In one embodiment the antigen-binding construct of the present invention has specificity for more than one antigen, for example where it is capable of binding two or more antigens which are implicated in cancer, for example where it is capable of binding two or more antigens selected from PDGFRα, FGFR1, FGFR3, VEGFR2, VEGFR3, IGF1R, EGFR and VEGF, VE cadherin, neuropilin, Flt-3, ron, Trp-1, CD-20 for example where it is capable of binding PDGFRα and FGFR1, or where it is capable of binding PDGFRα and VEGF, or where it is capable of binding PDGFRα and FGFR3, or where it is capable of binding PDGFRα and VEGFR2, or where it is capable of binding PDGFRα and VEGFR3, or where it is capable of binding PDGFRα and IGF1R, or where it is capable of binding PDGFRα and EGFR, or where it is capable of binding PDGFRα and VEGF, or where it is capable of binding PDGFRα and VE cadherin, or where it is capable of binding PDGFRα and neuropilin, or where it is capable of binding PDGFRα and Flt-3, or where it is capable of binding PDGFRα and ron, or where it is capable of binding PDGFRα and Trp1, or where it is capable of binding PDGFRα and CD-20, or where it is capable of binding FGFR1 and FGFR3, or where it is capable of binding FGFR1 and VEGFR2, or where it is capable of binding FGFR1 and VEGR3, or where it is capable of binding FGFR1 and IGF1R, or where it is capable of binding FGFR1 and EGFR, or where it is capable of binding FGFR1 and VEGF, or where it is capable of binding FGFR1 and VE cadherin, or where it is capable of binding FGFR1 and neuropilin, or where it is capable of binding FGFR1 and Flt-3, or where it is capable of binding FGFR1 and ron, or where it is capable of binding FGFR1 and Trp-1, or where it is capable of binding FGFR1 and CD-20, or where it is capable of binding FGFR3 and VEGFR2, or where it is capable of binding FGFR3 and VEGFR3, or where it is capable of binding FGFR3 and IGF1R, or where it is capable of binding FGFR3 and EGFR, or where it is capable of binding FGFR3 and VEGF, or where it is capable of binding FGFR3 and VE cadherin, or where it is capable of binding FGFR3 and neuropilin, or where it is capable of binding FGFR3 and Flt-3, or where it is capable of binding FGFR3 and ron, or where it is capable of binding FGFR3 and Trp-1, or where it is capable of binding FGFR3 and CD-20, or where it is capable of binding VEGFR2 and VEGFR3, or where it is capable of binding VEGFR2 and IGF1R, or where it is capable of binding VEGFR2 and EGFR, or where it is capable of binding VEGFR2 and VEGF, or where it is capable of binding VEGFR2 and VE cadherin, or where it is capable of binding VEGFR2 and neuropilin, or where it is capable of binding VEGFR2 and Flt-3, or where it is capable of binding VEGFR2 and ron, or where it is capable of binding VEGFR2 and Trp-1, or where it is capable of binding VEGFR2 and CD-20, or where it is capable of binding VEGFR3 and IGF-1R, or where it is capable of binding VEGFR3 and EGFR, or where it is capable of binding VEGFR3 and VEGF, or where it is capable of binding VEGFR3 and VE cadherin, or where it is capable of binding VEGFR3 and neuropilin, or where it is capable of binding VEGFR3 and Flt-3, or where it is capable of binding VEGFR3 and Trp-1, or where it is capable of binding VEGFR3 and CD-20, or where it is capable of binding IGF1R and EGFR, or where it is capable of binding IGF1R and VEGF, or where it is capable of binding IGF1R and VE cadherin, or where it is capable of binding IGF1R and neuropilin, or where it is capable of binding IGF1R and Flt-3, or where it is capable of binding IGF1R and ron, or where it is capable of binding IGF1R and Trp-1, or where it is capable of binding IGF1R and CD-20, or where it is capable of binding EGFR and VEGF, or where it is capable of binding EGFR and VE cadherin, or where it is capable of binding EGFR and neuropilin, or where it is capable of binding EGFR and Flt-3, or where it is capable of binding EGFR and ron, or where it is capable of binding EGFR and Trp-1, or where it is capable of binding EGFR and CD-20, or where it is capable of binding VEGF and VE cadherin, or where it is capable of binding VEGF and neuropilin, or where it is capable of binding VEGF and Flt-3, or where it is capable of binding VEGF and ron, or where it is capable of binding VEGF and Trp-1, or where it is capable of binding VEGF and CD-20, or where it is capable of binding VE cadherin and neuropilin, or where it is capable of binding VE cadherin and Flt-3, or where it is capable of binding VE cadherin and ron, or where it is capable of binding VE cadherin and Trp-1, or where it is capable of binding VE cadherin and CD-20, or where it is capable of binding neuropilin and Flt-3, or where it is capable of binding neuropilin and ron, or where it is capable of binding neuropilin and Trp-1, or where it is capable of binding neuropilin and CD-20, or where it is capable of binding Flt-3 and ron, or where it is capable of binding Flt-3 and Trp-1, or where it is capable of binding Flt-3 and CD-20, or where it is capable of binding ron and Trp-1, or where it is capable of binding ron and CD-20, and or where it is capable of binding Trp-1 and CD-20.

The antigen binding constructs of the invention include those which have specificity for beta-amyloid, for example which comprise an epitope-binding domain which is capable of binding to beta-amyloid, or which comprises a paired VH/VL which binds to beta-amyloid.

The antigen binding construct may comprise an antibody which is capable of binding to beta-amyloid. The antigen binding construct may comprise a dAb which is capable of binding to beta-amyloid.

The antigen binding constructs of the invention include those which have specificity for CD-3, for example which comprise an epitope-binding domain which is capable of binding to CD-3, or which comprises a paired VH/VL which binds to CD-3.

The antigen binding construct may comprise an antibody which is capable of binding to CD-3. The antigen binding construct may comprise a dAb which is capable of binding to CD-3.

The antigen binding constructs of the invention include those which have specificity for gpIIIb/IIa, for example which comprise an epitope-binding domain which is capable of binding to gpIIIb/IIa, or which comprises a paired VH/VL which binds to gpIIIb/IIa.

The antigen binding construct may comprise an antibody which is capable of binding to gpIIIb/IIa. The antigen binding construct may comprise a dAb which is capable of binding to gpIIIb/IIa.

The antigen binding constructs of the invention include those which have specificity for TGFbeta, for example which comprise an epitope-binding domain which is capable of binding to TGFbeta, or which comprises a paired VH/VL which binds to TGFbeta.

The antigen binding construct may comprise an antibody which is capable of binding to TGFbeta. The antigen binding construct may comprise a dAb which is capable of binding to TGFbeta.

In one embodiment of the present invention there is provided an antigen binding construct according to the invention described herein and comprising a constant region such that the antibody has reduced ADCC and/or complement activation or effector functionality. In one such embodiment the heavy chain constant region may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. One example comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering).

In one embodiment the antigen-binding constructs of the present invention will retain Fc functionality for example will be capable of one or both of ADCC and CDC activity. Such antigen-binding constructs may comprise an epitope-binding domain located on the light chain, for example on the c-terminus of the light chain.

The invention also provides a method of maintaining ADCC and CDC function of antigen-binding constructs by positioning of the epitope binding domain on the light chain of the antibody in particular, by positioning the epitope binding domain on the c-terminus of the light chain. Such ADCC and CDC function can be measured by any suitable assay, for example the ADCC assay set out in Example 15.3 and the CDC assay set out in Example 15.4.

The invention also provides a method of reducing CDC function of antigen-binding constructs by positioning of the epitope binding domain on the heavy chain of the antibody, in particular, by positioning the epitope binding domain on the c-terminus of the heavy chain. Such CDC function can be measured by any suitable assay, for example the CDC assay set out in Example 15.4.

In a further embodiment the antigen-binding construct of the present invention is capable of binding two or more antigens selected from VEGF, IGF-1R and EGFR, for example it is capable of binding EGFR and VEGF, or EGFR and IGF1R, or IGF1R and VEGF, or for example it is capable of binding to TNF and IL1-R. In embodiments of the invention which comprise an IGF-1R binding site, the IGF-1R binding site of the antigen-binding construct of the invention may comprise a paired VH/VL domain in the protein scaffold, which paired VH/VL domain may comprise one or more of the CDRs selected from those set out in SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, for example it may comprise at least CDRH3 as set out in SEQ ID NO:80, for example it may comprise all the CDRs set out in SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 85, and SEQ ID NO: 86.

In embodiments of the invention which comprise an EGFR binding site, the antigen-binding construct of the present invention may bind to an epitope comprising residues 273-501 of the mature or normal or wild type EGFR sequence, for example it may bind to an epitope comprising residues 287-302 of the mature or normal or wildtype EGFR (SEQ ID NO:103).

In one embodiment, the EGFR binding site of the antigen-binding construct of the invention may comprise a paired VH/VL domain in the protein scaffold, which paired VH/VL domain may comprise one or more of the CDRs selected from those set out in SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, for example, it may comprise CDRH3 as set out in SEQ ID NO: 106, or it may comprise all six CDRs set out in SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102. Such paired VH/VL domain may further comprise additional residues, particularly in the heavy chain CDRs, and in one embodiment, CDRH1 may comprise SEQ ID NO: 104 plus up to five additional residues, for example one or more of the five additional residues which are set out in SEQ ID NO: 97, CDRH2 may comprise SEQ ID NO: 105 plus up to two additional residues, for example one or both of the two additional residues which are set out in SEQ ID NO: 98 and SEQ ID NO: 107, and CDRH3 may comprise SEQ ID NO: 106 plus up to two additional residues, for example one or both of the two additional residues which are set out in SEQ ID NO: 99. In one such embodiment, the paired VH/VL comprises one or more of the CDRs set out in SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, for example it may comprise at least CDRH3 as set out in SEQ ID NO:99, for example it may comprise all six CDRs set out in SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102 (more detail of suitable antibodies can be found in WO02/092771 and WO2005/081854).

In one embodiment, the antigen binding constructs comprise an epitope-binding domain which is a domain antibody (dAb), for example the epitope binding domain may be a human VH or human VL, or a camelid V_HHor a shark dAb (NARV).

In one embodiment the antigen binding constructs comprise an epitope-binding domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a ligand other than the natural ligand.

The antigen binding constructs of the present invention may comprise a protein scaffold attached to an epitope binding domain which is an adnectin, for example an IgG scaffold with an adnectin attached to the c-terminus of the heavy chain, or it may comprise a protein scaffold attached to an adnectin, for example an IgG scaffold with an adnectin attached to the n-terminus of the heavy chain, or it may comprise a protein scaffold attached to an adnectin, for example an IgG scaffold with an adnectin attached to the c-terminus of the light chain, or it may comprise a protein scaffold attached to an adnectin, for example an IgG scaffold with an adnectin attached to the n-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is CTLA-4, for example an IgG scaffold with CTLA-4 attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with CTLA-4 attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with CTLA-4 attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with CTLA-4 attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a lipocalin, for example an IgG scaffold with a lipocalin attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a lipocalin attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a lipocalin attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a lipocalin attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is an SpA, for example an IgG scaffold with an SpA attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an SpA attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an SpA attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with an SpA attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is an affibody, for example an IgG scaffold with an affibody attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an affibody attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an affibody attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with an affibody attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is an affimer, for example an IgG scaffold with an affimer attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an affimer attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with an affimer attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with an affimer attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a GroEI, for example an IgG scaffold with a GroEI attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a GroEI attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a GroEI attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a GroEI attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a transferrin, for example an IgG scaffold with a transferrin attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a transferrin attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a transferrin attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a transferrin attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a GroES, for example an IgG scaffold with a GroES attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a GroES attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a GroES attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a GroES attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a DARPin, for example an IgG scaffold with a DARPin attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a DARPin attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a DARPin attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a DARPin attached to the c-terminus of the light chain.

In other embodiments it may comprise a protein scaffold, for example an IgG scaffold, attached to an epitope binding domain which is a peptide aptamer, for example an IgG scaffold with a peptide aptamer attached to the n-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a peptide aptamer attached to the c-terminus of the heavy chain, or it may comprise for example an IgG scaffold with a peptide aptamer attached to the n-terminus of the light chain, or it may comprise an IgG scaffold with a peptide aptamer attached to the c-terminus of the light chain.

In one embodiment of the present invention there are four epitope binding domains, for example four domain antibodies, two of the epitope binding domains may have specificity for the same antigen, or all of the epitope binding domains present in the antigen-binding construct may have specificity for the same antigen.

Protein scaffolds of the present invention may be linked to epitope-binding domains by the use of linkers. Examples of suitable linkers include amino acid sequences which may be from 1 amino acid to 150 amino acids in length, or from 1 amino acid to 140 amino acids, for example, from 1 amino acid to 130 amino acids, or from 1 to 120 amino acids, or from 1 to 80 amino acids, or from 1 to 50 amino acids, or from 1 to 20 amino acids, or from 1 to 10 amino acids, or from 5 to 18 amino acids. Such sequences may have their own tertiary structure, for example, a linker of the present invention may comprise a single variable domain. The size of a linker in one embodiment is equivalent to a single variable domain. Suitable linkers may be of a size from 1 to 20 angstroms, for example less than 15 angstroms, or less than 10 angstroms, or less than 5 angstroms.

In one embodiment of the present invention at least one of the epitope binding domains is directly attached to the Ig scaffold with a linker comprising from 1 to 150 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids. Such linkers may be selected from any one of those set out in SEQ ID NO: 6 to 11, ‘STG’ (serine, threonine, glycine), ‘GSTG’ or ‘RS’, for example the linker may be TVAAPS', or the linker may be ‘GGGGS’. Linkers of use in the antigen binding constructs of the present invention may comprise alone or in addition to other linkers, one or more sets of GS residues, for example ‘GSTVAAPS’ or TVAAPSGS' or ‘GSTVAAPSGS’. In another embodiment there is no linker between the epitope binding domain, for example the dAb, and the Ig scaffold. In another embodiment the epitope binding domain, for example a dAb, is linked to the Ig scaffold by the linker TVAAPS'. In another embodiment the epitope binding domain, for example a dAb, is linked to the Ig scaffold by the linker ‘TVAAPSGS’. In another embodiment the epitope binding domain, for example a dAb, is linked to the Ig scaffold by the linker ‘GS’.

In one embodiment, the antigen-binding construct of the present invention comprises at least one epitope binding domain which is capable of binding human serum albumin.

In one embodiment, there are at least 3 antigen binding sites, for example there are 4, or 5 or 6 or 8 or 10 antigen binding sites and the antigen binding construct is capable of binding at least 3 or 4 or 5 or 6 or 8 or 10 antigens, for example it is capable of binding 3 or 4 or 5 or 6 or 8 or 10 antigens simultaneously.

The invention also provides the antigen-binding constructs for use in medicine, for example for use in the manufacture of a medicament for treating cancer or inflammatory diseases such as asthma, rheumatoid arthritis, or osteoarthritis.

The invention provides a method of treating a patient suffering from cancer or inflammatory diseases such as asthma, rheumatoid arthritis, or osteoarthritis, comprising administering a therapeutic amount of an antigen-binding construct of the invention.

The antigen-binding constructs of the invention may be used for the treatment of cancer or inflammatory diseases such as asthma, rheumatoid arthritis, or osteoarthritis.

The antigen-binding constructs of the invention may have some effector function. For example if the protein scaffold contains an Fc region derived from an antibody with effector function, for example if the protein scaffold comprises CH2 and CH3 from IgG1. Levels of effector function can be varied according to known techniques, for example by mutations in the CH2 domain, for example wherein the IgG1 CH2 domain has one or more mutations at positions selected from 239 and 332 and 330, for example the mutations are selected from S239D and 1332E and A330L such that the antibody has enhanced effector function, and/or for example altering the glycosylation profile of the antigen-binding construct of the invention such that there is a reduction in fucosylation of the Fc region.

Protein scaffolds of use in the present invention include full monoclonal antibody scaffolds comprising all the domains of an antibody, or protein scaffolds of the present invention may comprise a non-conventional antibody structure, such as a monovalent antibody. Such monovalent antibodies may comprise a paired heavy and light chain wherein the hinge region of the heavy chain is modified so that the heavy chain does not homodimerise, such as the monovalent antibody described in WO2007059782. Other monovalent antibodies may comprise a paired heavy and light chain which dimerises with a second heavy chain which is lacking a functional variable region and CH1 region, wherein the first and second heavy chains are modified so that they will form heterodimers rather than homodimers, resulting in a monovalent antibody with two heavy chains and one light chain such as the monovalent antibody described in WO2006015371. Such monovalent antibodies can provide the protein scaffold of the present invention to which epitope binding domains can be linked, for example such as the antigen binding constructs describe in Example 32.

Epitope-binding domains of use in the present invention are domains that specifically bind an antigen or epitope independently of a different V region or domain, this may be a domain antibody or may be a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxinkunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a ligand other than the natural ligand. In one embodiment this may be an domain antibody or other suitable domains such as a domain selected from the group consisting of CTLA-4, lipocallin, SpA, an Affibody, an avimer, GroEI, transferrin, GroES and fibronectin. In one embodiment this may be selected from a dAb, an Affibody, an ankyrin repeat protein (DARPin) and an adnectin. In another embodiment this may be selected from an Affibody, an ankyrin repeat protein (DARPin) and an adnectin. In another embodiment this may be a domain antibody, for example a domain antibody selected from a human, camelid or shark (NARV) domain antibody.

Epitope-binding domains can be linked to the protein scaffold at one or more positions. These positions include the C-terminus and the N-terminus of the protein scaffold, for example at the C-terminus of the heavy chain and/or the C-terminus of the light chain of an IgG, or for example the N-terminus of the heavy chain and/or the N-terminus of the light chain of an IgG.

In one embodiment, a first epitope binding domain is linked to the protein scaffold and a second epitope binding domain is linked to the first epitope binding domain, for example where the protein scaffold is an IgG scaffold, a first epitope binding domain may be linked to the c-terminus of the heavy chain of the IgG scaffold, and that epitope binding domain can be linked at its c-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the c-terminus of the light chain of the IgG scaffold, and that first epitope binding domain may be further linked at its c-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the n-terminus of the light chain of the IgG scaffold, and that first epitope binding domain may be further linked at its n-terminus to a second epitope binding domain, or for example a first epitope binding domain may be linked to the n-terminus of the heavy chain of the IgG scaffold, and that first epitope binding domain may be further linked at its n-terminus to a second epitope binding domain. Examples of such antigen binding constructs are described in Example 31.

When the epitope-binding domain is a domain antibody, some domain antibodies may be suited to particular positions within the scaffold.

Domain antibodies of use in the present invention can be linked at the C-terminal end of the heavy chain and/or the light chain of conventional IgGs. In addition some dAbs can be linked to the C-terminal ends of both the heavy chain and the light chain of conventional antibodies.

In constructs where the N-terminus of dAbs are fused to an antibody constant domain (either C_H3 or CL), a peptide linker may help the dAb to bind to antigen. Indeed, the N-terminal end of a dAb is located closely to the complementarity-determining regions (CDRS) involved in antigen-binding activity. Thus a short peptide linker acts as a spacer between the epitope-binding, and the constant domain for the protein scaffold, which may allow the dAb CDRs to more easily reach the antigen, which may therefore bind with high affinity.

The surroundings in which dAbs are linked to the IgG will differ depending on which antibody chain they are fused to:

When fused at the C-terminal end of the antibody light chain of an IgG scaffold, each dAb is expected to be located in the vicinity of the antibody hinge and the Fc portion. It is likely that such dAbs will be located far apart from each other. In conventional antibodies, the angle between Fab fragments and the angle between each Fab fragment and the Fc portion can vary quite significantly. It is likely that—with mAbdAbs—the angle between the Fab fragments will not be widely different, whilst some angular restrictions may be observed with the angle between each Fab fragment and the Fc portion.

When fused at the C-terminal end of the antibody heavy chain of an IgG scaffold, each dAb is expected to be located in the vicinity of the C_H3 domains of the Fc portion. This is not expected to impact on the Fc binding properties to Fc receptors (e.g. FcγRI, II, III an FcRn) as these receptors engage with the C_H2 domains (for the FcγRI, II and III class of receptors) or with the hinge between the C_H2 and C_H3 domains (e.g. FcRn receptor). Another feature of such antigen-binding constructs is that both dAbs are expected to be spatially close to each other and provided that flexibility is provided by provision of appropriate linkers, these dAbs may even form homodimeric species, hence propagating the ‘zipped’ quaternary structure of the Fc portion, which may enhance stability of the construct.

Such structural considerations can aid in the choice of the most suitable position to link an epitope-binding domain, for example a dAb, on to a protein scaffold, for example an antibody.

The size of the antigen, its localization (in blood or on cell surface), its quaternary structure (monomeric or multimeric) can vary. Conventional antibodies are naturally designed to function as adaptor constructs due to the presence of the hinge region, wherein the orientation of the two antigen-binding sites at the tip of the Fab fragments can vary widely and hence adapt to the molecular feature of the antigen and its surroundings. In contrast dAbs linked to an antibody or other protein scaffold, for example a protein scaffold which comprises an antibody with no hinge region, may have less structural flexibility either directly or indirectly.

Understanding the solution state and mode of binding at the dAb is also helpful. Evidence has accumulated that in vitro dAbs can predominantly exist in monomeric, homo-dimeric or multimeric forms in solution (Reiter et al. (1999) J Mol Biol 290 p 685-698; Ewert et al (2003) J Mol Biol 325, p 531-553, Jespers et al (2004) J Mol Biol 337 p 893-903; Jespers et al (2004) Nat Biotechnol 22 p 1161-1165; Martin et al (1997) Protein Eng. 10 p 607-614; Sepulvada et al (2003) J Mol Biol 333 p 355-365). This is fairly reminiscent to multimerisation events observed in vivo with Ig domains such as Bence-Jones proteins (which are dimers of immunoglobulin light chains (Epp et al (1975) Biochemistry 14 p 4943-4952; Huan et al (1994) Biochemistry 33 p 14848-14857; Huang et al (1997) Mol immuno) 34 p 1291-1301) and amyloid fibers (James et al. (2007) J Mol. Biol. 367:603-8).

For example, it may be desirable to link domain antibodies that tend to dimerise in solution to the C-terminal end of the Fc portion in preference to the C-terminal end of the light chain as linking to the C-terminal end of the Fc will allow those dAbs to dimerise in the context of the antigen-binding construct of the invention.

The antigen-binding constructs of the present invention may comprise antigen-binding sites specific for a single antigen, or may have antigen-binding sites specific for two or more antigens, or for two or more epitopes on a single antigen, or there may be antigen-binding sites each of which is specific for a different epitope on the same or different antigens.

The antigen-binding sites can each have binding specificity for an antigen, such as human or animal proteins, including cytokines, growth factors, cytokine receptors, growth factor receptors, enzymes (e.g., proteases), co-factors for enzymes, DNA binding proteins, lipids and carbohydrates. Suitable targets, including cytokines, growth factors, cytokine receptors, growth factor receptors and other proteins include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA, CD40, CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, FAPα, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-131, human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-1 receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, c-fms, v-fmsMDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, IgE, and other targets disclosed herein. It will be appreciated that this list is by no means exhaustive.

In some embodiments, the protease resistant peptide or polypeptide binds a target in pulmonary tissue, such as a target selected from the group consisting of TNFR1, IL-1, IL-1R, IL-4, IL-4R, IL-5, IL-6, IL-6R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-12 IL-12R, IL-13, IL-13Rα1, IL-13Rα2, IL-15, IL-15R, IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4, CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138, ALK5, EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase, FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2, Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, I-309, integrins, L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase, osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF, amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

In particular, the antigen-binding constructs of the present invention may be useful in treating diseases associated with IL-13, IL-5 and IL-4, for example atopic dermatitis, allergic rhinitis, crohn's disease, COPD, fibrotic diseases or disorders such as idiopathic pulmonary fibrosis, progressive systemic sclerosis, hepatic fibrosis, hepatic granulomas, schistosomiasis, leishmaniasis, diseases of cell cycle regulation such as Hodgkins disease, B cell chronic lymphocytic leukaemia, for example the constructs may be useful in treating asthma.

Antigen-binding constructs of the present invention may be useful in treating diseases associated with growth factors such as IGF-1R, VEGF, and EGFR, for example cancer or rheumatoid arthritis, examples of types of cancer in which such therapies may be useful are breast cancer, prostrate cancer, lung cancer and myeloma.

Antigen-binding constructs of the present invention may be useful in treating diseases associated with TNF, for example arthritis, for example rheumatoid arthritis or osteoarthritis.

Antigen-binding constructs of the present invention may be useful in treating diseases associated with IL1-R, for example arthritis, for example rheumatoid arthritis or osteoarthritis.

Antigen-binding constructs of the present invention may be useful in treating diseases associated with CD-20, for example autoimmune diseases such as psoriasis, inflammatory bowel disease, ulcerative colitis, crohns disease, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, neurodegenerative diseases, for example multiple sclerosis, neutrophil driven diseases, for example COPD, Wegeners vasculitis, cystic fibrosis, Sjogrens syndrome, chronic transplant rejection, type 1 diabetes graft versus host disease, asthma, allergic diseases atoptic dermatitis, eczematous dermatitis, allergic rhinitis, autoimmune diseases other including thyroiditis, spondyloarthropathy, ankylosing spondylitis, uveitis, polychonritis or scleroderma, or cancer e.g. B-cell lymphomas or mature B cell neoplasm such as CLL or SLL.

Antigen-binding constructs of the present invention may be useful in treating diseases associated with IL-17 and IL-23, for example psoriasis, inflammatory bowel disease, ulcerative colitis, crohns disease, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, neurodegenerative diseases, for example multiple sclerosis, neutrophil driven diseases, for example COPD, Wegeners vasculitis, cystic fibrosis, Sjogrens syndrome, chronic transplant rejection, type 1 diabetes graft versus host disease, asthma, allergic diseases atoptic dermatitis, eczematous dermatitis, allergic rhinitis, autoimmune diseases other including thyroiditis, spondyloarthropathy, ankylosing spondylitis, uveitis, polychonritis or scleroderma.

The antigen binding constructs of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antigen binding construct of the invention. An expression vector or recombinant plasmid is produced by placing these coding sequences for the antigen binding construct in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies. Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antigen binding construct light or heavy chain. In certain embodiments this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the antigen binding construct may reside on a single vector, for example in two expression cassettes in the same vector.

A selected host cell is co-transfected by conventional techniques with both the first and second vectors (or simply transfected by a single vector) to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antigen binding construct of the invention. The antigen binding construct which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antigen binding constructs.

Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden). Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.

The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art.

The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.

- The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antigen binding constructs of the present invention. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. coli may be used for replication of the cloning vectors and other steps in the construction of antigen binding constructs of this invention. Suitable host cells or cell lines for the expression of the antigen binding constructs of the invention include mammalian cells such as NS0, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., cited above.

Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs or other embodiments of the present invention (see, e.g., Plückthun, A., Immunol. Rev., 130:151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule may express in the bacterial host and then be subsequently re-folded. For example, various strains of E. coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.

The general methods by which the vectors may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding construct of the invention from such host cell may all be conventional techniques. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension. Likewise, once produced, the antigen binding constructs of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention. For example, preparation of altered antibodies are described in WO 99/58679 and WO 96/16990.

Yet another method of expression of the antigen binding constructs may utilize expression in a transgenic animal, such as described in U.S. Pat. No. 4,873,316. This relates to an expression system using the animal's casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.

In a further aspect of the invention there is provided a method of producing an antibody of the invention which method comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and/or heavy chain of the antibody of the invention and recovering the antibody thereby produced.

In accordance with the present invention there is provided a method of producing an antigen binding construct of the present invention which method comprises the steps of;

- (a) providing a first vector encoding a heavy chain of the antigen binding construct;
- (b) providing a second vector encoding a light chain of the antigen binding construct;
- (c) transforming a mammalian host cell (e.g. CHO) with said first and second vectors;
- (d) culturing the host cell of step (c) under conditions conducive to the secretion of the antigen binding construct from said host cell into said culture media;
- (e) recovering the secreted antigen binding construct of step (d).

Once expressed by the desired method, the antigen binding construct is then examined for in vitro activity by use of an appropriate assay. Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the antigen binding construct to its target. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antigen binding construct in the body despite the usual clearance mechanisms.

The dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient. It is envisaged that repeated dosing (e.g. once a week or once every two weeks) over an extended time period (e.g. four to six months) maybe required to achieve maximal therapeutic efficacy.

The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host. The antigen binding constructs, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.), intravenously (i.v.), or intranasally.

Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding construct of the invention as an active ingredient in a pharmaceutically acceptable carrier. In the prophylactic agent of the invention, an aqueous suspension or solution containing the antigen binding construct, preferably buffered at physiological pH, in a form ready for injection is preferred. The compositions for parenteral administration will commonly comprise a solution of the antigen binding construct of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like. These solutions may be made sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antigen binding construct of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an antigen binding construct of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 and preferably 5 mg to about 25 mg of an antigen binding construct of the invention per ml of Ringer's solution. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. For the preparation of intravenously administrable antigen binding construct formulations of the invention see Lasmar U and Parkins D “The formulation of Biopharmaceutical products”, Pharma. Sci. Tech. today, page 129-137, Vol. 3 (3, Apr. 2000), Wang, W “Instability, stabilisation and formulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999) 129-188, Stability of Protein Pharmaceuticals Part A and B ed Ahern T. J., Manning M. C., New York, N.Y.: Plenum Press (1992), Akers, M. J. “Excipient-Drug interactions in Parenteral Formulations”, J. Pharm Sci 91 (2002) 2283-2300, Imamura, K et al “Effects of types of sugar on stabilization of Protein in the dried state”, J Pharm Sci 92 (2003) 266-274, Izutsu, Kkojima, S. “Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying”, J. Pharm. Pharmacol, 54 (2002) 1033-1039, Johnson, R, “Mannitol-sucrose mixtures-versatile formulations for protein lyophilization”, J. Pharm. Sci, 91 (2002) 914-922.

Ha, E Wang W, Wang Y. j. “Peroxide formation in polysorbate 80 and protein stability”, J. Pharm Sci, 91, 2252-2264,(2002) the entire contents of which are incorporated herein by reference and to which the reader is specifically referred.

It is preferred that the therapeutic agent of the invention, when in a pharmaceutical preparation, be present in unit dose forms. The appropriate therapeutically effective dose will be determined readily by those of skill in the art. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of 0.01 to 20 mg/kg, for example 0.1 to 20 mg/kg, for example 1 to 20 mg/kg, for example 10 to 20 mg/kg or for example 1 to 15 mg/kg, for example 10 to 15 mg/kg. To effectively treat conditions of use in the present invention in a human, suitable doses may be within the range of 0.01 to 1000 mg, for example 0.1 to 1000 mg, for example 0.1 to 500 mg, for example 500 mg, for example 0.1 to 100 mg, or 0.1 to 80 mg, or 0.1 to 60 mg, or 0.1 to 40 mg, or for example 1 to 100 mg, or 1 to 50 mg, of an antigen binding construct of this invention, which may be administered parenterally, for example subcutaneously, intravenously or intramuscularly. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.

The antigen binding constructs described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed.

There are several methods known in the art which can be used to find epitope-binding domains of use in the present invention.

The term “library” refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each of which has a single polypeptide or nucleic acid sequence. To this extent, “library” is synonymous with “repertoire.” Sequence differences between library members are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids. In one example, each individual organism or cell contains only one or a limited number of library members. Advantageously, the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids. In a one aspect, therefore, a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member. Thus, the population of host organisms has the potential to encode a large repertoire of diverse polypeptides.

A “universal framework” is a single antibody framework sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat (“Sequences of Proteins of Immunological Interest”, US Department of Health and Human Services) or corresponding to the human germline immunoglobulin repertoire or structure as defined by Chothia and Lesk, (1987) J. Mol. Biol. 196:910-917. There may be a single framework, or a set of such frameworks, which has been found to permit the derivation of virtually any binding specificity though variation in the hypervariable regions alone.

Amino acid and nucleotide sequence alignments and homology, similarity or identity, as defined herein are in one embodiment prepared and determined using the algorithm BLAST 2 Sequences, using default parameters (Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)).

The epitope binding domain(s) and antigen binding sites can each have binding specificity for a generic ligand or any desired target ligand, such as human or animal proteins, including cytokines, growth factors, cytokine receptors, growth factor receptors, enzymes (e.g., proteases), co-factors for enzymes, DNA binding proteins, lipids and carbohydrates. Suitable targets, including cytokines, growth factors, cytokine receptors, growth factor receptors and other proteins include but are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA, CD40, CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, FAPα, FGF-acidic, FGF-basic, fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-1 receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein, M-CSF, c-fms, v-fmsMDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, IgE, and other targets disclosed herein. It will be appreciated that this list is by no means exhaustive.

In some embodiments, binding is to a target in pulmonary tissue, such as a target selected from the group consisting of TNFR1, IL-1, IL-1R, IL-4, IL-4R, IL-5, IL-6, IL-6R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-12, IL-12R, IL-13, IL-13Rα1, IL-13Ra2, IL-15, IL-15R, IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4, CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138, ALK5, EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase, FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2, Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, I-309, integrins, L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase, osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF, amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

When a display system (e.g., a display system that links coding function of a nucleic acid and functional characteristics of the peptide or polypeptide encoded by the nucleic acid) is used in the methods described herein, eg in the selection of a dAb or other epitope binding domain, it is frequently advantageous to amplify or increase the copy number of the nucleic acids that encode the selected peptides or polypeptides. This provides an efficient way of obtaining sufficient quantities of nucleic acids and/or peptides or polypeptides for additional rounds of selection, using the methods described herein or other suitable methods, or for preparing additional repertoires (e.g., affinity maturation repertoires). Thus, in some embodiments, the methods of selecting epitope binding domains comprises using a display system (e.g., that links coding function of a nucleic acid and functional characteristics of the peptide or polypeptide encoded by the nucleic acid, such as phage display) and further comprises amplifying or increasing the copy number of a nucleic acid that encodes a selected peptide or polypeptide. Nucleic acids can be amplified using any suitable methods, such as by phage amplification, cell growth or polymerase chain reaction.

In one example, the methods employ a display system that links the coding function of a nucleic acid and physical, chemical and/or functional characteristics of the polypeptide encoded by the nucleic acid. Such a display system can comprise a plurality of replicable genetic packages, such as bacteriophage or cells (bacteria). The display system may comprise a library, such as a bacteriophage display library. Bacteriophage display is an example of a display system.

A number of suitable bacteriophage display systems (e.g., monovalent display and multivalent display systems) have been described. (See, e.g., Griffiths et al., U.S. Pat. No. 6,555,313 B1 (incorporated herein by reference); Johnson et al., U.S. Pat. No. 5,733,743 (incorporated herein by reference); McCafferty et al., U.S. Pat. No. 5,969,108 (incorporated herein by reference); Mulligan-Kehoe, U.S. Pat. No. 5,702,892 (Incorporated herein by reference); Winter, G. et al., Annu. Rev. Immunol. 12:433-455 (1994); Soumillion, P. et al., Appl. Biochem. Biotechnol. 47(2-3):175-189 (1994); Castagnoli, L. et al., Comb. Chem. High Throughput Screen, 4(2):121-133 (2001).) The peptides or polypeptides displayed in a bacteriophage display system can be displayed on any suitable bacteriophage, such as a filamentous phage (e.g., fd, M13, F1), a lytic phage (e.g., T4, T7, lambda), or an RNA phage (e.g., MS2), for example.

Generally, a library of phage that displays a repertoire of peptides or phagepolypeptides, as fusion proteins with a suitable phage coat protein (e.g., fd pIII protein), is produced or provided. The fusion protein can display the peptides or polypeptides at the tip of the phage coat protein, or if desired at an internal position. For example, the displayed peptide or polypeptide can be present at a position that is amino-terminal to domain 1 of pIII. (Domain 1 of pIII is also referred to as N1.) The displayed polypeptide can be directly fused to pIII (e.g., the N-terminus of domain 1 of pIII) or fused to pIII using a linker. If desired, the fusion can further comprise a tag (e.g., myc epitope, His tag). Libraries that comprise a repertoire of peptides or polypeptides that are displayed as fusion proteins with a phage coat protein, can be produced using any suitable methods, such as by introducing a library of phage vectors or phagemid vectors encoding the displayed peptides or polypeptides into suitable host bacteria, and culturing the resulting bacteria to produce phage (e.g., using a suitable helper phage or complementing plasmid if desired). The library of phage can be recovered from the culture using any suitable method, such as precipitation and centrifugation.

The display system can comprise a repertoire of peptides or polypeptides that contains any desired amount of diversity. For example, the repertoire can contain peptides or polypeptides that have amino acid sequences that correspond to naturally occurring polypeptides expressed by an organism, group of organisms, desired tissue or desired cell type, or can contain peptides or polypeptides that have random or randomized amino acid sequences. If desired, the polypeptides can share a common core or scaffold. For example, all polypeptides in the repertoire or library can be based on a scaffold selected from protein A, protein L, protein G, a fibronectin domain, an anticalin, CTLA4, a desired enzyme (e.g., a polymerase, a cellulase), or a polypeptide from the immunoglobulin superfamily, such as an antibody or antibody fragment (e.g., an antibody variable domain). The polypeptides in such a repertoire or library can comprise defined regions of random or randomized amino acid sequence and regions of common amino acid sequence. In certain embodiments, all or substantially all polypeptides in a repertoire are of a desired type, such as a desired enzyme (e.g., a polymerase) or a desired antigen-binding fragment of an antibody (e.g., human V_Hor human V_L). In some embodiments, the polypeptide display system comprises a repertoire of polypeptides wherein each polypeptide comprises an antibody variable domain. For example, each polypeptide in the repertoire can contain a V_H, a V_Lor an Fv (e.g., a single chain Fv). Amino acid sequence diversity can be introduced into any desired region of a peptide or polypeptide or scaffold using any suitable method. For example, amino acid sequence diversity can be introduced into a target region, such as a complementarity determining region of an antibody variable domain or a hydrophobic domain, by preparing a library of nucleic acids that encode the diversified polypeptides using any suitable mutagenesis methods (e.g., low fidelity PCR, oligonucleotide-mediated or site directed mutagenesis, diversification using NNK codons) or any other suitable method. If desired, a region of a polypeptide to be diversified can be randomized. The size of the polypeptides that make up the repertoire is largely a matter of choice and uniform polypeptide size is not required. The polypeptides in the repertoire may have at least tertiary structure (form at least one domain).

Selection/Isolation/Recovery

An epitope binding domain or population of domains can be selected, isolated and/or recovered from a repertoire or library (e.g., in a display system) using any suitable method. For example, a domain is selected or isolated based on a selectable characteristic (e.g., physical characteristic, chemical characteristic, functional characteristic). Suitable selectable functional characteristics include biological activities of the peptides or polypeptides in the repertoire, for example, binding to a generic ligand (e.g., a superantigen), binding to a target ligand (e.g., an antigen, an epitope, a substrate), binding to an antibody (e.g., through an epitope expressed on a peptide or polypeptide), and catalytic activity. (See, e.g., Tomlinson et al., WO 99/20749; WO 01/57065; WO 99/58655.)

In some embodiments, the protease resistant peptide or polypeptide is selected and/or isolated from a library or repertoire of peptides or polypeptides in which substantially all domains share a common selectable feature. For example, the domain can be selected from a library or repertoire in which substantially all domains bind a common generic ligand, bind a common target ligand, bind (or are bound by) a common antibody, or possess a common catalytic activity. This type of selection is particularly useful for preparing a repertoire of domains that are based on a parental peptide or polypeptide that has a desired biological activity, for example, when performing affinity maturation of an immunoglobulin single variable domain. Selection based on binding to a common generic ligand can yield a collection or population of domains that contain all or substantially all of the domains that were components of the original library or repertoire. For example, domains that bind a target ligand or a generic ligand, such as protein A, protein L or an antibody, can be selected, isolated and/or recovered by panning or using a suitable affinity matrix. Panning can be accomplished by adding a solution of ligand (e.g., generic ligand, target ligand) to a suitable vessel (e.g., tube, petri dish) and allowing the ligand to become deposited or coated onto the walls of the vessel. Excess ligand can be washed away and domains can be added to the vessel and the vessel maintained under conditions suitable for peptides or polypeptides to bind the immobilized ligand. Unbound domains can be washed away and bound domains can be recovered using any suitable method, such as scraping or lowering the pH, for example.

Suitable ligand affinity matrices generally contain a solid support or bead (e.g., agarose) to which a ligand is covalently or noncovalently attached. The affinity matrix can be combined with peptides or polypeptides (e.g., a repertoire that has been incubated with protease) using a batch process, a column process or any other suitable process under conditions suitable for binding of domains to the ligand on the matrix. domains that do not bind the affinity matrix can be washed away and bound domains can be eluted and recovered using any suitable method, such as elution with a lower pH buffer, with a mild denaturing agent (e.g., urea), or with a peptide or domain that competes for binding to the ligand. In one example, a biotinylated target ligand is combined with a repertoire under conditions suitable for domains in the repertoire to bind the target ligand. Bound domains are recovered using immobilized avidin or streptavidin (e.g., on a bead).

In some embodiments, the generic or target ligand is an antibody or antigen binding fragment thereof. Antibodies or antigen binding fragments that bind structural features of peptides or polypeptides that are substantially conserved in the peptides or polypeptides of a library or repertoire are particularly useful as generic ligands. Antibodies and antigen binding fragments suitable for use as ligands for isolating, selecting and/or recovering protease resistant peptides or polypeptides can be monoclonal or polyclonal and can be prepared using any suitable method.

Libraries/Repertoires

Libraries that encode and/or contain protease epitope binding domains can be prepared or obtained using any suitable method. A library can be designed to encode domains based on a domain or scaffold of interest (e.g., a domain selected from a library) or can be selected from another library using the methods described herein. For example, a library enriched in domains can be prepared using a suitable polypeptide display system.

Libraries that encode a repertoire of a desired type of domain can readily be produced using any suitable method. For example, a nucleic acid sequence that encodes a desired type of polypeptide (e.g., an immunoglobulin variable domain) can be obtained and a collection of nucleic acids that each contain one or more mutations can be prepared, for example by amplifying the nucleic acid using an error-prone polymerase chain reaction (PCR) system, by chemical mutagenesis (Deng et al., J. Biol. Chem., 269:9533 (1994)) or using bacterial mutator strains (Low et al., J. Mol. Biol., 260:359 (1996)).

In other embodiments, particular regions of the nucleic acid can be targeted for diversification. Methods for mutating selected positions are also well known in the art and include, for example, the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of PCR. For example, synthetic antibody libraries have been created by targeting mutations to the antigen binding loops. Random or semi-random antibody H3 and L3 regions have been appended to germline immunoblulin V gene segments to produce large libraries with unmutated framework regions (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra; Griffiths et al. (1994) supra; DeKruif et al. (1995) supra). Such diversification has been extended to include some or all of the other antigen binding loops (Crameri et al. (1996) Nature Med., 2:100; Riechmann et al. (1995) Bio/Technology, 13:475; Morphosys, WO 97/08320, supra). In other embodiments, particular regions of the nucleic acid can be targeted for diversification by, for example, a two-step PCR strategy employing the product of the first PCR as a “mega-primer.” (See, e.g., Landt, O. et al., Gene 96:125-128 (1990).) Targeted diversification can also be accomplished, for example, by SOE PCR. (See, e.g., Horton, R. M. et al., Gene 77:61-68 (1989).)

Sequence diversity at selected positions can be achieved by altering the coding sequence which specifies the sequence of the polypeptide such that a number of possible amino acids (e.g., all 20 or a subset thereof) can be incorporated at that position. Using the IUPAC nomenclature, the most versatile codon is NNK, which encodes all amino acids as well as the TAG stop codon. The NNK codon may be used in order to introduce the required diversity. Other codons which achieve the same ends are also of use, including the NNN codon, which leads to the production of the additional stop codons TGA and TAA. Such a targeted approach can allow the full sequence space in a target area to be explored.

Some libraries comprise domains that are members of the immunoglobulin superfamily (e.g., antibodies or portions thereof). For example the libraries can comprise domains that have a known main-chain conformation. (See, e.g., Tomlinson et al., WO 99/20749.) Libraries can be prepared in a suitable plasmid or vector. As used herein, vector refers to a discrete element that is used to introduce heterologous DNA into cells for the expression and/or replication thereof. Any suitable vector can be used, including plasmids (e.g., bacterial plasmids), viral or bacteriophage vectors, artificial chromosomes and episomal vectors. Such vectors may be used for simple cloning and mutagenesis, or an expression vector can be used to drive expression of the library. Vectors and plasmids usually contain one or more cloning sites (e.g., a polylinker), an origin of replication and at least one selectable marker gene. Expression vectors can further contain elements to drive transcription and translation of a polypeptide, such as an enhancer element, promoter, transcription termination signal, signal sequences, and the like. These elements can be arranged in such a way as to be operably linked to a cloned insert encoding a polypeptide, such that the polypeptide is expressed and produced when such an expression vector is maintained under conditions suitable for expression (e.g., in a suitable host cell).

Cloning and expression vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micron plasmid origin is suitable for yeast, and various viral origins (e.g. SV40, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication is not needed for mammalian expression vectors, unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.

Cloning or expression vectors can contain a selection gene also referred to as selectable marker. Such marker genes encode a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.

Suitable expression vectors can contain a number of components, for example, an origin of replication, a selectable marker gene, one or more expression control elements, such as a transcription control element (e.g., promoter, enhancer, terminator) and/or one or more translation signals, a signal sequence or leader sequence, and the like. Expression control elements and a signal or leader sequence, if present, can be provided by the vector or other source. For example, the transcriptional and/or translational control sequences of a cloned nucleic acid encoding an antibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell. Promoters can be constitutive or inducible. For example, a promoter can be operably linked to a nucleic acid encoding an antibody, antibody chain or portion thereof, such that it directs transcription of the nucleic acid. A variety of suitable promoters for procaryotic (e.g., the β-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system, lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g., simian virus 40 early or late promoter, Rous sarcoma virus long terminal repeat promoter, cytomegalovirus promoter, adenovirus late promoter, EG-1a promoter) hosts are available.

In addition, expression vectors typically comprise a selectable marker for selection of host cells carrying the vector, and, in the case of a replicable expression vector, an origin of replication. Genes encoding products which confer antibiotic or drug resistance are common selectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) and eucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes). Dihydrofolate reductase marker genes permit selection with methotrexate in a variety of hosts. Genes encoding the gene product of auxotrophic markers of the host (e.g., LEU2, URA3, HIS3) are often used as selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, and vectors which are capable of integrating into the genome of the host cell, such as retroviral vectors, are also contemplated.

Suitable expression vectors for expression in prokaryotic (e.g., bacterial cells such as E. coli) or mammalian cells include, for example, a pET vector (e.g., pET-12a, pET-36, pET-37, pET-39, pET-40, Novagen and others), a phage vector (e.g., pCANTAB 5 E, Pharmacia), pRIT2T (Protein A fusion vector, Pharmacia), pCDM8, pcDNA1.1/amp, pcDNA3.1, pRc/RSV, pEF-1 (Invitrogen, Carlsbad, Calif.), pCMV-SCRIPT, pFB, pSG5, pXT1 (Stratagene, La Jolla, Calif.), pCDEF3 (Goldman, L. A., et al., Biotechniques, 21:1013-1015 (1996)), pSVSPORT (GibcoBRL, Rockville, Md.), pEF-Bos (Mizushima, S., et al., Nucleic Acids Res., 18:5322 (1990)) and the like. Expression vectors which are suitable for use in various expression hosts, such as prokaryotic cells (E. coli), insect cells (Drosophila Schnieder S2 cells, Sf9), yeast (P. methanolica, P. pastoris, S. cerevisiae) and mammalian cells (eg, COS cells) are available.

Some examples of vectors are expression vectors that enable the expression of a nucleotide sequence corresponding to a polypeptide library member. Thus, selection with generic and/or target ligands can be performed by separate propagation and expression of a single clone expressing the polypeptide library member. As described above, a particular selection display system is bacteriophage display. Thus, phage or phagemid vectors may be used, for example vectors may be phagemid vectors which have an E. coli. origin of replication (for double stranded replication) and also a phage origin of replication (for production of single-stranded DNA). The manipulation and expression of such vectors is well known in the art (Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra). Briefly, the vector can contain a β-lactamase gene to confer selectivity on the phagemid and a lac promoter upstream of an expression cassette that can contain a suitable leader sequence, a multiple cloning site, one or more peptide tags, one or more TAG stop codons and the phage protein pIII. Thus, using various suppressor and non-suppressor strains of E. coli and with the addition of glucose, iso-propyl thio-β-D-galactoside (IPTG) or a helper phage, such as VCS M13, the vector is able to replicate as a plasmid with no expression, produce large quantities of the polypeptide library member only or product phage, some of which contain at least one copy of the polypeptide-pIII fusion on their surface.

Antibody variable domains may comprise a target ligand binding site and/or a generic ligand binding site. In certain embodiments, the generic ligand binding site is a binding site for a superantigen, such as protein A, protein L or protein G. The variable domains can be based on any desired variable domain, for example a human VH (e.g., V_H1a, V_H1b, V_H2, V_H3, V_H4, V_H5, V_H6), a human Vλ (e.g., VλI, VλII, VλIIl, VλIV, VλV, VλVI or Vκ1) or a human Vκ (e.g., Vκ2, Vκ3, Vκ4, Vκ5, Vκ6, Vκ7, Vκ8, Vκ9 or Vκ10).

A still further category of techniques involves the selection of repertoires in artificial compartments, which allow the linkage of a gene with its gene product. For example, a selection system in which nucleic acids encoding desirable gene products may be selected in microcapsules formed by water-in-oil emulsions is described in WO99/02671, WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol 16(7), 652-6. Genetic elements encoding a gene product having a desired activity are compartmentalised into microcapsules and then transcribed and/or translated to produce their respective gene products (RNA or protein) within the microcapsules. Genetic elements which produce gene product having desired activity are subsequently sorted. This approach selects gene products of interest by detecting the desired activity by a variety of means.

Characterisation of the Epitope Binding Domains.

The binding of a domain to its specific antigen or epitope can be tested by methods which will be familiar to those skilled in the art and include ELISA. In one example, binding is tested using monoclonal phage ELISA.

Phage ELISA may be performed according to any suitable procedure: an exemplary protocol is set forth below.

Populations of phage produced at each round of selection can be screened for binding by ELISA to the selected antigen or epitope, to identify “polyclonal” phage antibodies. Phage from single infected bacterial colonies from these populations can then be screened by ELISA to identify “monoclonal” phage antibodies. It is also desirable to screen soluble antibody fragments for binding to antigen or epitope, and this can also be undertaken by ELISA using reagents, for example, against a C- or N-terminal tag (see for example Winter et al. (1994) Ann. Rev. Immunology 12, 433-55 and references cited therein.

The diversity of the selected phage monoclonal antibodies may also be assessed by gel electrophoresis of PCR products (Marks et al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson et al., 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.

E. Structure of dAbs

In the case that the dAbs are selected from V-gene repertoires selected for instance using phage display technology as herein described, then these variable domains comprise a universal framework region, such that is they may be recognised by a specific generic ligand as herein defined. The use of universal frameworks, generic ligands and the like is described in WO99/20749.

Where V-gene repertoires are used variation in polypeptide sequence may be located within the structural loops of the variable domains. The polypeptide sequences of either variable domain may be altered by DNA shuffling or by mutation in order to enhance the interaction of each variable domain with its complementary pair. DNA shuffling is known in the art and taught, for example, by Stemmer, 1994, Nature 370: 389-391 and U.S. Pat. No. 6,297,053, both of which are incorporated herein by reference. Other methods of mutagenesis are well known to those of skill in the art.

Scaffolds for Use in Constructing dAbs

i. Selection of the Main-Chain Conformation

The members of the immunoglobulin superfamily all share a similar fold for their polypeptide chain. For example, although antibodies are highly diverse in terms of their primary sequence, comparison of sequences and crystallographic structures has revealed that, contrary to expectation, five of the six antigen binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited number of main-chain conformations, or canonical structures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia et al. (1989) Nature, 342: 877). Analysis of loop lengths and key residues has therefore enabled prediction of the main-chain conformations of H1, H2, L1, L2 and L3 found in the majority of human antibodies (Chothia et al. (1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et al. (1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399: 1).

The dAbs are advantageously assembled from libraries of domains, such as libraries of V_Hdomains and/or libraries of V_Ldomains. In one aspect, libraries of domains are designed in which certain loop lengths and key residues have been chosen to ensure that the main-chain conformation of the members is known. Advantageously, these are real conformations of immunoglobulin superfamily molecules found in nature, to minimise the chances that they are non-functional, as discussed above. Germline V gene segments serve as one suitable basic framework for constructing antibody or T-cell receptor libraries; other sequences are also of use. Variations may occur at a low frequency, such that a small number of functional members may possess an altered main-chain conformation, which does not affect its function.

Canonical structure theory is also of use to assess the number of different main-chain conformations encoded by ligands, to predict the main-chain conformation based on ligand sequences and to chose residues for diversification which do not affect the canonical structure. It is known that, in the human V_κdomain, the L1 loop can adopt one of four canonical structures, the L2 loop has a single canonical structure and that 90% of human V_κdomains adopt one of four or five canonical structures for the L3 loop (Tomlinson et al. (1995) supra); thus, in the V_κdomain alone, different canonical structures can combine to create a range of different main-chain conformations. Given that the Vλ domain encodes a different range of canonical structures for the L1, L2 and L3 loops and that V_κand Vλ domains can pair with any V_Hdomain which can encode several canonical structures for the H1 and H2 loops, the number of canonical structure combinations observed for these five loops is very large. This implies that the generation of diversity in the main-chain conformation may be essential for the production of a wide range of binding specificities. However, by constructing an antibody library based on a single known main-chain conformation it has been found, contrary to expectation, that diversity in the main-chain conformation is not required to generate sufficient diversity to target substantially all antigens. Even more surprisingly, the single main-chain conformation need not be a consensus structure—a single naturally occurring conformation can be used as the basis for an entire library. Thus, in a one particular aspect, the dAbs possess a single known main-chain conformation.

The single main-chain conformation that is chosen may be commonplace among molecules of the immunoglobulin superfamily type in question. A conformation is commonplace when a significant number of naturally occurring molecules are observed to adopt it. Accordingly, in one aspect, the natural occurrence of the different main-chain conformations for each binding loop of an immunoglobulin domain are considered separately and then a naturally occurring variable domain is chosen which possesses the desired combination of main-chain conformations for the different loops. If none is available, the nearest equivalent may be chosen. The desired combination of main-chain conformations for the different loops may be created by selecting germline gene segments which encode the desired main-chain conformations. In one example, the selected germline gene segments are frequently expressed in nature, and in particular they may be the most frequently expressed of all natural germline gene segments.

In designing libraries the incidence of the different main-chain conformations for each of the six antigen binding loops may be considered separately. For H1, H2, L1, L2 and L3, a given conformation that is adopted by between 20% and 100% of the antigen binding loops of naturally occurring molecules is chosen. Typically, its observed incidence is above 35% (i.e. between 35% and 100% and, ideally, above 50% or even above 65%. Since the vast majority of H3 loops do not have canonical structures, it is preferable to select a main-chain conformation which is commonplace among those loops which do display canonical structures. For each of the loops, the conformation which is observed most often in the natural repertoire is therefore selected. In human antibodies, the most popular canonical structures (CS) for each loop are as follows: H1—CS 1 (79% of the expressed repertoire), H2—CS 3 (46%), L1—CS 2 of V_κ (39%), L2—CS 1 (100%), L3—CS 1 of V_κ (36%) (calculation assumes a κ:λ ratio of 70:30, Hood et al. (1967) Cold Spring Harbor Symp. Quant. Biol., 48: 133). For H3 loops that have canonical structures, a CDR 3 length (Kabat et al. (1991) Sequences of proteins of immunological interest, U.S. Department of Health and Human Services) of seven residues with a salt-bridge from residue 94 to residue 101 appears to be the most common. There are at least 16 human antibody sequences in the EMBL data library with the required H3 length and key residues to form this conformation and at least two crystallographic structures in the protein data bank which can be used as a basis for antibody modelling (2cgr and 1tet). The most frequently expressed germline gene segments that this combination of canonical structures are the V_Hsegment 3-23 (DP-47), the J_Hsegment JH4b, the V_κ segment O2/O12 (DPK9) and the J_κ segment J_κ1. V_Hsegments DP45 and DP38 are also suitable. These segments can therefore be used in combination as a basis to construct a library with the desired single main-chain conformation.

Alternatively, instead of choosing the single main-chain conformation based on the natural occurrence of the different main-chain conformations for each of the binding loops in isolation, the natural occurrence of combinations of main-chain conformations is used as the basis for choosing the single main-chain conformation. In the case of antibodies, for example, the natural occurrence of canonical structure combinations for any two, three, four, five, or for all six of the antigen binding loops can be determined. Here, the chosen conformation may be commonplace in naturally occurring antibodies and may be observed most frequently in the natural repertoire. Thus, in human antibodies, for example, when natural combinations of the five antigen binding loops, H1, H2, L1, L2 and L3, are considered, the most frequent combination of canonical structures is determined and then combined with the most popular conformation for the H3 loop, as a basis for choosing the single main-chain conformation.

Diversification of the Canonical Sequence

Having selected several known main-chain conformations or a single known main-chain conformation, dAbs can be constructed by varying the binding site of the molecule in order to generate a repertoire with structural and/or functional diversity. This means that variants are generated such that they possess sufficient diversity in their structure and/or in their function so that they are capable of providing a range of activities.

The desired diversity is typically generated by varying the selected molecule at one or more positions. The positions to be changed can be chosen at random or they may be selected. The variation can then be achieved either by randomisation, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants.

Various methods have been reported for introducing such diversity. Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol., 226: 889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem., 269: 9533) or bacterial mutator strains (Low et al. (1996) J. Mol. Biol., 260: 359) can be used to introduce random mutations into the genes that encode the molecule. Methods for mutating selected positions are also well known in the art and include the use of mismatched oligonucleotides or degenerate oligonucleotides, with or without the use of PCR. For example, several synthetic antibody libraries have been created by targeting mutations to the antigen binding loops. The H3 region of a human tetanus toxoid-binding Fab has been randomised to create a range of new binding specificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457). Random or semi-random H3 and L3 regions have been appended to germline V gene segments to produce large libraries with unmutated framework regions (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381; Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al. (1995) J. Mol. Biol., 248: 97). Such diversification has been extended to include some or all of the other antigen binding loops (Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995) Bio/Technology, 13: 475; Morphosys, WO97/08320, supra).

Since loop randomisation has the potential to create approximately more than 10¹⁵structures for H3 alone and a similarly large number of variants for the other five loops, it is not feasible using current transformation technology or even by using cell free systems to produce a library representing all possible combinations. For example, in one of the largest libraries constructed to date, 6×10¹⁰different antibodies, which is only a fraction of the potential diversity for a library of this design, were generated (Griffiths et al. (1994) supra).

In a one embodiment, only those residues which are directly involved in creating or modifying the desired function of the molecule are diversified. For many molecules, the function will be to bind a target and therefore diversity should be concentrated in the target binding site, while avoiding changing residues which are crucial to the overall packing of the molecule or to maintaining the chosen main-chain conformation.

In one aspect, libraries of dAbs are used in which only those residues in the antigen binding site are varied. These residues are extremely diverse in the human antibody repertoire and are known to make contacts in high-resolution antibody/antigen complexes. For example, in L2 it is known that positions 50 and 53 are diverse in naturally occurring antibodies and are observed to make contact with the antigen. In contrast, the conventional approach would have been to diversify all the residues in the corresponding Complementarity Determining Region (CDR1) as defined by Kabat et al. (1991, supra), some seven residues compared to the two diversified in the library. This represents a significant improvement in terms of the functional diversity required to create a range of antigen binding specificities.

In nature, antibody diversity is the result of two processes: somatic recombination of germline V, D and J gene segments to create a naive primary repertoire (so called germline and junctional diversity) and somatic hypermutation of the resulting rearranged V genes. Analysis of human antibody sequences has shown that diversity in the primary repertoire is focused at the centre of the antigen binding site whereas somatic hypermutation spreads diversity to regions at the periphery of the antigen binding site that are highly conserved in the primary repertoire (see Tomlinson et al. (1996) J. Mol. Biol., 256: 813). This complementarity has probably evolved as an efficient strategy for searching sequence space and, although apparently unique to antibodies, it can easily be applied to other polypeptide repertoires. The residues which are varied are a subset of those that form the binding site for the target. Different (including overlapping) subsets of residues in the target binding site are diversified at different stages during selection, if desired.

In the case of an antibody repertoire, an initial ‘naive’ repertoire is created where some, but not all, of the residues in the antigen binding site are diversified. As used herein in this context, the term “naive” or “dummy” refers to antibody molecules that have no pre-determined target. These molecules resemble those which are encoded by the immunoglobulin genes of an individual who has not undergone immune diversification, as is the case with fetal and newborn individuals, whose immune systems have not yet been challenged by a wide variety of antigenic stimuli. This repertoire is then selected against a range of antigens or epitopes. If required, further diversity can then be introduced outside the region diversified in the initial repertoire. This matured repertoire can be selected for modified function, specificity or affinity.

It will be understood that the sequences described herein include sequences which are substantially identical, for example sequences which are at least 90% identical, for example which are at least 91%, or at least 92%, or at least 93%, or at least 94% or at least 95%, or at least 96%, or at least 97% or at least 98%, or at least 99% identical to the sequences described herein.

For nucleic acids, the term “substantial identity” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial identity exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

For nucleotide and amino acid sequences, the term “identical” indicates the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions. Alternatively, substantial identity exists when the DNA segments will hybridize under selective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions times 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 122, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 122 by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO: 122, or:

nn≦xn−(xn·y),

wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO: 122, and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of the polynucleotide sequence of SEQ ID NO: 122 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, in another example, a polypeptide sequence of the present invention may be identical to the reference sequence encoded by SEQ ID NO: 26, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 26 by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 26, or:

na≦xa−(xa·y),

wherein na is the number of amino acid alterations, xa is the total number of amino acids in the polypeptide sequence encoded by SEQ ID NO: 26, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.

EXAMPLES

The following methods were used in the examples described herein.

Method 1
Binding to E. Coli-Expressed Recombinant Human IL-13 by ELISA

mAbdAb molecules were assessed for binding to recombinant E. coil-expressed human IL-13 in a direct binding ELISA. In brief, 5 μg/ml recombinant E. coli-expressed human IL-13 (made and purified at GSK) was coated to a 96-well ELISA plate. The wells were blocked for 1 hour at room temperature, mAbdAb constructs were then titrated out across the plate (usually from around 100 nM in 3-fold dilutions to around 0.01 nM). Binding was detected using an appropriate dilution of anti-human kappa light chain peroxidase conjugated antibody (catalogue number A7164, Sigma-Aldrich) or an appropriate dilution of anti-human IgGγ chain specific peroxidase conjugated detection antibody (catalogue number A6029, Sigma-Aldrich).

Method 2
Binding to E. Coli-Expressed Recombinant Human IL-4 by ELISA

mAbdAb constructs were assessed for binding to recombinant E. coil-expressed human IL-4 in a direct binding ELISA. In brief, 5 μg/ml recombinant E. coli-expressed human IL-4 (made and purified at GSK) was coated to a 96-well ELISA plate. The wells were blocked for 1 hour at room temperature, mAbdAb constructs were then titrated out across the plate (usually from around 100 nM in 3-fold dilutions to around 0.01 nM). Binding was detected using an appropriate dilution of goat anti-human kappa light chain peroxidase conjugated antibody (catalogue number A7164, Sigma-Aldrich) or an appropriate dilution of anti-human IgGγ chain specific peroxidase conjugated detection antibody (catalogue number A6029, Sigma-Aldrich).

Method 3
Binding to E. Coli-Expressed Recombinant Human IL-18 by ELISA

mAbdAb constructs were assessed for binding to recombinant E. coli-expressed human IL-18 in a direct binding ELISA. In brief, 5 μg/ml recombinant E. coli-expressed human IL-18 (made and purified at GSK) was coated to a 96-well ELISA plate. The wells were blocked for 1 hour at room temperature, mAbdAb constructs were then titrated out across the plate (usually from around 100 nM in 3-fold dilutions to around 0.01 nM). Binding was detected using a dilution of 11n 2000 of anti-human kappa light chain peroxidase conjugated antibody (catalogue number A7164, Sigma-Aldrich) or using a dilution of 1 in 2000 of anti-human IgGγ chain specific peroxidase conjugated detection antibody (catalogue number A6029, Sigma-Aldrich).

Method 4
Biacore™ Binding Affinity Assessment for Binding to E. Coli-Expressed Recombinant Human IL-13

The binding affinity of mAbdAb constructs for recombinant E. Coli-expressed human IL-13 were assessed by Biacore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb constructs were then captured onto this surface and human IL-13 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions (such as 100 mM phosphoric acid), this did not significantly affect the ability to capture antibody for a subsequent IL-13 binding event. The anti-human IgG surface was regenerated either using similar conditions to the Protein A surface or by using 3M MgCl₂. The work was carried out on the Biacore™ 3000 and/or the T100 machine, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. Biacore™ runs were carried out at 25° C. or 37° C.

Method 5
Biacore™ Binding Affinity Assessment for Binding to E. Coli-Expressed Recombinant Human IL-4

The binding affinity of mAbdAb constructs for recombinant E. Coli-expressed human IL-4 were assessed by Biacore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb constructs were then captured onto this surface and human IL-4 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions (such as 100 mM phosphoric acid), this did not significantly affect the ability to capture antibody for a subsequent IL-4 binding event. The anti-human IgG surface was regenerated either using similar conditions to the Protein A surface or by using 3M MgCl₂. The work was carried out on Biacore™ 3000 and/or the T100 and/or the A100 machine, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. Biacore™ runs were carried out at 25° C. or 37° C.

Method 6
Biacore™ Binding Affinity Assessment for Binding to E. Coli-Expressed Recombinant Human IL-18

The binding affinity of mAbdAb constructs for recombinant E. Coli-expressed human IL-18 was assessed by Biacore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb constructs were then captured onto this surface and human IL-18 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions (such as 100 mM phosphoric acid), this did not significantly affect the ability to capture antibody for a subsequent IL-18 binding event. The anti-human IgG surface was regenerated either using similar conditions to the Protein A surface or by using 3M MgCl₂. The work was carried out on Biacore™ 3000 and/or the T100 and/or the A100 machine, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. The Biacore™ run was carried out at 25° C.

Method 7

Stoichiometry Assessment of mAbdAb Bispecific Antibodies or Trispecific Antibody for IL-13, IL-4 or IL-18 (Using Biacore™)

Anti-human IgG was immobilised onto a CM5 biosensor chip by primary amine coupling. mAbdAb constructs were captured onto this surface after which a single concentration of IL-13, IL-4 or IL-18 cytokine was passed over, this concentration was enough to saturate the binding surface and the binding signal observed reached full R-max. Stoichiometries were then calculated using the given formula:

Stoich=Rmax*Mw(ligand)/Mw(analyte)*R(ligand immobilised or captured)

Where the stoichiometries were calculated for more than one analyte binding at the same time, the different cytokines were passed over sequentially at the saturating cytokine concentration and the stoichometries calculated as above. The work was carried out on the Biacore 3000, at 25° C. using HBS-EP running buffer.

Method 8
Neutralisation of E. Coli-Expressed Recombinant Human IL-13 in a TF-1 Cell Proliferation Bioassay

TF-1 cells proliferate in response to a number of different cytokines including human IL-13. The proliferative response of these cells for IL-13 can therefore be used to measure the bioactivity of IL-13 and subsequently an assay has been developed to determine the IL-13 neutralisation potency (inhibition of IL-13 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 14 ng/ml recombinant E. Coli-expressed human IL-13 was pre-incubated with various dilutions of mAbdAb constructs (usually from 200 nM titrated in 3-fold dilutions to 0.02 nM) in a total volume of 50 μl for 1 hour at 37° C. These samples were then added to 50 μl of TF-1 cells (at a concentration of 2×10⁵cells per ml) in a sterile 96-well tissue culture plate. Thus the final 100 μl assay volume contained various dilutions of mAbdAb constructs (at a final concentration of 100 nM titrated in 3-fold dilutions to 0.01 nM), recombinant E. Coli-expressed human IL-13 (at a final concentration of 7 ng/ml) and TF-1 cells (at a final concentration of 1×10⁵cells per ml). The assay plate was incubated at 37° C. for approximately 3 days in a humidified CO₂incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions. The absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.

The capacity of the mAbdAb constructs to neutralise recombinant E. Coli-expressed human IL-13 bioactivity was expressed as that concentration of the mAbdAb construct required to neutralise the bioactivity of the defined amount of human IL-13 (7 ng/ml) by 50% (=ND₅₀). The lower the concentration of the mAbdAb construct required, the more potent the neutralisation capacity. The ND₅₀data provided herein were calculated manually or by using the Robosage software package which is inherent within microsoft excel.

Method 9
Neutralisation of E. Coli-Expressed Recombinant Human IL-4 in a TF-1 Cell Proliferation Bioassay

TF-1 cells proliferate in response to a number of different cytokines including human IL-4. The proliferative response of these cells for IL-4 can therefore be used to measure the bioactivity of IL-4 and subsequently an assay has been developed to determine the IL-4 neutralisation potency (inhibition of IL-4 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 2.2 ng/ml recombinant E. Coli-expressed human IL-4 was pre-incubated with various dilutions of mAbdAb constructs (usually from 200 nM titrated in 3-fold dilutions to 0.02 nM) in a total volume of 50 μl for 1 hour at 37° C. These samples were then added to 50 μl of TF-1 cells (at a concentration of 2×10⁵cells per ml) in a sterile 96-well tissue culture plate. Thus the final 100 μl assay volume contained various dilutions of mAbdAb constructs (at a final concentration of 100 nM titrated in 3-fold dilutions to 0.01 nM), recombinant E. Coli-expressed human IL-4 (at a final concentration of 1.1 ng/ml) and TF-1 cells (at a final concentration of 1×10⁵cells per ml). The assay plate was incubated at 37° C. for approximately 3 days in a humidified CO₂incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions. The absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.

The capacity of the mAbdAb constructs to neutralise recombinant E. Coli-expressed human IL-4 bioactivity was expressed as that concentration of the mAbdAb construct required to neutralise the bioactivity of the defined amount of human IL-4 (1.1 ng/ml) by 50% (=ND₅₀). The lower the concentration of the mAbdAb construct required, the more potent the neutralisation capacity. The ND₅₀data provided herein were calculated manually or by using the Robosage software package which is inherent within microsoft excel.

Method 10

Dual Neutralisation of E. Coli-Expressed Recombinant Human IL-13 and E. Coli-Expressed Recombinant Human IL-4 in a TF-1 cell Proliferation Bioassay

TF-1 cells proliferate in response to a number of different cytokines including human IL-13 and human IL-4. The proliferative response of these cells for IL-13 and IL-4 can therefore be used to measure the bioactivity of IL-13 and IL-4 simultaneously and subsequently an assay has been developed to determine the dual IL-13 and IL-4 neutralisation potency (dual inhibition of IL-13 and IL-4 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 14 ng/ml recombinant E. Coli-expressed human IL-13 and approximately 2.2 ng/ml recombinant E. Coli-expressed human IL-4 were pre-incubated with various dilutions of mAbdAb constructs (usually from 200 nM titrated in 3-fold dilutions to 0.02 nM) in a total volume of 50 μl for 1 hour at 37° C. These samples were then added to 50 μl of TF-1 cells (at a concentration of 2×10⁵cells per ml) in a sterile 96-well tissue culture plate. Thus the final 100 μl assay volume, contained various dilutions of mAbdAb constructs (at a final concentration of 100 nM titrated in 3-fold dilutions to 0.01 nM), recombinant E. Coli-expressed human IL-13 (at a final concentration of 7 ng/ml), recombinant E. Coli-expressed human IL-4 (at a final concentration of 1.1 ng/ml) and TF-1 cells (at a final concentration of 1×10⁵cells per ml). The assay plate was incubated at 37° C. for approximately 3 days in a humidified CO₂incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions. The absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.

Method 11
Biacore™ Binding Affinity Assessment for Binding to Sf21-Expressed Recombinant Human IL-5

The binding affinity of mAbdAb molecules for recombinant Sf21-expressed human IL-5 was assessed by Biacore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb molecules were then captured onto this surface and human IL-5 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions (such as 100 mM phosphoric acid), this did not significantly affect the ability to capture antibody for a subsequent IL-5 binding event. The anti-human IgG surface was regenerated either using similar conditions to the Protein A surface or by using 3M MgCl₂. The work was carried out on Biacore™ 3000, T100 and A100 machines, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. The Biacore™ run was carried out at 25° C.

Method 12
VEGF Receptor Binding Assay.

This assay measures the binding of VEGF₁₆₅to VEGF R2 (VEGF receptor) and the ability of test molecules to block this interaction. ELISA plates were coated overnight with VEGF receptor (R&D Systems, Cat No: 357-KD-050) (0.5 μg/ml final concentration in 0.2M sodium carbonate bicarbonate pH9.4), washed and blocked with 2% BSA in PBS. VEGF (R&D Systems, Cat No: 293-VE-050) and the test molecules (diluted in 0.1% BSA in 0.05% Tween 20™ PBS) were pre-incubated for one hour prior to addition to the plate (3 ng/ml VEGF final concentration). Binding of VEGF to VEGF receptor was detected using biotinylated anti-VEGF antibody (0.5 μg/ml final concentration) (R&D Systems, Cat No: BAF293) and a peroxidase conjugated anti-biotin secondary antibody (1:5000 dilution) (Stratech, Cat No: 200-032-096) and visualised at OD450 using a colorimetric substrate (Sure Blue TMB peroxidase substrate, KPL) after stopping the reaction with an equal volume of 1M HCl.

Method 13
EGFR Kinase Assay

Activation of EGFR expressed on the surface of A431 cells through its interaction with EGF leads to tyrosine kinase phosphorylation of the receptor. Reduction of EGFR tyrosine kinase phosphorylation was measured to determine potency of test molecules. A431 cells were allowed to adhere to 96 well tissue culture plates overnight then the test molecule was added and left for 1 hour and then incubated for 10 min with EGF (at 300 ng/ml) (R&D Systems catalogue number 236-EG). The cells were lysed and the lysed preparation transferred to ELISA plates coated with anti-EGFR antibody (at 1 ug/ml) (R&D Systems, cat #AF231). Both phosphorylated and non-phosphorylated EGFR present in the lysed cell solution was captured. After washing away unbound material phosphorylated EGFR was specifically detected using a HRP conjugated anti-phosphotyrosine antibody (1:2000 dilution) (Upstate Biotechnology, cat #16-105). Binding was visualised using a colorimetric substrate.

Method 14
MRC-5/TNF Assay

The ability of test molecules to prevent human TNF-a binding to human TNFR1 and neutralise IL-8 secretion was determined using human lung fibroblast MRC-5 cells. A dilution series of test samples was incubated with TNF-a (500 pg/ml) (Peprotech) for 1 hour. This was then diluted 1 in 2 with a suspension of MRC-5 cells (ATCC, Cat.# CCL-171) (5×10³cells/well). After an overnight incubation, samples were diluted 1 in 10, and IL-8 release was determined using an IL-8 ABI 8200 cellular detection assay (FMAT) where the IL-8 concentration was determined using anti-IL-8 (R&D systems, Cat# 208-IL) coated polystyrene beads, biotinylated anti-IL-8 (R&D systems, Cat# BAF208) and streptavidin Alexafluor 647 (Molecular Probes, Cat#532357). The assay readout was localised fluorescence emission at 647 nm and unknown IL-8 concentrations were interpolated using an IL-8 standard curve included in the assay. Method 15 MRC-5/IL-1 Assay.

The ability of test molecules to prevent human IL-1a binding to human IL1-R and neutralise IL-8 secretion was determined using human lung fibroblast MRC-5 cells. MRC-5 cells (ATCC, Cat.# CCL-171) were trypsinised then incubated with the test samples for one hour as a suspension. IL-1a (200 pg/ml final concentration) (R&D Systems cat no: 200-LA) was then added. After an overnight incubation IL-8 release was determined using an IL-8 quantification ELISA kit (R&D Systems) with anti-IL-8 coated ELISA plates, biotinylated anti-IL-8 and streptavidin-HRP. The assay readout is colourimetric absorbance at 450 nm and unknown IL-8 concentrations are interpolated using an IL-8 standard curve included in the assay.

Method 16
Neutralisation Potency of E. Coli-Expressed Recombinant Human IL-13 or IL-4 in a Whole Blood Phospho-STAT6 Bioassay

Whole blood cells can be stimulated ex-vivo with recombinant E. Coli-expressed human IL-4 (rhIL-14) or IL-13 (rhIL-13) to express phospho STAT6 (pSTAT6). This assay was developed to quantitatively measure pSTAT6 and consequently determine the neutralisation potency (inhibition of IL-4 or IL-13 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. 12 ng/ml of rhIL-13 or rhIL-4 was prepared in serum free cell culture medium and 31.24 added to wells of a 96-well plate. A 9 point dilution curve of mAbdAb constructs or isotype control was prepared at 6× the final assay concentration and 31.2 μL of each dilution added to wells containing either rhIL-4 or rhIL-13. 125 μL of heparinized human whole blood was added to all wells and mixed on a shaker for 30 seconds. The final assay volume contained various dilutions of mAbdAb constructs together with rhIL-13 or rhIL-4 at a final concentration of 2 ng/mL. The assay plate was incubated at 37° C., 5% CO₂for 60 minutes.

The cells were then lysed by the addition of 62.54 of 4× lysis buffer. The lysis buffer contained final assay concentrations of 50 mM Tris hydrochloride, 300 mM sodium chloride, 1% NP40, 0.5% sodium deoxycholate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM EDTA, and protease inhibitor cocktail. The plates were placed on ice for 30 minutes then frozen at −80° C. until assayed for pSTAT6.

The measurement of pSTAT6 in the whole blood samples was performed using an electro-chemiluminescent immuno-assay (Meso-Scale-Discovery, MSD). In brief, avidin coated 96-well MSD plates were blocked with 150 μL per well of 5% MSD blocker A for 1 hour at room temperature on a shaker at 750 rpm. The plate was washed 3 times with 150 μL per well of MSD Tris wash buffer. 254 per well of capture antibody (biotinylated mouse anti-human STAT6 monoclonal antibody) was added and the plates incubated overnight at 4° C. The capture antibody had been diluted to 4 μg/mL in assay buffer consisting of 50 mM Tris, 150 mM sodium chloride, 0.2% BSA, 0.5% Tween 20, 1 mM EDTA. The plate was washed 3 times with MSD tris wash buffer then blocked with 150 μL of 5% MSD blocker A for 1 hour at room temperature on a shaker. Plates were washed 3 times as stated previously, then 25 μL of whole blood lysate or pSTAT6 calibrator added per well. Plates were incubated for 3 hours at room temperature on a shaker. Plates were washed 3 times then 25 μL of rabbit anti human pSTAT6 antibody (diluted 1 in 800 in assay buffer) was added and then incubated for 1 hour at room temperature. After further washing, 25 μL per well of a 1 in 500 dilution of MSD TAG goat anti-rabbit IgG antibody was added and then incubated for 1 hour at room temperature on a shaker. Plates were washed again before addition of 150 μL per well of 2×MSD read buffer T. Plates were read immediately on a MSD SECTOR imager.

The ability of the mAbdAb constructs to neutralise rhIL-13 or rhIL-4 bioactivity was expressed as the concentration of the mAbdAb construct required to neutralise 2 ng/mL of human IL-4 or human IL-13 by 50% (IC₅₀). The lower the concentration of the mAbdAb construct required, the more potent the neutralisation capacity.

Method 17
Binding to E. Coli-Expressed Recombinant Cynomolgus IL-13 by ELISA

mAbdAb molecules were assessed for binding to recombinant E. coli-expressed cynomolgus IL-13 in a direct binding ELISA. In brief, 5 μg/ml recombinant E. coli-expressed cynomolgus IL-13 (made and purified at GSK) was coated to a 96-well ELISA plate. The wells were blocked for 1 hour at room temperature, mAbdAb molecules were then titrated out across the plate (usually from around 100 nM in 3-fold dilutions to around 0.01 nM). Binding was detected using an appropriate dilution of anti-human kappa light chain peroxidase conjugated antibody (catalogue number A7164, Sigma-Aldrich) or an appropriate dilution of anti-human IgGγ chain specific peroxidase conjugated detection antibody (catalogue number A6029, Sigma-Aldrich).

Method 18

Not used

Method 19
Inhibition of Human IL-4 Binding to Human IL4 Receptor Alpha (IL4Rα) by ELISA

Unless otherwise stated all reagents were diluted to the required concentration in block solution (4% bovine serum albumin in tris-buffered saline and 0.05% Tween20) just prior to use. An ELISA plate was coated over-night at 4° C. with 5 μg/ml of recombinant human IL4Rα-Fc chimaera (R&D Systems, Cat. No. 604-4R) in phosphate buffered saline. All subsequent steps were carried out at room temperature. The plate was blocked for 2 hours in block solution before addition of 50 μl of various concentrations of mAbdAb (or the positive control mAbs or dAbs) which had been pre-mixed with 0.02 m/ml of recombinant human IL-4 (made at GSK). Plates were incubated for 1 hour before washing 4 times in wash buffer (Tris buffered saline and 0.05% Tween20). 50 μl of a 0.5 μg/ml solution of biotinylated anti-human IL-4 (R&D Systems, Cat. No. BAF 204) was added to each well and incubated for 1 hour. The plate was washed ×4 in wash buffer before addition of 50 μl/well of a 1/2000 dilution of Extravadin (Sigma, Cat. No. E2886). After one hour the plate was washed 4 times and a colourimetric signal was detected by incubating with OPD peroxidase substrate (from Sigma), the reaction was stopped with the stop solution (3M H₂SO₄acid) and absorbance data obtained by reading on a plate-reader at 490 nm. Mean absorbance and standard error was plotted in GraphPad Prism and IC₅₀values were calculated using Cambridge Soft BioAssay.

Method 20
Neutralisation of E. Coli-Expressed Recombinant Cynomolgus IL-13 in a TF-1 Cell Proliferation Bioassay

TF-1 cells proliferate in response to a number of different cytokines including cynomolgus IL-13. The proliferative response of these cells for IL-13 can therefore be used to measure the bioactivity of IL-13 and subsequently an assay has been developed to determine the IL-13 neutralisation potency (inhibition of IL-13 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 14 ng/ml recombinant E. Coli-expressed cynomolgus IL-13 was pre-incubated with various dilutions of mAbdAb constructs (usually from 1000 nM or 200 nM titrated in 3-fold dilutions to 1 nM or 0.02 nM) in a total volume of 50 μl for 1 hour at 37° C. These samples were then added to 50 μl of TF-1 cells (at a concentration of 2×10⁵cells per ml) in a sterile 96-well tissue culture plate. Thus the final 100 μl assay volume contained various dilutions of mAbdAb constructs (at a final concentration of 500 nM or 100 nM titrated in 3-fold dilutions to 0.5 nM or 0.01 nM), recombinant E. Coli-expressed cynomolgus IL-13 (at a final concentration of 7 ng/ml) and TF-1 cells (at a final concentration of 1×10⁵cells per ml). The assay plate was incubated at 37° C. for approximately 3 days in a humidified CO₂incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions. The absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.

The capacity of the mAbdAb constructs to neutralise recombinant E. Coli-expressed cynomolgus IL-13 bioactivity was expressed as that concentration of the mAbdAb construct required to neutralise the bioactivity of the defined amount of cynomolgus IL-13 (7 ng/ml) by 50% (=ND₅₀). The lower the concentration of the mAbdAb construct required, the more potent the neutralisation capacity. The ND₅₀data provided herein were calculated manually or by using the Robosage software package which is inherent within microsoft excel.

Method 21
Neutralisation of E. Coli-Expressed Recombinant Cynomolgus IL-4 in a TF-1 Cell Proliferation Bioassay

TF-1 cells proliferate in response to a number of different cytokines including cynomolgus IL-4. The proliferative response of these cells for IL-4 can therefore be used to measure the bioactivity of IL-4 and subsequently an assay has been developed to determine the IL-4 neutralisation potency (inhibition of IL-4 bioactivity) of mAbdAb constructs.

The assay was performed in sterile 96-well tissue culture plates under sterile conditions and all test wells were performed in triplicate. Approximately 2.2 ng/ml recombinant E. Coli-expressed cynomolgus IL-4 was pre-incubated with various dilutions of mAbdAb constructs (usually from 200 nM titrated in 3-fold dilutions to 0.02 nM) in a total volume of 50 μl for 1 hour at 37° C. These samples were then added to 50 μl of TF-1 cells (at a concentration of 2×10⁵cells per ml) in a sterile 96-well tissue culture plate. Thus the final 100 μl assay volume contained various dilutions of mAbdAb constructs (at a final concentration of 100 nM titrated in 3-fold dilutions to 0.01 nM), recombinant E. Coli-expressed cynomolgus IL-4 (at a final concentration of 1.1 ng/ml) and TF-1 cells (at a final concentration of 1×10⁵cells per ml). The assay plate was incubated at 37° C. for approximately 3 days in a humidified CO₂incubator. The amount of cell proliferation was then determined using the ‘CellTitre 96® Non-Radioactive Cell Proliferation Assay’ from Promega (catalogue number G4100), as described in the manufacturers instructions. The absorbance of the samples in the 96-well plate was read in a plate reader at 570 nm.

The capacity of the mAbdAb constructs to neutralise recombinant E. Coli-expressed cynomolgus IL-4 bioactivity was expressed as that concentration of the mAbdAb construct required to neutralise the bioactivity of the defined amount of cynomolgus IL-4 (1.1 ng/ml) by 50% (=ND₅₀). The lower the concentration of the mAbdAb construct required, the more potent the neutralisation capacity. The ND₅₀data provided herein were calculated manually or by using the Robosage software package which is inherent within microsoft excel.

Method 22
Inhibition of Human IL-13 Binding to Human IL13 Receptor Alpha 2 (IL13Rα2) by ELISA

Unless otherwise stated all reagents were diluted to the required concentration in block solution (1% bovine serum albumin in tris-buffered saline and 0.05% Tween20) just prior to use. An ELISA plate was coated overnight at 4° C. with 5 μg/ml of recombinant human IL13Rα2/Fc chimera expressed in Sf21 cells (R&D Systems, Cat. No. 614-IR) in a solution of coating buffer (0.05M bicarbonate pH9.6, Sigma C-3041). The plate was blocked for 1 hour at room temperature in block solution (1% BSA in TBST) before addition of various concentrations of mAbdAb (or the positive control mAbs or dAbs) which had been pre-incubated with 30 ng/ml of recombinant human IL-13 (made at GSK) for 30 mins at 37° C. Plates were incubated for 1 hour at room temperature before washing 3 times in wash buffer (Tris buffered saline and 0.05% Tween20). 50 μl of a 0.5 μg/ml solution of biotinylated anti-human IL-13 (R&D Systems, Cat. No. BAF 213) was added to each well and incubated for 1 hour at room temperature. The plate was washed three times in wash buffer before addition of an appropriate dilution of Extravadin (Sigma, Cat. No. E2886). After one hour the plate was washed and a colourimetric signal was detected by incubating with OPD peroxidase substrate (from Sigma), the reaction was stopped with the stop solution (3M acid) and absorbance data obtained by reading on a plate-reader at 490 nm. Mean absorbance and standard error was plotted in Excel sheet and IC₅₀values were calculated using the Robosage software from Microsoft Excel.

Method 23
Biacore™ Binding Affinity Assessment for Binding to E. Coli-Expressed Recombinant Cynomolgus IL-13

The binding affinity of mAbdAb (or mAb) molecules for recombinant E. Coli-expressed cynomolgus IL-13 was assessed by Biacore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb (or mAb) molecules were then captured onto this surface and cynomolgus IL-13 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions, this did not significantly affect the ability to capture antibody for a subsequent IL-13 binding event. The work was carried out on BIAcore™ 3000 and/or the T100 machine, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. BIAcore™ runs were carried out at 25° C. or 37° C.

Method 24
BIAcore™ Binding Affinity Assessment for Binding to E. Coli-Expressed Recombinant Cynomolgus IL-4

The binding affinity of mAbdAb (or mAb) molecules for recombinant E. Coli-expressed cynomolgus IL-4 were assessed by BIAcore™ analysis. Analyses were carried out using Protein A or anti-human IgG capture. Briefly, Protein A or anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb (or mAb) molecules were then captured onto this surface and cynomolgus IL-4 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Protein A surface using mild acid elution conditions (such as 100 mM phosphoric acid), this did not significantly affect the ability to capture antibody for a subsequent IL-4 binding event. The anti-human IgG surface was regenerated either using similar conditions to the Protein A surface or by using 3M MgCl₂. The work was carried out on BIAcore™ 3000 and/or the T100 and/or the A100 machine, data were analysed using the evaluation software in the machines and fitted to the 1:1 model of binding. BIAcore™ runs were carried out at 25° C. or 37° C.

Method 25
IL-13 Cell-Based Neutralisation Assay

The potency of mAbdAbs having specificity for IL13 was assayed in an IL-13 cell assay using the engineered reporter cell line HEK Blue-STAT6.

The transcription factor STAT6 is activated primarily by two cytokines with overlapping biologic functions, IL-4 and IL-13 which bind a receptor complex composed of the IL-4Ralpha and IL-13Ralpha1. Upon ligand binding, the receptor complex activates the receptor-associated Janus kinases (JAK1 and Tyk2) leading to the recruitment of STAT6 and its phosphorylation. Activated STAT6 forms a homodimer that translocates to the nucleus, inducing transcription of genes under the control of the responsive promoter. The HEK Blue-STAT6 line is engineered to express Secreted Embryonic Alkaline Phosphatase (SEAP) under the control of such a promoter.

Cells were plated into 96 well plates and incubated for 20-24 hours with pre-equilibrated human IL-13 and test molecules. After this incubation period, the amount of SEAP produced by the cells as a result of IL-13 stimulation was then measured using the Quanti-blue system (Invivogen).

Example 1
1. Generation of Dual Targeting mAbdAbs

The dual targeting mAbdAbs set out in Tables 1-4 were constructed in the following way. Expression constructs were generated by grafting a sequence encoding a domain antibody on to a sequence encoding a heavy chain or a light chain (or both) of a monoclonal antibody such that when expressed the dAb is attached to the C-terminus of the heavy or light chain. For some constructs, linker sequences were used to join the domain antibody to heavy chain CH3 or light chain CK. In other constructs the domain antibody is joined directly to the heavy or light chain with no linker sequence. A general schematic diagram of these mAbdAb constructs is shown in FIG. 8 (the mAb heavy chain is drawn in grey; the mAb light chain is drawn in white; the dAb is drawn in black).

An example of mAbdAb type 1 as set out in FIG. 8 would be PascoH-G4S-474. An example of mAbdAb type 2 as set out in FIG. 8 would be PascoL-G4S-474. An example of mAbdAb type 3 as set out in FIG. 8 would be PascoHL-G4S-474. mAbdAb types 1 and 2 are tetravalent constructs, mAbdAb type 3 is a hexavalent construct.

A schematic diagram illustrating the construction of a mAbdAb heavy chain (top illustration) or a mAbdAb light chain (bottom illustration) is shown below. Unless otherwise stated, these restriction sites were used to construct the mAbdAbs described in Tables 1-4

Note that for the heavy chain the term ‘V_H’ is the monoclonal antibody variable heavy chain sequence; ‘CH1, CH2 and CH3’ are human IgG1 heavy chain constant region sequences; ‘linker’ is the sequence of the specific linker region used; ‘dAb’ is the domain antibody sequence. For the light chain the term ‘V_L’ is the monoclonal antibody variable light chain sequence; ‘CK’ is the human light chain constant region sequence; ‘linker’ is the sequence of the specific linker region used; ‘dAb’ is the domain antibody sequence.

Some DNA expression constructs were made de novo by oligo build. And other DNA expression constructs were derived from existing constructs (which were made as described above) by restriction cloning or site-directed mutagenesis.

These constructs (mAbdAb heavy or light chains) were cloned into mammalian expression vectors (Rln, Rld or pTT vector series) using standard molecular biology techniques. A mammalian amino acid signal sequence (as shown in SEQ ID NO: 64) was used in the construction of these constructs.

For expression of mAbdAbs where the dAb is joined to the C-terminal end of the heavy chain of the monoclonal antibody, the appropriate heavy chain mAbdAb expression vector was paired with the appropriate light chain expression vector for that monoclonal antibody. For expression of mAbdAbs where the dAb is joined to the C-terminal end of the light chain of the monoclonal antibody, the appropriate light chain mAbdAb expression vector was paired with the appropriate heavy chain expression vector for that monoclonal antibody.

For expression of mAbdAbs where the dAb is joined to the C-terminal end of the heavy chain of the monoclonal antibody and where the dAb is joined to the C-terminal end of the light chain of the monoclonal antibody, the appropriate heavy chain mAbdAb expression vector was paired with the appropriate light chain mAbdAb expression vector.

1.1 NOMENCLATURE AND ABBREVIATIONS USED

Monoclonal antibody (mAb)

Monoclonal antibodies (mAbs)

Domain antibody (dAb)

Domain antibodies (dAbs)

Heavy Chain (H chain)

Light chain (L chain)

Heavy chain variable region (V_H)

Light chain variable region (V_L)

Human IgG1 constant heavy region 1 (CH1)

Human IgG1 constant heavy region 2 (CH2)

Human IgG1 constant heavy region 3 (CH3)

Human kappa light chain constant region (CK)

1.2 Anti-IL13 mAb-Anti-IL4dAbs

Bispecific anti-IL13 mAb-anti-IL4dAbs were constructed by as described above. A number of different linkers were used to join the anti-IL4 domain antibodies to the monoclonal antibody. Some constructs had no linker.

Note that a BamHI cloning site (which codes for amino acid residues G and S) was used to clone the linkers and dAbs either to CH3 of the mAb heavy chain or to CK of the mAb light chain. Thus in addition to the given linker sequence, additional G and S amino acid residues are present between the linker sequence and the domain antibody for both heavy chain and light chain expression constructs or between CH3 and the linker sequence in some but not all heavy chain expression constructs. However, when the G4S linker was placed between the mAb and dAb in the mAbdAb format, the BamHI cloning site was already present (due to the G and S amino acid residues inherent within the G4S linker sequence) and thus additional G and S amino acid residues were not present between CH3 or CK and the domain antibody in the constructs using this linker. When no linker sequence was between used in the mAb and dAb in the mAbdAb format, the BamHI cloning site (and hence the G and S amino acid residues) was still present between CH3 or CK and the domain antibody. Full details on the amino acid sequences of mAbdAb heavy and light chains are set out in Table 1.

Several of the following examples use an IL-4 mAb as a control antibody. The control IL-4 mAb used in these examples will either be the antibody having the heavy chain sequence of SEQ ID NO: 14 and the light chain sequence of SEQ ID NO: 15, or will be the antibody having the heavy chain sequence of SEQ ID NO: 166 and the light chain sequence of SEQ ID NO: 15. Both of these IL-4 mAbs share the same CDRs, and are expected to bind in the same way hence both of these antibodies are referred to as ‘Pascolizumab’ or ‘IL-4 mAb’ in the following examples.

Several of the following examples use an IL-5 mAb as a control antibody. The control IL-5 mAb used in these examples will either be the antibody having the heavy chain sequence of SEQ ID NO: 65 and the light chain sequence of SEQ ID NO: 66, or the antibody having the heavy chain sequence of SEQ ID NO: 191 and the light chain sequence of SEQ ID NO: 66. Both of these IL-5 antibodies share the same CDRs, and are expected to bind in the same way hence both of these antibodies are referred to as ‘Mepolizumab’ or ‘IL-5 mAb’ in the following examples.

The mAbdAbs set out in table 1 were expressed transiently in CHOK1 cell supernatants. Following mAbdAb quantification these mAbdAb containing supernatants were analysed for activity in IL-13 and IL-4 binding ELISAs.

TABLE 1

Name
Description
Sequence ID No.

586H-25
H chain = Anti-human IL-13 mAb heavy
16 (= H chain)

chain-GS-DOM9-155-25 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-G4S-25
H chain = Anti-human IL-13 mAb heavy
20 (= H chain)

chain-G4S linker-DOM9-155-25 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-TVAAPS-25
H chain = Anti-human IL-13 mAb heavy
24 (= H chain)

chain-TVAAPS linker-GS-DOM9-155-25
13 (= L chain)

dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ASTKG-25
H chain = Anti-human IL-13 mAb heavy
28 (= H chain)

chain-GS-ASTKGPT linker-GS-DOM9-
13 (= L chain)

155-25 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-EPKSC-25
H chain = Anti-human IL-13 mAb heavy
32 (= H chain)

chain-GS-EPKSCDKTHTCPPCP linker-
13 (= L chain)

GS-DOM9-155-25 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ELQLE-25
H chain = Anti-human IL-13 mAb heavy
36 (= H chain)

chain-ELQLEESCAEAQDGELDG linker-
13 (= L chain)

GS-DOM9-155-25 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-147
H chain = Anti-human IL-13 mAb heavy
17 (= H chain)

chain-GS-DOM9-155-147 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-G4S-147
H chain = Anti-human IL-13 mAb heavy
21 (= H chain)

chain-G4S linker-DOM9-155-147 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-TVAAPS-147
H chain = Anti-human IL-13 mAb heavy
25 (= H chain)

chain-TVAAPS linker-GS-DOM9-155-147
13 (= L chain)

dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ASTKG-147
H chain = Anti-human IL-13 mAb heavy
29 (= H chain)

chain-GS-ASTKGPT linker-DOM9-155-
13 (= L chain)

147 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-EPKSC-147
H chain = Anti-human IL-13 mAb heavy
33 (= H chain)

chain-GS-EPKSCDKTHTCPPCP linker-
13 (= L chain)

GS-DOM9-155-147 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ELQLE-147
H chain = Anti-human IL-13 mAb heavy
37 (= H chain)

chain-GS-ELQLEESCAEAQDGELDG
13 (= L chain)

linker-GS-DOM9-155-147 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-154
H chain = Anti-human IL-13 mAb heavy
18 (= H chain)

chain-GS-DOM9-155-154 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-G4S-154
H chain = Anti-human IL-13 mAb heavy
22 (= H chain)

chain-G4S linker-DOM9-155-154 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-TVAAPS-154
H chain = Anti-human IL-13 mAb heavy
26 (= H chain)

chain-TVAAPS linker-GS-DOM9-155-154
13 (= L chain)

dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ASTKG-154
H chain = Anti-human IL-13 mAb heavy
30 (= H chain)

chain-GS-ASTKGPT linker-GS-DOM9-
13 (= L chain)

155-154 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-EPKSC-154
H chain = Anti-human IL-13 mAb heavy
34 (= H chain)

chain-GS-EPKSCDKTHTCPPCP linker-
13 (= L chain)

GS-DOM9-155-154 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ELQLE-154
H chain = Anti-human IL-13 mAb heavy
38 (= H chain)

chain-GS-ELQLEESCAEAQDGELDG
13 (= L chain)

linker-GS-DOM9-155-154 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-210
H chain = Anti-human IL-13 mAb heavy
19 (= H chain)

chain-GS-DOM9-112-210 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-G4S-210
H chain = Anti-human IL-13 mAb heavy
23 (= H chain)

chain-G4S linker-DOM9-112-210 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-TVAAPS-210
H chain = Anti-human IL-13 mAb heavy
27 (= H chain)

chain-TVAAPS linker-GS-DOM9-112-210
13 (= L chain)

dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ASTKG-210
H chain = Anti-human IL-13 mAb heavy
31 (= H chain)

chain-GS-ASTKGPT linker-GS-DOM9-
13 (= L chain)

112-210 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-EPKSC-210
H chain = Anti-human IL-13 mAb heavy
35 (= H chain)

chain-GS-EPKSCDKTHTCPPCP linker-
13 (= L chain)

GS-DOM9-112-210 dAb

L chain = Anti-human IL-13 mAb light

chain

586H-ELQLE-210
H chain = Anti-human IL-13 mAb heavy
39 (= H chain)

chain-GS-ELQLEESCAEAQDGELDG
13 (= L chain)

linker-GS-DOM9-112-210 dAb

L chain = Anti-human IL-13 mAb light

chain

586H
H chain = Anti-human IL-13 mAb heavy
40 (= H chain)

chain-GS-
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-ASTKG
H chain = Anti-human IL-13 mAb heavy
41 (= H chain)

chain-GS-ASTKGPT linker-GS
13 (= L chain)

L chain = Anti-human IL-13 mAb light

chain

586H-EPKSC
H chain = Anti-human IL-13 mAb heavy
42 (= H chain)

chain-GS-EPKSCDKTHTCPPCP linker-
13 (= L chain)

GS

L chain = Anti-human IL-13 mAb light

chain

586H-ELQLE
H chain = Anti-human IL-13 mAb heavy
43 (= H chain)

chain-GS-ELQLEESCAEAQDGELDG
13 (= L chain)

linker-GS

L chain = Anti-human IL-13 mAb light

chain

The mAbdAbs set out in table 2 were expressed in one or both of CHOK1 or CHOE1a cell supernatants, purified and analysed in a number of IL-13 and IL-4 activity assays.

TABLE 2

Sequence ID

Name
Description
No.

586H-TVAAPS-25
H chain = Anti-human IL-13 mAb heavy chain-
24 (= H chain)

TVAAPS linker-GS-DOM9-155-25 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light chain

586H-TVAAPS-154
H chain = Anti-human IL-13 mAb heavy chain-
26 (= H chain)

TVAAPS linker-GS-DOM9-155-154 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light chain

586H-TVAAPS-210
H chain = Anti-human IL-13 mAb heavy chain-
27 (= H chain)

TVAAPS linker-GS-DOM9-112-210 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light chain

1.3 Anti-IL4 mAb-anti-IL13dAbs

Bispecific anti-IL4 mAb-anti-IL13dAbs were constructed as described above. A number of different linkers were used to join the anti-IL13 domain antibody to the monoclonal antibody. Some constructs had no linker.

The mAbdAbs set out in table 3 were expressed transiently in CHOK1 cell supernatants. Following mAbdAb quantification these mAbdAb containing supernatants were analysed for activity in IL-13 and IL-4 binding ELISAs.

TABLE 3

Sequence ID

Name
Description
No.

PascoH-474
H chain = Pascolizumab heavy chain-GS-
48 (= H chain)

DOM10-53-474 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-G4S-474
H chain = Pascolizumab heavy chain-G4S
49 (= H chain)

linker-DOM10-53-474 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-TVAAPS-474
H chain = Pascolizumab heavy chain-TVAAPS
50 (= H chain)

linker-GS-DOM10-53-474 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-ASTKG-474
H chain = Pascolizumab heavy chain-GS-
51 (= H chain)

ASTKGPT linker-GS-DOM10-53-474 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-EPKSC-474
H chain = Pascolizumab heavy chain-GS-
52 (= H chain)

EPKSCDKTHTCPPCP linker-GS-DOM10-53-
15 (= L chain)

474 dAb

L chain = Pascolizumab light chain

PascoH-ELQLE-474
H chain = Pascolizumab heavy chain-GS-
53 (= H chain)

ELQLEESCAEAQDGELDG linker-GS-DOM10-
15 (= L chain)

53-474 dAb

L chain = Pascolizumab light chain

PascoL-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-GS-DOM10-
54 (= L chain)

53-474 dAb

PascoL-G4S-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-G4S
55 (= L chain)

linker-DOM10-53-474 dAb

PascoL-TVAAPS-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-TVAAPS
56 (= L chain)

linker-GS-DOM10-53-474 dAb

PascoL-ASTKG-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-ASTKGPT
57 (= L chain)

linker-GS-DOM10-53-474 dAb

PascoL-EPKSC-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-
58 (= L chain)

EPKSCDKTHTCPPCP linker-GS-DOM10-53-

474 dAb

PascoL-ELQLE-474
H chain = Pascolizumab heavy chain
14 (= H chain)

L chain = Pascolizumab light chain-
59 (= L chain)

ELQLEESCAEAQDGELDG linker-GS-DOM10-

53-474 dAb

The mAbdAbs set out in Table 4 were expressed in one or more of CHOK1, CHOE1a or HEK293-6E cells.

TABLE 4

Sequence ID

Name
Description
No.

PascoH-
H chain = Pascolizumab heavy chain-G4S
49 (=H chain)

G4S-474
linker-DOM10-53-474 dAb
15 (=L chain)

L chain = Pascolizumab light chain

PascoH-474
H chain = Pascolizumab heavy chain-GS-
48 (=H chain)

DOM10-53-474 dAb
15 (=L chain)

L chain = Pascolizumab light chain

PascoL-
H chain = Pascolizumab heavy chain
14 (=H chain)

G4S-474
L chain = Pascolizumab light chain-G4S
55 (=L chain)

linker-DOM10-53-474 dAb

PascoHL-
H chain = Pascolizumab heavy chain-G4S
49 (=H chain)

G4S-474
linker-DOM10-53-474 dAb
55 (=L chain)

L chain = Pascolizumab light chain-G4S

linker-DOM10-53-474 dAb

1.4 Sequence ID Numbers for Monoclonal Antibodies, Domain Antibodies and Linkers

Sequence IDs numbers for the monoclonal antibodies (mAb), domain antibodies (dAb) and linkers used to generate the mAbdAbs are shown below in table 5.

TABLE 5

Name
Specificity
Sequence ID

Anti-human IL-13 monoclonal antibody
Human IL-13
12 (H chain)

(also known as 586)

13 (L chain)

Anti-human IL-4 monoclonal antibody
Human IL-4
14 (H chain)

(also known as Pascolizumab)

15 (L chain)

DOM10-53-474 domain antibody
Human IL-13
5

Anti-human IL-13 monoclonal antibody
Human IL-13
161 (H chain)

(also known as 656)

156 (L chain)

DOM9-112-210 domain antibody
Human IL-4
4

DOM10-53-616 domain antibody
Human IL-13
148

DOM9-155-25 domain antibody
Human IL-4
1

DOM9-155-147 domain antibody
Human IL-4
2

DOM9-155-154 domain antibody
Human IL-4
3

ASTKGPS linker sequence
Derived from human IgG1
9

H chain (VH-CH1)

ASTKGPT linker sequence
Derived from human IgG1
8

H chain (VH-CH1), where

the last amino acid residue

in the native sequence (S)

has been substituted for T

EPKSCDKTHTCPPCP linker sequence
Derived from human IgG1
10

H chain (CH1-CH2)

TVAAPS linker sequence
Derived from human K L
7

chain (VL-CK)

ELQLEESCAEAQDGELDG linker
Derived from human IgG1
11

sequence
CH3 tether

GGGGS linker sequence
A published linker
6

sequence

Mature human IL-13 amino acid sequence (without signal sequence) is given in SEQ ID NO: 63.

Mature human IL-4 amino acid sequence (without signal sequence) is given in SEQ ID NO: 62.

1.5 Expression and Purification of mAbdAbs

The mAbdAb expression constructs described in Example 1 were transfected into one or more of CHOK1 cells, CHOE1a cells or HEK293-6E cells, expressed at small (approximately 3 mls) or medium (approximately 50 mls to 100 mls) or large (approximately 1 litre) scale and then some of the constructs were purified using immobilised Protein A columns and quantified by reading absorbance at 280 nm.

1.6 Size Exclusion Chromatography Analyses of Purified mAbdAbs

A number of mAbdAbs were analysed by size exclusion chromatography (SEC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Representative data for some of these molecules (PascoH-G4S-474, PascoL-G4S-474, PascoH-474 and PascoHL-G45-474.) are shown in FIGS. 9, 10, 11 and 12 respectively. Representative data showing SEC and SDS Page analysis for these molecules with the ‘GS’ motif removed are shown in FIGS. 90-98.

In some cases SEC was used to further purify these molecules to remove aggregates.

Example 2
Binding of mAbdAbs to Human IL-13 and Human IL-4 by ELISA
2.1 Binding of anti-IL13 mAb-anti-IL4dAbs to IL-13 and IL-4

mAbdAb supernatants, were tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 13.

FIG. 13 shows that all of these anti-IL13 mAb-anti-IL4dAbs bound IL-13. The binding activity of these mAbdAbs was also approximately equivalent (within 2-fold to 3-fold) to purified anti-human IL13 mAb alone, which was included in this assay as a positive control for IL-13 binding and in order to directly compare to the mAbdAbs. Purified anti-human IL4 mAb (Pascolizumab) was included as a negative control for IL-13 binding.

These molecules were also tested for binding to human IL-4 in a direct binding ELISA (as described in method 2). These data are shown in FIG. 14.

FIG. 14 shows that all of these anti-IL13 mAb-anti-IL4dAbs bound IL-4, but some variation in IL-4 binding activity was observed. No binding to IL-4 was observed when no anti-IL4 dAb was present in the mAbdAb construct. Purified anti-human IL13 mAb was also included as a negative control for binding to IL-4. Note that the anti-IL-4 dAbs alone were not tested in this assay as the dAbs are not detected by the secondary detection antibody; instead, purified anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay.

Purified samples of mAbdAbs, were also tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 15.

These purified anti-IL13 mAb-anti-IL4dAbs bound IL-13. The binding activity of these mAbdAbs for IL-13 was equivalent to that of purified anti-human IL13 mAb alone. An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-13 in this assay.

These purified mAbdAbs were also tested for binding to human IL-4 in a direct binding ELISA (as described in method 2). These data are shown in FIG. 16.

All of these anti-IL13 mAb-anti-IL4dAbs bound IL-4. Note that the anti-IL-4 dAbs alone were not tested in this assay as the dAbs are not detected by the secondary detection antibody; instead, purified anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay. An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-4 in this assay.

2.2 Binding of Anti-IL4 mAb-anti-IL13dAbs to IL-13 and IL-4

mAbdAb supernatants were tested for binding to human IL-4 in a direct binding ELISA (as described in method 2). These data are shown in FIG. 17 (some samples were prepared and tested in duplicate and this has been annotated as sample 1 and sample 2).

FIG. 17 shows that all of these mAbdAbs bound IL-4. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay but did not generate a binding curve as an error was made when diluting this mAb for use in the assay (Pascolizumab has been used successfully in all other subsequent IL-4 binding ELISAs). Purified anti-human IL13 mAb was included as a negative control for IL-4 binding.

The same mAbdAb supernatants were also tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 18 (some samples were prepared and tested in duplicate and this has been annotated as sample 1 and sample 2).

FIG. 18 shows that all of these anti-IL4 mAb-anti-IL13dAbs bound IL-13. Purified anti-human IL13 mAb alone was included in this assay but did not generate a binding curve as an error was made when diluting this mAb for use in the assay (purified anti-human IL13 mAb has been used successfully in all other subsequent IL-13 binding ELISAs). Purified anti-IL4 mAb (Pascolizumab) was included as a negative control for binding to IL-13. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as this dAb is not detected by the secondary detection antibody.

The purified anti-IL4 mAb-anti-IL13dAbs, ‘PascoH-G4S-474’, ‘PascoH-474’, ‘PascoL-G4S-474’ and ‘PascoHL-G4S-474’, were also tested for binding to human IL-4 in a direct binding ELISA (as described in method 2). These data are shown in FIG. 19.

These purified anti-IL4 mAb-anti-IL13dAbs bound IL-4. The binding activity of these mAbdAbs was approximately equivalent (within 2-fold) to purified anti-IL4 mAb alone (Pascolizumab). An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-4 in this assay.

These same purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, were also tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 20A.

These purified anti-IL4 mAb-anti-IL13dAbs bound IL-13. An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-13 in this assay. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb is not detected by the secondary detection antibody; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

Purified PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-ELQLE-474 were also tested for binding to cynomolgus IL-13 in a direct binding ELISA, as described in method 17 (PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed were also included in this assay, the construction of these molecules is described in Example 18). A graph showing representative data is shown in FIG. 20B.

Purified PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-ELQLE-474 all bound cynomolgus IL-13. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay as a negative control for binding to IL-13. Purified anti-human IL13 mAb was included as a positive control for cynomolgus IL-13 binding. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb is not detected by the secondary detection antibody; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

Example 3
Binding of mAbdAbs to Human IL-13 and Human IL-4 by Surface Plasmon Resonance (BIAcore™)

3.1 Binding of anti-IL13 mAb-anti-IL4dAbs to IL-13 and IL-4 by BIAcore™

mAbdAbs (in CHO cell supernatants, prepared as described in section 1.5) were tested for binding to human IL-13 using BIAcore™ at 25° C. (as described in method 4). For this data set, two IL-13 concentration curves (100 nM and 1 nM) were assessed and relative response capture levels of between 1000 and 1300 (approximately) were achieved for each mAbdAb construct. Due to the limited number of concentrations of IL-13 used, the data generated are more suitable for ranking of constructs rather than exact kinetic measurements. These data are shown in Table 6.

TABLE 6

Antibody
Binding affinity KD (nM)

586H-25
0.39

586H-G4S-25
0.41

586H-TVAAPS-25
0.5

586H-ASTKG-25
0.54

586H-EPKSC-25
0.55

586H-ELQLE-25
0.42

586H-147
0.46

586H-G4S-147
0.45

586H-TVAAPS-147
0.56

586H-ASTKG-147
0.44

586H-EPKSC-147
0.46

586H-ELQLE-147
0.51

586H-154
0.46

586H-G4S-154
0.37

586H-TVAAPS-154
0.56

586H-ASTKG-154
0.44

586H-EPKSC-154
0.42

586H-ELQLE-154
0.44

586H-210
0.44

586H-G4S-210
0.42

586H-TVAAPS-210
0.4

586H-ASTKG-210
0.4

586H-EPKSC-210
0.43

586H-ELQLE-210
0.43

586H
0.44

586H-ASTKG
0.32

586H-ELQLE
0.47

586H-EPKSC
0.45

Anti-human IL-13 mAb (purified)
0.38

Pascolizumab (purified)
no binding

All of these anti-IL13 mAb-anti-IL4dAbs bound IL-13 with similar binding affinities which were approximately equivalent to the binding affinity of purified anti-human IL13 mAb alone. These data suggested that the addition of linkers and/or anti-IL4 dAbs to the heavy chain of the anti-IL13 mAb, did not affect the IL-13 binding affinity of the mAb component within these mAbdAb constructs.

These mAbdAbs were also tested for binding to human IL-4 using BIAcore™ at 25° C. (as described in method 5). These data are shown in Table 7. For this data set, four IL-4 concentration curves (512, 128, 32 and 8 nM) were assessed and approximate relative response capture levels for each mAbdAb tested are indicated in the table. Note that the anti-IL-4 dAbs alone were not tested in this assay as the dAbs cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay.

TABLE 7

Binding

Capture
On rate
Off rate
affinity

Antibody
Level
(ka, Ms⁻¹)
(kd, s⁻¹)
KD (nM)

586H-25
864
6.13e3
4.11e−4
67

586H-G4S-25
1818
6.3e3
9.54e−4
151

586H-TVAAPS-25
673
1.27e5
1.2e−4
0.95

586H-ASTKG-25
809
5.4e5
1.20e−3
21.8

586H-EPKSC-25
748
4.79e4
1.42e−3
29.6

586H-ELQLE-25
603
1.26e6
1.63e−6
0.001*

586H-147
1095
3.42e3
1.18e−3
344.8

586H-G4S-147
1200
4.21e3
4.57e−4
108.5

586H-TVAAPS-147
433
6.62e4
6.69e−7
0.011**

586H-ASTKG-147
1248
3.67e4
6.9e−4
18.8

586H-EPKSC-147
878
2.54e4
6.71e−4
26.4

586H-ELQLE-147
676
7.01e5
1.52e−5
0.027*

586H-154
436
6.1e3
1.74e−3
285

586H-G4S-154
1437
5.00e3
6.85e−4
137.8

586H-TVAAPS-154
1530
6.44e4
1.15e−7
0.002**

586H-ASTKG-154
1373
3.26e4
2.84e−4
8.7

586H-EPKSC-154
794
3.03e4
5.7e−4
18.8

586H-ELQLE-154
795
1.25e6
3.57e−6
0.003*

586H-210
1520
not
not
—

determined
determined

586H-G4S-210
1448
not
not
—

determined
determined

586H-TVAAPS-210
1693
not
not
—

determined
determined

586H-ASTKG-210
1768
not
not
—

determined
determined

586H-EPKSC-210
1729
not
not
—

determined
determined

586H-ELQLE-210
1350
not
not
—

determined
determined

586H
1500
no binding
no binding
—

586H-ASTKG
1615
no binding
no binding
—

586H-ELQLE
343
no binding
no binding
—

586H-EPKSC
1416
no binding
no binding
—

Pascolizumab
1092
2.04e6
1.23e−4
0.060

(purified)

Caveats were observed for some of the above data sets. Poor curve fits were observed for some data sets (*), the actual binding affinity values that have been determined for these data should therefore be treated with caution. Positive dissociation was seen for some curves (**), the actual binding affinity values that have been determined for these data should therefore be treated with caution. In addition, BIAcore™ was unable (ie. not sensitive enough) to determine on and off rates for all mAbdAb constructs containing the DOM9-112-210 dAb, due to the exceptionally tight binding of these mAbdAbs to IL-4. Determination of binding kinetics for these mAbdAbs for IL-4 was further hampered by observed positive dissociation effects. These data are shown in FIG. 21.

Similar data was obtained in an additional experiment. These data are shown in FIG. 22.

These 2 independent data sets indicated that all of the anti-IL13 mAb-anti-IL4dAbs bound IL-4, but the binding affinities varied depending on the linker used to join the anti-IL4 dAb to the anti-IL13 mAb heavy chain. In this experiment, the presence of a linker was found to enhance the binding affinity for IL-4 of the anti-IL4 dAb component (when placed on the heavy chain) in the mAbdAb format. For example the molecules having TVAAPS or ELQLEESCAEAQDGELDG linkers appear to be more potent binders. No binding to IL-4 was observed when no anti-IL4 dAb was present in the mAbdAb construct. It was not possible to measure the binding affinity of the 586-linker-210 mAbdAbs for IL-4, due to the fact that the DOM9-112-210 component of these mAbdAbs binds very tightly and hence the off-rate is too small to determine using BIAcore™.

Purified anti-IL13 mAb-anti-IL4dAbs were also tested for binding to human IL-13 and human IL-4 using BIAcore™ at 25° C. (as described in methods 4 and 5). These data are shown in Table 8.

TABLE 8

Binding affinity, KD (nM)

Construct
Human IL-13
Human IL-4

586H-TVAAPS-25
0.38
1.1

586H-TVAAPS-154
0.41
0.49

586H-TVAAPS-210
0.38
very tight binder

(unable to determine KD due

to positive dissociation effects

and sensitivity level of

BIAcore ™ technique)

Anti-human IL-13 mAb
0.43
—

(purified)

Pascolizumab
—
0.03

(purified)

586H-TVAAPS-25, 586H-TVAAPS-154 and 586H-TVAAPS-210 all bound IL-13 with similar binding affinities and this was approximately equivalent to the binding affinity of purified anti-human IL13 mAb alone. 586H-TVAAPS-25, 586H-TVAAPS-154 and 586H-TVAAPS-210 all bound IL-4. It was not possible to measure the binding affinity of 586-TVAAPS-210 for IL-4, due to the fact that the DOM9-112-210 component of this mAbdAb bound very tightly and hence the off-rate was too small to determine using BIAcore™. Note that the anti-IL-4 dAbs alone (DOM9-155-25, DOM9-155-154 and DOM9-112-210) were not tested in this assay format as the dAbs cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay.

3.2 Binding of Anti-IL4 mAb-anti-IL13dAbs to IL-4 and IL-13 by BIAcore™

mAbdAbs (in CHO cell supernatants prepared as described in section 1.5) were tested for binding to human IL-4 using BIAcore™ at 25° C. (as described in method 5). These data are shown in Table 9 (some samples were prepared and tested in duplicate—this has been annotated as sample 1 and sample 2). For this data set, four IL-4 concentrations curves (100 nM, 10 nM, 1 nM and 0.1 nM) were assessed and approximate relative response capture levels for each mAbdAb tested are indicated in the table. An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-4 in this assay.

TABLE 9

Capture
On rate
Off rate
Binding affinity

Antibody
Level
(ka, Ms⁻¹)
(kd, s⁻¹)
KD (nM)

Experiment 1

PascoH-G4S-474
~500
5.1e6
8.6e−5
0.02

PascoH-TVAAPS-474
~500
5.5e6
9.7e−5
0.02

PascoH-474
~500
4.8e6
9.4e−5
0.02

PascoH-ASTKG-474
~500
5.3e6
8.6e−5
0.02

PascoH-ELQLE-474
~500
5.1e6
1.1e−4
0.02

PascoH-EPKSC-474
~500
4.9e6
9.8e−5
0.02

Pascolizumab (purified)
~700
5.3e6
1.6e−4
0.03

Experiment 2

PascoL-G4S-474 (sample 1)
1871
2.14e6
1.35e−4
0.063

PascoL-G4S-474 (sample 2)
1921
2.13e6
1.11e−4
0.052

PascoL-TVAAPS-474 (sample 1)
2796
2.48e6
2.12e−4
0.085

PascoL-TVAAPS-474 (sample 2)
3250
3.04e6
2.79e−4
0.092

PascoL-474 (sample 1)
3254
2.8e6
1.84e−4
0.065

PascoL-474 (sample 2)
2756
2.53e6
1.22e−4
0.048

PascoL-ASTKG-474 (sample 1)
3037
2.95e6
1.21e−4
0.041

PascoL-ASTKG-474 (sample 2)
3784
2.54e6
1.52e−4
0.060

PascoL-EPKSC-474 (sample 1)
3238
1.86e6
2.58e−4
0.139

PascoL-EPKSC-474 (sample 2)
3276
2.51e6
3.18e−4
0.127

Pascolizumab (purified)
1152
2.04e6
1.23e−4
0.060

Negative control mAb
2976
no binding
no binding
—

All of the anti-IL4 mAb-anti-IL13dAbs tested bound IL-4 with similar binding affinities and this was approximately equivalent to the binding affinity of the anti-human IL4 mAb alone (Pascolizumab). PascoL-EPKSC-474 bound IL-4 approximately 2-fold less potently than Pascolizumab. These data suggested that the addition of linkers and the anti-IL13 dAb to either the heavy chain or the light chain of Pascolizumab, did not overtly affect the IL-4 binding affinity of the mAb component within the mAbdAb construct.

These mAbdAbs were also tested for binding to human IL-13 using BIAcore™ at 25° C. (as described in method 4). These data are shown in Table 10 (some samples were prepared and tested in duplicate—this has been annotated as sample 1 and sample 2). For this data set, four IL-13 concentrations curves (128 nM, 32 nM, 8 nM and 2 nM) were assessed and approximate relative response capture levels for each mAbdAb tested are indicated in the table.

TABLE 10

Capture
On rate
Off rate
Binding affinity

Antibody
Level
(ka, Ms⁻¹)
(kd, s⁻¹)
KD (nM)

Experiment 1

PascoH-474
~500
3.6e5
3.1e−4
0.84

PascoH-G4S-474
~500
3.9e5
2.6e−4
0.67

PascoH-TVAAPS-474
~500
4.5e5
4.2e−4
0.94

PascoH-ASTKG-474
~500
3.1e5
4.6e−4
1.5

PascoH-ELQLE-474
~500
3.4e5
6.2e−4
1.8

PascoH-EPKSC-474
~500
3.5e5
4.0e−4
1.1

Anti-human IL-13 mAb
~650
8.6e5
4.9e−4
0.57

(purified)

Experiment 2

PascoL-474 (sample 1)
3254
2.86e5
3.82e−4
1.34

PascoL-474 (sample 2)
2756
3.12e5
3.86e−4
1.24

PascoL-G4S-474 (sample 1)
1871
5.63e5
4.25e−4
0.756

PascoL-G4S-474 (sample 2)
1921
5.59e5
3.47e−4
0.621

PascoL-TVAAPS-474 (sample 1)
2796
7.42e5
2.58e−4
0.348

PascoL-TVAAPS-474 (sample 2)
3250
6.22e5
1.71e−4
0.275

PascoL-ASTKG-474 (sample 1)
3037
5.26e5
2.38e−4
0.451

PascoL-ASTKG-474 (sample 2)
3784
5.38e5
3.20e−4
0.595

PascoL-EPKSC-474 (sample 1)
3238
4.17e5
3.34e−4
0.801

PascoL-EPKSC-474 (sample 2)
3276
3.51e5
2.86e−4
0.815

Anti-human IL-13 mAb
1373
9.12e4
6.11e−4
0.67

(purified)

Pascolizumab (purified)
1152
no binding
no binding
—

Negative control mAb
2976
no binding
no binding
—

Binding affinity data for constructs tested in experiment 2 are also shown in FIG. 23.

All of the anti-IL4 mAb-anti-IL13dAbs bound IL-13. In most cases the presence of a linker did not appear to enhance the binding affinity for IL-13 of the anti-IL13 dAb component when placed on the heavy chain of the anti-IL4 mAb. However, the presence of a linker did appear to enhance the binding affinity for IL-13 of the anti-IL13 dAb component when placed on the light chain of the anti-IL4 mAb. PascoL-TVAAPS-474 appeared to have the most potent IL-13 binding affinity in this experiment.

Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, purified anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay. An isotype-matched mAb (with specificity for an irrelevant antigen) was also included as a negative control for binding to IL-13 in this assay.

Purified anti-IL4 mAb-anti-IL13dAbs were also tested for binding to human IL-4 and human IL-13 using BIAcore™ at 25° C. (as described in methods 4 and 5). These data are shown in Table 11.

TABLE 11

Binding affinity, KD (nM)

Construct
Human IL-4
Human IL-13

PascoH-G4S-474
0.036
0.58

PascoH-474
0.037
0.71

PascoL-G4S-474
0.028
1.2

PascoHL-G4S-474
0.035
0.87

Anti-human IL-13 mAb (purified)
—
0.41

Pascolizumab (purified)
0.037
—

In this experiment PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474 all bound IL-4 with similar binding affinities and this was approximately equivalent to the binding affinity of the anti-human IL4 mAb alone (Pascolizumab). They also all bound IL-13. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

3.3 Stoichiometry of Binding of IL-13 and IL-4 to the anti-IL4 mAb-Anti-IL13dAbs using BIAcore™

Purified anti-IL4 mAb-anti-IL13dAbs were evaluated for stoichiometry of binding for IL-13 and IL-4 using BIAcore™ (as described in method 7). These data are shown in Table 12.

TABLE 12

Stoichiometry

Construct
Human IL-4
Human IL-13

PascoL-G4S-474
1.8
1.8

PascoH-G4S-474
1.8
1.9

Pasco-474
1.8
1.9

PascoHL-G4S-474
1.7
3.5

Anti-human IL-13 mAb (purified)
—
1.8

Pascolizumab (purified)
1.8
—

PascoH-G4S-474, PascoH-474 and PascoL-G4S-474 were able to bind to nearly two molecules of IL-13 and two molecules of IL-4. PascoHL-G4S-474 was able to bind nearly two molecules of IL-4 and nearly four molecules of IL-13. These data indicated that the constructs tested could be fully occupied by the expected number of IL-13 or IL-4 molecules.

Example 4
Neutralisation Potency of Mabdabs in Il-13 and Il-4 Bioassays

4.1 Anti-IL13 mAb-anti-IL4dAbs

Purified anti-IL13 mAb-anti-IL4dAbs were tested for neutralisation of human IL-13 in a TF-1 cell bioassay (as described in method 8). These data are shown in FIG. 24.

Purified anti-IL13 mAb-anti-IL4dAbs, 586H-TVAAPS-25, 586H-TVAAPS-154 and 586H-TVAAPS-210, fully neutralised the bioactivity of IL-13 in a TF-1 cell bioassay. The neutralisation potencies of these mAbdAbs were within 2-fold of purified anti-human IL-13 mAb alone. The purified anti-human IL-4 mAb (Pascolizumab) and purified anti-IL4 dAbs (DOM9-155-25, DOM9-155-154 or DOM9-112-210) were included as negative controls for neutralisation of IL-13 in this assay.

The purified anti-IL13 mAb-anti-IL4dAbs, 586H-TVAAPS-25, 586H-TVAAPS-154 and 586H-TVAAPS-210, were also tested for neutralisation of human IL-4 in a TF-1 cell bioassay (as described in method 9). These data are shown in FIG. 25.

586H-TVAAPS-210 fully neutralised the bioactivity of IL-4 in this TF-1 cell bioassay. The neutralisation potency of this mAbdAb was within 2-fold of purified anti-human IL-4 dAb alone (DOM9-112-210). 586H-TVAAPS-25 and 586H-TVAAPS-154 did not neutralise the bioactivity of IL-4 and this was in contrast to the purified anti-human IL-4 dAbs alone (DOM9-155-25 and DOM9-155-154). As demonstrated by BIAcore™, purified 586H-TVAAPS-25 and 586H-TVAAPS-154 had 1.1 nM and 0.49 nM binding affinities (respectively) for IL-4. IL-4 binds the IL-4 receptor very tightly (binding affinities of approximately 50 μM have been reported in literature publications) and thus the observation that both 586H-TVAAPS-25 or 586H-TVAAPS-154 were unable to effectively neutralise the bioactivity of IL-4 in the TF-1 cell bioassay maybe a result of the relative lower affinity of these mAbdAbs for IL-4 compared to the potency of IL-4 for the IL-4 receptor.

Purified anti-human IL-4 mAb (Pascolizumab) was included as a positive control for neutralisation of IL-4 in this bioassay. Purified anti-human IL-13 mAb was included as a negative control for neutralisation of IL-4 in this bioassay.

4.2 Anti-IL4 mAb-Anti-IL13dAbs

The purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, were tested for neutralisation of human IL-4 in a TF-1 cell bioassay (as described in method 9). These data are shown in FIG. 26.

Purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, fully neutralised the bioactivity of IL-4 in a TF-1 cell bioassay. The neutralisation potencies of these mAbdAbs were approximately equivalent to that of purified anti-human IL4 mAb alone (Pascolizumab), Purified anti-human IL-13 mAb, purified DOM10-53-474 dAb and a dAb with specificity for an irrelevant antigen (negative control dAb) were also included as negative controls for neutralisation of IL-4 in this bioassay.

The purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, were tested for neutralisation of human IL-13 in a TF-1 cell bioassay (as described in method 8). These data are shown in FIG. 27.

Purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, fully neutralised the bioactivity of IL-13 in a TF-1 cell bioassay. The neutralisation potencies of these mAbdAbs were within 3-fold of purified anti-IL13 dAb alone (DOM10-53-474). Purified anti-human IL-13 mAb was also included as a positive control for IL-13 neutralisation in this bioassay. A dAb with specificity for an irrelevant antigen (negative control dAb) and purified anti-human IL4 mAb alone (Pascolizumab) were also included as negative controls for neutralisation of IL-4 in this bioassay.

The purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, were also tested for simultaneous neutralisation of human IL-4 and human IL-13 in a dual neutralisation TF-1 cell bioassay (as described in method 10). These data are shown in FIG. 28.

Purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474, fully neutralised the bioactivity of both IL-4 and IL-13 in a dual neutralisation TF-1 cell bioassay. The neutralisation potencies of these mAbdAbs were approximately equivalent to that of a combination of purified anti-human IL4 mAb (Pascolizumab) and purified anti-IL13 dAb (DOM10-53-474). Purified anti-human IL-13 mAb alone, purified anti-human IL-4 mAb alone (Pascolizumab) and the anti-human IL-13 dAb (DOM10-53-474) alone (which were included as negative controls) did not fully neutralise the bioactivity of both IL-4 and IL-13 in this dual IL-4 and IL-13 neutralisation bioassay.

Example 5
SEC-MALLS Analysis of dAbs

Antigen-specific dAbs were characterized for their solution state by SEC-MALLS (size-exclusion chromatography—multi-angle laser light scattering) and the results are shown in Table 13: the DOM10-53-474, dAb exists as a monomer in solution whilst all DOM9 dAbs (DOM9-112-210, DOM9-155-25, DOM9-155-147 and DOM9-155-154) can form stable dimers (and in some instances tetramers).

5.1. Preparation of the Proteins

Samples were purified and dialysed into appropriate buffer e.g. PBS. Samples were filtered after dialysis and the concentration determined (0.43 mg/ml DOM-155-25), (1.35 mg/ml DOM9-155-147) and 1.4 mg/ml DOM9-155-159). DOM10-53-474 and DOM9-112-210 were adjusted to 1 mg/ml. BSA was purchased from Sigma and used without further purification.

5.2. Size-Exclusion Chromatography and Detector Set-Up

Shimadzu LC-20AD Prominence HPLC system with an autosampler (SIL-20A) and SPD-20A Prominence UV/Vis detector was connected to Wyatt Mini Dawn Treos (MALLS, multi-angle laser light scattering detector) and Wyatt Optilab rEX DRI (differential refractive index) detector. The detectors were connected in the following order—LS-UV-RI. Both RI and LS instruments operated at a wavelength of 488 nm. TSK2000 (Tosoh corporation)) column were used (silica-based HPLC column) with mobile phase of 50 mM phosphate buffer (without salt), or 1×PBS, both at pH7.4. The flow rate used is 0.5 or 1 ml/min, the time of the run was adjusted to reflect different flow rates (45 or 23 min) and was not expected to have significant impact onto separation of the molecules. Proteins were prepared in buffer to a concentration of 1 mg/ml and injection volume was 100u1.

5.3. Detector Calibration

The light-scattering detector was calibrated with toluene according to manufacturer's instructions.

5.4. Detector Calibration with BSA

The UV detector output and RI detector output were connected to the light scattering instrument so that the signals from all three detectors could be simultaneously collected with the Wyatt ASTRA software. Several injections of BSA in a mobile phase of PBS (1 ml/min) are run over a Tosoh TSK2000 column with UV, LS and RI signals collected by the Wyatt software. The traces are then analysed using ASTRA software, and the signals are normalised aligned and corrected for band broadening following manufacturer's instructions. Calibration constants are then averaged and input into the template which is used for future sample runs.

5.5. Absolute Molar Mass Calculations

100 ul of each sample were injected onto appropriate pre-equilibrated column. After SEC column the sample passes through 3 on-line detectors—UV, MALLS (multi-angle laser light scattering) and DRI (differential refractive index) allowing absolute molar mass determination. The dilution that takes place on the column is about 10 fold, and the concentration at which in-solution state was determined as appropriate.

The basis of the calculations in ASTRA as well as of the Zimm plot technique, which is often implemented in a batch sample mode is the equation from Zimm [J. Chem. Phys. 16, 1093-1099 (1948)]:

$\begin{matrix} \frac{R_{q}}{K_{c}^{*}} = M P (θ) - 2 A_{2} c M^{2} P^{2} (θ) & (Eq . 1) \end{matrix}$

where

- c is the mass concentration of the solute molecules in the solvent (g/mL)
- M is the weight average molar mass (g/mol)
- A₂is the second virial coefficient (mol mL/g²)
- K*=4p²n₀²(dn/dc)²1₀⁻⁴N_A⁻¹is an optical constant where n_ois the refractive index of the solvent at the incident radiation (vacuum) wavelength, 1₀is the incident radiation (vacuum) wavelength, expressed in nanometers, N_Ais Avogadro's number, equal to 6.022×10²³mol⁻¹, and do/dc is the differential refractive index increment of the solvent-solute solution with respect to a change in solute concentration, expressed in mL/g (this factor must be measured independently using a dRI detector).
- P(q) is the theoretically-derived form factor, approximately equal to 1-2μ²<r²>/3!+ . . . , where μ=(4π/λ) sin (θ/2), and <r²> is the mean square radius. P(q) is a function of the molecules' z-average size, shape, and structure.
- R_qis the excess Rayleigh ratio (cm⁻¹)

This equation assumes vertically polarized incident light and is valid to order c².

To perform calculations with the Zimm fit method, which is a fit to R_q/K^*c vs. sin²(q/2), we need to expand the reciprocal of Eq. 1 first order in c:

To perform calculations with the Zimm fit method, which is a fit to

Rq/K*c vs. sin 2(q/2), we need to expand the reciprocal of Eq. 1 to first order in c:

$\begin{matrix} \frac{K_{c}^{*}}{R_{q}} = \frac{1}{MP (θ)} + 2 A_{2} c & Eq . 2 \end{matrix}$

The appropriate results in this case are

$\begin{matrix} M = {(\frac{K_{c}^{*}}{R_{0}} - 2 A_{2} c)}^{- 1} and & Eq . 3 \\ 〈 r^{2} 〉 = \frac{3 m_{0} λ^{2} M}{16 π^{2}} & Eq . 4 \\ where m_{0} \equiv d [K_{c}^{*} / R_{θ}] / {d [\sin^{2} (θ / 2)]}_{θ -> 0} & Eq . 5 \end{matrix}$

The calculations are performed automatically by ASTRA software, resulting in a plot with molar mass determined for each of the slices [Astra manual].

Molar mass obtained from the plot for each of the peaks observed on chromatogram was compared with expected molecular mass of a single unit of the protein. This provides a basis to form conclusions about in-solution state of the protein. Representative data is shown in Table 13.

TABLE 13

Summary

Column

dAb
SEC-MALLS
MW
& mobile phase

DOM10-53-474
monomer
14
kDa
TSK2000, PBS,

pH 7.4, 0.5 ml/min

DOM9-112-210
dimer
30
kDa
TSK2000, PBS,

pH 7.4, 0.5 ml/min

DOM9-155-25
dimer
28
kDa
TSK2000, 50 mM,

phosphate buffer

pH 7.4, 1 ml/min

DOM9-155-147
dimer-tetramer
26-51
kDa
TSK2000, 50 mM

equilibrium

phosphate buffer,

pH 7.4, 1 ml/min

DOM9-155-159
dimer
28
kDa
TSK2000, 50 mM

phosphate Buffer,

pH 7.4, 1 ml/min

DOM10-53-474

Single peak with the average molar mass defined as ˜14 kDa indicating a monomeric state in solution, shown in FIG. 29

DOM9-112-210

Single peak with the molar mass defined as 30 kDa indicating a dimeric state in solution, shown in FIG. 30

DOM9-155-25

Nice symmetrical peak but running at the buffer front. The mid part of the peak has been used for molar mass determination (see figure below with all three signals overlaid). Molar mass is 28 kDa which represents a dimeric dAb, shown in FIG. 31. Overlay of all three signals (FIG. 32)

DOM9-155-147

The main peak is assigned with molar mass of 26 kDa over the right part of the peak and increasing steeply over the left part of the peak up to 53 kDa. The peak most likely represents a dimer and a smaller fraction of tetramer in a rapid equilibrium. A much smaller peak eluting at 7.6 min, represents tetrameric protein with molar mass of 51 kDa (FIG. 33).

DOM9-155-159

The protein runs as a single symmetric peak, with average molar mass assigned at ˜28 kDa indicating a dimeric state in solution (FIG. 34)

Control for MW Assignment by SEC-MALLS: BSA

Each BSA run for each of the experiments set out above resulted in the expected MW, e.g. 2 peaks with molar mass of 67 and 145 kDa (monomer and dimer) (FIG. 35).

Example 6
Generation of Trispecific mAbdAbs

Trispecific mAb-dAbs were constructed either by generating VH and VL sequences by assembly PCR which were then cloned into existing mAbdAb expression vectors or by sub-cloning existing VH and VL regions from mAb expression vectors into existing mAbdAb expression vectors, such that when expressed, the trispecific mAbdAb has dAbs attached to both the C-terminus of the heavy and light chains.

A linker sequence was used to join the domain antibody to heavy chain CH3 or light chain CK. A schematic diagram of a trispecific mAbdAb molecule is shown in FIG. 36 (the mAb heavy chain is drawn in grey; the mAb light chain is drawn in white; the dAbs are drawn in black).

A schematic diagram illustrating the construction of a trispecific mAbdAb heavy chain (top illustration) and a trispecific mAbdAb light chain (bottom illustration) is shown below

For the heavy chain the term ‘V_H’ is the monoclonal antibody variable heavy chain sequence; ‘CH1, CH₂and CH₃’ are human IgG1 heavy chain constant region sequences; ‘linker’ is the sequence of the specific linker region used; ‘dAb’ is the domain antibody sequence. For the light chain: ‘V_L’ is the monoclonal antibody variable light chain sequence; ‘CK’ is the human light chain constant region sequence; ‘linker’ is the sequence of the specific linker region used; ‘dAb’ is the domain antibody sequence.

A mammalian amino acid signal sequence (as shown in SEQ ID NO: 64) was used in the generation of these constructs.

6.1 Trispecific Anti-IL18 mAb-anti-IL4dAb-anti-IL13dAb

A trispecific anti-IL18 mAb-anti-IL4dAb-anti-IL13dAb (also known as IL18 mAb-210-474) was constructed by grafting a sequence encoding an anti-human IL-4 domain antibody (DOM9-112-210) onto a sequence encoding the heavy chain and a sequence encoding an anti-IL13 domain antibody (DOM10-53-474) onto a sequence encoding the light chain of an anti-human IL-18 humanised monoclonal antibody. A G4S linker was used to join the anti-IL4 domain antibody onto the heavy chain of the monoclonal antibody. A G4S linker was also used to join the anti-IL13 domain antibody onto the light chain of the monoclonal antibody.

IL18 mAb-210-474 was expressed transiently in CHOK1 cell supernatants, and following quantification of IL18 mAb-210-474 in the cell supernatant, analysed in a number of IL-18, IL-4 and IL-13 binding assays.

Name
Description
Sequence ID No.

IL18mAb-210-474
H chain = Anti-human IL-18
69 (=H chain)

mAb heavy chain-G4S linker-
70 (=L chain)

DOM9-112-210 dAb L chain =

Anti-human IL-18 mAb light

chain-G4S linker-DOM10-53-

474 dAb

6.2 Trispecific Anti-IL5 mAb-anti-IL4dAb-anti-IL13dAb

A trispecific anti-IL5 mAb-anti-IL4dAb-anti-IL13dAb (also known as Mepo-210-474) was constructed by grafting a sequence encoding an anti-human IL-4 domain antibody DOM9-112-210 (SEQ ID NO: 4) onto a sequence encoding the heavy chain of an anti-human IL-5 humanised monoclonal antibody (SEQ ID NO: 65), and grafting a sequence encoding an anti-IL13 domain antibody DOM10-53-474 (SEQ ID NO: 5) onto a sequence encoding the light chain of an anti-human IL-5 humanised monoclonal antibody (SEQ ID NO: 66). A G4S linker was used to join the anti-IL4 domain antibody onto the heavy chain of the monoclonal antibody. A G4S linker was also used to join the anti-IL13 domain antibody onto the light chain of the monoclonal antibody.

This mAbdAb was expressed transiently in CHOK1 and HEK293-6E cell supernatants, and following quantification in the cell supernatant, analysed in a number of IL-4, IL-5 and IL-13 binding assays.

Name
Description
Sequence ID No.

Mepo-210-474
H chain = Anti-human IL-5 mAb
71 (=H chain)

heavy chain-G4S linker-DOM9-112-
72 (=L chain)

210 dAb L chain = Anti-human IL-5

mAb light chain-G4S linker-DOM10-

53-474 dAb

6.3 Sequences of Monoclonal Antibodies, Domain Antibodies and Linkers

The sequences for the monoclonal antibodies, domain antibodies and linkers used to generate the trispecific mAbdAbs (or used as control reagents in the following exemplifications) are shown below in table 14.

TABLE 14

Name
Specificity
Sequence ID

DOM9-112-210 domain antibody
Human IL-4
4

DOM10-53-474 domain antibody
Human IL-13
5

GGGGS linker sequence

6

Pascolizumab (Anti-human IL-4
Human IL-4
14 (=H chain)

monoclonal antibody)

15 (=L chain)

Mepolizumab (Anti-human IL-5
Human IL-5
65 (=H chain)

monoclonal antibody)

66 (=L chain)

Anti-human IL-13 (humanised)
Human IL-13
12 (=H chain)

monoclonal antibody

13 (=L chain)

Anti-human IL-18 (humanised)
Human IL-18
67 (=H chain)

monoclonal antibody

68 (=L chain)

Mature human IL-4 amino acid sequence (without signal sequence) is given in SEQ ID NO: 62.

Mature human IL-13 amino acid sequence (without signal sequence) is given in SEQ ID NO: 63.

6.4 Expression and Purification of Trispecific mAbdAbs

DNA sequences encoding trispecific mAbdAb molecules were cloned into mammalian expression vectors (Rln, Rld or pTT) using standard molecular biology techniques. The trispecific mAbdAb expression constructs were transiently transfected into one or both of CHOK1 or HEK293-6E cells, expressed at small scale (3 mls to 150 mls). The expression procedures used to generate the trispecfic mAbdAbs were the same as those routinely used to express and monoclonal antibodies.

Some of the constructs were purified using immobilised Protein A columns and quantified by reading absorbance at 280 nm.

Example 7
Binding of Trispecific mAbdAbs to Human IL-4, Human IL-13 and Human IL-18 by ELISA

7.1 Binding of IL-18 mAb-210-474 to IL-4, IL-13 and IL-18 by ELISA Trispecific mAbdAb IL18 mAb-210-474 (supernatants) prepared as described in Example 6 (SEQ ID NO: 69 and 70), was tested for binding to human IL-18, human IL-13 and human IL-4 in direct binding ELISAs (as described in methods 112 and 3) and these data are shown in FIGS. 37, 38 and 39 respectively (IL18 mAb-210-474 was prepared and tested a number of times and this has been annotated in the figures as sample 1, sample 2, sample 3, etc).

These figures show that IL18 mAb-210-474 bound IL-4, IL-13 and IL-18 by ELISA. Purified anti-human IL18 mAb was included in the IL-18 binding ELISA as a positive control for IL-18 binding. The anti-IL-4 dAb (DOM9-112-210) was not tested in the IL-4 binding ELISA as this dAb is not detected by the secondary detection antibody; instead, purified anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this ELISA. The anti-IL-13 dAb (DOM10-53-474) was not tested in the IL-13 binding ELISA as this dAb is not detected by the secondary detection antibody; instead, purified anti-human IL-13 mAb was included as a positive control to demonstrate IL-13 binding in this ELISA. As shown in the figures, negative control mAbs to an irrelevant antigen were included in each binding ELISA.

In each ELISA the binding curve for IL18 mAb-210-474 sample 5 sits apart from the binding curves for the other IL18 mAb-210-474 samples. The reason for this is unknown however, it maybe due to a quantification issue in the human IgG quantification ELISA for this particular IL18 mAb-210-474 sample 5.

7.2 Binding of Mepo-210-474 to IL-4 and IL-13 by ELISA

Trispecific mAbdAbs Mepo-210-474 (supernatant) prepared as described in section 1 (sequence ID numbers 71 and 72), were tested for binding to human IL-13 and human IL-4 in direct binding ELISAs (as described in methods 1 and 2 respectively) and these data are shown in FIGS. 40 and 41 respectively (Mepo-210-474 was prepared and tested in quadruplicate and this has been annotated as sample 1, sample 2, sample 3 and sample 4).

These figures illustrate that Mepo-210-474 bound IL-4 and IL-13 by ELISA. The anti-IL-4 dAb (DOM9-112-210) was not tested in the IL-4 binding ELISA as this dAb is not detected by the secondary detection antibody; instead, purified anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this ELISA. The anti-IL-13 dAb (DOM10-53-474) was not tested in the IL-13 binding ELISA as this dAb is not detected by the secondary detection antibody; instead, purified anti-human IL-13 mAb was included as a positive control to demonstrate IL-13 binding in this ELISA. As shown in FIGS. 40 and 41, negative control mAbs to an irrelevant antigen were included in each binding ELISA.

Mepo-210-474 sample 1 and sample 2 were prepared in one transient transfection experiment and Mepo-210-474 sample 3 and sample 4 were prepared in another separate transient transfection experiment. All four samples bound IL-13 and IL-4 in IL-13 and IL-4 binding ELISAs. However, the reason for the different binding profiles of samples 1 and 2 verses samples 3 and 4 is unknown, but may reflect a difference in the quality of the mAbdAb (in the supernatant) generated in each transfection experiment.

Example 8
Binding of Trispecific mAbdAbs to Human IL-4, Human IL-5, Human IL-13 and Human IL-18 by Surface Plasmon Resonance (BIAcore™)

8.1 Binding of IL-18 mAb-210-474 to IL-4, IL-13 and IL-18 by BIAcore™

Trispecific mAbdAb IL18 mAb-210-474 (supernatants) prepared as described in Example 6.1 (SEQ ID NO: 69 and 70), was tested for binding to human IL-4, human IL-13 and human IL-18 using BIAcore™ at 25° C. (as described in methods 4, 5 and 6 respectively). Capture levels were within the range of approximately 400 to 850 Response Units. Three concentrations of each analyte were tested (256, 32 and 4 nM). The resulting data are shown in Table 15 (samples were prepared and tested in triplicate, this has been annotated as sample 1, sample 2 and sample 3).

TABLE 15

Binding affinity, KD

Construct
On rate (ka)
Off rate (kd)
(nM)

Binding to IL-18

IL18mAb-210-474 (sample 1)
2.1e6
2.3e−5
0.011

IL18mAb-210-474 (sample 2)
2.1e6
2.8e−5
0.014

IL18mAb-210-474 (sample 3)
2.1e6
2.9e−5
0.014

Anti-human IL-18 mAb (purified)
1.9e6
6.8e−5
0.035

Binding to IL-13

IL18mAb-210-474 (sample 1)
5.8e5
5.7e−4
0.99

IL18mAb-210-474 (sample 2)
6.2e5
6.1e−4
0.99

IL18mAb-210-474 (sample 3)
7.4e5
7.4e−4
1.0

Anti-human IL-13 mAb (purified)
1.2e6
5.0e−4
0.41

Binding to IL-4

IL18mAb-210-474 (sample 1)
—
—
very tight binder*

IL18mAb-210-474 (sample 2)
—
—
very tight binder*

IL18mAb-210-474 (sample 3)
—
—
very tight binder*

Pascolizumab (purified)
4.6e6
1.7e−4
0.037

*unable to determine KD due to positive dissociation effects and sensitivity level of BIAcore ™ technique

The trispecific mAbdab bound IL-4, IL-13 and IL-18 using BIAcore™. The binding affinity of the mAbdAb for IL-18 was approximately equivalent to that of purified anti-human IL18 mAb alone, which was included in this assay as a positive control for IL-18 binding and in order to directly compare to the binding affinity of the mAbdab. It was not possible to determine the absolute binding affinity of the mAbdab for IL-4, due to the fact that the DOM9-112-210 component of this trispecific mAbdAb bound very tightly to IL-4 and hence the off-rate was too small to determine using BIAcore™. The anti-IL-4 dAb alone (DOM9-112-210) was not tested in this assay as this dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was included as a positive control to demonstrate IL-4 binding in this assay. The anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as this dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was included as a positive control to demonstrate IL-13 binding in this assay.

8.2 Binding of Mepo-210-474 to IL-4, IL-5 and IL-13 by BIAcore™

Trispecific mAbdAb Mepo-210-474 (supernatants) prepared as described in Example 6.2 (SEQ ID NO: 71 and 72), was tested for binding to human IL-4, human IL-5 and human IL-13 using BIAcore™ at 25° C. (as described in methods 5, 11 and 4 respectively). Capture levels were within the range of approximately 550 to 900 Response Units. For IL-4 and IL-13 binding five concentrations of each analyte were tested (256, 64, 16, 4 and 1 nM). For IL-5 binding four concentrations of each analyte were tested (64, 16, 4 and 1 nM). The resulting data are shown in Table 16.

TABLE 16

Binding affinity,

Construct
On rate (ka)
Off rate (kd)
KD (nM)

Binding to IL-5

Mepo-210-474
3.34e5
1.50e−4
0.45

Mepolizumab
3.78e4
1.30e−4
0.34

(purified)

Binding to IL-13

Mepo-210-474
6.38e5
1.03e−3
1.62

Anti-human IL-13
1.51e6
5.68e−4
0.38

mAb (purified)

Binding to IL-4

Mepo-210-474
—
—
very tight binder

(unable to determine KD

due to positive

dissociation effects and

sensitivity level of

BIAcore ™ technique)

Pascolizumab
6.26e6
1.43e−4
0.02

(purified)

Mepo-210-474 bound IL-4, IL-5 and IL-13 using BIAcore™. The binding affinity of Mepo-210-474 for IL-5 was approximately equivalent to that of purified anti-human IL5 mAb (Mepolizumab) alone, which was included in this assay as a positive control for IL-5 binding and in order to directly compare to the binding affinity of Mepo-210-474. It was not possible to determine the absolute binding affinity of Mepo-210-474 for IL-4, due to the fact that the DOM9-112-210 component of this trispecific mAbdAb bound very tightly to IL-4 and hence the off-rate was too small to determine using BIAcore™. The anti-IL-4 dAb alone (DOM9-112-210) was not tested in this assay as this dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was included as a positive control to demonstrate IL-4 binding in this assay. The anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as this dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was included as a positive control to demonstrate IL-13 binding in this assay.

Example 9
Stoichiometry

9.1 Stoichiometry of Binding of IL-4, IL-13 and IL-18 to IL-18 mAb-210-474 using BIAcore™

IL18 mAb-210-474 (in CHO cell supernatants prepared as described in section 1) (SEQ ID NO: 69 and 70), were evaluated for stoichiometry of binding for IL-4, IL-13 and IL-18 using BIAcore™ (as described in method 7). These data are shown in Table 17 (R-max is the saturated binding response and this is required to calculate the stoichiometry, as per the given formulae in method 7). The concentration of each of the cytokines was 500 nM. The injection position refers to the order in which each of the cytokines was added.

TABLE 17

Cytokine
Injection position
R-max
Stoichiometry

IL-4
1st
59
0.9

IL-4
2nd
56
0.9

IL-4
3rd
51
0.8

IL-13
1st
74
1.6

IL-13
2nd
77
1.7

IL-13
3rd
80
1.8

IL-18
1st
112
1.8

IL-18
2nd
113
1.8

IL-18
3rd
110
1.7

The stoichiometry data indicated that IL18 mAb-210-474 bound approximately two molecules of IL-18, two molecules of IL-13 and only one molecule of IL-4. The anti-IL4 dAb alone (DOM9-112-210) is known to be a dimer in solution state and is only able to bind one molecule of IL-4. It is therefore not unexpected that IL18 mAb-210-474 would bind only one molecule of IL-4. These data indicated that the molecules tested could be fully occupied by the expected number of IL-18, IL-13 and IL-4 molecules. The stoichiometry data also indicated that the order of capture of the cytokines appears to be independent of the order of addition of the cytokines.

Example 10
10.1 Generation of a Dual Targeting Anti-TNF/Anti-EGFR mAbdAb

This dual targeting mAbdAb was constructed by fusion of a dAb to the C-terminus of the mAb heavy chain. The anti-TNF mAb heavy and light chain expression cassettes had been previously constructed. The restriction sites which were used for cloning are shown below (FIG. 42).

To introduce restriction sites for dAb insertion in the heavy chain, site directed mutagenesis was used to create SaII and HindIII cloning sites using the mAb heavy chain expression vector as a template. DNA coding an anti-EGFR dAb (DOM16-39-542) was then amplified by PCR (using primers coding SalI and HindIII ends) and inserted into the modified 3′ coding region, resulting in a linker of ‘STG’ (serine, threonine, glycine) between the mAb and the dAb.

Sequence verified clones (SEQ ID NO: 170 and 169) for light and heavy chains respectively) were selected and large scale were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 73 and 74)

10.2 Purification and SEC Analysis of the Dual Targeting Anti-TNF/Anti-EGFR mAbdAb

The anti-TNF/anti-EGFR mAbdAb was purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis (FIG. 43) of the purified sample shows non-reduced sample running at ˜170 kDa whilst reduced sample shows two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-TNF/anti-EGFR mAbdAb was applied onto a Superdex-200 10/30 HR column (attached to an Akta Express FPLC system) pre-equilibrated and running in PBS at 0.5 ml/min. The SEC profile shows a single species running as a symmetrical peak (FIG. 44).

10.3 Binding Affinities of the Dual Targeting Anti-TNF/Anti-EGFR mAbdAb

Binding affinities to EGFR and TNF were determined as described in methods 13 and 14 respectively. Assay data were analysed using Graph Pad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 45) of this mAbdAb was calculated to be 39.1 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 3.4 nM. In the anti-TNF bioassay (FIG. 46) the potency was of the mAbdAb was 3 pM (0.0028 nM) whilst an anti-TNF control mAb produced an EC50 of 104 pM. In conclusion, assay data shows that the construct of example 10, a dual targeting anti-TNF/anti-EGFR mAbdAb is potent against both antigens.

10.4 Rat PK of the Dual Targeting Anti-TNF/Anti-EGFR mAbdAb

This molecule was tested for its in vivo pharmacokinetic properties in the rat. The anti-TNF/anti-EGFR mAbdAb was administered i.v. to three rats, and serum samples collected over a period of 7 days (168 hours). The concentration of drug remaining at various time points post-dose was assessed by ELISA against both TNF & EGFR. Results are shown in FIG. 125.

The PK parameters confirmed that this molecule had a long half life, in the same region as that previously observed for unmodified adalimumab (125 hours). Further details are shown in Table 17.1

TABLE 17.1

Assay
Half Life
Cmax
AUC (0-inf)
Clearance
% AUC

Antigen
(hr)
(ug/mL)
(hr*ug/mL)
(mL/hr/kg)
Extrapolated

TNF
157.2
149.8
10301.3
0.5
40.6

EGFR
140.8
123.6
7986.7
0.7
35

Example 11
11.1 Generation of a Dual Targeting Anti-TNF/Anti-VEGF mAbdAb

An anti-TNF/anti-VEGF mAbdAb was produced employing a similar strategy described for example 10. For construction of the heavy chain expression cassette, vector DNA encoding the heavy chain of example 10 was taken as a starting point. The anti-EGFR dAb was excised using the restriction enzymes SalI and HindIII. DOM15-26-593, an anti-VEGF dAb was amplified by PCR (using primers coding SalI and HindIII ends) and ligated into the vector backbone which previously had the anti-EGFR dAb excised using the same restriction sites, resulting in a linker of ‘STG’ (serine, threonine, glycine) between the mAb and the dAb.

Sequence verified clones (SEQ ID NO: 169 and 168 for light and heavy chains respectively) were selected and large scale DNA preparations were made and the anti-TNF/anti-VEGF mAbdAb was expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 73 and 75).

11.2 Purification and SEC Analysis of the Dual Targeting Anti-TNF/Anti-VEGF mAbdAb

The anti-TNF/anti-VEGF mAbdAb (designated DMS4000) was purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis (FIG. 47) of the purified sample shows non-reduced sample running at ˜170 kDa whilst reduced sample shows two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-TNF/anti-VEGF mAbdAb was applied onto a Superdex-200 10/30 HR column (attached to an Akta Express FPLC system) pre-equilibrated and running in PBS at 0.5 ml/min. The SEC profile shows a single species running as a symmetrical peak (FIG. 48).

11.3 Binding Affinities of the Dual Targeting Anti-TNF/Anti-VEGF mAbdAb

Binding affinities to VEGF and TNF were determined as described in methods 12 and 14 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-VEGF potency (FIG. 49) of this mAbdAb was calculated to be 57 pM whilst the control, an anti-VEGF mAb gave an EC50 value of 366 pM. In the anti-TNF bioassay (FIG. 50) the potency was 10 pM whilst an anti-TNF control mAb produced an EC50 of 22 pM. In conclusion, assay data shows that the molecule of Example 11, a dual targeting anti-TNF/anti-VEGF mAbdAb is potent against both antigens.

11.4 Rat PK of the Dual Targeting Anti-TNF/Anti-VEGF mAbdAb

This molecule was tested for its in vivo pharmacokinetic properties in the rat. The anti-TNF/anti-VEGF mAbdAb was administered i.v. to three rats, and serum samples collected over a period of 10 days (240 hours). The concentration of drug remaining at various time points post-dose was assessed by ELISA against both TNF & VEGF. The results are shown in FIG. 126

The PK parameters confirmed that this molecule had in vivo pharmacokinetic properties that compared with those of unmodified adalimumab. The shorter observed t_1/2β for the VEGF component is not considered to be significant and may be an assay artifact. Further details are shown in Table 17.2

TABLE 17.2

Half Life
Cmax
AUC (0-inf)
Clearance
% AUC

Antigen
(hr)
(μg/mL)
(hr* μg/mL)
(mL/hr/kg)
Extrapolated

TNF
180.1
89.9
7286.3
0.7
35.8

VEGF
94.2
102.8
4747.1
1.1
14.3

11.5 Generation of an Alternative Anti-TNF/Anti-VEGF mAbdAb

An alternative anti-TNF/anti-VEGF mAbdAb was constructed in a similar way to that described above in Example 11.1, using the same anti-TNF mAb linked to a VEGF dAb on the C-terminus of the heavy chain using an STG linker. The anti-VEGF dAb used in this case was DOM15-10-11. This molecule was expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 73 and 185). This molecule expressed to give a mAbdAb of similar expression levels to that described in Example 11.2, however when tested for potency in the same VEGF assay as described in Example 11.3 it was found to have undetectable levels of inhibition of VEGF binding to VEGF receptor in this assay.

Example 12
12.1 Generation of a Dual Targeting anti-VEGF/anti-IL1R1 dAb-Extended IgG

Two dual targeting dAb-extended IgG molecules were constructed using standard molecular biology techniques following a strategy of insertion of Dummy V domain coding regions in between dAb and constant regions of both chains.

For the light chain, the anti-IL1R1 dAb DOM4-130-54 was previously cloned into an expression cassette with SalI and BsiWI sites (FIG. 51) to produce a dAb-Ck chain. To produce the dAb-extended IgG light chain, Dummy Vk region was amplified by PCR with primers coding BsiWI on both ends. Plasmid containing the dAb-Ck expression cassette was digested with BsiWI. BsiWI digested Dummy Vk domain was ligated into this to produce the dAb-extended IgG light chain, with a linker of TVAAPS' between the two variable domains.

An identical strategy was followed to produce the dAb-extended IgG heavy chain where the PCR amplified Dummy VH domain with NheI ends was ligated into an NheI digested dAb heavy chain in between the dAb DOM15-26 and CH1 (FIG. 51), with a linker of ‘ASTKGPS’ between the two variable domains. This is designated DMS2090 and has the sequence set out in SEQ ID NO: 163 (DNA sequence SEQ ID NO: 162). This was paired with the light chain set out in SEQ ID NO: 77 (DNA sequence SEQ ID NO: 171).

A second heavy chain was constructed in the same way but using the dAb DOM15-26-593. This is designated DMS2091, and has the sequence set out in SEQ ID NO: 76 (DNA sequence SEQ ID NO: 172). This was paired with the light chain set out in SEQ ID NO: 77 (DNA sequence SEQ ID NO: 171).

Sequence verified clones were selected and large scale DNA preparations were made using Qiagen Maxi or Mega Prep Kits following the manufacturer's protocols. The resulting construct was expressed in mammalian cells using transient transfection techniques by co-transfection of light and heavy chains.

12.2 Purification and SEC Analysis of the Dual Targeting Anti-VEGF/Anti-IL1R1 mAbdAb

Both anti-IL1R1/anti-VEGF dAb-extended IgG molecules were purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis for DMS2090 is shown in FIG. 52, and for DMS2091 is shown in FIG. 53. Both purified samples show non-reduced samples running at 190 kDa whilst the reduced samples show two bands running at 35 and 60 kDa corresponding to dAb-extended light chain and heavy chains respectively.

For size exclusion chromatography (SEC) analysis the anti-VEGF/anti-IL1R1 dAb-extended-IgG was applied onto a Superdex-200 10/30 HR column (attached to an Akta Express FPLC system) pre-equilibrated and running in PBS at 0.5 ml/min. The SEC profiles for DMS2090 (FIG. 54) and DMS2091 (FIG. 55) both show a single species running as a symmetrical peak.

12.3 Binding Affinities of the Dual Targeting Anti-VEGF/anti-IL1R1 mAbdAb

Binding affinities to VEGF and IL1R1 were determined as described in methods 12 and 15 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-VEGF potency (FIG. 57) of DMS2090 was calculated to be 158.4 pM whilst the control, an anti-VEGF mAb gave an EC50 value of 689.2 pM. Anti-VEGF potency (FIG. 56) of DMS2091 was calculated to be 55 pM whilst the control, an anti-VEGF mAb gave an EC50 value of 766 pM.

In the anti-IL1R1 bioassay the potency of DMS2090 (FIG. 58) was 32 pM whilst an anti-IL1R1 control mAb produced an EC50 of 35 pM. The potency of DMS2091 (FIG. 59) was 17.47 pM whilst an anti-IL1-R1 control mAb produced an EC50 of 35.02 pM.

In conclusion, assay data shows that example 12, a dual targeting anti-IL1R1/anti-VEGF dAb-extended IgG is potent against both antigens.

12.4 Rat PK of the Dual Targeting Anti-VEGF/Anti-IL1R1 mAbdAb

This molecule was tested for its in vivo pharmacokinetic properties in the rat. The anti-IL1R1/anti-VEGF dAb-extended IgG A was administered i.v. to three rats, and serum samples collected over a period of 7 days (168 hours). The concentration of drug remaining at various time points post-dose was assessed by ELISA against both IL1R1 & VEGF. The results are shown in FIG. 127

The PK parameters are shown in Table 17.3

TABLE 17.3

Assay
Half Life
Cmax
AUC (0-inf)
Clearance
% AUC

Molecule
Antigen
(hr)
(ug/mL)
(hr*ug/mL)
(mL/hr/kg)
Extrapolated

DMS2090
VEGF
72.1
100.4
4811.6
1.1
19

DMS2090
IL-1R1
86.3
87.7
3467.4
1.6
23.7

Example 13
13.1 Generation of a Triple Targeting Anti-TNF/Anti-VEGF/Anti-EGFR mAbdAb

A triple targeting mAb was constructed using standard molecular biology techniques and following a strategy of insertion of mAb V domain coding regions in between dAb and constant regions of both chains.

For the light chain, the anti-EGFR dAb DOM16-39-542 was previously cloned into an expression cassette with SalI and BsiWI sites (FIG. 15) to produce a dAb-Ck chain. To produce the mAbdAb light chain, the mAb VL region was amplified by PCR with primers coding BsiWI on both ends. Plasmid containing the dAb-Ck expression cassette was digested with BsiWI. BsiWI digested mAb VL domain was ligated into this to produce the mAbdAb light chain, resulting in a linker of TVAAPS' between the two variable domains.

An identical strategy was followed to produce the mAbdAb heavy chain where the PCR amplified mAb VH region with NheI ends was ligated into an NheI digested dAb heavy chain vector in between the dAb (DOM15-26) and CH1 (FIG. 60), resulting in a linker of ‘ASTKGPS’ between the two variable domains.

Sequence verified clones (amino acid SEQ ID NO: 78 and 79 for heavy and light chains respectively) were selected and large scale DNA preparations were made using Qiagen Mega Prep Kits following the manufacturer's protocols. mAbdAbs were expressed in mammalian cells using transient transfection techniques by co-transfection of light and heavy chains.

13.2 Purification of the Triple Targeting Anti-TNF/Anti-VEGF/Anti-EGFR mAbdAb

The anti-TNF/anti-VEGF/anti-EGFR mAbdAb was purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis (FIG. 61) of the purified sample shows non-reduced sample running at 190 kDa whilst reduced sample shows two bands running at 35 and 60 kDa corresponding to dAb-extended light chain and heavy chains respectively.

13.3 Binding Affinities of the Triple Targeting Anti-TNF/Anti-VEGF/Anti-EGFR mAbdAb

Binding affinities to VEGF, EGFR and TNF were determined as described in methods 12, 13 and 14 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-VEGF potency (FIG. 62) of this mAbdAb was calculated to be 1.885 mM whilst the control, an anti-VEGF mAb gave an EC50 value of 0.145 nM.

In the anti-TNF bioassay (FIG. 63) the potency was 87 pM whilst an anti-TNF control mAb produced an EC50 of 104 pM. In the anti-EGFR assay (FIG. 64) the triple targeting mAbdAb produced ˜20% inhibition at ˜300 nM. Whilst EC50 values were calculated for VEGF and TNF binding, for EGFR binding a full curve was not produced to calculate an EC50 value.

Example 14
IGF-1R/VEGF mAbdAb
14.1 Construction of IGF-1R/VEGF mAbdAb

Anti IGF-1R variable heavy and variable light gene sequences were originally built de novo from PCR of overlapping oligonucleotides. These regions were fused to human IgG1 or kappa constant regions in a mammalian expression vector using standard molecular biology techniques. The gene sequence for an anti VEGF domain antibody was likewise constructed by PCR using overlapping oligonucleotides and fused to the 3′ end of either the heavy or light chain genes of the anti IGF-1R components described above. The fusion incorporated either a two amino acid (GS) linker or an 8 amino acid (TVAAPSGS) linker between the antibody and the domain antibody components. Antibody heavy and light chains were also constructed without the domain antibody and linker sequences. Sequence verified clones were selected for large scale DNA preparations using Endofree Qiagen Maxiprep kit following the manufacturer's protocols.

In sequences SEQ ID NO: 108, 109, 111 and 112, the position of the linker sequence between the antibody and domain antibody is underlined. Alternative variants could be constructed by removing the linker entirely or by using different linkers. Examples of other suitable linkers are provided in SEQ ID NO: 6 and 8 to 11. Table 18 provides a list of the antibodies constructed and expressed.

TABLE 18

Antibodies constructed and tested

SEQ ID
SEQ ID

NO
NO

for
for

Alternative

Domain

heavy
light
Site of

Identifier
names
Antibody
antibody
Linker
chain
chain
fusion

BPC1603
381H
Anti-IGF-
Dom15-
TVAAPSGS
108
113
Heavy

TVAAPSGS
1R
26-593

chain C

593,
antibody
anti-VEGF

terminus

DMS4019
H0L0

BPC1604
381H GS
Anti-IGF-
Dom15-
GS
109
113
Heavy

593,
1R
26-593

chain C

DMS4020
antibody
anti-VEGF

terminus

H0L0

BPC1605
381L
Anti-IGF-
Dom15-
TVAAPSGS
110
111
Light

TVAAPSGS
1R
26-593

chain C

593,
antibody
anti-VEGF

terminus

DMS4021
H0L0

BPC1606
381L GS
Anti-IGF-
Dom15-
GS
110
112
Light

593,
1R
26-593

chain C

DMS4022
antibody
anti-VEGF

terminus

H0L0

Example 14.2
Expression, Purification and SEC Profile of IGF-1R/VEGF mAbdAb

Combinations of the heavy and light chain vectors expressed in transient transfections of HEK293-6E. Briefly, 50 ml of HEK293-6E cells at 1.5×10⁶cells/ml were transfected with 25 μg of heavy and 25 μg of light chain plasmid previously incubated with 293fectin reagent (Invitrogen # 51-0031). Cells were placed in a shaking incubator at 37° C., 5% CO₂, and 95% relative humidity. After 24 hours, tryptone feeding media was added and the cells grown for a further 72 hours. Supernatant was harvested by centrifugation followed by filtration using a 0.22 μm filter. The expressed protein was purified using a Protein A sepharose column and dialysed into PBS. Purified protein was analysed by size exclusion chromatography (SEC) and is shown in FIG. 65.

An IGF-1R antibody (H0L0) was used as a comparator in the following assays. This molecule has the heavy chain sequence set out in SEQ ID NO: 110, and the light chain sequence set out in SEQ ID NO: 113.

Another mAbdAb with irrelevant specificity was used as a comparator in the following assays. This molecule has the heavy chain sequence set out in SEQ ID NO: 87, and the light chain sequence set out in SEQ ID NO: 13 and is designated BPC1601.

Example 14.3
IGF-1R Binding ELISA

A binding ELISA was carried out to test the binding of the purified anti-IGF-1R/VEGF mAbdAbs to IGF-1R. Briefly, ELISA plates coated with anti-polyhistidine (AbCam AB9108) at 1 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline) were loaded with 400 ng/ml recombinant human IGF-1R-his tag (R&D Systems 305-GR) in PBS. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Various concentrations of the purified mAbdAbs were added as well as an anti IGF-1R monoclonal antibody (HOLO) and an irrelevant mAbdAb (BPC1601), diluted in blocking solution. The plate was incubated for 1 hour at room temperature before washing in TBST. Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results of the binding ELISA are presented in FIG. 66 and confirm that all the IGF1R-VEGF mAbdAb variants tested (BPC1603-1606) show binding to IGF-1R at levels comparable to the anti-IGF-1R antibody H0L0. EC50 values were calculated using Cambridgesoft Bioassay software and are as follows: H0L0 (0.1797 μg/ml)), BPC1603 (0.1602 μg/ml), BPC1604 (0.1160 μg/ml), BPC1605 (0.1975 μg/ml), BPC1606 (0.1403 μg/ml). The irrelevant control bispecific antibody BPC1601 showed now detectable binding to IGF-1R.

Example 14.4
VEGF Binding ELISA

A binding ELISA was carried out to test the binding of the purified anti IGF-1R/VEGF bispecific antibodies to VEGF. ELISA plates were coated with recombinant human VEGF (GSK) at 400 ng/ml in PBS and then blocked in blocking solution (4% BSA in TBS). Various concentrations of the purified mAbdAbs diluted in blocking solution were added and mAbdAb BPC1601 was included as a negative control. The plate was incubated for 1 hour at room temperature before washing in TBST. Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 40 minutes at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results of the binding ELISA are presented in FIG. 67 and confirm that all four anti-IGF-1R/VEGF mAbdAbs (BPC1603-1606) can bind to immobilised VEGF. The apparent lower binding activity of BPC1605 and BPC1606 may be attributable to interference between the domain antibody (located at the C-terminus of the light chain) and the detection antibody. EC50 values were calculated using Cambridgesoft Bioassay software and are as follows: BPC1603 (0.044 μg/ml), BPC1604 (0.059 μg/ml), BPC1605 (0.571 μg/ml). It was not possible to calculate an accurate EC50 value for BPC1606 due to the lower response values. The anti-IGF-1R antibody H0L0 and the irrelevant control mAbdAb BPC1601 showed now detectable binding to VEGF.

Example 14.5
Kinetics of Binding to VEGF

A mouse monoclonal against human IgG (Biacore BR-1008-39) was immobilised by primary amine coupling to a CM5 biosensor chip. The antibody constructs were captured using this surface. After capture VEGF was passed over the surface which was then regenerated using 3M MgCl₂. The concentrations of VEGF used to generate kinetics were 256, 64, 16, 4, 1 and 0.25 nM, with a buffer only injection over the captured surface used for double referencing. The experiments were carried out on the Biacore T100 machine, using 1×HBS-EP buffer (BR-1006-69) at 25° C. The data were fitted to the 1:1 model inherent to the machine in its analysis software. The data shown in Table 19 is from two independent experiments.

TABLE 19

Kinetics of binding to human VEGF

Experiment 1
Experiment 2

KD

KD

Construct
ka
kd
(nM)
ka
kd
(nM)

BPC1603
2.398E+6
2.762E−4
0.115
1.334E+6
3.497E−4
0.262

BPC1604
9.933E+5
3.092E−4
0.311
5.806E+5
3.266E−4
0.563

BPC1605
1.599E+6
2.161E−4
0.135
1.089E+6
2.727E−4
0.251

BPC1606
4.343E+5
1.573E−4
0.362
2.717E+5
1.607E−4
0.591

Example 14.6
Inhibition of VEGF Binding to Receptor

The activity of the mAbdAbs was measured using a VEGF receptor binding assay as described in Method 12. The IC50s obtained in this assay for inhibition of the binding of VEGF to VEGFR2 are:

BPC1603 (0.037 nM)
BPC1604 (0.010 nM)
BPC1605 (0.167 nM)
BPC1606 (0.431 nM)

These results confirm that all four antigen binding constructs inhibit ligand binding to receptor.

Example 14.7
Inhibition of IGF-1R Receptor Phosphorylation

3T3/LISN c4 cells were plated at a density of 10 000 cells/well into 96 well plates and incubated overnight in complete DMEM (DMEM-Hepes modification+10% FCS). Purified mAbdAbs were added to the cells and incubated for 1 hour. rhIGF-1 was added to the treated cells to achieve a final concentration of 50 ng/ml and incubated for a further 30 mins to stimulate receptor phosphorylation. The media was aspirated and then the cells lysed by the addition of RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl, 6 mM Na Deoxycholate, 1% Tween 20) plus protease inhibitor cocktail (Roche 11 697 498 001). The plate was frozen overnight. After thawing, lysate from each well was transferred to a 96 well ELISA plate pre-coated with an anti IGF-1R capture antibody 2B9 (GSK) at 2 μg/ml and blocked with 4% BSA/TBS. The plate was washed with TBST (TBS+0.1% Tween 20) and a Europium labelled anti Phosphotyrosine antibody (PerkinElmer DELFIA Eu-N1 PT66) diluted 1/2500 in 4% BSA/TBS was added to each well. After 1 hour incubation the plate was washed and DELFIA Enhancement (PerkinElmer 1244-105) solution added. After 10 min incubation the level of receptor phosphorylation was determined using a plate reader set up to measure Europium time resolved fluorescence (TRF).

The results of the experiment are presented in FIGS. 68 and 69. The results confirm that the mAbdAbs BPC1603-1606 can inhibit IGF-I mediated receptor phosphorylation at levels comparable to the anti-IGF-1R monoclonal antibody H0L0. an irrelevant antibody (labelled as IgG1, Sigma I5154) showed no activity in this assay.

Example 15 anti-CD20/IL-13 Antigen Binding Protein
Example 15.1
Molecular Biology

The mammalian expression vectors encoding the heavy and light chain sequences of an anti-CD20 mAb set out in SEQ ID NO: 117 and 120 were constructed de novo using a PCR based approach and standard molecular biology techniques. Bispecific anti-CD20 mAb-anti-IL13dAb heavy and light chains were constructed by cloning the sequences encoding anti-CD20 mAb heavy and light variable regions into mammalian expression vectors containing human antibody constant regions fused to an anti-human IL-13 domain antibody (DOM10-53-474).

The mAbdAb expression constructs were transfected into CHOE1a cells. The supernatant was harvested and then the antibody purified using immobilised Protein A and quantified by reading absorbance at 280 nm. The mAbdAbs (and the anti-CD20 control mAb) constructed and tested are listed in Table 20.

TABLE 20

Seq ID:
Seq ID:

Alternative
Antibody
Domain

for heavy
for light
Site of

Identifier
names
Target
antibody
Linker
chain
chain
fusion

BPC1401
RituxanH-
CD20
DOM10-
TVAAPSGS
116
117
Heavy chain

TVAAPSGS-

53-474

C -term

DOM474

BPC1402
RituxanH-
CD20
DOM10-
GS
118
117
Heavy chain

GS-DOM474

53-474

C -term

BPC1403
RituxanL-
CD20
DOM10-
TVAAPSGS
120
119
Light chain

TVAAPSGS-

53-474

C -term

DOM474

BPC1404
RituxanL-
CD20
DOM10-
GS
120
121
Light chain

GS-DOM474

53-474

C -term

anti-CD20
Rituxan
CD20
—
—
120
117
—

mAb

Example 15.2
Kinetics of Binding to Human IL-13

The binding affinity of mAbdAb constructs for human IL-13 were assessed by BIAcore™ analysis. Analyses were carried out anti-human IgG capture. Briefly, Anti-human IgG (Biacore BR-1008-39) was coupled onto a CM5 chip by primary amine coupling. MAbdAb constructs were then captured onto this surface and human IL-13 (made and purified at GSK) passed over at defined concentrations. The surface was regenerated back to the Anti-human IgG surface using 3M MgCl₂. This treatment did not significantly affect the ability to capture antibody for a subsequent IL-13 binding event. The runs were carried out at 25° C. using HBS-EP buffer, on the BIAcore™ T100 machine. Data were analysed using the evaluation software in the machine and fitted to the 1:1 binding model. The results of the analysis are presented in Table 48, confirming that for all mAbdAb constructs, the kinetics of binding to IL-13 are comparable.

TABLE 48

Surface plasmon resonance (BIAcore ™) data

Antibody name
Ka (M⁻¹· s⁻¹)
Kd (s⁻¹)
KD (nM)

BPC1401
5.81E+5
1.82E−4
0.313

BPC1402
8.52E+5
3.05E−4
0.358

BPC1403
1.07E+6
2.95E−4
0.277

BPC1404
4.99E+5
5.08E−4
1.02

PascoH-474 GS removed
6.29E+5
2.66E−4
0.423

anti-CD20 mAb
No binding detected

Binding of the mAb-dAbs to CD20 was assessed by flow cytometry using a CD20 positive cell line (Wein133). All mAb-dAbs (BPC1401-BPC1404) and the anti-CD20 control antibody showed a dose dependent increase in mean fluorescence intensity (MFI) (data not shown).

Example 15.3
ADCC Assay with Anti-CD20/1L-13 Bispecific Antibody

The ADCC assay was based on the published method of Boyd et al. (1995) J. Imm. Meth. 184:29-38. Briefly, Raji cells (targets) were labelled with Europium as follows. Cells were harvested, counted and prepared to a final density of 1×10⁷in a 15 ml falcon tube, wash once with Hepes buffer (50 mM HEPES, 83 mM NaCl, 5 mM KCl, 2 mM MgCl₂.H₂0, pH7.4). The cells were pelleted and 1 ml of ice cold Europium labelling buffer (HEPES buffer plus 600 μM EuCl₃, 3 mM DTPA and 25 mg/ml Dextran sulphate) was added to each tube. The cell suspension was flicked vigorously at the start of the labelling and then every 10 minutes during the 30 minute incubation period on ice. 10 ml ice cold repair buffer (Hepes buffer containing 294 mg/l CaCl₂.2H₂O, 1.8 g/l D-Glucose, pH7.4) was added and the cells incubated on ice for a further 10 minutes. The cells were then centrifuged, the supernatant decanted and washed twice with repair buffer and then once with complete medium. The labelled cells were then counted and resuspended in serum free medium at 2×10⁵cells/ml and stored on ice.

Human purified blood mononuclear cells (PBMCs or effector cells) were prepared as follows. 150 mls of whole blood was centrifuged at 2000 rpm for 10 mins to remove the serum. The cells were the diluted to twice the original volume with PBS (Invitrogen/Gibco, #14190). Accuspin density gradient tubes (Sigma, #A2055-10EA) were prepared by adding 15 ml lymphoprep (Axis shield, #NYC1114547) and centrifuged for 1 min at 1500 rpm. 25 ml of blood suspension was added to the density gradient tubes and centrifuged for 20 min at 2500 rpm with the centrifuge brake off. The top 10 ml of supernatant was discarded. The remainder (including the “buffy” layer) was poured into a clean tube, topped up with PBS and centrifuged at 1500 rpm for 5 mins. The supernatant was discarded, the cell pellets pooled, wash once in RPMI medium, recentrifuged and counted. Effector cells at were prepared at 5×10⁶/ml in serum free RPMI medium.

The assay plates were set up in 96-well round bottom plates (Nunc 96 maxisorb plate, #735-0199) as follows. Antibody dilutions were made in serum free RPMI medium at a starting concentration of approximately 12 m/ml and eleven further 3-fold dilutions. Using the plate layout below, 50 μl antibody sample was added to the appropriate wells (rows B-G only), allowing 6 replicates per dilution). 50 μl RPMI medium was added to all wells in rows A and H. 50 μl of RPMI medium was added to all wells in plates labelled medium. 50 μl recombinant human IL-13 diluted in RPMI medium to 4 μg/ml (1 μg/ml final concentration, GSK in-house material) was added to all wells in plates labelled+IL13. All plates were incubated at 4° C. for minimum 30 minutes. 50 μl of Europium labelled target cells were added to all plates. 20 μl of a 10× triton was added to all wells in row H on all plates. Plates were incubated 4° C. for a minimum of 30 minutes. 50 μl RPMI medium was added to all wells in columns labelled targets alone. 50 μl PBMCs was added to all wells in columns labelled effector:targets to give a 25:1 ratio. The plates were centrifuged at 1500 rpm for 3 mins and incubated at 37° C. for 3-4 hrs. 200 μl of enhancement solution (Wallac/Perkin Elmer, Catalogue# 1244-105) was added to each well of a nunc immunosorbant ELISA plates (one ELISA plate for each assay plate). 20 μl of supernatant was transferred from assay plate to ELISA plate. The ELISA plates were incubated at room temperature on plate shaker for a minimum 30 minutes or stored over night at 4° C. Europium release is measured using time-delayed fluorimetry (Wallac Victor plate reader). Spontaneous lysis=measurement of Europium released from cells and medium alone. Maximum lysis=non-specific lysis of target cells by addition of Triton-X100 (non-ionic detergent).

Effector:Targets
Targets
Effector:Targets
Targets

1
2
3
4
5
6
7
8
9
10
11
12

A
Spontaneous

Spontaneous

release

release

B
3 μg/ml

0.003 μg/ml

C
1 μg/ml

0.001 μg/ml

D
0.3 μg/ml

0.0003 μg/ml

E
0.1 μg/ml

0.0001 μg/ml

F
0.03 μg/ml

0.00003 μg/ml

G
0.01 μg/ml

0.00001 μg/ml

H
Maximum

Maximum

release

release

The ADCC assay was performed on two separate occasions using two different donor PBMCs. The results from one representative assay are presented in FIGS. 70 and 71. In addition, a similar ADCC assay using a shorter dose range was performed on a separate occasion using different donor PBMCs. The results from this assay are presented in FIGS. 72 and 73.

Example 15.4
CDC Assay with Anti-CD20/IL-13 Bispecific antibody

WEIN cells (targets) were labelled with Europium as follows. Briefly, cells were harvested, counted and prepared to a final density of 1×10⁷in a 15 ml falcon tube, wash once with Hepes buffer (50 mM HEPES, 83 mM NaCl, 5 mM KCl, 2 mM MgCl₂.H₂O, pH7.4). The cells were pelleted and 1 ml of ice cold Europium labelling buffer (HEPES buffer plus 600 μM EuCl₃, 3 mM DTPA and 25 mg/ml Dextran sulphate) was added to each tube. The cell suspension was flicked vigorously at the start of the labelling and then every 10 minutes during the 30 minute incubation period on ice. 10 ml ice cold repair buffer (Hepes buffer containing 294 mg/l CaCl₂.2H₂O, 1.8 g/l D-Glucose, pH7.4) was added and the cells incubated on ice for a further 10 minutes. The cells were then centrifuged, the supernatant decanted and washed twice with repair buffer and then once with complete medium. The labelled cells were then counted and resuspended in serum free medium at 2×10⁵cells/ml and stored on ice.

Serum was removed from whole blood collected from in house donors by centrifugation. Half of the sample was inactivated by heat treatment at 56° C. for 30 mins. Antibodies samples were diluted in serum free RPMI medium, starting a 12 μg/ml with five further 3-fold dilutions. 50 μl antibody sample was added to appropriate wells in rows B-G only (as per the plate layout below). 50 μl RPMI medium was added to all wells in columns 1 to 6. Where indicated, 50 μl recombinant human IL-13 (at 4 μg/ml in RPMI medium) was added to all wells in columns 7 to 12. The plates were incubated at 4° C. for a minimum 30 minutes. 50 μl of Europium labelled target cells were added to all the plates and the plates incubated at 4° C. for minimum of 30 minutes. 50 μl of serum (active or heat-inactivated) was added to the appropriate wells (see plate layout below). The plates were incubated at 37° C. incubator for 2-3 hrs, after which time the plates were centrifuged at 1500 rpm for 3 mins. 200 μl of enhancement solution (Wallac/Perkin Elmer, Catalogue# 1244-105) was added to each well of a Nunc immunosorbant ELISA plates (one ELISA plate for each assay plate). 20 μl of supernatant was transferred from assay plate to ELISA plate. The ELISA plates were incubated at room temperature on plate shaker for a minimum 30 minutes or stored over night at 4° C. Europium release is measured using time-delayed fluorimetry (Wallac Victor plate reader). Spontaneous lysis=measurement of Europium released from cells and medium alone. Maximum lysis=non-specific lysis of target cells by addition of Triton-X100 (non-ionic detergent).

MEDIUM
IL13

Active

Active

complement
Heat treated
complement
Heat treated

1
2
3
4
5
6
7
8
9
10
11
12

A
Spontaneous

release

B
3 μg/ml

C
1 μg/ml

D
0.3 μg/ml

E
0.1 μg/ml

F
0.03 μg/ml

G
0.01 μg/ml

H
Maximum

release

The CDC assay was performed on three separate occasions using three different donor sera. The results from one representative assay are presented in FIGS. 74 and 75 and show that the CDC activity of the antibody samples is comparable in the absence of IL-13. In the presence of excess IL-13, the CDC activity of antibody samples BPC1401 and BPC1402 (domain antibody fused to the heavy chain) is reduced whilst the CDC activity of BPC1403 and BPC1404 (domain antibody fused to the light chain) is largely unaffected by the presence of IL-13.

Example 16
16.1 Design and Construction of Antigen Binding Proteins Comprising Epitope Binding Domains Composed of Alternative Scaffolds

Five alternative scaffolds, listed below, were combined with monoclonal antibodies to provide mAb—alternative scaffold bispecific molecules:

- anti VEGF tear lipocalin (TLPC)
- anti HER2Affibody (AFFI)
- anti HER2DARPin (DRPN)
- anti hen egg white lysozyme (NARV)
- anti-RNaseA Camelid VHH

The protein sequences of TLPC (for further information see US2007/0224633), AFFI (for further information see WO2005003156A1), DRPN (for further information see Zahnd, C. et al. (2007), J. Mol. Biol., 369, 1015-1028) and NARV (for further information see US20050043519A) were reverse-translated to DNA and codon optimised. A BamHI site at the N-terminus and EcoR1 site at the C-terminus were included on each of these four alternative scaffolds to facilitate cloning.

DNA fragments encoding the four final alternative scaffold DNA sequences were constructed de novo using a PCR-based strategy and overlapping oligonucleotides. The TLPC, AFFI and DRPN PCR products were cloned into mammalian expression vectors containing the heavy chain of H0L0, an anti-hIGF-1R antibody. The resulting DNA sequences encode the alternative scaffolds fused onto the C-terminus of the heavy chain via a TVAAPSGS linker or GS linker. The NAR V PCR product was cloned into mammalian expression vectors containing DNA encoding the heavy chain of Pascolizumab (an anti-IL-4 antibody). The resulting DNA sequence encodes the NAR V fused onto the C-terminus of the heavy chain via a GS linker.

An anti-RNAse A camelid VHH DNA sequence was modified by PCR to include a BamHI site at the 5′ end and an EcoR1 site at the 3′ end in order to facilitate cloning. The PCR product was cloned into mammalian expression vectors containing the heavy chain of Pascolizumab, an anti-IL4 antibody. The resulting DNA sequence encodes a camelid VHH fused onto the C-terminus of the heavy chain via a GS linker.

Table 21 below is a summary of the antigen binding proteins that have been constructed.

TABLE 21

SEQ ID

NO:

amino

Antibody

acid

ID
Description
sequence

BPC1803
antiIGF1R Heavy Chain-GS-TLPC
123

antiIGF1R Light Chain
113

BPC1804
antiIGF1R Heavy Chain-
125

TVAAPSGS-TLPC

antiIGF1R Light Chain
113

BPC1805
antiIGF1R Heavy Chain-GS-AFFI
126

antiIGF1R Light Chain
113

BPC1806
antiIGF1R Heavy Chain-
127

TVAAPSGS-AFFI

antiIGF1R Light Chain
113

BPC1807
antiIGF1R Heavy Chain-GS-DRPN
128

antiIGF1R Light Chain
113

BPC1808
antiIGF1R Heavy Chain-
129

TVAAPSGS-DRPN

antiIGF1R Light Chain
113

BPC1809
Anti IL-4 heavy Chain-GS-anti
130

RNAse A camelidVHH

Anti IL-4 Light Chain
15

BPC1816
Anti IL-4 heavy Chain-GS-NARV
131

Anti IL-4 Light Chain
15

Expression plasmids encoding the heavy and light chains of the antigen binding proteins set out in Table 21 were transiently co-transfected into HEK 293-6E cells using 293fectin (Invitrogen, 12347019). A tryptone feed was added to each of the cell cultures the same day or the following day and the supernatant material was harvested after about 2 to 6 days from initial transfection. The antigen binding protein was purified from the supernatant using a Protein A column before being tested in binding assays.

16.2: rhIGF-1R Binding ELISA

96-well high binding plates were coated with 1 μg/ml of anti-his-tag antibody (Abcam, ab9108) in PBS and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. 0.4 μg/mL of rhIGF-1R(R&D systems) was added to each well at 50 μL per well. The plates were incubated for an hour at room temperature and then washed. The purified antigen binding proteins/antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIGS. 76, 78 and 80 show the ELISA results and confirm that antigen binding proteins BPC1803-BPC1808 bind to recombinant human IGF-1R. The anti-IGF-1R monoclonal antibody H0L0 also showed binding to recombinant human IGF-1R whereas the negative control antibody (sigma I5154) showed no binding to IGF-1R.

16.3: VEGF Binding ELISA

96-well high binding plates were coated with 0.4 μg/mL of hVEGF165 (R&D Systems) and incubated at +4° C. overnight. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added to each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. The purified antigen binding proteins/antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 77 shows the results of the VEGF binding ELISA and confirms that bispecific antibodies BPC1803 and BPC1804 bind to human VEGF. An anti VEGF bispecific antibody (BPC1603) was used as a positive control in this assay and showed binding to VEGF. In contrast the anti-IGF-1R monoclonal antibody H0L0 showed no binding to human VEGF.

16.4: HER2Binding ELISA

96-well high binding plates were coated with 1 μg/mL of HER2 (R&D Systems) and incubated at +4° C. overnight. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added to each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. The purified antigen binding proteins/antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIGS. 79 and 81 show the results of the HER2 binding ELISA and confirms that the antigen binding proteins BPC1805, BPC1806, BPC1807 and BPC1808 bind to recombinant human HER2. Herceptin was used as a positive control in this assay and showed binding to HER2. In contrast the anti-IGF-1R monoclonal antibody H0L0 showed no binding to human HER2.

16.5: IL-4 Binding ELISA

96-well high binding plates were coated with 5 μg/ml of human IL-4 in PBS and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. The purified antigen binding proteins/antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 82 shows the results of the ELISA and confirms that antigen binding protein BPC1809 binds to human IL-4 at levels comparable to the anti IL-4 monoclonal antibody, Pascolizumab. The negative control antibody (Sigma I5154) showed no binding to IL-4.

FIG. 84 shows the results of the ELISA and confirms that antigen binding protein BPC1816 binds to human IL-4 at levels comparable to the anti IL-4 monoclonal antibody, Pascolizumab. The negative control antibody (Sigma I5154) showed no binding to IL-4.

16.6: RNAse A Binding ELISA

50 uL of 1 ug/mL RNAse A (Qiagen, 19101) that had been diluted in PBS was added to each well of a 96 well Costar plate. The plate was incubated for 2 hours at room temperature then washed with PBST before addition of 200 uL of 4% BSA/PBS block to each well. The plate was incubated for an hour and washed before addition of the samples. Purified antibodies and antigen binding protein BPC1809 were added at a concentration of 2 ug/mL in wells of column 1 then serially diluted 1 in 2 across the plate in block. The plate was incubated for an hour then washed. 50 ul/well of Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was added at a 1 in 1000 dilution. The plate was incubated for an hour then washed. 50 uL of OPD was added to each well and the reaction was stopped with 3M sulphuric acid after 15-30 minutes. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 83 shows the results of the RNAse A binding ELISA and confirms that purified human monoclonal antibody-camelid VHH bispecific antibody BPC1809 shows binding to RNAse A. In contrast both the IL-4 monoclonal antibody Pascolizumab and the negative control (sigma I5154) showed no binding to RNAse A.

16.7: HEL Binding ELISA

A 96-well high binding plate was coated with 5 μg/ml of HEL (Hen Egg Lysozyme, Sigma L6876) in PBS and stored overnight at 4° C. The plate was washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plate was incubated for at least 1 hour at room temperature. Another wash step was then performed. The purified antibodies were successively diluted across the plate in blocking solution. After 1 hour incubation, the plate was washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plate was incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 85 shows the results of the HEL binding ELISA and confirms that purified human monoclonal antibody—NAR V bispecific antibody BPC1816 binds to HEL. In contrast the IL-4 monoclonal antibody Pascolizumab showed no binding to HEL.

Example 17
17.1 Design and Construction of Antigen-Binding Proteins Comprising Epitope Binding Domains Composed of Adnectin

CT01 adnectin is specific for VEGFR2 (for further information see WO2005/056764). The CT01 adnectin protein sequence was reverse-translated to DNA and codon optimised. A BamHI site at the N-terminus and EcoR1 site at the C-terminus were included to facilitate cloning.

DNA fragments encoding the final CT01 DNA sequence were constructed de novo using a PCR-based strategy and overlapping oligonucleotides. The PCR product was cloned into mammalian expression vectors containing the heavy chain of H0L0 (an anti-hIGF-1R antibody) allowing the adnectin to be fused onto the C-terminus of the heavy chain via either a GS linker or a TVAAPSGS linker. Protein sequences of the heavy and light chains of the IGF-1R—VEGFR2 bispecific are given in SEQ ID numbers 124, 113 and 133.

Another adnectin protein sequence coding for an anti-TNF-α adnectin (for further information see US20080139791) was reverse translated to DNA, codon optimised and modified to include terminal BamHI and EcoR1 sites before being constructed using the overlapping oligonucleotide PCR method described previously. The PCR product was cloned into mammalian expression vectors containing DNA encoding the heavy chain of Pascolizumab (an anti-IL-4 antibody) allowing DNA encoding the anti-TNF-α adnectin to be fused onto the C-terminus of the heavy chain via either a GS linker or a TVAAPSGS linker. Protein sequences of the heavy and light chains of the IL-4—TNF-α bispecific are given in SEQ ID NO: 146, 147 and 15.

Antigen binding proteins using the TNF-α specific adnectin fused at the C-terminus of the heavy chain of an IL-13 monoclonal antibody have also been designed. Example protein sequences are given in SEQ ID's 134, 13 and 135. In addition, bispecific molecules based on the fusion of CT01 at either the C-terminus or the N-terminus of the heavy or light chain of anti-EGFR antibodies Erbitux and IMC-11F8 have been designed. Examples of protein sequences which have been designed are given in SEQ ID NO: 136-145.

Of these example sequences, a number were constructed. DNA sequences encoding SEQ ID NO 136 (CT01 fused onto the C-terminus of the Erbitux heavy chain), SEQ ID NO 144 (CT01 fused onto the N-terminus of Erbitux heavy chain) and SEQ ID NO 138 (CT01 fused onto the C-terminus of the Erbitux light chain) were constructed. All three sequences were constructed using PCR-based cloning methods and cloned into mammalian expression vectors. Table 22 below is a summary of the antigen binding proteins that have been designed and/or constructed.

TABLE 22

SEQ ID NO:

amino acid

Antibody ID
Description
sequence

BPC1801
antiIGF1R Heavy Chain-
124

(constructed)
GS-CT01 adnectin

antiIGF1R Light Chain
113

BPC1802
antiIGF1R Heavy Chain-
133

(constructed)
TVAAPSGS-CT01 adnectin

antiIGF1R Light Chain
113

BPC1810
antiIL13-Heavy Chain-
134

(designed)
GS-antiTNFα adnectin

antiIL13-Light Chain
13

BPC1811
antiIL13-Heavy Chain-
135

(designed)
TVAAPSGS-antiTNFα adnectin

antiIL13-Light Chain
13

BPC1812
Erbitux Heavy chain-RS-
136

(constructed)
CT01 adnectin

Erbitux Light Chain
137

BPC1813
Erbitux Light chain-RS-
138

(constructed)
CT01 adnectin

Erbitux Heavy Chain
139

BPC1814
11F8 Heavy Chain-GS-CT01
140

(designed)
adnectin

11F8 Light Chain
141

BPC1815
11F8 Light Chain-GS-CT01
142

(designed)
adnectin

11F8 Heavy Chain
143

BPC1818
GS-CT01 adnectin-GSTG-
144

(constructed)
Erbitux Heavy Chain

Erbitux Light Chain
137

BPC1819
CT01 adnectin-STG-Erbitux
145

(designed)
Light Chain

Erbitux Heavy Chain
139

BPC1823
Anti IL-4 Heavy Chain-GS-
146

(constructed)
anti TNF-α adnectin

Anti IL-4 Light Chain
15

BPC1822
Anti IL-4 Heavy Chain-
147

(constructed)
TVAAPSGS- anti TNF-α

adnectin

Anti IL-4 Light Chain
15

Expression plasmids encoding the heavy and light chains of BPC1801, BPC1802, BPC1822, BPC1823, BPC1812, BPC1813 and BPC1818 were transiently co-transfected into HEK 293-6E cells using 293fectin (Invitrogen, 12347019). A tryptone feed was added to the cell culture the same day or the following day and the supernatant material was harvested after about 2 to 6 days from initial transfection. In some instances the supernatant material was used as the test article in binding assays. In other instances, the antigen binding protein was purified using a Protein A column before being tested in binding assays.

17.2: rhIGF-1R Binding ELISA

96-well high binding plates were coated with 1 μg/ml of anti-his-tag antibody (Abcam, ab9108) in PBS and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. 0.4 μg/mL of rhIGF-1R (R&D systems) was added to each well at 50 μL per well. The plates were incubated for an hour at room temperature and then washed. The purified antigen binding proteins/antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride)

SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 86 shows the results of the IGF-1R binding ELISA and confirms that purified human monoclonal antibody-adnectin bispecific antibodies (BPC1801 and BPC 1802) bind to recombinant human IGF-1R at levels comparable to the anti-IGF-1R monoclonal antibody H0L0. The negative control antibody (Sigma I5154) showed no binding to IGF-1R.

17.3: VEGFR2Binding ELISA

96-well high binding plates were coated with 0.4 μg/mL of VEGFR2 (R&D Systems) and incubated at +4° C. overnight. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added to each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. The supernatants or purified antibodies were successively diluted across the plates in blocking solution. After 1 hour incubation, the plate was washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 87 shows the results of the VEGFR2 binding ELISA and confirms that purified human monoclonal antibody-adnectin bispecific antibodies (BPC1801 and BPC1802) bind to recombinant human VEGFR2. In contrast the anti-IGF-1R monoclonal antibody H0L0 showed no binding to human VEGFR2.

FIG. 172 shows the results of the VEGFR2 binding ELISA and confirms that antigen binding proteins BPC1818 and BPC1813 bind to recombinant human VEGFR2. In contrast Erbitux showed no binding to human VEGFR2. For the antigen binding proteins BPC1813 and BPC1818, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 172 is represented as a dilution factor of the neat supernatant material. For Erbitux, purified material was used in the assay aT the starting concentration of 2 μg/ml, which is equivalent to dilution factor of 1 FIG. 172.

FIG. 175 shows the results of the VEGFR2 binding ELISA and confirms that antigen binding protein BPC1812 binds to recombinant human VEGFR2. In contrast Erbitux and the negative control Sigma IgG I5154 antibody showed no binding to human VEGFR2. For the antigen binding protein BPC1812, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 175 is represented as a dilution factor of the neat supernatant material. For Erbitux and Sigma IgG I5154 purified material was used in the assay at the starting concentration of 2 μg/ml, which is equivalent to dilution factor of 1 FIG. 175.

17.4: IL-4 Binding ELISA

96-well high binding plates were coated with 5 μg/ml of human IL-4 in PBS and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plates were incubated for at least 1 hour at room temperature. Another wash step was then performed. The supernatant or purified antibodies/antigen binding proteins were successively diluted across the plate in blocking solution. After 1 hour incubation, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plates were incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 88 shows the results of the IL-4 binding ELISA and confirms that human monoclonal antibody-adnectin bispecific antibodies (BPC1823 and BPC 1822) bind to recombinant human IL-4. The positive control anti-IL-4 monoclonal antibody Pascolizumab showed binding to IL-4 and the negative control antibody (Sigma I5154) showed no binding to IL-4.

The HEK transfection for this experiment was repeated to obtain supernatant material with a higher antibody concentration. FIG. 88b shows binding of this higher concentration supernatant human monoclonal antibody-adnectin bispecific antibody (BPC1823) to human IL-4 as determined by ELISA

For the antigen binding proteins BPC1823 and BPC1822, the amount of antibody in the supernatant was not quantified thus the data presented in FIGS. 88 and 88b is represented as a dilution factor of the neat supernatant material. For Pascolizumab and the negative control antibody (Sigma I5154), purified material was used in the assay and the starting concentration of 1 μg/ml, which is equivalent to dilution factor of 1 in FIGS. 88 and 88b.

17.5: TNF-α Binding ELISA

A 96-well high binding plate was coated with 0.4 μg/ml of recombinant human TNFα (RnD Systems 210-TA-050/CF) in PBS and stored overnight at 4° C. The plate was washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plate was incubated for at least 1 hour at room temperature. Another wash step was then performed. The supernatant or purified antibodies were successively diluted across the plate in blocking solution. After 1 hour incubation, the plate was washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plate was incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 15 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 89 shows the results of the TNF-α binding ELISA and confirms that human monoclonal antibody-adnectin bispecific antibodies (BPC1823 and BPC1822) bind to recombinant human TNF-α. In contrast the anti-IL-4 monoclonal antibody Pascolizumab showed no binding to recombinant human TNF-α.

The HEK transfection for this experiment was repeated to obtain supernatant material with a higher antibody concentration. FIG. 89b shows binding of this higher concentration supernatant human monoclonal antibody-adnectin bispecific antibody (BPC1823) to recombinant human TNF-α as determined by ELISA. The IgG control showed no binding to recombinant human TNF-α.

For the antigen binding proteins BPC1822 and BPC1823, the amount of antibody in the supernatant was not quantified thus the data presented in FIGS. 89 and 89b is represented as a dilution factor of the neat supernatant material. For Pascolizumab, purified material was used in the assay at the starting concentration of 1 μg/ml, which is equivalent to dilution factor of 1 in FIGS. 89 and 89b.

17.6: EGFR Binding ELISA

A 96-well high binding plate was coated with 0.67 μg/ml of recombinant human EGFR protein in PBS and stored overnight at 4° C. The plate was washed twice with Tris-Buffered Saline with 0.05% of Tween-20. 200 μL of blocking solution (5% BSA in DPBS buffer) was added in each well and the plate was incubated for at least 1 hour at room temperature. Another wash step was then performed. The antigen binding proteins/antibodies were successively diluted across the plate in blocking solution. After 1 hour incubation, the plate was washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma, A7164) was diluted in blocking solution to 1 μg/mL and 50 μL was added to each well. The plate was incubated for one hour. After another wash step, 50 μl of OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 25 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 171 shows the results of the EGFR binding ELISA and confirms that bispecific antibodies BPC1818 and BPC1813 bind to recombinant human EGFR. The positive control antibody, Erbitux, also showed binding to recombinant human EGFR. In contrast the Sigma IgG I5154 showed no binding to recombinant human EGFR. For the bispecific antibodies BPC1813 and BPC1818, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 171 is represented as a dilution factor of the neat supernatant material. For Erbitux and the negative control antibody (Sigma I5154), purified material was used in the assay and the starting concentration of 2 μg/ml and 1 μg/ml respectively, which is equivalent to dilution factor of 1 in FIG. 171.

FIG. 176 shows the results of the EGFR binding ELISA and confirms that bispecific antibodies BPC1812 binds to recombinant human EGFR. The positive control antibody, Erbitux, also showed binding to recombinant human EGFR. In contrast the Sigma IgG I15154 showed no binding to recombinant human EGFR. For the bispecific antibodies BPC1812, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 176 is represented as a dilution factor of the neat supernatant material. For Erbitux and the negative control antibody (Sigma I5154), purified material was used in the assay at the starting concentration of 2 μg/ml and 1 μg/ml respectively, which is equivalent to dilution factor of 1 in FIG. 176.

Example 18
Binding Activity Data of IL-13/IL-4 mAbdAbs where the ‘G and S’ Amino Acid Residues have been Removed
18.1 Construction of mAbdAbs

mAbdAbs were constructed in which the G and S amino acid residues (next to the linker sequence) were removed. Expression plasmids were constructed using standard molecular biology techniques. These mAbdAbs are described in Table 23. They were cloned, then expressed in one or more of HEK293-6E cells, CHOK1 cells, or CHOE1a cells, they were purified (as described in examples 1, 1.3 and 1.5 respectively) and analysed in a number of IL-13 and IL-4 activity assays.

TABLE 23

Sequence ID

Name
Description
No.

PascoH-474 GS
H chain = Pascolizumab heavy chain-DOM10-53-474
91 (= H chain)

removed
dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-TVAAPS-
H chain = Pascolizumab heavy chain-TVAAPS-DOM10-
92 (= H chain)

474 GS removed
53-474 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-GS-
H chain = Pascolizumab heavy chain-GS-ASTKGPT-
96 (= H chain)

ASTKGPT-474 2^nd
DOM10-53-474 dAb
15 (= L chain)

GS removed
L chain = Pascolizumab light chain

586H-210 GS
H chain = Anti-human IL-13 mAb heavy chain-DOM9-
87 (= H chain)

removed
112-210 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light chain

586 H-TVAAPS-210
H chain = Anti-human IL-13 mAb heavy chain-
88 (= H chain)

GS removed
TVAAPS-DOM9-112-10 dAb
13 (= L chain)

L chain = Anti-human IL-13 mAb light chain

18.2 Expression and Purification These mAbdAbs were purified and analysed by SEC and SDS PAGE. A number of purified preparations were made and the SEC and SDS PAGE data shown in FIGS. 90 to 98 are representative of these preparations.
18.3 Binding to Human IL-4 in a Direct Binding ELISA

Purified PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed were tested for binding to human IL-4 in a direct binding ELISA as described in method 2 (PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-ELQLE-474 were also tested for binding in this assay). A number of ELISA assays have been completed for these molecules, the data shown in FIG. 99 is representative of these assays.

PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, both bound human IL-4. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay as a positive control for binding to IL-4. Purified anti-human IL13 mAb was included as a negative control for IL-4 binding. The binding activity of PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed was similar to purified anti-IL4 mAb alone (Pascolizumab), PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-ELQLE-474.

18.4 Binding to Human IL-13 in a Direct Binding ELISA

Purified PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed were also tested for binding to human IL-13 in a direct binding ELISA as described in method 1 (PascoH-616 and PascoH-TVAAPS-616 were also tested for binding in this assay, the generation of these molecules is described in Example 19). A number of ELISA assays have been completed for these molecules, the data shown in FIG. 100 is representative of all assays.

PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-616 and PascoH-TVAAPS-616 all bound to human IL-13. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay as a negative control for binding to IL-13. Purified anti-human IL13 mAb was included as a positive control for IL-13 binding. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb is not detected by the secondary detection antibody; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

18.5 Binding to Cynomolgus IL-13 in a Direct Binding ELISA

Purified PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-616 and PascoH-TVAAPS-616 mAbdAbs were also tested for binding to cynomolgus IL-13 in a direct binding ELISA (as described in method 17). A number of ELISA assays have been completed for these molecules, the data shown in FIG. 101 is representative of all assays.

PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-616 and PascoH-TVAAPS-616 all bound cynomolgus IL-13. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay as a negative control for binding to IL-13. Purified anti-human IL13 mAb was included as a positive control for cynomolgus IL-13 binding. Note that the anti-IL-13 dAbs alone (DOM10-53-474 and DOM10-53-616) were not tested in this assay as the dAb is not detected by the secondary detection antibody; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

18.6 Biacore Analysis for Binding to Human IL-4 and Human IL-13

Purified mAbdAbs were tested for binding to human IL-4 and human IL-13 using the BIAcore™ T100 at 25° C. (as described in methods 4 and 5). These data are shown in Table 24.

In experiment 1, a mAbdAb capture level of approximately 600 relative response units was achieved and six IL-13 and IL-4 concentration curves (256 nM, 64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM) were assessed. Only one IL-13 (256 nM) and IL-4 (256 nM) concentration curve was assessed for the mAbs in experiment 1.

In experiment 2, a mAbdAb a capture level of approximately 400 relative response units was achieved and six IL-4 (64 nM, 16 nM, 4 nM, 1 nM, 0.25 nM and 0.0625 nM) and six IL-13 concentration curves (256 nM, 64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM) were assessed. In experiment 2, only one IL-13 concentration curve (256 nM) was assessed for the anti-IL13 mAb and five IL-4 concentration curves (64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM) were assessed for Pascolizumab.

In experiment 3, a mAbdAb or mAb capture level of approximately 700 relative response units was achieved and six IL-4 concentration curves (256 nM, 64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM) and six IL-13 concentration curves (256 nM, 64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM) were assessed.

TABLE 24

Binding affinity, KD (nM)

Molecule
Human IL-4
Human IL-13

(purified
Experiment
Experiment
Experiment
Experiment
Experiment
Experiment

material)
1
2
3
1
2
3

PascoH-474
not done
0.005
not done
0.3307
0.351
not done

GS removed

PascoH-TVAAPS-474
not done
0.011
not done
0.2677
0.305
not done

GS removed

586H-210
not done
not done
very tight
not done
not done
0.438

GS removed

binder *

586H-TVAAPS-210
not done
not done
very tight
not done
not done
0.501

GS removed

binder *

Anti-human
does not
does not
does not
0.2799
0.31
0.547

IL-13 mAb
bind
bind
bind

Pascolizumab
0.0137
0.011
0.013
does not
does not
does not

bind
bind
bind

In experiments 1 and 2, PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, both bound IL-4 with similar binding affinities and this was approximately equivalent to the binding affinity of the anti-human IL4 mAb alone (Pascolizumab). PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, also bound IL-13 with similar binding affinities. Note that the anti-IL-13 dAb alone (DOM10-53-474) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

In experiment 3, 586H-210 GS removed and 586H-TVAAPS-210 GS removed, both bound IL-13 with similar binding affinities and this was approximately equivalent to the binding affinity of the anti-human IL13 mAb. 586H-210 GS removed and 586H-TVAAPS-210 GS removed, also bound IL-4 very tightly, however this method was unable to determine the binding affinity due to positive dissociation effects and the sensitivity level of the BIAcore™ technique (*). Note that the anti-IL-4 dAb alone (DOM9-112-210) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay.

18.7 Biacore Analysis for Binding to Cynomolgus IL-4 and Cynomolgus IL-13

Purified mAbdAbs were tested for binding to cynomolgus IL-4 and cynomolgus IL-13 using the BIAcore™ T100 at 25° C. (as described in methods 24 and 23). These data are shown in Table 25. A mAbdAb capture level of approximately 600 relative response units was achieved and six IL-13 concentration curves (256, 64, 16, 4, 1, 0.25 nM) and five IL-4 concentration curves (64, 16, 4, 1, 0.25 nM) were assessed.

TABLE 25

Binding affinity, KD

Molecule
Cynomolgus IL-13
Cynomolgus IL-4

(purified
on rate
off rate
KD
on rate
off rate
KD

material)
(ka, Ms⁻¹)
(kd, s⁻¹)
(nM)
(ka, Ms⁻¹)
(kd, s⁻¹)
(pM)

PascoH-474
6.62E+5
1.10E−2
16.6
1.87E+6
6.38E−5
34.2

GS removed

PascoH-TVAAPS-474
4.83E+5
1.29E−2
26.7
1.83E+6
5.30E−5
29.0

GS removed

PascoH-ASTKGPT-474
4.79E+5
1.14E−2
23.8
1.83E+6
5.30E−5
29.0

2^ndGS removed

PascoH-474
5.86E+5
1.09E−2
18.6
1.85E+6
8.14E−5
43.9

PascoH-TVAAPS-474
4.33E+5
1.17E−2
27.1
1.80E+6
8.85E−5
49.1

PascoH-ASTKG-474
3.64E+5
1.07E−2
29.5
1.78E+6
7.90E−5
44.5

PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-ASTKGPT-474 2^ndGS removed, PascoH-474, PascoH-TVAAPS-474 and PascoH-ASTKG-474 all bound cynomolgus IL-4 with similar binding affinities. PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-ASTKGPT-474 2^ndGS removed, PascoH-474, PascoH-TVAAPS-474 and PascoH-ASTKG-474, also all bound IL-13 with similar binding affinities.

Purified mAbdAbs were also tested for binding to cynomolgus IL-4 and cynomolgus IL-13 using the BIAcore™ T100 at 25° C. (as described in methods 24 and 23). These data are shown in Table 26. A mAbdAb capture level of approximately 600 relative response units was achieved and six IL13 and six IL-4 concentration curves (256, 64, 16, 4, 1 and 0.25 nM) were assessed.

TABLE 26

Binding affinity, KD

Molecule
Cynomolgus IL-13
Cynomolgus IL-4

(purified
on rate
off rate
KD
on rate
off rate
KD

material)
(ka, Ms⁻¹)
(kd, s⁻¹)
(pM)
(ka, Ms⁻¹)
(kd, s⁻¹)
(pM)

586H-TVAAPS-210
4.92E+5
2.86E−5
58
very tight
very tight
very tight

GS removed

binder *
binder *
binder *

586H-210
5.07E+5
2.24E−5
44
very tight
very tight
very tight

GS removed

binder *
binder *
binder *

Anti-IL13 mAb
4.74E+5
1.05E−4
222
does not
does not
does not

bind
bind
bind

Pascolizumab
does not
does not
does not
2.34E+6
1.08E−4
46

bind
bind
bind

586H-210 GS removed and 586H-TVAAPS-210 GS removed, both bound cynomolgus IL-13 with similar binding affinities; these mAbdAbs appeared to bind IL-13 more potently than the anti-human IL13 mAb, however in the case of the mAb only one concentration curve was completed which is inherently less accurate than a full concentration range assessment. 586H-210 GS removed and 586H-TVAAPS-210 GS removed, also bound IL-4 very tightly, however this method was unable to determine the binding affinity due to positive dissociation effects and the sensitivity level of the BIAcore™ technique (*). Note that the anti-IL-4 dAb alone (DOM9-112-210) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL4 mAb (Pascolizumab) was used as a positive control to demonstrate IL-4 binding in this assay.

18.8 Biacore Analysis of Effect of IL-4 Binding to mAbdAbs on Subsequent IL-13 Binding Kinetics and Vice Versa; and the Effect of IL-13 Binding to mAbdAbs on Subsequent IL-4 Binding Kinetics and Vice Versa

The IL-13 and IL-4 BIAcore™ binding assays were also used to investigate the effect of IL-4 binding to PascoH-474 GS removed on subsequent IL-13 binding kinetics and vice versa; and the effect of IL-13 binding to 586H-TVAAPS-210 on subsequent IL-4 binding kinetics and vice versa. Analyses were carried out on the BIAcore™ T100 machine at 25° C., using anti-human IgG capture of the mAbdAb (or positive control mAb). Briefly, anti-human IgG was coupled onto a CM5 chip by primary amine coupling in accordance with the manufactures recommendations. mAbdAb constructs (or positive control mAb) were then captured onto this surface (at approximately 250 to 750RUs) and the first analyte (either human IL-13 or human IL-4) was passed over at 256 nM for 4 minutes. The second analyte (human IL-4 or human IL-13 respectively) was then passed over at concentrations of 256 nM, 64 nM, 16 nM, 4 nM, 1 nM and 0.25 nM, and for double referencing a buffer injection was passed over the capture antibody or mAbdAb surface. The data was analysed (fitted to the 1:1 model of binding) using the evaluation software in the machine. The surface was then regenerated using 3M magnesium chloride. The data from these experiments are shown in Tables 27 and 28.

TABLE 27

IL-13 Binding Kinetics

with human IL-4 bound to
without human IL-4 bound to

molecule
molecule

on rate
off rate
KD
on rate
off rate
KD

Molecule
(ka, Ms⁻¹)
(kd, s⁻¹)
(pM)
(ka, Ms⁻¹)
(kd, s⁻¹)
(pM)

PascoH-474
5.66E+5
2.31E−4
407.7
6.09E+5
2.59E−4
425.2

GS removed

586H-TVAAPS-210
9.68E+5
3.15E−4
325.2
9.09E+5
3.47E−4
381.3

Anti-IL13 mAb
not tested
1.01E+6
3.68E−4
366.1

The binding affinity of PascoH-474 GS removed for human IL-13 was similar, irrespective of whether human IL-4 was bound to this molecule or not. In addition, the binding affinity of 586H-TVAAPS-210 for human IL-13 was similar, irrespective of whether human IL-4 was bound to this molecule or not and it was also similar to the binding affinity of the anti-IL13 mAb for human IL-13.

The off-rates (kd) for IL-4 binding obtained for PascoH-474 GS removed and 586H-TVAAPS-210, are very slow and out of the sensitivity range of the BIAcore™ T100 hence could not be used as an accurate determination of the binding affinity (data not shown). However, the data do indicate that all of the constructs tested bind very tightly to human IL-4. Thus the binding affinity of PascoH-474 GS removed for human IL-4 was very tight, irrespective of whether human IL-13 was bound to this molecule or not. In addition, the binding affinity of 586H-TVAAPS-210 for human IL-4 was very tight, irrespective of whether human IL-13 was bound to this molecule or not.

18.9 Potency of mAbdAbs

mAbdAbs were tested for inhibition of human IL-4 binding to human IL-4Rα by ELISA, as described in Method 19. All of the molecules shown in Table 28 were tested in one experiment, however the data have been plotted on two graphs to distinguish between the curve plots (586H-TVAAPS-210 was run twice, this is labelled as sample 1 and sample 2 in table 25). These data are shown in FIGS. 102 and 103.

PascoH-474 GS removed, inhibited binding of human IL-4 to human IL4Rα similarly to Pascolizumab. 586H-210 GS removed, 586H-TVAAPS-210 GS removed, 586H-TVAAPS-210, 586H-210, 586H-G4S-210 and 586H-ASTKG-210 all inhibited binding of human IL-4 to human IL4Rα similarly to DOM9-112-210. Pascolizumab and DOM9-112-210 were included as positive controls for the inhibition of IL-4 binding to IL4Rα. DOM10-53-474 and an isotype-matched mAb (with specificity for an irrelevant antigen) were included as negative controls for the inhibition of IL-4 binding to IL4Rα.

These data were also used to determine IC₅₀values for each molecule. The IC₅₀value is the concentration of mAbdAb or mAb or dAb, which is able to inhibit binding of human IL-4 to human IL4Rα by 50%. The IC₅₀values are shown in Table 28.

TABLE 28

Molecule
IC₅₀(nM)

PascoH-474 GS removed
5.14

Pascolizumab
3.45

DOM9-112-210
36.77

586H-210
26.79

586H-210 GS removed
36.18

586H-TVAAPS-210 (sample 1)
23.77

586H-TVAAPS-210 (sample 2)
21.03

586H-TVAAPS-210 GS
19.21

removed

586H-ASTKG-210
27.32

586H-G4S-210
29.85

DOM10-53-474
No inhibition at concentration

range tested

Negative control mAb
No inhibition at concentration

range tested

These data confirm that PascoH-474 GS removed behaves similarly to Pascolizumab and that all 586H-210 mAbdAb ‘family members’ behave similarly to the DOM9-112-210 dAb.

18.10 Neutralisation of Human and Cynomolgus IL-13 in TF-1 Cell Bioassays by mAbdAbs

A number of purified mAbdAbs were tested for neutralisation of human and cynomolgus IL-13 in TF-1 cell bioassays (as described in method 8 and method 20 respectively). Each molecule was tested between one and nine times in these assays, not all graphs are shown, but FIGS. 104 and 105 are representative graphs showing the neutralisation data for human IL-13 and cynomolgus IL-13 respectively. DOM10-53-474 was included as a positive control for neutralisation of human or cynomolgus IL-13 in the bioassays. A dAb with specificity for an irrelevant antigen (negative control dAb) was also included as a negative control for neutralisation of human or cynomolgus IL-13 in the bioassays.

PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed and PascoH-ASTKG-474 2^ndGS removed, as well as PascoH-616, PascoH-TVAAPS-616 and DOM10-53-616 (these are described in Example 19), fully neutralised the bioactivity of both human and cynomolgus IL-13 in TF-1 cell bioassays.

18.11 Neutralisation of Human and Cynomolgus IL-4 in TF-1 Cell Bioassays by mAbdAbs

A number of purified mAbdAbs were also tested for neutralisation of human and cynomolgus IL-4 in TF-1 cell bioassays (as described in method 9 and method 21 respectively). Each molecule was tested twice in these assays, not all graphs are shown, but FIGS. 106 and 107 are representative graphs (from the dataset) showing neutralisation data for human IL-4 and cynomolgus IL-4 respectively. The anti-IL13 mAb was included as a negative control and Pascolizumab was included as a positive control for neutralisation of human or cynomolgus IL-4 in the bioassays. In addition, PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-G4S-474 were also tested for neutralisation of human and cynomolgus IL-4 in these bioassays.

PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, fully neutralised the bioactivity of both human and cynomolgus IL-4 in TF-1 cell bioassays. In addition PascoH-474, PascoH-TVAAPS-474, PascoH-ASTKG-474 and PascoH-G4S-474 also fully neutralised the bioactivity of human IL-4 in the TF-1 cell bioassay.

ND₅₀values were derived based on data obtained from a number of different experiments. The ND₅₀value is the concentration of mAbdAb or mAb or dAb, which is able to neutralise the bioactivity of IL-13 or IL-4 by 50%. The mean ND₅₀value, the standard deviation (SD) and the number of times tested (n) are shown in table 29.

TABLE 29

Mean ND₅₀value & standard deviation (nM)

human IL-13
cyno IL-13
human IL-4
cyno IL-4

Molecule
mean (SD)
n
mean (SD)
n
mean (SD)
n
mean (SD)
n

PascoH-474
2.269 (0.763)
9
39.834 (14.675)
8
2.855 (1.464)
2
1.89 (0.325)
2

GS removed

PascoH-TVAAPS474-
2.114 (0.766)
9
47.882 (13.181)
9
3.015 (2.015)
2
1.36 (0.41)
2

GS removed

PascoH-ASTKGPT-474
1.37
1
21.49
1
not done
not done

2^ndGS removed

DOM 10-53-474
1.035 (0.741)
4
10.495 (6.958)
4
did not
did not

neutralise
neutralise

Negative control
did not
did not
not done
not done

dAb
neutralise
neutralise

Pascolizumab
did not
did not
1.36 (0.198)
2
2.615 (2.242)
2

neutralise
neutralise

PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, and PascoH-ASTKGPT-474 2^ndGS removed, all fully neutralised the bioactivity of human and cynomolgus IL-13 in TF-1 cell bioassays. In addition, the neutralisation potencies (ND₅₀values) of PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, and PascoH-ASTKGPT-474 2^ndGS removed, for human IL-13 were similar and within-fold of the ND₅₀value for purified anti-IL13 dAb alone (DOM10-53-474). PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, both fully neutralised the bioactivity of human and cynomolgus IL-4 in TF-1 cell bioassays. In addition, the neutralisation potencies (ND₅₀values) of PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed, for human IL-13 and cyno IL-13 were similar and withinfold of the ND₅₀value for Pascolizumab.

18.12 Ability of mAbdAbs to Inhibit Binding of Human IL-13 Binding to Human IL-13Rα2

The molecules listed in Table 30 were tested for inhibition of human IL-13 binding to human IL-13Rα2 by ELISA, as described in Method 22. All molecules were tested in one experiment. The data are shown in FIG. 108.

All mAbdAbs tested inhibited binding of human IL-13 to human IL13Rα2. The level of inhibition was similar to that of DOM10-53-474, DOM10-53-616 and the anti-IL13 mAb. Pascolizumab and a negative control dAb (with specificity for an irrelevant antigen), were included as negative controls for the inhibition of IL-13 binding to IL13Rα2.

These data were also used to determine IC₅₀values for each molecule. The IC₅₀value is the concentration of mAbdAb or mAb or dAb, which is able to inhibit binding of human IL-13 to human IL13Rα2 by 50%. The IC₅₀values are shown in Table 30.

TABLE 30

Molecule
IC₅₀(nM)

PascoH-474 GS removed
9.58

PascoH-TVAAPS-474 GS
7.41

removed

DOM10-53-474
7.61

PascoH-616
6.41

PascoH-TVAAPS-616
6.17

DOM10-53-616
5.76

Anti-IL13 mAb
6.43

Pascolizumab
No inhibition at concentration

range tested

Negative control dAb
No inhibition at concentration

range tested

These data confirm that PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-616 and PascoH-TVAAPS-616 behaved similarly to DOM10-53-474, DOM10-53-616 and the anti-IL13 mAb.

Example 19
mAbdAbs Containing the Anti-IL13 DOM10-53-616 dAb

19.1 Construction of mAbdAbs Containing the Anti-IL13 DOM10-53-616 dAb

Two anti-IL4 mAb-anti-IL13dAbs as set out in Table 31 were cloned from existing vectors by site-directed mutagenesis as described in example 1.

TABLE 31

Sequence ID

Name
Description
No.

PascoH-616
H chain = Pascolizumab heavy chain-
149 (= H chain)

DOM10-53-616 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-TVAAPS-
H chain = Pascolizumab heavy chain-
150 (= H chain)

616
TVAAPS-DOM10-53-616 dAb
15 (= L chain)

L chain = Pascolizumab light chain

19.2 Expression and Purification of mAbdAbs Containing the anti-IL13 DOM10-53-616 dAb

These mAbdAbs were expressed in HEK293-6E cells and CHOE1a cells as described in example 1.3.

The mAbdAbs were purified and analysed by SEC and SDS PAGE. A number of purified preparations of PascoH-616 and PascoH-TVAAPS-616 mAbdAbs were made, the SEC and SDS PAGE data shown in FIGS. 109 (SEC profile for PascoH-616), 110 (SEC profile for PascoH-TVAAPS_—616), 111 (SDS PAGE for PascoH-616) and 112 (SDS PAGE for PascoH-TVAAPS-616), are representative of these preparations.

19.3 Binding of mAbdAbs to Human IL-13 in a Direct Binding ELISA

PascoH-616 and PascoH-TVAAPS-616 purified mAb dAbs were tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 113.

Purified PascoH-616 and PascoH-TVAAPS-616 both bound human IL-13. Purified anti-human IL4 mAb alone (Pascolizumab) was included in this assay as a negative control for binding to IL-13. Purified anti-human IL13 mAb was included as a positive control for IL-13 binding. Note that the anti-IL-13 dAb alone (DOM10-53-616) was not tested in this assay as the dAb is not detected by the secondary detection antibody; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

19.4 Biacore Analysis for Binding to Human IL-4 and Human IL-13

Purified mAbdAbs were tested for binding to human IL-4 and human IL-13 using the BIAcore™ T100 at 25° C. (as described in methods 4 and 5). These data are shown in Table 32.

TABLE 32

Binding affinity, KD (nM)

Molecule
Human IL-4
Human IL-13

(purified material)
Experiment 1
Experiment 2
Experiment 1
Experiment 2

PascoH-616
0.00172
Very tight
0.1137
0.15

binder

PascoH-TVAAPS-616
0.003
0.005
0.0497
0.056

Anti-human IL-13 mAb
does not bind
does not bind
0.2799
0.31

Pascolizumab
0.0137
0.011
does not bind
does not bind

PascoH-616 and PascoH-TVAAPS-616 both bound IL-4 with similar binding affinities and this was similar to the binding affinity of the anti-human IL4 mAb alone (Pascolizumab). PascoH-616 and PascoH-TVAAPS-616 both bound IL-13. Note that the anti-IL-13 dAb alone (DOM10-53-616) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

19.5 Biacore Analysis for Binding to Cynomolgus IL-13

Purified mAbdAbs were also tested for binding to cynomolgus IL-13 using the BIAcore™ T100 at 25° C. (as described in method and 30). These data are shown in Table 33. A mAbdAb capture level of approximately 400 relative response units was achieved and six IL-13 concentration curves (256, 64, 16, 4, 1 and 0.25 nM) were assessed. There was only one IL-13 concentration curve (256 nM) for the anti-IL13 mAb.

TABLE 33

Molecule
Binding affinity for

(purified
cynomolgus IL-13 (KD)

material)
on rate (ka, Ms⁻¹)
off rate (kd, s⁻¹)
KD (nM)

PascoH-616
3.411E+5
1.842E−3
5.4

PascoH-
4.597E+5
4.514E−3
9.8

TVAAPS-616

Anti-IL13 mAb
5.498E+5
6.549E−5
0.119

Pascolizumab
does not bind
does not bind
does not bind

PascoH-616 and PascoH-TVAAPS-616 both bound cynomolgus IL-13 with similar binding affinities. Note that the anti-IL-13 dAb alone (DOM10-53-616) was not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

19.6 Neutralisation of Human and Cynomolgus IL-13 in TF-1 Cell Bioassays by mAbdAbs

Purified mAbdAbs were tested for neutralisation of human IL-13 and cynomolgus IL-13 in TF-1 cell bioassays (as described in method 8 and method 20 respectively). These molecules were tested 3 times in each assay, and FIG. 114 is a representative graph showing the neutralisation data for human IL-13. FIG. 114a is a representative graph showing the neutralisation data for cyno IL-13. DOM10-53-616 was included as a positive control for neutralisation of IL-13 in this bioassay. A dAb with specificity for an irrelevant antigen (negative control dAb) was also included as a negative control for neutralisation of IL-13 in this bioassay.

The mean ND₅₀value, the standard deviation (SD) and the number of times tested (n) are shown in table 34.

TABLE 34

Mean ND₅₀value &

standard deviation (nM)

human IL-13
cyno IL-13

Molecule
mean (SD)
n
mean (SD)
n

PascoH-616
0.7956
3
9.23
3

(0.129)

(1.422)

PascoH-TVAAPS-616
0.722
3
14.477
3

(0.245)

(2.847)

DOM10-53-616
0.416
3
4.81
3

(0.144)

(3.266)

Negative control dAb
did not neutralise
did not neutralise

Both PascoH-616 and PascoH-TVAAPS-616, as well as In addition mAbdAbs PascoH-TVAAPS-474 GS removed and PascoH-474 GS removed fully neutralised the bioactivity of human and cynomolgus IL-13 in TF-1 cell bioassays.

In addition, the neutralisation potencies (ND₅₀values) of PascoH-616 and PascoH-TVAAPS-616 for human IL-13 were similar and within 2-fold of the ND₅₀value for the purified anti-IL13 dAb alone (DOM10-53-616).

PascoH-616 and PascoH-TVAAPS-616 were also tested for inhibition of human IL-13 binding to human IL-13Rα2 by ELISA, as described in Method 22. These data are presented in Example 18.12

Example 20
Ability of mAbdAbs to Neutralise Human IL-13 or IL-4 in a Human Whole Blood Phospho STAT6 Bioassay

The ability of mAbdAbs to neutralise human IL-13 or IL-4 in a human whole blood phospho STAT6 bioassay was carried out as described in Method 16. The IL-4 or IL-13 neutralisation potencies (ie. inhibition of IL-4 or IL-13 bioactivity) of 2 mAbdAb constructs (the purified anti-IL13 mAb-anti-IL4dAb, 586H-TVAAPS-210; and the purified anti-IL4 mAb-anti-IL13dAb, PascoH-474 GS removed) were determined. Purified anti-human IL-4 mAb (Pascolizumab) and purified anti-IL4 dAb (DOM9-112-210) were included as positive controls for neutralisation of rhIL-4 in this assay. Purified anti-human IL-13 mAb and purified anti-IL13 dAb (DOM10-53-474) were included as positive controls for neutralisation of rhIL-13. An isotype matched mAb mixed with a dAb (both with specificities for irrelevant antigens), were included as a negative control for neutralisation of rhIL-4 or rhIL-13. Each molecule was tested at least twice, using blood from different donors. FIGS. 115 to 124 are graphs showing representative data.

The purified mAbdAbs fully neutralised the bioactivity of rhIL-13 and rhIL-4.

As described in method 16, the ability of the test molecules to neutralise rhIL-13 or rhIL-4 bioactivity was expressed as the concentration of the molecules (e.g. mAbdAbs) required to neutralise 2 ng/mL of human IL-4 or human IL-13 by 50% (IC₅₀). These data are shown in Table 35. The combined mean IC₅₀from all donors for each molecule is presented, along with the standard deviation.

TABLE 35

Mean IC₅₀

Target in
(standard deviation)
Number of

Molecule
assay
nM
donors

586-TVAAPS-210
IL-4
1.23 (0.6)
3

IL-13
2.68 (1.2)
3

PascoH-474 GS
IL-4
7.95 (7.8)
3

removed
IL-13
2.78 (0.7)
3

Anti-IL13 mAb
IL-13
1.47 (0.4)
3

Pascolizumab
IL-4
2.44 (1.2)
3

DOM10-53-474
IL-13
6.83 (2.2)
2

DOM9-112-210
IL-4
3.51 (0.8)
2

Negative control
—
No inhibition shown
4

mAb

Negative control
—
No inhibition shown
4

dAb

Comparison of IC₅₀values indicated that 586-TVAAPS-210 inhibited IL-13 and IL-4 induced pSTAT-6 similarly to the anti-IL13 mAb and DOM9-112-210 (in the IL-13 and IL-4 whole blood assays respectively). Comparison of IC₅₀data also indicated that PascoH-474 GS removed, inhibited IL-13 and IL-4 induced pSTAT-6 similarly to DOM10-53-474 and Pascolizumab (in the IL-13 and IL-4 whole blood assays respectively). The control mAb showed no inhibition up to the maximum concentration tested of 661 nM in all donors, and the control dAb showed no neutralisation up to the maximum concentration tested of 2291 nM in all donors.

Example 21
Rat PK Studies of the Dual Targeting Anti-IL4/Anti-IL13 mAbdAb

PascoH-G4S-474, PascoL-G4S-474, 586H-TVAAPS-210 and 586H-TVAAPS-154 were assessed in rat PK studies (as summarised in Table 35.1). In brief, male Sprague-Dawley rats (approximately 200 grams to 220 grams in weight) were given a single intravenous (i/v) administration of mAbdAb at a target dose level of 2 mg/kg. At allotted time points (0 hours through to 312 hours) 104.1 blood samples were withdrawn and processed for plasma. The rat plasma samples were evaluated for the presence of the test molecule in a human IgG detection assay, and/or an IL-13 ligand binding assay, and/or an IL-4 ligand binding assay. In addition, the PK profile in plasma for Pascolizumab (in rat) was also evaluated: in this case the rat plasma samples were evaluated for the presence of Pascolizumab in a human IgG detection assay and an IL-4 ligand binding assay.

In a first study, pascolizumab was given to 4 rats. In a second study, there were four treatment groups (2 mg/kg PascoH-G4S-474, 2 mg/kg PascoL-G4S-474, 2 mg/kg 586H-TVAAPS-210 and 2 mg/kg 586H-TVAAPS-154), with 4 rats in each group.

The PK parameters (shown in Table 35.1) were derived from plasma concentration-time profile data (which are not shown). Note that, some plasma samples were analysed more than once in these assays and the PK parameters in Table 35.1 were derived from only one of these datasets. Also note that the PK parameters have not been normalised for individual animal doses, instead nominal doses of 2 mg/kg have been assumed. Note that some technical difficulties were encountered with the IgG PK assay for PascoH-G4S-474 hence the concentrations may be overestimated. In addition, analysis of the IgG plasma concentration-time profiles was performed twice in some cases (at analysis 1 and analysis 2, as annotated in Table 35.1). Analysis of the plasma concentration-time profile data generated from the IL-13 and IL-4 ligand binding PK assays was only performed at analysis 2. The plasma concentration-time profile data generated from the IL-13 and IL-4 ligand binding assays for PascoL-G4S-474 and 586H-TVAAPS-154 have not been used to derive PK parameters for these molecules.

TABLE 35.1

AUC
AUC

Mean

T1/2
Cmax
(last)
(extrap)
Clearance
residenc

Molecule
Assay
Analysis
(hr)
(ng/mL)
(ng · h/mL)
(%)
(mL/hr/kg)
time (hr)

PascoH-G4S-474
IgG
1
129
60950
3430345
18.7
0.49
83

IL-4
2
117
21975
1231243
17.2
1.42
83

IL-13
2
87
22050
1328651
13.2
1.36
85

586H-TVAAPS-210
IgG
1
134
42400
1932615
21.9
0.81
88

IgG
2
92
42400
2215579
16.1
0.76
82

IL-4
2
60
41350
1674740
20.9
1.08
77

IL-13
2
101
31125
1515155
16.0
1.13
83

Pascolizumab
IgG
1
188
45150
3528174
31.9
0.39
110

IgG
2
193
45150
3496503
34.9
0.38
109

IL-4
2
136
42825
2590114
30.6
0.54
110

PascoL-G4S-474
IgG
1
69
42550
2539978
6.8
0.75
87

586H-TVAAPS-154
IgG
1
166
43900
3315164
41.7
0.36
93

indicates data missing or illegible when filed

Plasma concentration-time profile data, generated in the IL-13 and IL-4 assays for PascoH-G4S-474 (and subsequent derived PK parameters), tended to be comparable; this was also the case for the plasma concentration-time profile data generated in the IL-13, IL-4 and IgG PK assays for 586H-TVAAPS-210 (and subsequent derived PK parameters). This suggests that these mAbdAbs were ‘intact’ in the rat plasma throughout the course of the study. The derived PK parameters for 586H-TVAAPS-154 appeared to be more similar to the derived PK parameters for Pascolizumab than any of the other mAbdAbs.

Example 22
Cyno PK Studies

PK studies were carried out in cynomolgus monkeys. The animals were given a single intravenous administration at a target dose level of 1 mg/kg. At allotted time points, 500 μl blood samples were withdrawn and processed for plasma. The cynomolgus plasma samples were then evaluated for the presence of the test molecule in an IL-13 ligand binding assay, an IL-4 ligand binding assay and an IL-13/IL-4 bridging assay.

Preliminary data from these studies with the mAbdAbs ‘PascoH-474 Gremoved’ and ‘586H-TVAAPS-210’ are consistent with the rat PK data shown above and indicated that these molecules are cleared from the systemic circulation more rapidly than a mAb but less rapidly than a dAb.

Example 23
23.1 Generation of a Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb

This dual targeting mAbdAb was constructed by fusion of a dAb to the C-terminus of the mAb heavy chain. The anti-EGFR mAb heavy and light chain expression cassettes had been previously constructed. The restriction sites which were used for cloning are the same as those set out in Example 10 (SalI and HindIII).

DNA coding an anti-VEGF dAb (DOM15-26-593) was then amplified by PCR (using primers coding SalI and HindIII ends) and inserted into the modified 3′ coding region, resulting in a linker of ‘STG’ (serine, threonine, glycine) between the mAb and the dAb.

Sequence verified clones (SEQ ID NO: 164 and 243) for light and heavy chains respectively) were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 165 and 137).

23.2 Purification and SEC Analysis of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb

This dual targeting mAbdAb was purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis (FIG. 128) of the purified sample (designated DMS4010) shows non-reduced sample running at ˜170 kDa whilst reduced sample shows two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-EGFR/anti-VEGF mAbdAb was applied onto a S-200 10/300 GL column (attached to an HPLC system) pre-equilibrated and running in PBS at 1 ml/min. The SEC profile shows a single species running as a symmetrical peak (FIG. 129).

23.3 Potency of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb

The ability of the molecule to neutralise VEGF and EGFR were determined as described in methods 12 and 13 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 130) of this mAbdAb (designated DMS4010) was calculated to be 4.784 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 4.214 nM. In the anti-VEGF receptor binding assay (FIG. 131) the EC50 of the mAbdAb (designated DMS4010) was 58 pM (0.058 nM) whilst an anti-VEGF control mAb produced an EC50 of 214.1 pM (0.2141 nM). In conclusion, assay data shows that the construct of example 23, a dual targeting anti-EGFR/anti-VEGF mAbdAb is potent against both antigens.

23.4 PK of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb

The pharmacokinetic profile of the dual targeting anti-EGFR/anti-VEGF mAbdAb (designated DMS4010) was determined after administration to cynomolgus monkeys. The compound was administered at a dose of 5 mg/kg i.v. and the serum levels of drug at multiple time points post-administration was determined by binding to both EGFR and VEGF in separate ELISA assays. FIG. 132 shows the results for this assay in which the data was compared with historical data that had been generated for the mAbs cetuximab (anti-EGFR) and bevacizumab (anti-VEGF). Further details are shown in table 36.

TABLE 36

Half Life
Cmax
AUC (0-inf)
Clearance
% AUC

Antigen
(hr)
(ug/mL)
(hr*ug/mL)
(mL/hr/kg)
Extrapolated

cetuximab
EGFR
43.6
151.4
5684.3
0.9
18.4

bevacizumab
VEGF
238.7
167.4
24201.9
0.2
13.4

DMS4010
EGFR
7.5
89.7
623.2
8.1
5.2

DMS4010
VEGF
6.7
125.5
733.8
7
7.2

23.5 Generation of an Alternative Anti-EGFR/Anti-VEGF mAbdAb

An alternative anti-EGFR/anti-VEGF mAbdAb was constructed in a similar way to that described above in Example 11.1, using the same anti-EGFR mAb linked to a VEGF dAb on the C-terminus of the heavy chain using an STG linker. The anti-VEGF dAb used in this case was DOM15-10-11. This molecule was expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 165 and 186), however significantly reduced levels of expression were achieved in comparison to the expression of the molecule described in Example 23.2. When tested for potency in the same VEGF assay as described in Example 23.3 it was found to have undetectable levels of inhibition of VEGF binding to VEGF receptor in this assay.

Example 24
24.1 Generation of a Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with No Linker

A derivative of the mAbdAb described above in Example 23 was made where the ‘STG’ linker between the dAb and the CH3 domain of the mAb was removed. SDM was used to delete the residues encoding the STG linker from the plasmid encoding the heavy chain. Sequence verified clones for light and heavy chains (SEQ ID NO: 243 and SEQ ID NO: 174) respectively were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 175 and 137).

24.2 Purification and SEC Analysis of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with No Linker

This dual targeting mAbdAb was purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis (FIG. 133) of the purified sample (designated DMS4011) shows non-reduced sample running at ˜170 kDa whilst reduced sample shows two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

24.3 Potency of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with No Linker

The ability of the molecule to neutralise VEGF and EGFR were determined as described in methods 12 and 13 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 135) of this mAbdAb (designated DMS4011) was calculated to be 3.529 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 3.647 nM. In the anti-VEGF receptor binding assay (FIG. 136) the EC50 of the mAbdAb (designated DMS4011) was 342.9 pM (0.3429 nM) whilst an anti-VEGF control mAb produced an EC50 of 214.1 pM (0.2141 nM). In conclusion, assay data shows that the construct of example 24, a dual targeting anti-EGFR/anti-VEGF mAbdAb with no linker is potent against both antigens.

Example 25
25.1 Generation of a Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with Longer Linkers

Derivatives of the mAbdAb described above in Example 23 were made where the linker between the dAb and the CH3 domain of the mAb was lengthened by the insertion of one or two repeats of a flexible “GGGGS” motif into the plasmid encoding the heavy chain.

The first molecule with a heavy chain sequence as set out in SEQ ID NO: 175 has one repeat of this motif, hence having a linker of ‘STGGGGGS’.

The second molecule with a heavy chain sequence as set out in SEQ ID NO: 177 has two repeats of this motif, hence having a linker of ‘STGGGGGSGGGGS’.

These were both indepentyl paired with the same light chain as used in Example 23 (SEQ ID NO: 243)

Sequence verified clones for light and heavy chains were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 176 and 137 which is designated DMS4023; and SEQ ID NO: 178 and 137 which is designated DMS4024).

25.2 Purification and SEC Analysis of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with Longer Linkers

These dual targeting mAbdAbs were purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis of the purified samples DMS4023 and DMS4024 (FIG. 137) shows non-reduced samples running at ˜170 kDa whilst reduced samples show two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-EGFR/anti-VEGF mAbdAb was applied onto a S-200 10/300 GL column (attached to an HPLC system) pre-equilibrated and running in PBS at 1 ml/min. The SEC profile for both DMS4023 (FIG. 138) and DMS4024 (FIG. 139) show a single species with a slightly trailing peak.

25.3 Potency of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAb with Longer Linkers

The ability of the molecule to neutralise VEGF and EGFR were determined as described in methods 12 and 13 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 140) of the mAbdAb DMS4023 was calculated to be 7.066 nM and the potency of mAbdAb DMS4024 was calculated to be 6.420 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 7.291 nM. In the anti-VEGF receptor binding assay (FIG. 141) the EC50 of the mAbdAb DMS4023 was 91.79 pM (0.091 nM) and the EC50 of the mAbdAb DMS4024 was 90 pM (0.0906 nM) whilst an anti-VEGF control mAb produced an EC50 of 463.2 pM (0.4632 nM). In conclusion, assay data shows that the constructs of example 25, dual targeting anti-EGFR/anti-VEGF mAbdAbs with longer linkers are potent against both antigens.

Example 26
26.1 Generation of a Dual Targeting Anti-VEGF/Anti-EGFR mAbdAb

This dual targeting mAbdAb was constructed by fusion of a dAb to the C-terminus of the mAb heavy chain. The anti-VEGF mAb heavy and light chain expression cassettes had been previously constructed. The restriction sites which were used for cloning are the same as those set out in Example 10 (SalI and HindIII).

DNA coding an anti-EGFR dAb (DOM16-39-542) was then amplified by PCR (using primers coding SalI and HindIII ends) and inserted into the modified 3′ coding region, resulting in a linker of ‘STG’ (serine, threonine, glycine) between the mAb and the dAb.

Sequence verified clones (SEQ ID NO: 179 and 181) for light and heavy chains respectively) were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 180 and 182).

26.2 Purification and SEC Analysis of the Dual Targeting Anti-VEGF/Anti-EGFR mAbdAb

These dual targeting mAbdAbs were purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis of the purified sample (designated DMS4009) (FIG. 142) shows non-reduced samples running at ˜170 kDa whilst reduced samples show two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-EGFR/anti-VEGF mAbdAb was applied onto a S-200 10/300 GL column (attached to an HPLC system) pre-equilibrated and running in PBS at 1 ml/min. The SEC profile for this molecule (FIG. 143) shows a single species with a symmetrical peak.

26.3 Potency of the Dual Targeting Anti-VEGF/Anti-EGFR mAbdAb

The ability of the molecule to neutralise VEGF and EGFR were determined as described in methods 12 and 13 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 144) of the mAbdAb DMS4009 was calculated to be 132.4 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 6.585 nM. In the anti-VEGF receptor binding assay (FIG. 145) the EC50 of the mAbdAb was 539.7 pM (0.5397 nM) whilst an anti-VEGF control mAb produced an EC50 of 380.5 pM (0.3805 nM). In conclusion, assay data shows that the construct of example 26, a dual targeting anti-VEGF/anti-EGFR mAbdAb is potent against both antigens.

Example 27
27.1 Generation of a Dual Targeting Anti-EGFR/Anti-IL-13 mAbdAb

DNA coding an anti-IL-13 dAb (DOM10-53-474) was then amplified by PCR (using primers coding SalI and HindIII ends) and inserted into the modified 3′ coding region, resulting in a linker of ‘STG’ (serine, threonine, glycine) between the mAb and the dAb.

Sequence verified clones (SEQ ID NO: 243 and 183) for light and heavy chains respectively) were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 137 and 184).

27.2 Purification and SEC Analysis of the Dual Targeting Anti-EGFR/anti-IL-13 mAbdAb

These dual targeting mAbdAbs were purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis of the purified sample (designated DMS4029) (FIG. 146) shows non-reduced samples running at ˜170 kDa whilst reduced samples show two bands running at ˜25 and ˜60 kDa corresponding light chain and dAb-fused heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-EGFR/anti-IL-13 mAbdAb was applied onto a S-200 10/300 GL column (attached to an HPLC system) pre-equilibrated and running in PBS at 0.5 ml/min. The SEC profile for this molecule (FIG. 147) shows a single species with a symmetrical peak.

27.3 Potency of the Dual Targeting Anti-EGFR/Anti-IL-13 mAbdAb

The ability of the molecule to neutralise EGFR and IL-13 were determined as described in methods 13 and 25 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 148) of the mAbdAb DMS4029 was calculated to be 9.033 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 8.874 nM. In the IL-13 cell-based neutralisation assay (FIG. 149) the EC50 of the mAbdAb was 1.654 nM whilst an anti-IL-13 control dAb produced an EC50 of 0.996 nM. In conclusion, assay data shows that the construct of example 27, a dual targeting anti-EGFR/anti-IL-13 mAbdAb is potent against both antigens.

Example 28
28.1 Generation of a Dual Targeting Anti-EGFR/Anti-VEGF mAbdAbs where the dAb is Located on the Light Chain

Dual targeting anti-EGFR/anti-VEGF mAbdAbs were constructed by fusion of a dAb to the C-terminus of the mAb light chain. The anti-EGFR mAb heavy and light chain expression cassettes had been previously constructed.

To introduce restriction sites for dAb insertion in the light chain, site directed mutagenesis was used to create BamHI and HindIII cloning sites using the mAb light chain expression vector as a template. DNA coding an anti-VEGF dAb (DOM15-26-593) was then amplified by PCR (using primers coding BamHI and HindIII ends) and inserted into the modified 3′ coding region, resulting in a linker of either ‘GSTG’ or ‘GSTVAAPS’ between the mAb and the dAb.

The first molecule with a light chain sequence as set out in SEQ ID NO: 187 has a linker of ‘GSTG’.

The second molecule with a light chain sequence as set out in SEQ ID NO: 189 has a linker of ‘GSTVAAPS’.

These were both independently paired with the heavy chain of SEQ ID NO: 245.

Sequence verified clones for light and heavy chains were selected and large scale DNA preparations were made using Qiagen Mega Prep Kit following the manufacturer's protocols. mAbdAbs were expressed in mammalian HEK293-6E cells using transient transfection techniques by co-transfection of light and heavy chains (SEQ ID NO: 188 and 139 which is designated DMS4013; and SEQ ID NO: 190 and 139 which is designated DMS4027).

28.2 Purification and SEC Analysis of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAbs where the dAb is Located on the Light Chain

These dual targeting mAbdAbs were purified from clarified expression supernatant using Protein-A affinity chromatography according to established protocols. Concentrations of purified samples were determined by spectrophotometry from measurements of light absorbance at 280 nm. SDS-PAGE analysis of the purified samples DMS4013 and DMS4027 (FIG. 150) shows non-reduced samples running at ˜170 kDa whilst reduced samples show two bands running at ˜38 and ˜50 kDa corresponding to dAb-fused light chain and heavy chain respectively.

For size exclusion chromatography (SEC) analysis the anti-EGFR/anti-VEGF mAbdAb was applied onto a S-200 10/300 GL column (attached to an HPLC system) pre-equilibrated and running in PBS at 1 ml/min. The SEC profile for both DMS4013 (FIG. 151) and DMS4027 (FIG. 152) show a single species with a symmetrical peak.

28.3 Potency of the Dual Targeting Anti-EGFR/Anti-VEGF mAbdAbs where the dAb is Located on the Light Chain

The ability of the molecule to neutralise VEGF and EGFR were determined as described in methods 12 and 13 respectively. Assay data were analysed using GraphPad Prism. Potency values were determined using a sigmoidal dose response curve and the data fitted using the best fit model. Anti-EGFR potency (FIG. 153) of the mAbdAb DMS4013 was calculated to be 7.384 nM and the potency of mAbdAb DMS4027 was calculated to be 7.554 nM whilst the control, an anti-EGFR mAb gave an EC50 value of 7.093 nM. In the anti-VEGF receptor binding assay (FIG. 154) the EC50 of the mAbdAb DMS4013 was 1.179 nM and the EC50 of the mAbdAb DMS4027 was 0.1731 nM whilst an anti-VEGF control mAb produced an EC50 of 0.130 nM. In conclusion, assay data shows that the constructs of example 28, dual targeting anti-EGFR/anti-VEGF mAbdAbs where the dAb is located on the light chain are potent against both antigens.

Example 29
Biacore Analysis of Dual Targeting Anti-EGFR/Anti-VEGF and Anti-TNF/Anti-VEGF mAbdAbs

The mAbdAbs described in example 11 (anti-TNF/anti-VEGF mAbdAb) and examples 23, 24, 25 and 28 (anti-EGFR/anti-VEGF mAbdAbs) were subjected to BIAcore analysis to determine kinetic association and dissociation constants for binding to their corresponding antigens. Analysis was performed on BIAcore™ 3000 instrument. The temperature of the instrument was set to 25° C. HBS-EP buffer was used as running buffer. Experimental data were collected at the highest possible rate for the instrument. One flow cell on a research grade CM5 chip was coated with protein A using standard amine coupling chemistry according to manufacturers instructions, and a second flow cells was treated equally but buffer was used instead of protein A to generate a reference surface. The flow cell coated with protein A was then used to capture mAbdAbs. Antigen was injected as a series 2× serial dilutions as detailed in table 37. Several dilutions were run in duplicate. Injections of buffer alone instead of ligand were used for background subtraction. Samples were injected in random order using the kinetics Wizard inherent to the instrument software. The surface was regenerated at the end of each cycle by injecting 10 mM Glycine, pH 1.5. Both data processing and kinetic fitting were performed using BIAevaluation software 4.1. Data showing averages of duplicate results (from the same run) is shown in Table 37. The multiple values shown for DMS4010 represent two experiments run on separate occasions. The value of 787 nM probably overestimates the affinity due to the concentrations of ligand analysed

TABLE 37

Top

mAbdAb
Molecule

KD
concentration
#

Example
number
Antigen
Ka [1/Ms]
Kd [1/s]
[pM]
(nM)
dilutions

11
4000
TNF
3.65E+05
4.16E−05
112
10
6

23
4010
EGFR
1.47E+06
1.16E−03
787
1.25
5

23
4010
EGFR
3.14E+05
1.16E−03
3700
10
6

24
4011
EGFR
1.81E+05
1.11E−03
6120
5
6

28
4013
EGFR
2.20E+05
1.17E−03
5310
20
5

28
4013
EGFR
3.01E+05
1.40E−03
4650
10
7

25
4023
EGFR
2.38E+05
1.10E−03
4630
5
7

25
4024
EGFR
2.34E+05
1.10E−03
4700
10
6

28
4027
EGFR
2.80E+05
1.14E−03
4060
20
7

11
4000
VEGF
9.19E+05
4.78E−04
520
2.5
5

23
4010
VEGF
9.85E+05
1.90E−04
193
10
8

24
4011
VEGF
6.17E+05
1.26E−04
204
10
8

28
4013
VEGF
7.62E+05
3.64E−04
478
2
5

25
4023
VEGF
1.60E+06
2.40E−04
150
2
6

25
4024
VEGF
1.01E+06
2.30E−04
224
2
4

28
4027
VEGF
7.47E+05
2.23E−04
229
2
5

indicates data missing or illegible when filed

Example 30
Trispecific Antibodies which Comprise Single Domain Antibodies Fused onto a Bispecific Antibody Scaffold
30.1 Construction

Genes encoding variable heavy and light domains of a bispecific antibody molecule which has specificity for IL-18 and IL-12 antigens (for further information see WO 2007/024715) were constructed de-novo with appropriate restriction enzyme sites and signal sequence added. Using standard molecular biology techniques, the variable heavy domains were cloned into an expression vector containing the IgG1 heavy chain constant region fused to an anti-IL4 domain antibody DOM9-112-210 (SEQ ID NO: 4) via a TVAAPS linker at the c-terminus of the constant region. The light chain variable domain was similarly cloned into an expression vector containing the Ck constant region sequence. The antibodies constructed and expressed are listed in Table 38.

TABLE 38

SED ID NO: of

Antibody

amino acid

ID
Description
sequence

BPC1616
IL-12/18 DVDH TVAAPS-210
193

heavy chain

IL-12/18 DVD Kappa
194

light chain

30.2 Expression and Purification

Briefly, 25 ml of HEK293 cells at 1.5×10⁶cells/ml were co-transfected with heavy and light chain expression plasmids previously incubated with 293fectin reagent (Invitrogen # 51-0031). These were placed in a shaking incubator at 37° C., 5% CO₂, and 95% RH. After 24 hours Tryptone feeding media was added and the cells grown for a further 48 hours. Supernatant was harvested by centrifugation and IgG levels quantified by ELISA. The resulting mAbdAb was designated BPC1616 (SEQ ID NO: 193 and 194)

30.3 IL-12 Binding ELISA

The cell supernatant from the transfections were assessed for binding to recombinant human IL-12. Briefly, ELISA plates coated with anti-human IL-12 (R&D Systems AF219NA) at 2 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline). The plates were then loaded with 25 ng/ml recombinant human IL-12 (PeproTech #200-12) in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Various dilutions of the cell supernatant were added as well as irrelevant control antibodies (Pascolizumab and an isotyped matched control hIgG) diluted in blocking solution. The plate was incubated for 1 hour at room temperature before washing in TBST. Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results are presented in FIG. 155 and show that BPC1616 binds to recombinant human IL-12 whereas the two control antibodies showed no binding.

30.4 IL-18 Binding ELISA

The cell supernatants from the transfections were assessed for binding to recombinant human IL-18. Briefly, ELISA plates were coated with human IL-18 (made at GSK) at 1 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline). Various dilutions of the cell supernatant were added as well as irrelevant antibodies (Pascolizumab and an isotype matched control human IgG), diluted in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted. The results are presented in FIG. 156 and show that BPC1616 binds to recombinant human IL-18 whereas the two control antibodies show no binding.

30.5 IL-4 Binding ELISA

The cell supernatants from the transfections were assessed for binding to recombinant human IL-4. Briefly, ELISA plates were coated with human IL-4 (made at GSK) at 1 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline). Various dilutions of the cell supernatant were added as well as an anti IL-4 monoclonal antibody (Pascolizumab) and irrelevant antibody (Isotype matched control hIgG), diluted in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results are presented in FIG. 157 show that BPC1616 and Pascolizumab bind to recombinant human IL-4 whereas the control antibody shows no binding.

Example 31
Trispecific mAbdAbs Comprising Two Single Domains Antibodies Fused in-Line at the C-Terminus of a Monoclonal Antibody
31.1 Construction

Three trispecific antibodies (mAbdAb-dAb) were constructed where two single domain antibodies are fused in-line at the C-terminus of the heavy chain of a monoclonal antibody.

Briefly, a BglII restriction site at the N-terminus and BamHI restriction site at the C-terminus were introduced by PCR to flank the DNA sequences encoding the domain antibodies into the DOM10-53-474 (SEQ ID NO: 5), DOM9-155-154 (SEQ ID NO: 3) and DOM9-112-210 (SEQ ID NO: 4).

The DNA fragment encoding the DOM10-53-474 domain antibody was then cloned into a BamHI site of mammalian expression vector encoding the heavy chain of an anti IL-5 monoclonal antibody fused with an anti IL-4 domain antibody DOM9-112-210 (SEQ ID NO: 71). The DNA fragments encoding the DOM9-155-154 and DOM9-112-210 domain antibodies were both independently cloned into the BamHI site of a mammalian expression vector encoding the heavy chain of an anti-CD20 monoclonal antibody fused with an anti IL-13 domain antibody DOM10-53-474 (SEQ ID NO: 116). The resulting expression vectors encode a heavy chain with two single domain antibodies fused onto the C-terminus. The protein sequences of the heavy chains are given in SEQ ID NO: 195, 196 and 197 as set out in Table 39.

Table 39 is a summary of the mAbdAbs that have been constructed.

TABLE 39

SEQ ID NO: of

amino acid

Antibody ID
Description
sequence

BPC1008
Anti IL-5 Heavy Chain-
195

G4S-dAb474-TVAAPSGS-dAb210

Anti IL-5 Light Chain
66

BPC1009
Anti CD-20 Heavy Chain-
196

TVAAPSGS-dAb154-

TVAAPSGS-dAb474

Anti CD-20 Light Chain
117

BPC1010
Anti CD-20 Heavy Chain-
197

TVAAPSGS-dAb210-

TVAAPSGS-dAb474

Anti CD-20 Light Chain
117

31.2 Expression and Purification

Expression plasmids encoding the heavy and light chains of BPC1008, BPC1009 and BPC1010 were co-transfected into HEK 2936E cells using 293fectin (Invitrogen, 12347019). A tryptone feed was added to the cell culture the following day and the supernatant material was harvested after about 2 to 6 days from initial transfection.

The antibodies were purified using a Protein A column before being tested in binding assays.

31.3: IL-4 Binding ELISA

96-well high binding plates were coated with 5 μg/ml human IL-4 (GSK) in Coating buffer (0.05M bicarbonate pH9.6, Sigma C-3041)₃and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 μL of blocking solution (1% BSA in TBST buffer) was added in each well and the plates were incubated for at least one hour at room temperature. The purified antibodies were successively diluted across the plates in blocking solution. After one hour incubation, the plates were washed three times. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma A7164) was diluted 1 in 2000 in blocking solution and 50 μL was added to each well. The plates were incubated for one hour. The plates were washed three times, then OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 5 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

The results of the ELISA are shown in the FIG. 158 and confirm that antibodies BPC1008, 1009 and BPC1010 bind to recombinant human IL-4. The positive control Pascolizumab also showed binding to recombinant IL-4 whereas the negative control anti IL-13 mAb and Mepolizumab showed no binding to IL-4. Antibodies BPC1009 and BPC1010 were also tested in a separate experiment which gave similar result to those shown in FIG. 158.

31.4: IL-5 Binding ELISA

96-well high binding plates were coated with 5.9 μg/ml human IL-5 (GSK) in coating buffer (0.05M bicarbonate pH9.6) and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 μL of blocking solution (1% BSA in TBST buffer) was added in each well and the plates were incubated for at least one hour at room temperature. The purified antibodies were successively diluted across the plates in blocking solution. After one hour incubation, the plates were washed three times. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma A7164) was diluted 1 in 2000 in blocking solution and 50 μL was added to each well. The plates were incubated for one hour. This was washed three times, then OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 5 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 159 shows the results of the ELISA which confirms that antibodies BPC1008 bind to recombinant human IL-5 whereas BPC1009 and BPC1010 showed no binding to IL-5. The positive control Mepolizumab also showed binding to recombinant IL-5 whereas the negative control anti IL-13 mAb and Pascolizumab showed no binding to IL-5.

IL-13 Binding ELISA
96-well high binding plates were coated with 5 μg/ml human IL-13 (GSK) in Coating buffer (0.05M bicarbonate pH9.6, Sigma C-3041)₃and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 μL of blocking solution (1% BSA in TBST buffer) was added in each well and the plates were incubated for at least one hour at room temperature. The purified antibodies were successively diluted across the plates in blocking solution. After one hour incubation, the plates were washed three times. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma A7164) was diluted 1 in 2000 in blocking solution and 50 μL was added to each well. The plates were incubated for one hour. This was washed three times, then OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 5 minutes later by addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

The results of the ELISA are shown in the FIG. 160 and confirm that antibodies BPC1008, 1009 and BPC1010 bind to recombinant human IL-13. The positive control anti IL-13 mAb also showed binding to recombinant IL-13 whereas the negative control Pascolizumab and Mepolizumab showed no binding to IL-13. Antibodies BPC1009 and BPC1010 were also tested in a separate experiment which gave similar result to those shown in FIG. 160.

Example 32
mAbdAbs with Single Domain Antibodies Fused onto Monovalent Scaffold

32.1 Construction of mAbdAbs

Bispecific antibodies comprising a fusion of a monovalent antibody (for further information see WO2006015371 and WO2007059782) and a domain antibody DOM-15-26-293 were constructed as follows. DNA sequences encoding the anti-c-Met Knob-into-hole heavy chain (SEQ ID NO: 202 and 203) was constructed using a PCR-based strategy followed by site directed mutagenesis. The DNA sequence encoding the anti-c-Met Unibody heavy chain (SEQ ID NO: 204) was constructed using a PCR-based strategy followed by removal of the hinge region by a PCR-based approach. Additionally, for fusion constructs, BamHI and EcoRI restriction sites were included at the C-terminus of the heavy chain expression cassette to facilitate the subsequent cloning of the anti VEGF-A domain antibody (DOM-15-26-593) DNA sequence (encoding amino acids 455-570 of SEQ ID NO: 75) as a BamHI-EcoRI fragment from an existing vector. The resulting expression vectors encode an anti-VEGFA domain antibody fused onto the C-terminus of the heavy chain via a GS linker (SEQ ID NO: 198, 199 and 201). The DNA sequence encoding the anti-c-Met light chain (SEQ ID NO: 200) was constructed by a PCR-based strategy.

The construction of BPC1604 is described in Example 14. Table 40 below is a summary of the monovalent scaffold mAbdAbs and antibodies that have been generated and expressed.

TABLE 40

SED ID NO: of

amino acid

Antibody ID
Description
sequence

BPC1017
anti cMET 5D5v2 Heavy Chain (hole)-
198

GS-dAb593

anti cMET 5D5v2 Heavy Chain (knob)-
199

GS-dAb593

anti cMET 5D5v2 Light Chain
200

BPC1018
anti cMET 5D5v2 IgG4 Heavy Chain
201

(UNIBODY)-GS-dAb593

anti cMET 5D5v2 Light Chain
200

BPC1019
anti cMET 5D5v2 Heavy Chain (hole)
202

anti cMET 5D5v2 Heavy Chain (knob)
203

anti cMET 5D5v2 Light Chain
200

BPC1020
anti cMET 5D5v2 IgG4 Heavy Chain
204

(UNIBODY)

anti cMET 5D5v2 Light Chain
200

32.2 Expression and Purification

Expression plasmids encoding the heavy chain of BPC1017, BPC1018, BPC1019 and BPC1020 were co-transfected into HEK 2936E cells using 293fectin (Invitrogen, 12347019). A tryptone feed was added to the cell culture the following day and the supernatant material was harvested after about 2 to 6 days from initial transfection. The antibodies were purified using a Protein A column before being tested in binding assays.

32.3 HGF Receptor Binding ELISA

96-well high binding plates were coated with 5 μg/ml Recombinant Human HGF R (c-MET)/Fc Chimera (R&D system, Catalog Number: 358-MT/CF) in Coating buffer (0.05M bicarbonate pH9.6, Sigma C-3041)₃and stored overnight at 4° C. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 μL of blocking solution (1% BSA in TBST buffer) was added in each well and the plates were incubated for at least 30 minutes at room temperature. The plates were washed three times. Then the purified antibodies were successively diluted across the plates in blocking solution. After one hour incubation at room temp, the plates were washed three times. Goat anti-human kappa light chain specific peroxidase conjugated antibody (Sigma A7164) was diluted in blocking solution to 1 in 2000 and was added to each well. The plates were incubated for one hour. This was washed three times and then OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 5 minutes later by the addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

The results of the ELISA are shown in the FIG. 161 and confirm that mAbdAbs BPC1017 and BPC1018 bind to recombinant human c-MET with comparable activity to the antibodies BPC1019 and BPC1020. The negative control Pascolizumab and BPC1604 (an IGF-1R/VEGF mAbdAb) showed no binding to c-MET.

32.4 VEGF Binding ELISA

96-well high binding plates were coated with 0.4 μg/mL of human VEGF (GSK) in PBS and incubated at 4° C. overnight. The plates were washed twice with Tris-Buffered Saline with 0.05% of Tween-20 (TBST). 100 μL of blocking solution (4% BSA in TBST buffer) was added to each well and the plates were incubated for at least one hour at room temperature. Another wash step was then performed. The purified antibodies were successively diluted across the plates in blocking solution. After one hour incubation at room temp, the plates were washed. Goat anti-human kappa light chain specific peroxidase conjugated antibody was diluted in blocking solution to 1 in 2000 and was added to each well. The plates were incubated for one hour at room temp. After another wash step, OPD (o-phenylenediamine dihydrochloride) SigmaFast substrate solution was added to each well and the reaction was stopped 5 minutes later by the addition of 25 μL of 3M sulphuric acid. Absorbance was read at 490 nm using the VersaMax Tunable Microplate Reader (Molecular Devices) using a basic endpoint protocol.

FIG. 162 shows the results of the ELISA which confirms that mAbdAbs BPC1017 and BPC1018 bind to recombinant human VEGF. The positive control BPC1604 also showed binding to recombinant human VEGF whereas Pascolizumab, BPC1019 and BPC1020 showed no binding to VEGF.

Example 33
mAbdAbs Containing the Anti-IL13 dAbs DOM10-53-546 dAb and DOM10-53-567
33.1 Construction, Expression and Purification

The anti-IL4 mAb-anti-IL13dAbs shown in Table 41 were cloned and expressed transiently in HEK2936E cells, purified (as described in examples 1, 1.3 and 1.5 respectively).

TABLE 41

Sequence ID

Name
Description
No.

PascoH-TVAAPS-546
H chain = Pascolizumab heavy chain-
157 (= H chain)

TVAAPS-DOM10-53-546 dAb
15 (= L chain)

L chain = Pascolizumab light chain

PascoH-TVAAPS-567
H chain = Pascolizumab heavy chain-
159 (= H chain)

TVAAPS-DOM10-53-567 dAb
15 (= L chain)

L chain = Pascolizumab light chain

Purified PascoH-TVAAPS-546 and PascoH-TVAAPS-567 mAbdAbs were analysed by size exclusion chromatography (SEC) and sodium dodecyl sulphate poly acrylamide gel electrophoresis (SDS PAGE), under reducing conditions. The SEC and SDS PAGE data are shown in FIGS. 163, 164, 165 and 166.

33.2 Biacore Analysis of Binding to IL-13 and IL-4

Purified PascoH-TVAAPS-546 and PascoH-TVAAPS-567 were tested for binding to human IL-13 and human IL-4 using the BIAcore™ T100 at 25° C. (as described in methods 4 and 5). These data are shown in Table 42.

TABLE 42

Binding affinity, KD (nM)

Human IL-4
Human IL-13

Molecule
on rate
off rate

on rate
off rate

(purified material)
(ka, Ms⁻¹)
(kd, s⁻¹)
KD (nM)
(ka, Ms⁻¹)
(kd, s⁻¹)
KD (nM)

PascoH-TVAAPS-546
4.92E+6
2.37E−5
0.00482
2.26E+5
1.69E−4
0.747

PascoH-TVAAPS-567
—
—
Tight binding
4.46E+5
1.70E−5
0.038

observed

Anti-human IL-13
—
—
Does not
1.00E+6
3.78E−4
0.377

bind

mAb

Pascolizumab
4.25E+6
2.43E−5
0.00572
—
—
Does

not bind

The mAbdAbs tested in this assay both bound IL-4 with very high affinity (NB, for PascoH-TVAAPS-567 this was beyond the sensitivity of the machine) and with similar binding affinity to that of the anti-human IL4 mAb alone (Pascolizumab). PascoH-TVAAPS-546 and PascoH-TVAAPS-567 both bound IL-13. Note that the anti-IL-13 dAbs alone (DOM10-53-546 and DOM10-53-567) were not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

These mAbdAbs were also tested for binding to cynomolgus IL-13 using the BIAcore™ T100 at 25° C. (as described in method 23). These data are shown in Table 43. A mAbdAb capture level between 500 and 750 relative response units was achieved, six IL-13 concentration curves (256, 64, 16, 4, 1 and 0.25 nM) were assessed for both the mAbdAbs and the anti-IL13 mAb.

TABLE 43

Binding affinity for

Molecule
cynomolgus IL-13 (KD)

(purified material)
on rate (ka, Ms⁻¹)
off rate (kd, s⁻¹)
KD (nM)

PascoH-TVAAPS-546
1.67E+6
2.09E−2
12.5

PascoH-TVAAPS-567
5.92E+5
5.22E−3
8.8

Anti-IL13 mAb
4.79E+5
8.22E−5
0.171

PascoH-TVAAPS-546 and PascoH-TVAAPS-567 both bound cynomolgus IL-13 with similar binding affinities. Note that the anti-IL-13 dAbs alone (DOM10-53-546 and DOM10-53-567) were not tested in this assay as the dAb cannot be captured onto the Protein A or anti-human IgG coated CM5 chip; instead, the anti-human IL13 mAb was used as a positive control to demonstrate IL-13 binding in this assay.

33.3 Neutralisation of Human IL-13 and Cynomolgus IL-13 in TF-1 Cell Bioassays

Purified PascoH-TVAAPS-546 and PascoH-TVAAPS-567 were tested for neutralisation of human IL-13 and cynomolgus IL-13 in TF-1 cell bioassays (as described in method 8 and method 20 respectively). FIGS. 167 and 168 show the neutralisation data for human IL-13 and cynomolgus IL-13 (in the TF-1 cell bioassays) respectively. DOM10-53-616 was included as a positive control for neutralisation of IL-13 in these bioassays. A dAb with specificity for an irrelevant antigen (negative control dAb) was also included as a negative control for neutralisation of IL-13. In addition, PascoH-616 and PascoH-TVAAPS-616 were also tested in these assays.

Both PascoH-TVAAPS-546 and PascoH-TVAAPS-567 fully neutralised the bioactivity of human and cynomolgus IL-13 in the TF-1 cell bioassays.

ND₅₀values were calculated from the dataset. The ND₅₀value is the concentration of mAbdAb or mAb or dAb, which is able to neutralise the bioactivity of IL-13 by 50%. The mean ND₅₀value, the standard deviation (SD) and the number of times tested (n) are shown in table 44.

TABLE 44

Mean ND₅₀value &

standard deviation (nM)

human IL-13

cyno IL-13

Molecule
mean (SD)
n
mean (SD)
n

PascoH-TVAAPS-546
1.522
1
33.88
1

PascoH-TVAAPS-567
2.06
1
38.25
1

DOM10-53-616
0.536
1
3.57
1

Negative control dAb
did not

did not

neutralise

neutralise

Example 34
mAbdAbs with IgG2, IgG4 and IgG4PE Heavy Chain Constant Regions
34.1 Construction of mAbdAbs

The heavy chain constant regions of human antibody isotypes IgG2, IgG4 and a variant IgG4 (IgG4PE) genes were amplified from existing constructs by PCR and cloned using standard molecular biology techniques into an expression vector encoding the PascoH-GS-474 heavy chain (SEQ ID NO: 48). The mAbdAbs antibodies designed and tested are listed in Table 45.

TABLE 45

SED ID NO: of amino

Antibody ID
Description
acid sequence

BPC1617
PascoH IgG2-GS-474
207

Pasco Kappa
15

BPC1618
PascoH IgG4-GS-474
208

Pasco Kappa
15

BPC1619
PascoH IgG4PE-GS-474
209

Pasco Kappa
15

34.2 Expression

The mAbdAbs set out in table 45 were expressed, along with PascoH-GS-474 (SEQ ID NO: 48 and 15) which is designated BPC1000. Briefly, 750 μl of HEK293 cells at 1.5×10⁶cells/ml were co-transfected with heavy and light chain expression plasmids previously incubated with 293fectin reagent (Invitrogen # 51-0031). These were placed in a shaking incubator at 37° C., 5% CO₂, and 95% RH. After 1 hour, Tryptone feeding media was added and the cells grown for a further 72 hours. Supernatant was harvested by centrifugation.

34.3 IL-4 Binding ELISA

The supernatants containing these mAbdAbs were assessed for binding to recombinant human IL-4. Briefly, ELISA plates were coated with human IL-4 (made at GSK) at 1 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline). Various dilutions of the cell supernatant, an anti IL-4 monoclonal antibody (Pascolizumab) and an antibody of irrelevant specificity (586 anti IL-13) were added. All samples were diluted in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results presented in FIG. 169 show that the mAbdAbs containing the alternative isotypes all bind to human IL-4. For the mAbdAbs BPC1000, BPC1617, BPC1618 and BPC1619, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 169 is represented as a dilution factor of the neat supernatant material. For the anti-IL4 and IL-13 control antibodies, purified material was used in the assay and the starting concentration of 1 μg/ml and 1 μg/ml was used respectively (which is equivalent to dilution factor of 1 in FIG. 169).

34.4 IL-13 Binding ELISA

The supernatants containing these mAbdAbs were assessed for binding to recombinant human IL-13. Briefly, ELISA plates were coated with human IL-13 (made at GSK) at 5 μg/ml and blocked with blocking solution (4% BSA in Tris buffered saline). Various dilutions of the cell supernatant, an anti IL-13 monoclonal antibody (586) and an antibody of irrelevant specificity (Pascolizumab anti IL-4) were added. All samples were diluted in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBS+0.05% Tween 20 (TBST). Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) at a dilution of 1/1000 in blocking solution. The plate was incubated for 1 hour at room temp before washing in TBST. The plate was developed by addition of OPD substrate (Sigma P9187) and colour development stopped by addition of 3M H₂SO₄. Absorbance was measured at 490 nm with a plate reader and the mean absorbance plotted.

The results presented in FIG. 170 show that the bispecific antibodies containing the alternative isotypes all bind to human IL-13. For the bispecific antibodies BPC1000, BPC1617, BPC1618 and BPC1619, the amount of antibody in the supernatant was not quantified thus the data presented in FIG. 170 is represented as a dilution factor of the neat supernatant material. For the anti-IL4 and IL-13 control antibodies, purified material was used in the assay at a starting concentration of 1 μg/ml and 1 μg/ml respectively (which is equivalent to dilution factor of 1 in FIG. 170).

Example 35
Alternative Anti-IL-13/IL-4 mAbdAbs

35.1 Construction of Anti-IL-13/IL-4 mAbdAbs with Alternative Variable Region Sequences

Using standard molecular biology techniques, DNA sequences encoding alternative heavy chain variable region anti-IL-13 mAb designated ‘C1’ and ‘D1’ were transferred from existing constructs to an expression vector containing DNA encoding the hIgG1 constant region fused to an anti IL-4 domain antibody (DOM9-112-210) via a TVAAPSGS linker at the c-terminus of the constant region. DNA sequences encoding alternative light chain variable region of IL-13 mAbs designated ‘M0’ and ‘N0’ were assembled de novo and cloned into expression vectors containing the human Ck constant region. These alternative heavy and light chain antibody variable regions comprise the same CDR regions as the anti-IL-13 antibody described in SEQ ID NO: 12 and 13 but with an alternative humanised variable framework region.

35.2 Construction of mAbdAbs using the variable regions of the anti IL-13 mAb ‘656’

Using standard molecular biology techniques, a DNA sequence encoding the heavy chain variable region of the humanised anti IL-13 mAb ‘656’, were transferred from an existing construct and cloned into an expression vector containing DNA encoding the hIgG1 constant region fused to an anti IL-4 domain antibody (DOM9-112-210) via a TVAAPS linker at the c-terminus of the constant region. A DNA sequence encoding the variable light region was transferred from an existing construct and cloned into an expression vector containing the human Ck constant region.

35.3 Expression of mAbdAbs

Briefly, 25 ml of HEK293 cells at 1.5×10⁶cells/ml were co-transfected with heavy and light chain expression plasmids previously incubated with 293fectin reagent (Invitrogen # 51-0031). These were placed in a shaking incubator at 37° C., 5% CO₂, and 95% RH. After 24 hours Tryptone feeding media was added and the cells grown for a further 72 hours. Supernatant was harvested by centrifugation and IgG levels quantified by ELISA. The antibodies constructed and expressed are listed in Table 46.

TABLE 46

SED ID NO: of

Antibody ID/

amino acid

Name
Description
sequence

BPC1607
H chain = C1-TVAAPSGS-210
151

L chain = M0 Kappa
154

BPC1608
H chain = C1-TVAAPSGS-210
151

L chain = N0 Kappa
153

BPC1609
H chain = C1-TVAAPSGS-210
151

L chain = 586 Kappa
13

(Anti-human IL-13 mAb light chain)

BPC1610
H chain = D1-TVAAPSGS-210
152

L chain = M0 Kappa
154

BPC1611
H chain = D1-TVAAPSGS-210
152

L chain = N0 Kappa
153

BPC1612
H chain = D1-TVAAPSGS-210
152

L chain = 586 Kappa
13

(Anti-human IL-13 mAb light chain)

BPC1613
H chain = 586H-TVAAPS-210
88

(Anti-human IL-13 mAb heavy chain-

TVAAPS-DOM9-112-210 dAb)

L chain = M0 Kappa
154

BPC1614
H chain = 586H-TVAAPS-210
88

(Anti-human IL-13 mAb heavy chain-

TVAAPS-DOM9-112-210 dAb)

L chain = N0 Kappa
153

BPC1615
H chain = 656H-TVAAPS-210
155

(Anti-human IL-13 mAb 2 heavy chain-

TVAAPS-DOM9-112-210 dAb)

L chain = 656 Kappa
156

(Anti-human IL-13 mAb 2 light chain)

BPC1602
H chain = 586H-TVAAPS-210
88

(586H-
(Anti-human IL-13 mAb heavy chain-

TVAAPS-210
TVAAPS-DOM9-112-210 dAb)

GS removed)
L chain = 586 Kappa
13

(Anti-human IL-13 mAb light chain)

35.4 Binding of the mAbdAbs to IL-13

The binding activity of the mAbdAbs to IL-13 was assessed by ELISA. In brief, 5 μg/ml recombinant E. coil-expressed human IL-13 (made and purified at GSK) was coated to a 96-well ELISA plate. The wells were blocked for 2 hours at room temperature, mAbdAb constructs were then titrated out down the plate. Binding was detected using a 1 in 1000 dilution of anti-human kappa light chain peroxidase conjugated antibody (catalogue number A7164, Sigma-Aldrich).

FIG. 177 shows that all of the tested molecules were able to bind to human IL-13. Although BPC1615 showed binding in this ELISA it was not possible to accurately quantify the concentration of this molecule and therefore the IL-13 binding ELISA data for this molecule is not plotted in FIG. 177. BPC1615 has also been shown to have high affinity binding to IL-13 in an independent Biacore assay (Table 47).

35.5 Binding of Anti-IL13 mAb-Anti-IL4dAbs to IL-13 by BIAcore™

Cell supernatants from the HEK cell transfections were also tested for binding to recombinant E. Coli-expressed human IL-13 using BIAcore™ at 25° C. (as described in method 4). BPC1601 was tested as a purified protein. Binding affinities, presented in Table 47, confirm that all antibodies show high affinity binding to human IL-13.

TABLE 47

ka
kd
KD (nM)

C1-TVAAPSGS-210 &
5.15E+5
8.89E−4
1.73

M0Kappa BPC1607

C1-TVAAPSGS-210 &
4.90E+5
8.83E−4
1.80

N0Kappa BPC1608

C1-TVAAPSGS-210 &
7.55E+5
7.61E−4
1.01

586kappa BPC1609

D1-TVAAPSGS-210 &
3.31E+5
4.66E−4
1.41

M0kappa BPC1610

D1-TVAAPSGS-210 &
2.59E+5
3.31E−4
1.28

N0kappa BPC1611

D1-TVAAPSGS-210 &
4.85E+5
2.74E−4
0.565

586kappa BPC1612

586H TVAAPS-210 &
5.54E+5
4.45E−4
0.804

M0kappa BPC1613

586H TVAAPS-210 &
5.49E+5
4.42E−4
0.805

N0kappa BPC1614

656H TVAAPS-210 &
4.89E+6
4.17E−4
0.085

656kappa BPC1615

586H-TVAAPS-210noGS
8.21E+5
4.62E−4
0.562

BPC1602

586H-nolinker-210noGS
8.93E+5
3.93E−4
0.440

BPC1601 purified

Example 36
Generation of mAbdAb with Specificity for Human IL-5 and Human IL-13

36.1 Construction and Expression of mAbdAb

A mAbdAb molecule having the heavy chain set out in SEQ ID NO: 65 and the light chain set out in SEQ ID NO: 72 was expressed in HEK2936E cells. This was designated MepolizumabL-G4S-474 or BPC1021.

36.2 Binding of Anti-IL5 mAb-Anti-IL13dAb to IL-5 and IL-13

This mAbdAb (in cell supernatants) was tested for binding to human IL-13 in a direct binding ELISA (as described in method 1). These data are shown in FIG. 173. The sample was transfected and tested in duplicate and this has been annotated as sample A and sample B.

This mAbdAb bound IL-13. Purified anti-human IL13 mAb alone was included in this assay as a positive control for IL-13 binding. Purified anti-human IL-4 mAb (Pascolizumab) and anti-human IL-5 mAb (Mepolizumab) were included as negative controls for IL-13 binding.

This mAbdAb was also tested for binding to human IL-5 in a direct binding ELISA (as described in Example 31.4) These data are illustrated in FIG. 174.

MepolizumabL-G4S-474 bound IL-5. Purified anti-human IL4 mAb (Pascolizumab) and purified anti human 13 mAb were included as negative controls for binding to IL-5. Purified anti-human IL5 mAb (Mepolizumab) was used as a positive control to demonstrate IL-5 binding in this assay.

Sequences

TABLE 49

DNA
Protein

Sequence
Sequence

Protein or polynucleotide description
(SEQ ID NO:)
(SEQ ID NO:)

DOM9-155-25
—
1

DOM9-155-147
60
2

DOM9-155-154
61
3

DOM9-112-210
—
4

DOM10-53-474
—
5

G4S Linker
—
6

Linker
—
7

Linker
—
8

Linker
—
9

Linker
—
10

Linker
—
11

Anti-human IL13 mAb Heavy chain &
205
12

alternative polynucleotide sequence
206

Anti-human IL13 mAb Light chain
210
13

Pascolizumab Heavy chain
211
14

Pascolizumab Light chain
212
15

Mepolizumab Heavy chain
213
65

Mepolizumab Light chain
214
66

Anti-human IL18 mAb Heavy chain
—
67

Anti-human IL18 mAb Light chain
—
68

586H-25 Heavy chain
—
16

586H-147 Heavy chain
—
17

586H-154 Heavy chain
—
18

586H-210 Heavy chain
—
19

586H-G4S-25 Heavy chain
—
20

586H-G4S-147 Heavy chain
—
21

586H-G4S-154 Heavy chain
—
22

586H-G4S-210 Heavy chain
—
23

586H-TVAAPS-25 Heavy chain
—
24

586H-TVAAPS-147 Heavy chain
—
25

586H-TVAAPS-154 Heavy chain
122
26

586H-TVAAPS-210 Heavy chain
167
27

586H-ASTKG-25 Heavy chain
—
28

586H-ASTKG-147 Heavy chain
—
29

586H-ASTKG-154 Heavy chain
—
30

586H-ASTKG-210 Heavy chain
192
31

586H-EPKSC-25 Heavy chain
—
32

586H-EPKSC-147 Heavy chain
—
33

586H-EPKSC-154 Heavy chain
—
34

586H-EPKSC-210 Heavy chain
—
35

586H-ELQLE-25 Heavy chain
—
36

586H-ELQLE-147 Heavy chain
—
37

586H-ELQLE-154 Heavy chain
—
38

586H-ELQLE-210 Heavy chain
—
39

586H Heavy chain-GS
—
40

586H-ASTKG Heavy chain
—
41

586H-EPKSC Heavy chain
—
42

586H-ELQLE Heavy chain
—
43

586L-G4S-25 Light chain
—
44

586L-G4S-147 Light chain
—
45

586L-G4S-154 Light chain
—
46

586L-G4S-210 Light chain
—
47

PascoH-474 Heavy chain
215
48

PascoH-G4S-474 Heavy chain
—
49

PascoH-TVAAPS-474 Heavy chain
216
50

PascoH-ASTKG-474 Heavy chain
—
51

PascoH-EPKSC-474 Heavy chain
—
52

PascoH-ELQLE-474 Heavy chain
—
53

PascoL-474 Light chain
217
54

PascoL-G4S-474 Light chain
—
55

PascoL-TVAAPS-474 Light chain
218
56

PascoL-ASTKG-474 Light chain
—
57

PascoL-EPKSC-474 Light chain
—
58

PascoL-ELQLE-474 Light chain
—
59

Interleukin-4
—
62

Interleukin-13
—
63

Mammalian signal sequence
—
64

IGF1R binding VH CDR3
—
80

IGF1R binding VH CDR2
—
81

IGF1R binding VH CDR1
—
82

IGF1R binding VL CDR1
—
83

IGF1R alternative VL CDR2
—
84

IGF1R binding VL CDR3
—
85

IGF1R binding VL CDR2
—
86

IL-18mAb-G4S-DOM9-112-210 heavy chain
—
69

IL-18mAb-G4S-DOM10-53-474 Light Chain
—
70

IL-5 mAb-G4S-DOM9-112-210 heavy chain
219
71

IL-5 mAb-G4S-DOM10-53-474 light chain
220
72

Anti-TNF mAb Light Chain
169
73

Anti-TNF mAb-DOM16-39-542 Heavy Chain
170
74

Anti-TNF mAb-DOM15-26-593 Heavy Chain
168
75

DOM 15-26-593-VHdUMMY Heavy Chain
172
76

DOM 4-130-54-VKdUMMY Light Chain
171
77

DOM 15-26-anti-TNFmAb Heavy Chain
—
78

DOM 16-39-542-anti-TNFmAb Light Chain
—
79

586H-210 Heavy chain (GS removed)
221
87

586H-TVAAPS-210 Heavy chain (GS removed)
222
88

586H-ASTKGPT-210 Heavy chain (both GS
—
89

removed)

586H-ASTKGPS-210 Heavy chain (both GS
—
90

removed, linker ASTKGPS)

PascoH-474 Heavy Chain (GS removed)
223
91

PascoH-TVAAPS-474 Heavy Chain (GS
224
92

removed)

PascoH-ASTKGPT-474 Heavy Chain (both GS
—
93

removed)

PascoH-ASTKGPS-474 Heavy Chain (both GS
—
94

removed, linker ASTKGPS)

PascoH-ASTKGPS-474 Heavy Chain (second
—
95

GS removed, linker ASTKGPS)

PascoH-ASTKGPT-474 Heavy Chain (second
225
96

GS removed)

EGFR binding VH CDR1
—
97

EGFR binding VH CDR2
—
98

EGFR binding VH CDR3
—
99

EGFR binding VL CDR1
—
100

EGFR binding VL CDR2
—
101

EGFR binding VL CDR3
—
102

EGFR epitope
—
103

EGFR binding VH alternative CDR1
—
104

EGFR binding VH alternative CDR2
—
105

EGFR binding VH alternative CDR3
—
106

EGFR binding VH alternative CDR2
—
107

Heavy chain of anti-IGF-1R antibody H0L0 with
226
108

DOM15-26-593 fused at C-terminus with

TVAAPSGS linker

IGF1RmAb-GS-DOM15-26-593 Heavy chain
227
109

anti-IGF-1R antibody Heavy chain
228
110

Light chain of anti-IGF-1R antibody H0L0 with
229
111

DOM15-26-593 fused at C-terminus with

TVAAPSGS linker

Light chain of anti-IGF-1R antibody H0L0 with
230
112

DOM15-26-593 fused at C-terminus with GS

linker

anti-IGF-1R antibody Light chain
231
113

Variable heavy domain of antibody 2B9
—
114

Variable light domain of antibody 2B9
—
115

Anti-CD20 mAb heavy chain with TVAAPSGS
—
116

linker and DOM10-53-474 domain antibody

fused at C-terminus

Anti-CD20 mAb Light Chain
—
117

Anti-CD20 mAb heavy chain with GS linker and
—
118

DOM10-53-474 domain antibody fused at C-

terminus

Anti-CD20 light chain with TVAAPSGS linker
—
119

and DOM10-53-474 domain antibody fused at

C-terminus

Anti-CD20 mAb heavy chain
—
120

Anti-CD20 mAb light chain with GS linker and
—
121

DOM10-53-474 domain antibody fused at C-

terminus

antiIGF1R Heavy Chain-GS-TLPC
232
123

antiIGF1R Heavy Chain-GS-CT01 adnectin
233
124

antiIGF1R Heavy Chain-TVAAPSGS-TLPC
234
125

antiIGF1R Heavy Chain-GS-AFFI
235
126

antiIGF1R Heavy Chain-TVAAPSGS-AFFI
236
127

antiIGF1R Heavy Chain-GS-DRPN
237
128

antiIGF1R Heavy Chain-TVAAPSGS-DRPN
238
129

Anti IL-4 heavy Chain-GS-anti RNAse A
239
130

camelidVHH

Anti IL-4 heavy Chain-GS-NARV
240
131

antiIGF1R Heavy Chain-TVAAPSGS-CT01
241
133

adnectin

antiIL13-Heavy Chain-GS-antiTNFα adnectin
—
134

antiIL13-Heavy Chain-TVAAPSGS-antiTNFα
—
135

adnectin

Erbitux Heavy chain-RS-CT01 adnectin
242
136

Anti-EGFR mAb Light chain (RS12)
243
137

Erbitux Light chain-RS-CT01 adnectin
244
138

Anti-EGFR mAb Heavy chain
245
139

11F8 Heavy Chain-GS-CT01 adnectin
—
140

11F8 Light Chain
—
141

11F8 Light Chain-GS-CT01 adnectin
—
142

11F8 Heavy Chain
—
143

CT01 adnectin-GSTG- Erbitux Heavy Chain
246
144

CT01 adnectin-STG-Erbitux Light Chain
247
145

Anti IL-4 Heavy Chain-GS-anti TNF-α adnectin
270
146

Anti IL-4 Heavy Chain-TVAAPSGS- anti TNF-α
132
147

adnectin

DOM10-53-616
—
148

PascoH-616 Heavy chain
248
149

PascoH-TVAAPS-616 Heavy chain
249
150

C1-TVAAPSGS-210 Heavy chain
—
151

D1-TVAAPSGS-210 Heavy chain
—
152

N0 Light chain
—
153

M0 Light chain
—
154

656H-TVAAPS-210 Heavy chain
250
155

656 Light chain
251
156

PascoH-TVAAPS-546_Heavy chain
252
157

PascoH-546_Heavy chain
253
158

PascoH-TVAAPS-567_Heavy chain
254
159

PascoH-567_Heavy chain
255
160

656 Heavy chain
256
161

DOM15-26-VHdUMMY Heavy Chain
162
163

Cetuximab-DOM15-26-593 Heavy Chain
164
165

Pascolizumab alternative Heavy chain

166

Cetuximab-DOM15-26-593 Heavy Chain no
173
174

linker

Cetuximab-DOM15-26-593 Heavy Chain
175
176

STGGGGGS linker

Cetuximab-DOM15-26-593 Heavy Chain
177
178

STGGGGGSGGGGS linker

Avastin-DOM16-39-542 Heavy Chain
179
180

Avastin Light chain
181
182

Cetuximab-DOM10-53-474 Heavy chain
183
184

Anti-TNF mAb-DOM15-10-11 Heavy Chain
—
185

Anti-EGFR mAb-DOM15-10-11 Heavy Chain
—
186

Anti-EGFR mAb-DOM15-26-593 Light Chain
187
188

GSTG

Anti-EGFR mAb-DOM15-26-593 Light Chain
189
190

GSTVAAPS

Mepolizumab alternative Heavy chain

191

IL-12/18 DVDH TVAAPS-210 heavy chain
—
193

IL-12/18 DVD Kappa light chain
—
194

Anti IL-5 Heavy Chain-G4S-dAb474-
257
195

TVAAPSGS-dAb210

Anti CD-20 Heavy Chain-TVAAPSGS-dAb154-
258
196

TVAAPSGS-dAb474

Anti CD-20 Heavy Chain-TVAAPSGS-dAb210-
259
197

TVAAPSGS-dAb474

anti cMET 5D5v2 Heavy Chain (hole)-GS-
260
198

dAb593

anti cMET 5D5v2 Heavy Chain (knob)-GS-
261
199

dAb593

anti cMET 5D5v2 Light Chain
262
200

anti cMET 5D5v2 IgG4 Heavy Chain
263
201

(UNIBODY)-GS-dAb593

anti cMET 5D5v2 Heavy Chain (hole)
264
202

anti cMET 5D5v2 Heavy Chain (knob)
265
203

anti cMET 5D5v2 IgG4 Heavy Chain
266
204

(UNIBODY)

PascoH IgG2-GS-474 heavy chain
267
207

PascoH IgG4-GS-474 heavy chain
268
208

PascoH IgG4PE-GS-474 heavy chain
269
209

BRIEF DESCRIPTION OF FIGURES

FIGS. 1 to 7: Examples of antigen-binding constructs

FIG. 8: Schematic diagram of mAbdAb constructs.

FIG. 9: SEC and SDS Page analysis of PascoH-G4S-474

FIG. 10: SEC and SDS Page analysis of PascoL-G4S-474

FIG. 11: SEC and SDS Page analysis of PascoH-474

FIG. 12: SEC and SDS Page analysis of PascoHL-G4S-474

FIG. 13: mAbdAb supernatants binding to human IL-13 in a direct binding ELISA

FIG. 14: mAbdAb supernatants binding to human IL-4 in a direct binding ELISA

FIG. 15: Purified mAbdAbs binding to human IL-13 in a direct binding ELISA

FIG. 16: purified mAbdAbs binding to human IL-4 in a direct binding ELISA

FIG. 17: mAbdAb supernatants binding to human IL-4 in a direct binding ELISA FIG. 18: mAbdAb supernatants binding to human IL-13 in a direct binding ELISA

FIG. 19: purified mAbdAb binding to human IL-4 in a direct binding ELISA

FIG. 20A: purified mAbdAb binding to human IL-13 in a direct binding ELISA

FIG. 20B: purified mAbdAb binding to cynomolgus IL-13 in a direct binding ELISA

FIG. 21: mAbdAb binding kinetics for IL-4 using BIAcore™

FIG. 22: mAbdAb binding kinetics for IL-4 using BIAcore™

FIG. 23: mAbdAbs binding kinetics for IL-13 using BIAcore™

FIG. 24: Purified anti-IL13 mAb-anti-IL4dAbs ability to neutralise human IL-13 in a TF-1 cell bioassay

FIG. 25: Purified anti-IL13 mAb-anti-IL4dAbs ability to neutralise human IL-4 in a TF-1 cell bioassay

FIG. 26: purified anti-IL4 mAb-anti-IL13dAbs PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474 ability to neutralise human IL-4 in a TF-1 cell bioassay

FIG. 27: purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G4S-474 ability to neutralise human IL-13 in a TF-1 cell bioassay

FIG. 28: purified anti-IL4 mAb-anti-IL13dAbs, PascoH-G4S-474, PascoH-474, PascoL-G4S-474 and PascoHL-G45-474 ability to simultaneously neutralise human IL-4 and human IL-13 in a dual neutralisation TF-1 cell bioassay

FIG. 29: DOM10-53-474 SEC-MALLS

FIG. 30: DOM9-112-210 SEC-MALLS

FIG. 31: DOM9-155-25 SEC-MALLS

FIG. 32: DOM9-155-25 SEC-MALLS Overlay of all three signals

FIG. 33: DOM9-155-147 SEC-MALLS

FIG. 34: DOM9-155-159 SEC-MALLS

FIG. 35: Control for MW assignment by SEC-MALLS: BSA

FIG. 36: schematic diagram of a trispecific mAbdAb molecule

FIG. 37: Trispecific mAbdAb IL18 mAb-210-474 (supernatants) binding to human IL-18 in direct binding ELISA

FIG. 38: Trispecific mAbdAb IL18 mAb-210-474 (supernatants) binding to human IL-13 in direct binding ELISA

FIG. 39: Trispecific mAbdAb IL18 mAb-210-474 (supernatants) binding to human IL-4 in direct binding ELISA

FIG. 40: Trispecific mAbdAb Mepo-210-474 (supernatant) binding to human IL-13 in direct binding ELISA

FIG. 41: Trispecific mAbdAb Mepo-210-474 (supernatant) binding to human IL-4 in direct binding ELISA

FIG. 42: Cloning of the anti-TNF/anti-EGFR mAb-dAb

FIG. 43. SDS-PAGE analysis of the anti-TNF/anti-EGFR mAb-dAb

FIG. 44. SEC profile of the anti-TNF/anti-EGFR mAb-dAb (Example 10)

FIG. 45: Anti-EGFR activity of Example 10 FIG. 46. Anti-TNF activity of Example 10

FIG. 47. SDS-PAGE analysis of the anti-TNF/anti-VEGF mAb-dAb (Example 11)

FIG. 48. SEC profile of the anti-TNF/anti-VEGF mAb-dAb (Example 11)

FIG. 49. Anti-VEGF activity of Example 11

FIG. 50. Anti-TNF activity of example 11 FIG. 51. Cloning of the anti-VEGF/anti-IL1R1 dAb-extended-IgG (Example 12)

FIG. 52. SDS-PAGE analysis of the anti-TNF/anti-VEGF dAb-extended IgG A (Example 12)

FIG. 53: SDS-PAGE analysis of the anti-TNF/anti-VEGF dAb-extended IgG B (Example 12)

FIG. 54. SEC profile of the anti-TNF/anti-VEGF dAb-extended IgG A (Example 12)

FIG. 55: SEC profile of the anti-TNF/anti-VEGF dAb-extended IgG B (Example 12)

FIG. 56. Anti-VEGF activity of Example 12 (DMS2091)

FIG. 57 Anti-VEGF activity of Example 12 (DMS2090)

FIG. 58. Anti-IL1R1 activity of Example 12 (DMS2090)

FIG. 59: Anti-IL1R1 activity of Example 12 (DMS2091)

FIG. 60: Cloning of the anti-TNF/anti-VEGF/anti-EGFR mAb-dAb (Example 13)

FIG. 61. SDS-PAGE analysis of the anti-TNF/anti-VEGF/anti-EGFR mAb-dAb (Example 13)

FIG. 62: Anti-VEGF activity of Example 13

FIG. 63: Anti-TNF activity of Example 13

FIG. 64: Anti-EGFR activity of Example 13

FIG. 65: SEC analysis of purified Bispecific antibodies, BPC1603 (A), BPC1604 (B), BPC1605 (C), BPC1606 (D)

FIG. 66. Binding of bispecific antibodies to immobilised IGF-1R

FIG. 67. Binding of Bispecific antibodies to immobilised VEGF

FIG. 68. Inhibition of ligand mediated receptor phosphorylation by various bispecific antibodies

FIG. 69: Inhibition of ligand mediated receptor phosphorylation by various bispecific antibodies

FIG. 70 ADCC assay with anti-CD20/IL-13 bispecific antibody

FIG. 71: ADCC assay with anti-CD20/IL-13 bispecific antibody

FIG. 72: ADCC assay with anti-CD20/IL-13 bispecific antibody using a shorter dose range

FIG. 73: ADCC assay with anti-CD20/IL-13 bispecific antibody using a shorter dose range

FIG. 74: CDC assay with anti-CD20/IL-13 bispecific antibody

FIG. 75: CDC assay with anti-CD20/IL-13 bispecific antibody

FIG. 76: BPC1803 and BPC1804 binding in recombinant human IGF-1R ELISA

FIG. 77: BPC1803 and BPC1804 binding in recombinant VEGF binding ELISA

FIG. 78: BPC1805 and BPC1806 binding in recombinant human IGF-1R ELISA

FIG. 79: BPC1805 and BPC1806 binding in recombinant human HER2ELISA

FIG. 80: BPC1807 and BPC1808 binding in recombinant human IGF-1R ELISA

FIG. 81: BPC1807 and BPC1808 binding in recombinant human HER2ELISA

FIG. 82: BPC1809 binding in recombinant human IL-4 ELISA

FIG. 83: BPC1809 binding in RNAse A ELISA.

FIG. 84: BPC1816 binding in recombinant human IL-4 ELISA

FIG. 85: BPC1816 binding in HEL ELISA

FIG. 86: BPC1801 and BPC 1802 binding in recombinant human IGF-1R ELISA

FIG. 87: BPC1801 and BPC1802 binding in recombinant human VEGFR2ELISA

FIG. 88 BPC1823 and BPC 1822 binding in recombinant human IL-4 ELISA

FIG. 88
b BPC1823 (higher concentration supernatant) binding in recombinant human IL-4 ELISA

FIG. 89: BPC1823 and BPC1822 binding in recombinant human TNF-α ELISA

FIG. 89
b: BPC1823 (higher concentration supernatant) binding in recombinant human TNF-α ELISA

FIG. 90: SEC profile for PascoH-474 GS removed

FIG. 91: SEC profile for PascoH-TVAAPS-474 GS removed

FIG. 92: SEC profile for PascoH-GS-ASTKGPT-474 2^ndGS removed

FIG. 93: SEC profile for 586H-210 GS removed

FIG. 94: SEC profile for 586H-TVAAPS-210 GS removed

FIG. 95: SDS PAGE for PascoH-474 GS removed (lane B) and PascoH-TVAAPS-474 GS removed (lane A)

FIG. 96: SDS PAGE for PascoH-GS-ASTKGPT-474 2^ndGS removed [A=non-reducing conditions, B=reducing conditions]

FIG. 97: SDS PAGE for 586H-210 GS removed (lane A)

FIG. 98: SDS PAGE for 586H-TVAAPS-210 GS removed (lane A)

FIG. 99: Purified PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed binding in human IL-4 ELISA

FIG. 100: Purified PascoH-474 GS removed and PascoH-TVAAPS-474 GS removed binding in human IL-13 ELISA

FIG. 101: Purified PascoH-474 GS removed, PascoH-TVAAPS-474 GS removed, PascoH-616 and PascoH-TVAAPS-616 binding in cynomolgus IL-13 ELISA

FIG. 102: mAbdAbs inhibition of human IL-4 binding to human IL-4Rα by ELISA FIG. 103: mAbdAbs inhibition of human IL-4 binding to human IL-4Rα by ELISA

FIG. 104 Neutralisation of human IL-13 in TF-1 cell bioassays by mAbdAbs

FIG. 105: Neutralisation of cynomolgus IL-13 in TF-1 cell bioassays by mAbdAbs

FIG. 106: Neutralisation of human IL-4 in TF-1 cell bioassays by mAbdAbs

FIG. 107: Neutralisation of cynomolgus IL-4 in TF-1 cell bioassays by mAbdAbs

FIG. 108: Ability of mAbdAbs to inhibit binding of human IL-13 binding to human IL-13Rα2

FIG. 109: SEC profile for PascoH-616

FIG. 110: SEC profile for PascoH-TVAAPS_—616

FIG. 111: SDS PAGE for PascoH-616 [E1=non-reducing conditions, E2=reducing conditions]

FIG. 112: SDS PAGE for PascoH-TVAAPS-616 [A=non-reducing conditions, B=reducing conditions]

FIG. 113: purified PascoH-616 and PascoH-TVAAPS-616 binding in human IL-13 ELISA

FIG. 114: Neutralisation of human IL-13 in TF-1 cell bioassays by mAbdAbs

FIG. 114
a: Neutralisation of cynomolgus IL-13 in TF-1 cell bioassays by mAbdAbs

FIG. 115: Inhibition of IL-4 activity by PascoH-474 GS removed

FIG. 116: Inhibition of IL-13 activity by PascoH-474 GS removed

FIG. 117: Inhibition of IL-4 activity by 586-TVAAPS-210

FIG. 118: Inhibition of IL-13 activity by 586-TVAAPS-210

FIG. 119: Inhibition of IL-4 activity by Pascolizumab

FIG. 120: Inhibition of IL-4 activity by DOM9-112-210

FIG. 121: Inhibition of IL-13 activity by anti-IL13 mAb

FIG. 122: Inhibition of IL-13 activity by DOM10-53-474

FIG. 123: Activity of control mAb and dAb in IL-4 whole blood assay

FIG. 124: Activity of control mAb and dAb in IL-13 whole blood assay

FIG. 125: The concentration of drug remaining at various time points post-dose assessed by ELISA against both TNF & EGFR.

FIG. 126: The concentration of drug remaining at various time points post-dose assessed by ELISA against both TNF & VEGF.

FIG. 127: The concentration of drug remaining at various time points post-dose assessed by ELISA against both IL1R1 & VEGF.

FIG. 128: SDS-PAGE of the purified DMS4010

FIG. 129: SEC profile of the purified DMS4010

FIG. 130: Anti-EGFR potency of DMS4010

FIG. 131: anti-VEGF receptor binding assay

FIG. 132: pharmacokinetic profile of the dual targeting anti-EGFR/anti-VEGF mAbdAb

FIG. 133: SDS-PAGE analysis purified DMS4011

FIG. 134: SEC profile of the purified DMS4011

FIG. 135: Anti-EGFR potency of DMS4011

FIG. 136: DMS4011 in anti-VEGF receptor binding assay

FIG. 137: SDS-PAGE analysis of the purified samples DMS4023 and DMS4024

FIG. 138: The SEC profile for DMS4023

FIG. 139: The SEC profile for DMS4024

FIG. 140: Anti-EGFR potency of the mAbdAb DMS4023

FIG. 141: DMS4023 and DMS4024 in anti-VEGF receptor binding assay

FIG. 142: SDS-PAGE analysis of the purified DMS4009

FIG. 143: The SEC profile for DMS4009

FIG. 144: Anti-EGFR potency of the mAbdAb DMS4009

FIG. 145: DMS4009 in anti-VEGF receptor binding assay

FIG. 146: SDS-PAGE analysis of the purified DMS4029

FIG. 147: The SEC profile for DMS4029

FIG. 148: Anti-EGFR potency of the mAbdAb DMS4029

FIG. 149: DMS4029 in the IL-13 cell-based neutralisation assay

FIG. 150: SDS-PAGE analysis of the purified samples DMS4013 and DMS4027

FIG. 151: The SEC profile for DMS4013

FIG. 152: The SEC profile for DMS4027

FIG. 153: Anti-EGFR potency of the mAbdAb DMS4013

FIG. 154: DMS4013 in anti-VEGF receptor binding assay

FIG. 155: BPC1616 binding in recombinant human IL-12 ELISA

FIG. 156: BPC1616 binding in recombinant human IL-18 ELISA

FIG. 157: BPC1616 binding in recombinant human IL-4 ELISA

FIG. 158: BPC1008, 1009 and BPC1010 binding in recombinant human IL-4 ELISA

FIG. 159: BPC1008 binding in recombinant human IL-5 ELISA

FIG. 160: BPC1008, 1009 and BPC1010 binding in recombinant human IL-13 ELISA

FIG. 161: BPC1017 and BPC1018 binding in recombinant human c-MET ELISA

FIG. 162: BPC1017 and BPC1018 binding in recombinant human VEGF ELISA

FIG. 163: SEC profile for PascoH-TVAAPS-546

FIG. 164: SEC profile for PascoH-TVAAPS-567

FIG. 165: SDS PAGE for PascoH-TVAAPS-546 [A=non-reducing conditions, B=reducing conditions]

FIG. 166: SDS PAGE for PascoH-TVAAPS-567 [A=non-reducing conditions, B=reducing conditions]

FIG. 167: neutralisation data for human IL-13 in the TF-1 cell bioassay

FIG. 168: neutralisation data for cynomolgus IL-13 in the TF-1 cell bioassay

FIG. 169: mAbdAbs containing alternative isotypes binding in human IL-4 ELISA

FIG. 170: mAbdAbs containing alternative isotypes binding in human IL-13 ELISA

FIG. 171: BPC1818 and BPC1813 binding in recombinant human EGFR ELISA FIG. 172: BPC1818 and BPC1813 binding in recombinant human VEGFR2ELISA

FIG. 173: anti-IL5 mAb-anti-IL13dAb binding in IL-13 ELISA

FIG. 174: anti-IL5 mAb-anti-IL13dAb binding in IL-5 ELISA

FIG. 175: BPC1812 binding in recombinant human VEGFR2ELISA

FIG. 176: BPC1812 binding in recombinant human EGFR ELISA

FIG. 177: mAbdAb binding in human IL-13 ELISA

1. Domain Antibodies

SEQ ID NO: 1 = DOM9-155-25

DIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQKPGKAPKLLIAWASTLDSGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 2 = DOM9-155-147

DIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQKPGKAPKLLIAWASSLYEGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 3 = DOM9-155-154

DIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQKPGKAPKLLIAWASSLQGGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 4 = DOM9-112-210

EVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQAPGKGLEWVSWIISSGTETYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 5 = DOM10-53-474

GVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 60 = DNA sequence of DOM9-155-147 (protein = SEQ ID NO: 2)

GACATCCAGATGACCCAATCACCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT

CACTtGCCGGGCAAGTCGCCCCATtAGCGACTGGTTACATtGGTATCAGCAGAAACCAGGGA

AAGCCCCCAAGCTCCTGATCGCCTGGGCGtCCTCGTTGTACGAGGGGGtCCCATCACGtTTC

AGTGGCAGTGGGTCGGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCCGAAGATTT

CGCTACGTACTACTGTTTGCAGGAGGGGTGGGGTCCTCCGACGTTCGGCCAAGGGACCAAGG

TGGAAATCAAACGG

SEQ ID NO: 61 = DNA sequence of DOM9-155-154 (protein = SEQ ID NO: 3)

GACATCCAGATGACCCAATCACCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT

CACTTGCCGGGCAAGTCGCCCCATTAGCGACTGGTTACATTGGTATCAGCAGAAACCAGGGA

AAGCCCCCAAGCTCCTGATCGCCTGGGCGTCCAGCTTGCAGGGGGGGGTCCCATCACGTTTC

AGTGGCAGTGGGTCGGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCCGAAGATTT

CGCTACGTACTACTGTTTGCAGGAGGGGTGGGGTCCTCCGACGTTCGGCCAAGGGACCAAGG

TGGAAATCAAACGG

2. Linkers

SEQ ID NO: 6 = G4S linker

GGGGS

SEQ ID NO: 7 = linker

TVAAPS

SEQ ID NO: 8 = linker

ASTKGPT

SEQ ID NO: 9 = linker

ASTKGPS

SEQ ID NO: 10 = linker

EPKSCDKTHTCPPCP

SEQ ID NO: 11 = linker

ELQLEESCAEAQDGELDG

3. Monoclonal Antibodies

SEQ ID NO: 12 = Anti-human IL13 mAb (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 13 = Anti-human IL13 mAb (L chain)

DIVMTQSPLSLPVTPGEPASISCRSSQNIVHINGNTYLEWYLQKPGQSPRLLIYKISDRFSG

VPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 14 = Pascolizumab (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

SEQ ID NO: 15 = Pascolizumab (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 65 = Mepolizumab (H chain)

QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSA

LMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK

SEQ ID NO: 66 = Mepolizumab (L chain)

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRES

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 67 = Anti-human IL-18 mAb (H chain)

QVQLVQSGAEVKKPGASVKVSCKVSGEISTGYYFHWVRQAPGKGLEWMGRIDPEDDSTKYAE

RFKDRVTMTEDTSTDTAYMELSSLRSEDTAVYYCTTWRIYRDSSGRPFYVMDAWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 68 = Anti-human IL-18 mAb (L chain)

DIQMTQSPSSVSASVGDRVTITCLASEDIYTYLTWYQQKPGKAPKLLIYGANKLQDGVPSRF

SGSGSGTDYTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGEC

4. Bispecific mAbdAbs

NB, the underlined portion of the sequence in “bold” text corresponds to the linker. ‘GS’ amino acid residues) the DNA coding sequence of which was used as the BamHI cloning site) are not in ‘bold’ text, but are underlined.

SEQ ID NO: 16 = 586H-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQK

PGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQG

TKVEIKR

SEQ ID NO: 17 = 586H-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQK

PGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQG

TKVEIKR

SEQ ID NO: 18 = 586H-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQQK

PGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFGQG

TKVEIKR

SEQ ID NO: 19 = 586H-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQ

APGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLG

RFDYWGQGTLVTVSS

SEQ ID NO: 20 = 586H-G4S-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWY

QQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTF

GQGTKVEIKR

SEQ ID NO: 21 = 586H-G4S-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWY

QQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTF

GQGTKVEIKR

SEQ ID NO: 22 = 586H-G4S-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWY

QQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTF

GQGTKVEIKR

SEQ ID NO: 23 = 586H-G4S-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGW

VRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

SLGRFDYWGQGTLVTVSS

SEQ ID NO: 24 = 586H-TVAAPS-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWL

HWYQQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGP

PTFGQGTKVEIKR

SEQ ID NO: 25 = 586H-TVAAPS-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWL

HWYQQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGP

PTFGQGTKVEIKR

SEQ ID NO: 26 = 586H-TVAAPS-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWL

HWYQQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGP

PTFGQGTKVEIKR

SEQ ID NO: 27 = 586H-TVAAPS-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFG

MGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

CAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 28 = 586H-ASTKG-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSASTKGPTGSDIQMTQSPSSLSASVGDRVTITCRASRPIS

DWLHWYQQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEG

WGPPTFGQGTKVEIKR

SEQ ID NO: 29 = 586H-ASTKG-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSASTKGPTGSDIQMTQSPSSLSASVGDRVTITCRASRPIS

DWLHWYQQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEG

WGPPTFGQGTKVEIKR

SEQ ID NO: 30 = 586H-ASTKG-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSASTKGPTGSDIQMTQSPSSLSASVGDRVTITCRASRPIS

DWLHWYQQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEG

WGPPTFGQGTKVEIKR

SEQ ID NO: 31 = 586H-ASTKG-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSASTKGPTGSEVQLLESGGGLVQPGGSLRLSCAASGFTFR

NFGMGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA

VYYCAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 32 = 586H-EPKSC-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGSDIQMTQSPSSLSASVGDRVTIT

CRASRPISDWLHWYQQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFA

TYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 33 = 586H-EPKSC-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGSDIQMTQSPSSLSASVGDRVTIT

CRASRPISDWLHWYQQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFA

TYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 34 = 586H-EPKSC-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGSDIQMTQSPSSLSASVGDRVTIT

CRASRPISDWLHWYQQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFA

TYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 35 = 586H-EPKSC-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGSEVQLLESGGGLVQPGGSLRLSC

AASGFTFRNFGMGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMN

SLRAEDTAVYYCAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 36 = 586H-ELQLE-25 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGSDIQMTQSPSSLSASVGDRV

TITCRASRPISDWLHWYQQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 37 = 586H-ELQLE-147 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGSDIQMTQSPSSLSASVGDRV

TITCRASRPISDWLHWYQQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 38 = 586H-ELQLE-154 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGSDIQMTQSPSSLSASVGDRV

TITCRASRPISDWLHWYQQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPE

DFATYYCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 39 = 586H-ELQLE-210 (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGSEVQLLESGGGLVQPGGSLR

LSCAASGFTFRNFGMGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYL

QMNSLRAEDTAVYYCAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 40 = 586H-GS (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGS

SEQ ID NO: 41 = 586H-ASTKG (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSASTKGPTGS

SEQ ID NO: 42 = 586H-EPKSC (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGS

SEQ ID NO: 43 = 586H-ELQLE (H chain)

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGS

SEQ ID NO: 44 = 586L-G4S-25 (L chain)

DIVMTQSPLSLPVTPGEPASISCRSSQNIVHINGNTYLEWYLQKPGQSPRLLIYKISDRFSG

VPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSDIQMTQSPSSLSASVGDRVTITCR

ASRPISDWLHWYQQKPGKAPKLLIAWASTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 45 = 586L-G4S-147 (L chain)

DIVMTQSPLSLPVTPGEPASISCRSSQNIVHINGNTYLEWYLQKPGQSPRLLIYKISDRFSG

VPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSDIQMTQSPSSLSASVGDRVTITCR

ASRPISDWLHWYQQKPGKAPKLLIAWASSLYEGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 46 = 586L-G4S-154 (L chain)

DIVMTQSPLSLPVTPGEPASISCRSSQNIVHINGNTYLEWYLQKPGQSPRLLIYKISDRFSG

VPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSDIQMTQSPSSLSASVGDRVTITCR

ASRPISDWLHWYQQKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATY

YCLQEGWGPPTFGQGTKVEIKR

SEQ ID NO: 47 = 586L-G4S-210 (L chain)

DIVMTQSPLSLPVTPGEPASISCRSSQNIVHINGNTYLEWYLQKPGQSPRLLIYKISDRFSG

VPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSEVQLLESGGGLVQPGGSLRLSCAA

SGFTFRNFGMGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSL

RAEDTAVYYCAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 48 = PascoH-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGK

GLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGY

DYWGQGTLVTVSS

SEQ ID NO: 49 = PascoH-G4S-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGGGGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQA

PGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDE

PGYDYWGQGTLVTVSS

SEQ ID NO: 50 = PascoH-TVAAPS-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWV

RQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATA

EDEPGYDYWGQGTLVTVSS

SEQ ID NO: 51 = PascoH-ASTKG-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSASTKGPTGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDM

GWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

ATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 52 = PascoH-EPKSC-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSEPKSCDKTHTCPPCPGSGVQLLESGGGLVQPGGSLRLSCAASG

FTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRA

EDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 53 = PascoH-ELQLE-474 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSELQLEESCAEAQDGELDGGSGVQLLESGGGLVQPGGSLRLSCA

ASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNS

LRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 54 = PascoL-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSGVQLLESGGGLVQPGGSLRLSCAASGFT

FAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED

TAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 55 = PascoL-G4S-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGVQLLESGGGLVQPGGSLRLSCAAS

GFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLR

AEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 56 = PascoL-TVAAPS-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECTVAAPSGSGVQLLESGGGLVQPGGSLRLSC

AASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMN

SLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 57 = PascoL-ASTKG-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECASTKGPTGSGVQLLESGGGLVQPGGSLRLS

CAASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQM

NSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 58 = PascoL-EPKSC-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECEPKSCDKTHTCPPCPGSGVQLLESGGGLVQ

PGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 59 = PascoL-ELQLE-474 (L chain)

DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGI

PSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGECELQLEESCAEAQDGELDGGSGVQLLESGGG

LVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

5. Cytokines

SEQ ID NO: 62 = IL-4 (Interleukin-4)

HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRA

ATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNS

CPVKEANQSTLENFLERLKTIMREKYSKCSS

SEQ ID NO: 63 = IL-13 (Interleukin-13)

GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESL

INVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL

HLKKLFREGRFN

6. Signal Sequence

SEQ ID NO: 64 = Mammalian amino acid signal

sequence

MGWSCIILFLVATATGVHS

7. IGF-1R Binding CDRs

SEQ ID 80 = VH CDR3

WILYYGRSKWYFDV

SEQ ID 81 = VH CDR2

NINPNNGGTNYNQKFKD

SEQ ID 82 = VH CDR1

DYYMN

SEQ ID 83 = VL CDR1

RSSQSIVQSNGDTYLE

SEQ ID 84 = alternative VL CDR2

RISNRFS

SEQ ID 85 = VL CDR3

FQGSHVPYT

SEQ ID 86 = VL CDR2

RVSNRFS

8. Trispecific mAbdAbs

SEQ ID NO: 69 = IL18mAb-G4S-DOM9-112-210 (H chain)

QVQLVQSGAEVKKPGASVKVSCKVSGEISTGYYFHWVRQAPGKGLEWMGRIDPEDDSTKYAE

RFKDRVTMTEDTSTDTAYMELSSLRSEDTAVYYCTTWRIYRDSSGRPFYVMDAWGQGTLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGKGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMG

WVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

KSLGRFDYWGQGTLVTVSS

SEQ ID NO: 70 = IL18mAb-G4S-DOM10-53-474 (L chain)

DIQMTQSPSSVSASVGDRVTITCLASEDIYTYLTWYQQKPGKAPKLLIYGANKLQDGVPSRF

SGSGSGTDYTLTISSLQPEDFATYYCLQGSKFPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGVQLLESGGGLVQPGGSLRLSCAASGFTF

AWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT

AVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 71 = IL-5 mAb-G4S-DOM9-112-210 heavy chain

QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSA

LMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGKGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQAPG

KGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLGRFD

YWGQGTLVTVSS

SEQ ID NO: 72 = IL-5 mAb-G4S-DOM10-53-474 light chain

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRES

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNVHSFPFTFGGGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGVQLLESGGGLVQPGGSLRLSCA

ASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNS

LRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

9. Dual Targeting Anti-TNF/Anti-EGFR mAbdAb

SEQ ID NO: 73 = anti-TNFmAb (L chain)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRF

SGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 74 = anti-TNFmAb-DOM16-39-542 (H chain)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD

SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGDIQMTQSPSSLSASVGDRVTITCRASQWIGNLLDWYQQKPGK

APKLLIYYASFLQSGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQANPAPLTFGQGTKV

EIKR

10. Dual Targeting Anti-TNF/Anti-VEGF mAbdAb

SEQ ID NO: 75 = anti-TNFmAb-DOM15-26-593 (H chain)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD

SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPG

KGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLD

YWGQGTLVTVSS

11. Dual Targeting Anti-IL1R1/Anti-VEGF dAb-Extended IgG

SEQ ID NO: 76 = DOM 15-26-593-VHdUMMY (H chain)

EVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSSASTKGPSE

VQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY

TQKSLSLSPGK

SEQ ID NO: 77 = DOM4-130-54-VkdUMMY (L chain)

DIQMTQSPSSLSASVGDRVTITCRASQDIYLNLDWYQQKPGKAPKLLINFGSELQSGVPSRF

SGSGYGTDFTLTISSLQPEDFATYYCQPSFYFPYTFGQGTKVEIKRTVAAPSDIQMTQSPSS

LSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT

LTISSLQPEDFATYYCQQSYSTPNTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

12. Triple Targeting Anti-TNF/Anti-EGFR/Anti-VEGF mAbdAb

SEQ ID NO: 78 = DOM 15-26-anti-TNFmAb (H chain)

EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWVSEISPSGSYTYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKFDYWGQGTLVTVSSASTKGPSE

VQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADS

VEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

SEQ ID NO: 79 = DOM 16-39-542-anti-TNFmAb (L chain)

DIQMTQSPSSLSASVGDRVTITCRASQWIGNLLDWYQQKPGKAPKLLIYYASFLQSGVPSRF

SGSGYGTDFTLTISSLQPEDFATYYCQQANPAPLTFGQGTKVEIKRTVAAPSDIQMTQSPSS

LSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFT

LTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO: 87 = 586H-210 (H chain) GS removed

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQAP

GKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLGRF

DYWGQGTLVTVSS

SEQ ID NO: 88 = 586H-TVAAPS-210 (H chain) GS removed

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMG

WVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

KSLGRFDYWGQGTLVTVSS

SEQ ID NO: 89 = 586H-ASTKGPT-210 (H chain) both GS removed

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKASTKGPTEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGM

GWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

AKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 90 = 586H-ASTKGPS-210 (H chain) both GS removed

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKASTKGPSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGM

GWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

AKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 91 = PascoH-474 (H chain) GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGL

EWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDY

WGQGTLVTVSS

SEQ ID NO: 92 = PascoH-TVAAPS-474 (H chain) GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQ

APGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAED

EPGYDYWGQGTLVTVSS

SEQ ID NO: 93 = PascoH-ASTKGPT-474 (H chain) Both GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKASTKGPTGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVR

QAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAE

DEPGYDYWGQGTLVTVSS

SEQ ID NO: 94 = PascoH-ASTKGPS-474 (H chain) Both GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKASTKGPSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVR

QAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAE

DEPGYDYWGQGTLVTVSS

SEQ ID NO: 95 = PascoH-ASTKGPS-474 (H chain) Second GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSASTKGPSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGW

VRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT

AEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 96 = PascoH-ASTKGPT-474 (H chain) Second GS removed

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSASTKGPTGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGW

VRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT

AEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 97 = CDRH1

GYSITSDFAWN

SEQ ID NO: 98 = CDRH2

GYISYSGNYNPSLK

SEQ ID NO: 99 = CDRH3

VTAGRGFPY

SEQ ID NO: 100 = CDRL1

HSSQDINSNIG

SEQ ID NO: 101 = CDRL2

HGINLDD

SEQ ID NO: 102 = CDRL3

VQYAQFPWT

SEQ ID NO: 103 = EGFR epitope

CGADSYEMEEDGVRKC

SEQ ID NO: 104 = CDRH1

SDFAWN

SEQ ID NO: 105 = CDRH2

YISYSGNYNPSLK

SEQ ID NO: 106 = CDRH3

AGRGFPY

SEQ ID NO: 107 = CDRH2

YISYSGNYNPSLKS

SEQ ID NO: 108 = Heavy chain of anti-IGF-1R antibody H0L0 with DOM15-26-

593 fused at C-terminus with TVAAPSGS linker

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKTVAAPSGSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMM

WVRQAPGKGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

KDPRKLDYWGQGTLVTVSS

SEQ ID NO: 109 = Heavy chain of anti-IGF-1R antibody H0L0 with DOM15-26-

593 fused at C-terminus with GS linker

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKGSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAP

GKGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKL

DYWGQGTLVTVSS

SEQ ID NO: 110 = Heavy chain of anti-IGF-1R antibody H0L0

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID NO: 111 = Light chain of anti-IGF-1R antibody H0L0 with DOM15-26-593

fused at C-terminus with TVAAPSGS linker

DIVMTQSPLSLPVTPGEPASISCRSSQSIVQSNGDTYLEWYLQKPGQSPQLLIYRVSNRFSG

VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTVAAPSGSEVQLLVSGGGLVQPGGSLRLS

CAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQM

NSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 112 = Light chain of anti-IGF-1R antibody H0L0 with DOM15-26-593

fused at C-terminus with GS linker

DIVMTQSPLSLPVTPGEPASISCRSSQSIVQSNGDTYLEWYLQKPGQSPQLLIYRVSNRFSG

VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGSEVQLLVSGGGLVQPGGSLRLSCAASGF

TFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE

DTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 113 = Light chain of anti-IGF-1R antibody H0L0

DIVMTQSPLSLPVTPGEPASISCRSSQSIVQSNGDTYLEWYLQKPGQSPQLLIYRVSNRFSG

VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 114 = Variable heavy domain of antibody 2B9

QVQLKQSGPGLVQSSQSLSITCTISGFSLTSHGIYWLRQSPGKGLEWLGVIWSGGSADYNAA

FISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARSPYYYRSSLYAMDYWGQGTSVTVSS

SEQ ID NO: 115 = Variable light domain of antibody 2B9

NIVLTQSPKSMSMSIGERVTLSCKASENVGTYVSWYQQKAEQSPKLLIYGASNRHTGVPDRF

TGSGSSTDFTLTISSVQAEDLADYHCGQSYSDPLTFGAGTKLELKRA

SEQ ID NO: 116 = Protein sequence of anti-CD20 mAb heavy chain with

TVAAPSGS linker and DOM10-53-474 domain antibody fused at C-terminus

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ

KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWV

RQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATA

EDEPGYDYWGQGTLVTVSS

SEQ ID NO: 117 = Protein sequence anti-CD20 mAb VL-human CK light chain

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS

GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 118 = Protein sequence of anti-CD20 mAb heavy chain with GS

linker and DOM10-53-474 domain antibody fused at C-terminus

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ

KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGK

GLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGY

DYWGQGTLVTVSS

SEQ ID NO: 119 = Protein sequence of anti-CD20 mAb light chain with

TVAAPSGS linker and DOM10-53-474 domain antibody fused at C-terminus

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS

GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGECTVAAPSGSGVQLLESGGGLVQPGGSLRLSCAASGF

TFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE

DTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 120 = Protein sequence of anti-CD20 mAb VH-human IgG1 heavy

chain

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ

KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

SEQ ID NO: 121 = Protein sequence of anti-CD20 mAb light chain with GS

linker and DOM10-53-474 domain antibody fused at C-terminus

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS

GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGECGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYD

MGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

CATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 122: 586H-TVAAPS-154 Heavy chain

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

GACCGTGGCCGCCCCCTCGGGATCCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCG

CCAGCGTGGGCGACAGGGTGACCATTACCTGCAGGGCCAGCAGGCCCATCAGCGACTGGCTG

CACTGGTACCAACAGAAGCCCGGCAAGGCTCCCAAGCTGCTGATCGCCTGGGCCAGCAGCCT

GCAGGGAGGCGTGCCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTCACCA

TCTCTTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGAGGGCTGGGGGCCC

CCTACTTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGG

SEQ ID NO: 123

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKGSDGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLK

GHNLEAKVTMLISGRCQEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHG

KPVRGVKLVGRDPKNNLEALEDFEKAAGARGLSTESILIPRQSETCSPG

SEQ ID NO: 124

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKGSEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFT

VPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO:125

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKTVAAPSGSDGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPM

TLTLLKGHNLEAKVTMLISGRCQEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYS

EGQLHGKPVRGVKLVGRDPKNNLEALEDFEKAAGARGLSTESILIPRQSETCSPG

SEQ ID NO: 126

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKGSVDNKFNKELRQAYWEIQALPNLNWTQSRAFIRSLYDDPSQS

ANLLAEAKKLNDAQAPK

SEQ ID NO: 127

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKTVAAPSGSVDNKFNKELRQAYWEIQALPNLNWTQSRAFIRSLY

DDPSQSANLLAEAKKLNDAQAPK

SEQ ID NO: 128

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYL

ATAHGHLEIVEVLLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKT

AFDISIGNGNEDLAEILQKL

SEQ ID NO: 129

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKTVAAPSGSDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYG

LTPLYLATAHGHLEIVEVLLKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQ

DKFGKTAFDISIGNGNEDLAEILQKL

SEQ ID NO: 130

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSQVQLVESGGGLVQAGGSLRLSCAASGYAYTYIYMGWFRQAPGK

EREGVAAMDSGGGGTLYADSVKGRFTISRDKGKNTVYLQMDSLKPEDTATYYCAAGGYELRD

RTYGQWGQGTQVTVSS

SEQ ID NO: 131

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSARVDQTPRSVTKETGESLTINCVLRDASYALGSTCWYRKKSGE

GNEESISKGGRYVETVNSGSKSFSLRINDLTVEDGGTYRCGLGVAGGYCDYALCSSRYAECG

DGTAVTVN

SEQ ID NO: 132: Anti IL-4 Heavy Chain-TVAAPSGS-anti TNF-a adnectin

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGC

CCCCTCGGGATCCGTGAGCGACGTGCCAAGGGACCTCGAGGTGGTGGCAGCCACTCCCACCT

CTCTGCTGATCAGCTGGGACACACACAACGCCTACAACGGCTACTACAGGATCACCTACGGA

GAGACCGGCGGCAATAGCCCCGTGAGGGAGTTCACCGTGCCCCACCCCGAGGTGACCGCCAC

CATTAGCGGCCTGAAGCCCGGCGTGGACGATACCATCACCGTCTACGCCGTGACCAACCACC

ACATGCCCCTGAGGATCTTCGGCCCCATCAGCATCAACCATAGGACC

SEQ ID NO: 133

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGNINPNNGGTNYNQ

KFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWILYYGRSKWYFDVWGRGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKTVAAPSGSEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNS

PVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO: 134

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGSVSDVPRDLEVVAATPTSLLISWDTHNAYNGYYRITYGET

GGNSPVREFTVPHPEVTATISGLKPGVDDTITVYAVTNHHMPLRIFGPISINHRT

SEQ ID NO: 135

QVQLVQSGAEVKKPGSSVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSVSDVPRDLEVVAATPTSLLISWDTHNAYNGYYR

ITYGETGGNSPVREFTVPHPEVTATISGLKPGVDDTITVYAVTNHHMPLRIFGPISINHRT

SEQ ID NO: 136

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTP

FTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGKRSEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQ

PPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO: 137

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF

SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 138

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRF

SGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGECRSEVVAATPTSLLISWRHPHFPTRYYRITYGETG

GNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO: 139

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTP

FTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK

SEQ ID NO: 140

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYN

PSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVP

LQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO: 141

EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 142

EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGECGSEVVAATPTSLLISWRHPHFPTRYYRITYGETG

GNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT

SEQ ID NO: 143

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYN

PSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

SEQ ID NO: 144

EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTI

TVYAVTDGRNGRLLSIPISINYRTGSTGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGV

HWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCA

RALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 145

EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTI

TVYAVTDGRNGRLLSIPISINYRTSTGDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW

YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTT

FGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 146

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGSVSDVPRDLEVVAATPTSLLISWDTHNAYNGYYRITYGETGGNS

PVREFTVPHPEVTATISGLKPGVDDTITVYAVTNHHMPLRIFGPISINHRT

SEQ ID NO: 147

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGSVSDVPRDLEVVAATPTSLLISWDTHNAYNGYYRITYG

ETGGNSPVREFTVPHPEVTATISGLKPGVDDTITVYAVTNHHMPLRIFGPISINHRT

SEQ ID NO: 148 = DOM10-53-616 (dAb)

GVQLLESGGGLVQPGGSLRLSCAASGFVFPWYDMGWVRQAPGKGLEWVSSIDWHGKITYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 149 = PascoH-616 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGVQLLESGGGLVQPGGSLRLSCAASGFVFPWYDMGWVRQAPGKGL

EWVSSIDWHGKITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDY

WGQGTLVTVSS

SEQ ID NO: 150 = PascoH-TVAAPS-616 (H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGVQLLESGGGLVQPGGSLRLSCAASGFVFPWYDMGWVRQ

APGKGLEWVSSIDWHGKITYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAED

EPGYDYWGQGTLVTVSS

SEQ ID NO: 151 = C1-TVAAPSGS-210 (H chain)

QVQLVQSGAEVKKPGASVKVSCKASGFYIKDTYMHWVRQAPGQGLEWMGTIDPANGNTKYVP

KFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFG

MGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

CAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 152 = D1-TVAAPSGS-210 (H chain)

EVQLVQSGAEVKKPGESLKISCKGSGFYIKDTYMHWVRQMPGKGLEWMGTIDPANGNTKYVP

KFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSIYDDYHYDDYYAMDYWGQGTLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV

FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKTVAAPSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFG

MGWVRQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

CAKSLGRFDYWGQGTLVTVSS

SEQ ID NO: 153 = N0 (L chain)

EIVLTQSPATLSLSPGERATLSCRSSQNIVHINGNTYLEWYQQKPGQAPRLLIYKISDRFSG

IPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 154 = M0 (L chain)

EIVLTQSPATLSLSPGERATLSCRSSQNIVHINGNTYLEWYQQKPGQAPRLLIYKISDRFSG

IPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 155 = 656H-TVAAPS-210_(H chain)

QVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQGLEWMGAIDPETGGTAYNQ

KFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYPMDYWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGKTVAAPSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQAP

GKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLGRF

DYWGQGTLVTVSS

SEQ ID NO: 156 = 656_(L chain)

EIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQAPRLLIYYASQSISGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

EKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 157 = PascoH-TVAAPS-546_(H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGVQLLESGGGLVQPGGSLRLSCAASGFVFPWYDMGWVRQ

APGKGLEWVSSIDWKGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAED

EPGYDYWGQGTLVTVSS

SEQ ID NO: 158 = PascoH-546_(H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGVQLLESGGGLVQPGGSLRLSCAASGFVFPWYDMGWVRQAPGKGL

EWVSSIDWKGGKTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDY

WGQGTLVTVSS

SEQ ID NO: 159 = PascoH-TVAAPS-567_(H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGVQLLESGGGLVQPGGSLRLSCAASGFVFAWYDMGWVRQ

APGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAED

EPGYDYWGQGTLVTVSS

SEQ ID NO: 160 = PascoH-567_(H chain)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKGVQLLESGGGLVQPGGSLRLSCAASGFVFAWYDMGWVRQAPGKGL

EWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDY

WGQGTLVTVSS

SEQ ID NO: 161 = 656_(H chain)

QVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQGLEWMGAIDPETGGTAYNQ

KFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYPMDYWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK

SEQ ID NO: 162

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC

CTGTGCAGCCTCCGGATTCACCTTTGGGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAG

GGAAGGGTCTAGAGTGGGTCTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGCAGAC

TCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAAT

GAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTTG

ACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCCAGCGAG

GTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTG

TGCAGCCTCCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGA

AGGGTCTAGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCC

GTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAA

CAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAGTTATGGTGCTTTTGACT

ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCCAGCGTGTTC

CCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAA

GGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGC

ACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTG

CCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACAC

CAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGCCCCCCCTGCC

CTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACC

CTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCACGAGGACCC

TGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCA

GAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAT

TGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCCTGCCCCTATCGA

GAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACACCCTGCCTCCCT

CCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC

AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC

CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCA

GATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 163

EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWVSEISPSGSYTYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKFDYWGQGTLVTVSSASTKGPSE

VQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGAFDYWGQGTLVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY

TQKSLSLSPGK

SEQ ID NO: 164

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCACCTGTACCGTGAGCG

GCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAGGCAAGGGCCTGGAATGGCTGGGAGTGAT

CTGGTCCGGCGGCAACACCGACTACAACACCCCCTTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGC

CAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACT

ACGACTACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGCCCCAGCGT

GTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC

CCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGA

GCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAA

CGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGC

CCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGA

TGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTG

GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTG

GTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCC

TGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACACCCTGCCTCC

CTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCT

TCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCA

CGAGGCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGAGGTGCAGCTG

TTGGTGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTA

AGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTTTCAGAGATTTCGCCTTCGGG

TTCTTATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTAT

CTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAGACTACT

GGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 165:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD

NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEIS

PSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 166

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTPVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

SEQ ID NO: 167 586H-TVAAPS-210 Heavy chain

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

GACCGTGGCCGCCCCCTCGGGATCCGAAGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGC

AGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGC

ATGGGCTGGGTCAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTC

CGGCACCGAGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACA

GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTAC

TGCGCCAAGAGCCTGGGCAGGTTCGACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAG

C

SEQ ID NO: 168

GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAG

CTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTG

GCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGAC

AGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCA

CCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTCTCGAGCGCTAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAG

CCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAA

CCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACA

ACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGG

TCTACACCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGA

GGTGCAGCTGTTGGTGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCT

GTGCAGCCTCCGGATTCACCTTTAAGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAGGG

AAGGGTCTAGAGTGGGTTTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGCAGACTC

CGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA

ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAGAC

TACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 169

GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCGGGCCA

GCCAGGGCATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGC

CAGCACCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGC

AGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAGAGCCCCTTACACCTTCGGCCAGGGCA

CCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTCAAGAG

CGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAAC

GCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA

CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAG

CCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 170

GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAG

CTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTG

GCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGAC

AGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGAT

GAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCA

CCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACCCTGGTGACAGTCTCGAGCGCTAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAG

CCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAA

CCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACA

ACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGG

TCTACACCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGA

CATCCAGATGACCCAGAGCCCTTCAAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCA

CCTGCCGGGCCAGCCAGTGGATCGGCAACCTGCTGGACTGGTATCAGCAGAAGCCCGGCAAG

GCCCCCAAGCTGCTGATCTACTACGCCAGCTTCCTGCAGAGCGGCGTGCCCAGCCGGTTTAG

CGGCAGCGGCTACGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCG

CCACCTACTACTGCCAGCAGGCCAACCCTGCCCCCCTGACCTTCGGCCAGGGTACCAAGGTG

GAAATCAAACGG

SEQ ID NO: 171

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT

CACTTGCCGGGCAAGTCAGGATATTTACCTGAATTTAGACTGGTATCAGCAGAAACCAGGGA

AAGCCCCTAAGCTCCTGATCAATTTTGGTTCCGAGTTGCAAAGTGGTGTCCCATCACGTTTC

AGTGGCAGTGGATATGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT

CGCTACGTACTACTGTCAACCGTCTTTTTACTTCCCTTATACGTTCGGCCAAGGGACCAAGG

TGGAAATCAAACGTACGGTGGCCGCCCCCAGCGACATCCAGATGACCCAGTCTCCATCCTCC

CTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAG

CTATTTAAATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAT

CCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACT

CTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACGTACTACTGTCAACAGAGTTACAG

TACCCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCCGCCCCCA

GCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTCAAGAGCGGCACCGCCAGCGTGGTGTGT

CTGCTGAACAACTTCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCA

GAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGA

GCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTG

ACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 172

GAGGTGCAGCTGTTGGTGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTC

CTGTGCAGCCTCCGGATTCACCTTTAAGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAG

GGAAGGGTCTAGAGTGGGTTTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGCAGAC

TCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAAT

GAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAG

ACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCCAGCGAG

GTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTG

TGCAGCCTCCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGA

AGGGTCTAGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCC

GTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAA

CAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAAGTTATGGTGCTTTTGACT

ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGCTAGCACCAAGGGCCCCAGCGTGTTC

CCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAA

GGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGC

ACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTG

CCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACAC

CAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGCCCCCCCTGCC

CTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACC

CTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCACGAGGACCC

TGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCA

GAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTGCTGCACCAGGAT

TGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCCTGCCCCTATCGA

GAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACACCCTGCCTCCCT

CCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC

AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCC

CCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCA

GATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 173

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCAC

CTGTACCGTGAGCGGCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAG

GCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGACTACAACACCCCC

TTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAA

CAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACTACGACT

ACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGA

CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA

GCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCA

AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACA

CCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGGAGGTGCAGCTGTTGGT

GTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAT

TCACCTTTAAGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGG

GTTTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGCAGACTCCGTGAAGGGCCGGTT

CACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCG

AGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAGACTACTGGGGTCAGGGA

ACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 174

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD

NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSG

SYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 175

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCACCTGTACCGTGAGCG

GCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAGGCAAGGGCCTGGAATGGCTGGGAGTGAT

CTGGTCCGGCGGCAACACCGACTACAACACCCCCTTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGC

CAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACT

ACGACTACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGCCCCAGCGT

GTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC

CCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGA

GCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAA

CGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGC

CCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGA

TGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTG

GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTG

GTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCC

TGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACACCCTGCCTCC

CTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCT

TCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCA

CGAGGCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGGTGGAGGTGGA

TCAGAGGTGCAGCTGTTGGTGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCT

CCGGATTCACCTTTAAGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTTTCAGA

GATTTCGCCTTCGGGTTCTTATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCC

AAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTC

GGAAGTTAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 176

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD

NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGGGGGSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEW

VSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 177

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCACCTGTACCGTGAGCG

GCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAGGCAAGGGCCTGGAATGGCTGGGAGTGAT

CTGGTCCGGCGGCAACACCGACTACAACACCCCCTTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGC

CAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACT

ACGACTACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGCCCCAGCGT

GTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC

CCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGA

GCAGCGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAA

CGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCTGC

CCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGA

TGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTG

GTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTG

GTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCC

TGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACACCCTGCCTCC

CTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCT

TCTTCCTGTACTCCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCA

CGAGGCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGGTGGAGGTGGA

TCAGGTGGAGGTGGATCAGAGGTGCAGCTGTTGGTGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTC

TCTCCTGTGCAGCCTCCGGATTCACCTTTAAGGCTTATCCGATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCT

AGAGTGGGTTTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC

TCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACT

GTGCGAAAGATCCTCGGAAGTTAGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 178

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD

NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGGGGGSGGGGSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPG

KGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTV

SS

SEQ ID NO: 179

GAGGTGCAGCTGGTCGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCAGCCTGAGACTGAG

CTGCGCCGCCAGCGGCTACACCTTCACCAACTACGGCATGAACTGGGTGCGGCAGGCCCCTG

GCAAGGGCCTGGAATGGGTGGGCTGGATCAACACCTACACCGGCGAGCCCACCTACGCCGCC

GACTTCAAGCGGCGGTTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGAT

GAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTACCCCCACTACTACG

GCAGCAGCCACTGGTACTTCGACTACTGGGGGCAGGGTACCCTGGTCACCGTCTCGAGCGCT

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATA

GCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

TCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAA

CGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAG

TGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCT

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGC

CCCAGGTCTACACCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACT

CCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGAC

CGGTGACATCCAGATGACCCAGAGCCCTTCAAGCCTGAGCGCCAGCGTGGGCGACAGAGTGA

CCATCACCTGCCGGGCCAGCCAGTGGATCGGCAACCTGCTGGACTGGTATCAGCAGAAGCCC

GGCAAGGCCCCCAAGCTGCTGATCTACTACGCCAGCTTCCTGCAGAGCGGCGTGCCCAGCCG

GTTTAGCGGCAGCGGCTACGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGG

ACTTCGCCACCTACTACTGCCAGCAGGCCAACCCTGCCCCCCTGACCTTCGGCCAGGGTACC

AAGGTGGAAATCAAACGG

SEQ ID NO: 180

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAA

DFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDYWGQGTLVTVSSA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKSTGDIQMTQSPSSLSASVGDRVTITCRASQWIGNLLDWYQQKP

GKAPKLLIYYASFLQSGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQANPAPLTFGQGT

KVEIKR

SEQ ID NO: 181

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCAT

CACCTGCAGCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCA

AGGCCCCCAAGGTGCTGATCTACTTCACCAGCTCCCTGCACAGCGGCGTGCCCAGCCGGTTT

AGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTT

CGCCACCTACTACTGCCAGCAGTACAGCACCGTGCCCTGGACCTTCGGCCAGGGTACCAAGG

TGGAGATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAG

CTGAAGAGCGGCACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAA

GGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAAAGCGTCACCGAGC

AGGACTCCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTAC

GAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA

GAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 182

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFT

LTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 183

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCAC

CTGTACCGTGAGCGGCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAG

GCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGACTACAACACCCCC

TTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAA

CAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACTACGACT

ACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGA

CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA

GCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCA

AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACA

CCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAGTCGACCGGTGGGGTGCA

GCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAG

CCTCCGGATTCACCTTCGCTTGGTATGATATGGGGTGGGTCCGCCAGGCTCCAGGGAAGGGT

CTAGAGTGGGTCTCAAGTATTGATTGGCATGGTGAGGTTACATACTACGCAGACTCCGTGAA

GGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC

TGCGTGCCGAGGACACCGCGGTATATTACTGTGCGACAGCGGAGGACGAGCCGGGGTATGAC

TACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 184

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKS

QVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF

PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSTGGVQL

LESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCATAEDEPGYDYWGQGTLVTVSS

SEQ ID NO: 185

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISR

DNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD

ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGKSTGDIQMTQSPSSLSASVGDRVTITCRASQWIGPELRWYQQKPGKAPKLLIYH

TSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPMTFGQGTKVEIKR

SEQ ID NO: 186

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKD

NSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKSTGDIQMTQSPSSLSASVGDRVTITCRASQWIGPELRWYQQKPGKAPKLLIYHTS

ILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPMTFGQGTKVEIKR

SEQ ID NO: 187

GACATCCTGCTGACCCAGAGCCCCGTGATCCTGAGCGTGAGCCCTGGCGAGAGAGTGAGCTTCAGCTGCCGGGCCA

GCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACGGCAGCCCCAGGCTGCTGATCAAGTACGC

CAGCGAGTCCATCAGCGGCATCCCCAGCCGGTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAAC

AGCGTGGAGAGCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCA

CCAAGCTGGAACTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTCAAGAG

CGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAAC

GCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA

CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAG

CCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGGATCCACCGGCGAGGTGCAGCTGTTGGTGTCTGGGGGAGGC

TTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAAGGCTTATCCGATGATGT

GGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTTTCAGAGATTTCGCCTTCGGGTTCTTATACATACTACGC

AGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG

CGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAGACTACTGGGGTCAGGGAACCCTGG

TCACCGTCTCGAGC

SEQ ID NO: 188

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFT

LSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGS

TGEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADSVKGRFTI

SRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 189

GACATCCTGCTGACCCAGAGCCCCGTGATCCTGAGCGTGAGCCCTGGCGAGAGAGTGAGCTTCAGCTGCCGGGCCA

GCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACGGCAGCCCCAGGCTGCTGATCAAGTACGC

CAGCGAGTCCATCAGCGGCATCCCCAGCCGGTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAAC

AGCGTGGAGAGCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCA

CCAAGCTGGAACTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTCAAGAG

CGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAAC

GCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCA

CCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAG

CCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGGATCCACGGTGGCCGCCCCCAGCGAGGTGCAGCTGTTGGTG

TCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTAAGGCTT

ATCCGATGATGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTTTCAGAGATTTCGCCTTCGGGTTCTTA

TACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAA

ATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGATCCTCGGAAGTTAGACTACTGGGGTC

AGGGAACCCTGGTCACCGTCTCGAGC

SEQ ID NO: 190

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFT

LSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGS

TVAAPSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADSVKG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 191

QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSALMSRLSI

SKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTPVTVSSASTKGPSVFPLAPSSKSTS

GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 192: 586H-ASTKG-210 Heavy chain

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

GGGATCAGCCAGCACCAAGGGCCCCACGGGATCCGAAGTGCAGCTCCTGGAGAGCGGCGGCG

GCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGG

AACTTCGGCATGGGCTGGGTCAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTCAGCTGGAT

CATCAGCTCCGGCACCGAGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCC

GCGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCC

GTCTACTACTGCGCCAAGAGCCTGGGCAGGTTCGACTACTGGGGACAGGGGACCCTGGTGAC

TGTGAGCAGC

SEQ ID NO: 193

EVTLRESGPALVKPTQTLTLTCTFSGFSLSKSVMGVSWIRQPPGKALEWLAHIYWDDDKYYN

PSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARRGIRSAMDYWGQGTTVTVSSASTKG

PEVQLVQSGTEVKKPGESLKISCKGSGYTVTSYWIGWVRQMPGKGLEWMGFIYPGDSETRYS

PTFQGQVTISADKSFNTAFLQWSSLKASDTAMYYCARVGSGWYPYTFDIWGQGTLVTVSSAS

TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH

EALHNHYTQKSLSLSPGKTVAAPSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVR

QAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSL

GRFDYWGQGTLVTVSS

SEQ ID NO: 194

DIVMTQSPDSLAVSLGERATINCKASQSVSNDVAWYQQKPGQPPKLLIYYASNRYTGVPDRF

SGSGSGTDFTLTISSLQAEDVAVYYCQQDYNSPWTFGGGTKVEIKRTVAAPEIVMTQSPATL

SVSPGERATLSCRASESASSNLAWYQQKPGQAPRLFIYTASTRATDIPARFSGSGSGTEFTL

TISSLQSEDFAVYYCQQYNNWPSITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO: 195: Anti IL-5 Heavy Chain-G4S-dAb474-TVAAPSGS-dAb210

QVTLRESGPALVKPTQTLTLTCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSA

LMSRLSISKDTSRNQVVLTMTNMDPVDTATYYCARDPPSSLLRLDYWGRGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGKGGGGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPG

KGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPG

YDYWGQGTLVTVSSTVAAPSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWVRQA

PGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSLGR

FDYWGQGTLVTVSS

SEQ ID NO: 196: Anti CD-20 Heavy Chain-TVAAPSGS-dAb154-TVAAPSGS-

dAb474

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ

KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGSDIQMTQSPSSLSASVGDRVTITCRASRPISDWLHWYQ

QKPGKAPKLLIAWASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQEGWGPPTFG

QGTKVEIKRTVAAPSGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGL

EWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDY

WGQGTLVTVSS

SEQ ID NO: 197: Anti CD-20 Heavy Chain-TVAAPSGS-dAb210-TVAAPSGS-

dAb474

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQ

KFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGKTVAAPSGSEVQLLESGGGLVQPGGSLRLSCAASGFTFRNFGMGWV

RQAPGKGLEWVSWIISSGTETYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKS

LGRFDYWGQGTLVTVSSTVAAPSGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWV

RQAPGKGLEWVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATA

EDEPGYDYWGQGTLVTVSS

SEQ ID NO: 198: anti cMET 5D5v2 Heavy Chain (hole)-GS-dAb593

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNP

NFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGKGSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGL

EWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWG

QGTLVTVSS

SEQ ID NO: 199: anti cMET 5D5v2 Heavy Chain (knob)-GS-dAb593

CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSEVQLLVSGGGLVQPGGSLRLSCAA

SGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSL

RAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 200: anti cMET 5D5v2 LightChain

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRES

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIF

PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 201: anti cMET 5D5v2 IgG4 Heavy Chain (UNIBODY)-GS-dAb593

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNP

NFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKG

PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

GSEVQLLVSGGGLVQPGGSLRLSCAASGFTFKAYPMMWVRQAPGKGLEWVSEISPSGSYTYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKLDYWGQGTLVTVSS

SEQ ID NO: 202: anti cMET 5D5v2 Heavy Chain (hole)

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNP

NFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL

HNHYTQKSLSLSPGK

SEQ ID NO: 203: anti cMET 5D5v2 Heavy Chain (knob)

CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 204: anti cMET 5D5v2 IgG4 Heavy Chain (UNIBODY)

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNP

NFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKG

PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC

VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

SEQ ID NO: 205: Anti-human IL13 mAb Heavy chain

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

G

SEQ ID NO: 206: Alternative Anti-human IL13 mAb Heavy chain

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTC

CTGCAAGGCTTCTGGATTCTACATTAAAGACACCTATATGCACTGGGTGCGACAGGCCCCTG

GACAAGGGCTTGAGTGGATGGGAACGATTGATCCTGCGAATGGTAATACTAAATATGTCCCG

AAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCT

GAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAGCATCTATGATGATT

ACCACTACGACGATTACTATGCTATGGACTACTGGGGCCAAGGGACACTAGTCACAGTCTCC

TCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGG

GGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT

GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA

CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT

CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT

GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC

TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG

CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG

GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC

GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGC

CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG

GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC

TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC

GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA

A

SEQ ID NO 207: PascoH IgG2-GS-474 heavy

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH

QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPGKGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLEW

VSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYWG

QGTLVTVSS

SEQ ID NO 208: PascoH IgG4-GS-474 heavy chain

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL

HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH

NHYTQKSLSLSLGKGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLE

WVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYW

GQGTLVTVSS

SEQ ID NO 209: PascoH IgG4PE-GS-474 heavy chain

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYN

PSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSAST

KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL

HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH

NHYTQKSLSLSLGKGSGVQLLESGGGLVQPGGSLRLSCAASGFTFAWYDMGWVRQAPGKGLE

WVSSIDWHGEVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATAEDEPGYDYW

GQGTLVTVSS

SEQ ID NO: 210: Anti-human IL13 mAb Light chain

GACATCGTGATGACCCAGTCTCCTCTGAGCCTCCCCGTGACCCCCGGCGAACCAGCCAGCAT

CTCCTGCAGAAGCAGCCAGAACATCGTGCACATCAACGGCAACACCTACCTGGAGTGGTACC

TGCAAAAGCCCGGCCAGAGCCCCAGGCTGCTGATCTACAAGATCAGCGACAGGTTCAGCGGC

GTGCCCGATAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGT

GGAGGCCGACGACGTGGGCATCTACTACTGCTTCCAGGGCAGCCACGTCCCCTGGACTTTCG

GACAGGGCACCAAGCTGGAGATTAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCC

CCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTA

CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGG

AGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG

AGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC

CAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 211: Pascolizumab Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 212: Pascolizumab Light chain

GACATCGTGCTGACCCAGAGCCCCTCTTCCCTGAGCGCAAGCGTGGGCGATAGGGTGACCAT

CACCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACATGAACTGGTACCAGC

AGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTCGAGTCAGGCATT

CCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACAATCAGCAGCCTGCA

GCCCGAGGACATCGCCACCTACTACTGCCAGCAGAGCAACGAGGACCCTCCCACCTTCGGAC

AGGGCACCAAGGTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCC

AGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCC

CCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGA

GCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGC

AAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAG

CCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 213: Mepolizumab Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACACAGACCCTCACTCTGAC

CTGCACCGTGAGCGGCTTCAGCCTGACCTCCTACAGCGTCCACTGGGTGAGGCAGCCCCCCG

GCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGCAAGCGGCGGCACCGACTACAACAGCGCC

CTGATGAGCAGGCTCTCCATCAGCAAGGACACCAGCCGGAACCAGGTGGTGCTGACCATGAC

CAACATGGACCCCGTGGACACCGCCACCTATTACTGCGCCAGGGACCCTCCCTCTAGCCTGC

TGAGGCTGGACTACTGGGGCAGGGGAACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGA

CCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAA

GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCA

AGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACA

CCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 214: Mepolizumab Light chain

GACATCGTGATGACCCAGTCTCCCGATTCACTGGCCGTGAGCCTGGGCGAGAGGGCCACCAT

CAACTGCAAGAGCAGCCAGAGCCTCCTGAACAGCGGCAACCAGAAGAACTACCTGGCCTGGT

ACCAGCAGAAACCCGGCCAGCCCCCCAAGCTGCTGATCTATGGCGCCTCCACCAGGGAGAGC

GGCGTGCCAGACAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACAATCAGCAG

CCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGTCCACAGCTTCCCCTTCACCT

TCGGCGGGGGAACCAAGCTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTC

CCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTT

CTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCC

AGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACC

CTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCT

GTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 215: PascoH-474 Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCGGCGT

GCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCG

CCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAG

GGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGT

GAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACA

GCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTAC

GACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 216: PascoH-TVAAPS-474 Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGC

CCCCTCGGGATCCGGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCA

GCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTG

AGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGAC

CTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCC

TGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCC

GAGGACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 217: PascoL-474 Light chain

GACATCGTGCTGACCCAGAGCCCCTCTTCCCTGAGCGCAAGCGTGGGCGATAGGGTGACC

ATCACCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACATGAACTGGTAC

CAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTCGAGTCA

GGCATTCCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACAATCAGC

AGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGAGCAACGAGGACCCTCCC

ACCTTCGGACAGGGCACCAAGGTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTC

ATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTG

AACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGC

GGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGC

AGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTG

ACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGTGGATCC

GGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTG

AGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCC

CCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTAC

GCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAG

GACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 218: PascoL-TVAAPS-474 Light chain

GACATCGTGCTGACCCAGAGCCCCTCTTCCCTGAGCGCAAGCGTGGGCGATAGGGTGACC

ATCACCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACATGAACTGGTAC

CAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTCGAGTCA

GGCATTCCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACAATCAGC

AGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGAGCAACGAGGACCCTCCC

ACCTTCGGACAGGGCACCAAGGTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTC

ATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTG

AACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGC

GGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGC

AGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTG

ACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCACCGTG

GCCGCCCCCTCGGGATCCGGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCC

GGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATG

GGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCAC

GGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC

AGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTAC

TACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTG

ACTGTGAGCAGC

SEQ ID NO: 219: IL-5 mAb-G4S-DOM9-112-210 heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACACAGACCCTCACTCTGAC

CTGCACCGTGAGCGGCTTCAGCCTGACCTCCTACAGCGTCCACTGGGTGAGGCAGCCCCCCG

GCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGCAAGCGGCGGCACCGACTACAACAGCGCC

CTGATGAGCAGGCTCTCCATCAGCAAGGACACCAGCCGGAACCAGGTGGTGCTGACCATGAC

CAACATGGACCCCGTGGACACCGCCACCTATTACTGCGCCAGGGACCCTCCCTCTAGCCTGC

TGAGGCTGGACTACTGGGGCAGGGGAACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGA

CCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAA

GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCA

AGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACA

CCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGGCGGCGGATCCGA

AGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCT

GCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGCTGGGTCAGGCAGGCCCCCGGC

AAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCACCGAGACCTACTACGCCGACAG

CGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGATGA

ACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCCAAGAGCCTGGGCAGGTTCGAC

TACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 220: IL-5 mAb-G4S-DOM10-53-474 light chain

GACATCGTGATGACCCAGTCTCCCGATTCACTGGCCGTGAGCCTGGGCGAGAGGGCCACC

ATCAACTGCAAGAGCAGCCAGAGCCTCCTGAACAGCGGCAACCAGAAGAACTACCTGGCC

TGGTACCAGCAGAAACCCGGCCAGCCCCCCAAGCTGCTGATCTATGGCGCCTCCACCAGG

GAGAGCGGCGTGCCAGACAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACA

ATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGTCCACAGCTTC

CCCTTCACCTTCGGCGGGGGAACCAAGCTGGAGATCAAGCGTACGGTGGCCGCCCCCAGC

GTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGT

CTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG

CAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGC

CTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGT

GAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

GGCGGCGGCGGATCCGGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGC

GGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGC

TGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGG

GAGGTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGC

AAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTACTAC

TGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTGACT

GTGAGCAGC

SEQ ID NO: 221: 586H-210 Heavy chain (GS removed)

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

GGAAGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGA

GCTGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGCTGGGTCAGGCAGGCCCCC

GGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCACCGAGACCTACTACGCCGA

CAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGA

TGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCCAAGAGCCTGGGCAGGTTC

GACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 222: 586H-TVAAPS-210 Heavy chain (GS removed)

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAG

CTGCAAGGCCTCCGGCTTCTACATCAAGGACACCTACATGCACTGGGTCAGGCAGGCTCCTG

GCCAGGGCCTGGAGTGGATGGGCACTATCGACCCCGCCAACGGCAACACCAAGTACGTGCCC

AAGTTCCAGGGCAGGGTGACCATCACCGCCGATGAGAGCACCAGCACCGCCTACATGGAACT

GAGCAGCCTGAGGTCTGAGGACACCGCCGTGTACTATTGCGCCAGGAGCATCTACGACGACT

ACCACTACGACGACTACTACGCCATGGACTACTGGGGACAGGGCACACTAGTGACCGTGTCT

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGG

CGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCT

GGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACAT

CTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT

GTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTG

TTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTG

TGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCG

TGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTG

GTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGT

GTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCA

GAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCC

CTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG

CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCC

GTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAA

GACCGTGGCCGCCCCCTCGGAAGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCG

GCGGCAGCCTGAGGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGC

TGGGTCAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCAC

CGAGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGA

ACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCC

AAGAGCCTGGGCAGGTTCGACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 223: PascoH-474 Heavy Chain (GS removed)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGTGCAGCTCCTGGAGAGCGGCGGA

GGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTC

GCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCC

AGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACC

ATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAG

GACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGC

CAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 224: PascoH-TVAAPS-474 Heavy Chain (GS removed)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGCGTGCAG

CTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCC

GCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAG

GGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGC

GTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATG

AACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCC

GGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 225: PascoH-ASTKGPT-474 Heavy Chain (second GS removed)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCAGCCAGCACCAAGGGCCCCACG

GGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTG

AGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCC

CCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTAC

GCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAG

GACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 226: Heavy chain of anti-IGF-1R antibody H0L0 with DOM15-26-593

fused at C-terminus with TVAAPSGS linker

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTgTAa

CgTgAACcACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGT

GGCCGCCCCCTCGGGATCCGAGGTGCAGCTCCTGGTCAGCGGCGGCGGCCTGGTCCAGCCCG

GAGGCTCACTGAGGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAAGGCCTACCCCATGATG

TGGGTCAGGCAGGCCCCCGGCAAAGGCCTGGAGTGGGTGTCTGAGATCAGCCCCAGCGGCAG

CTACACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGA

ACACCCTGTACCTGCAGATGAACTCTCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCC

AAGGACCCCAGGAAGCTGGACTATTGGGGCCAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 227: IGF1RmAb-GS-DOM15-26-593 Heavy chain

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTgTAa

CgTgAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATC

CGAGGTGCAGCTCCTGGTCAGCGGCGGCGGCCTGGTCCAGCCCGGAGGCTCACTGAGGCTGA

GCTGCGCCGCTAGCGGCTTCACCTTCAAGGCCTACCCCATGATGTGGGTCAGGCAGGCCCCC

GGCAAAGGCCTGGAGTGGGTGTCTGAGATCAGCCCCAGCGGCAGCTACACCTACTACGCCGA

CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGA

TGAACTCTCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGACCCCAGGAAGCTG

GACTATTGGGGCCAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 228: anti-IGF-1R antibody Heavy chain

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 229: Light chain of anti-IGF-1R antibody H0L0 with DOM15-26-593

fused at C-terminus with TVAAPSGS linker

GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCAT

CAGCTGCAGAAGCAGCCAGAGCATCGTCCAGAGCAACGGCGACACCTACCTGGAATGGTATC

TGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACAGAGTGAGCAACCGGTTCAGCGGC

GTGCCCGACAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCCGGGT

GGAGGCCGAGGACGTGGGCGTGTACTACTGCTTTCAAGGCAGCCACGTGCCCTACACCTTCG

GCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCC

CCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTA

CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGG

AGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG

AGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC

CAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCACCGTGGCCGCCCCCTCGGGATCCG

AGGTGCAGCTCCTGGTCAGCGGCGGCGGCCTGGTCCAGCCCGGAGGCTCACTGAGGCTGAGC

TGCGCCGCTAGCGGCTTCACCTTCAAGGCCTACCCCATGATGTGGGTCAGGCAGGCCCCCGG

CAAAGGCCTGGAGTGGGTGTCTGAGATCAGCCCCAGCGGCAGCTACACCTACTACGCCGACA

GCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATG

AACTCTCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGACCCCAGGAAGCTGGA

CTATTGGGGCCAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 230: Light chain of anti-IGF-1R antibody H0L0 with DOM15-26-593

fused at C-terminus with GS linker

GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCAT

CAGCTGCAGAAGCAGCCAGAGCATCGTCCAGAGCAACGGCGACACCTACCTGGAATGGTATC

TGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACAGAGTGAGCAACCGGTTCAGCGGC

GTGCCCGACAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCCGGGT

GGAGGCCGAGGACGTGGGCGTGTACTACTGCTTTCAAGGCAGCCACGTGCCCTACACCTTCG

GCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCC

CCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTA

CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGG

AGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG

AGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC

CAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGTGGATCCGAGGTGCAGCTCCTGGTCA

GCGGCGGCGGCCTGGTCCAGCCCGGAGGCTCACTGAGGCTGAGCTGCGCCGCTAGCGGCTTC

ACCTTCAAGGCCTACCCCATGATGTGGGTCAGGCAGGCCCCCGGCAAAGGCCTGGAGTGGGT

GTCTGAGATCAGCCCCAGCGGCAGCTACACCTACTACGCCGACAGCGTGAAGGGCAGGTTCA

CCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCTCTGAGGGCCGAG

GACACCGCCGTGTACTACTGCGCCAAGGACCCCAGGAAGCTGGACTATTGGGGCCAGGGCAC

TCTGGTGACCGTGAGCAGC

SEQ ID NO: 231: anti-IGF-1R antibody Light chain

GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCTGGCGAGCCCGCCAGCAT

CAGCTGCAGAAGCAGCCAGAGCATCGTCCAGAGCAACGGCGACACCTACCTGGAATGGTATC

TGCAGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACAGAGTGAGCAACCGGTTCAGCGGC

GTGCCCGACAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCAGCCGGGT

GGAGGCCGAGGACGTGGGCGTGTACTACTGCTTTCAAGGCAGCCACGTGCCCTACACCTTCG

GCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCC

CCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTA

CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGG

AGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTG

AGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTC

CAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 232: anti-IGF1R Heavy Chain-GS-TLPC

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATC

CGACGGCGGCGGCATTAGGAGGAGCATGAGCGGCACCTGGTACCTGAAGGCCATGACCGTGG

ATAGGGAGTTCCCCGAGATGAACCTGGAGAGCGTGACCCCCATGACACTGACCCTGCTCAAG

GGCCACAACCTGGAGGCCAAGGTCACCATGCTGATCTCAGGCAGGTGCCAGGAGGTGAAGGC

AGTGCTGGGCAGGACCAAGGAGAGGAAGAAGTACACCGCCGACGGGGGCAAGCACGTGGCCT

ATATCATCCCCAGCGCCGTGAGGGACCACGTGATCTTCTACAGCGAGGGCCAGCTCCACGGA

AAGCCCGTGAGAGGCGTGAAGCTGGTGGGCAGGGACCCCAAGAACAACCTGGAGGCCCTGGA

GGACTTCGAAAAAGCCGCAGGCGCCAGGGGCCTGTCCACTGAGAGCATCCTGATCCCTAGGC

AGAGCGAGACCTGCAGCCCCGGC

SEQ ID NO: 233: anti-IGF1R Heavy Chain-GS-CT01 adnectin

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATC

CGAGGTGGTGGCCGCCACCCCCACCAGCCTGCTGATTTCCTGGAGGCACCCCCACTTCCCCA

CACGCTACTACAGGATCACCTACGGCGAGACCGGCGGCAACAGCCCCGTGCAGGAGTTCACC

GTGCCCCTGCAGCCTCCCACTGCCACCATCAGCGGCCTCAAGCCCGGCGTGGACTACACCAT

CACCGTGTACGCCGTCACCGACGGAAGGAACGGCAGGCTGCTGAGCATCCCCATCAGCATCA

ACTACAGGACC

SEQ ID NO: 234: anti-IGF1R Heavy Chain-TVAAPSGS-TLPC

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGT

GGCCGCCCCCTCGGGATCCGACGGCGGCGGCATTAGGAGGAGCATGAGCGGCACCTGGTACC

TGAAGGCCATGACCGTGGATAGGGAGTTCCCCGAGATGAACCTGGAGAGCGTGACCCCCATG

ACACTGACCCTGCTCAAGGGCCACAACCTGGAGGCCAAGGTCACCATGCTGATCTCAGGCAG

GTGCCAGGAGGTGAAGGCAGTGCTGGGCAGGACCAAGGAGAGGAAGAAGTACACCGCCGACG

GGGGCAAGCACGTGGCCTATATCATCCCCAGCGCCGTGAGGGACCACGTGATCTTCTACAGC

GAGGGCCAGCTCCACGGAAAGCCCGTGAGAGGCGTGAAGCTGGTGGGCAGGGACCCCAAGAA

CAACCTGGAGGCCCTGGAGGACTTCGAAAAAGCCGCAGGCGCCAGGGGCCTGTCCACTGAGA

GCATCCTGATCCCTAGGCAGAGCGAGACCTGCAGCCCCGGC

SEQ ID NO: 235: anti-IGF1R Heavy Chain-GS-AFFI

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATC

CGTGGACAACAAGTTCAACAAGGAGCTGAGGCAGGCCTACTGGGAGATCCAGGCCCTGCCCA

ATCTGAACTGGACCCAGAGCAGGGCCTTCATCAGGAGCCTGTACGACGACCCCAGCCAGAGC

GCTAACCTCCTGGCCGAGGCCAAAAAGCTGAACGACGCCCAGGCCCCCAAG

SEQ ID NO: 236 anti-IGF1R Heavy Chain-TVAAPSGS-AFFI

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGT

GGCCGCCCCCTCGGGATCCGTGGACAACAAGTTCAACAAGGAGCTGAGGCAGGCCTACTGGG

AGATCCAGGCCCTGCCCAATCTGAACTGGACCCAGAGCAGGGCCTTCATCAGGAGCCTGTAC

GACGACCCCAGCCAGAGCGCTAACCTCCTGGCCGAGGCCAAAAAGCTGAACGACGCCCAGGC

CCCCAAG

SEQ ID NO: 237: anti-IGF1R Heavy Chain-GS-DRPN

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATC

CGACCTGGGGAAAAAGCTGCTTGAAGCCGCTAGGGCAGGACAGGATGACGAGGTGAGGATTC

TGATGGCAAATGGCGCCGACGTCAATGCCAAAGACGAGTACGGCCTCACCCCTCTTTATCTG

GCCACTGCACACGGACACTTGGAGATCGTGGAGGTGCTGCTCAAGAACGGAGCTGATGTGAA

CGCTGTGGACGCTATTGGGTTCACACCCCTTCACCTCGCAGCCTTTATTGGCCACCTGGAGA

TCGCCGAAGTTCTCCTGAAACACGGCGCAGACGTCAACGCACAGGATAAGTTCGGGAAGACC

GCCTTCGACATCAGCATCGGCAATGGGAACGAGGATCTGGCCGAGATCCTGCAGAAGCTG

SEQ ID NO: 238: anti-IGF1R Heavy Chain-TVAAPSGS-DRPN

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGT

GGCCGCCCCCTCGGGATCCGACCTGGGGAAAAAGCTGCTTGAAGCCGCTAGGGCAGGACAGG

ATGACGAGGTGAGGATTCTGATGGCAAATGGCGCCGACGTCAATGCCAAAGACGAGTACGGC

CTCACCCCTCTTTATCTGGCCACTGCACACGGACACTTGGAGATCGTGGAGGTGCTGCTCAA

GAACGGAGCTGATGTGAACGCTGTGGACGCTATTGGGTTCACACCCCTTCACCTCGCAGCCT

TTATTGGCCACCTGGAGATCGCCGAAGTTCTCCTGAAACACGGCGCAGACGTCAACGCACAG

GATAAGTTCGGGAAGACCGCCTTCGACATCAGCATCGGCAATGGGAACGAGGATCTGGCCGA

GATCCTGCAGAAGCTG

SEQ ID NO: 239: Anti IL-4 heavy Chain-GS-anti RNAse A camelidV_HH

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCCAGGT

GCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTG

CAGCCTCTGGATACGCATACACTTACATCTACATGGGCTGGTTCCGCCAGGCTCCAGGGAAA

GAGCGTGAGGGGGTCGCAGCTATGGATAGTGGTGGTGGTGGCACACTCTACGCCGACTCCGT

GAAGGGCCGATTCACCATCTCCCGCGACAAAGGCAAGAACACGGTGTATCTGCAAATGGACA

GCCTGAAACCTGAGGACACGGCCACGTATTACTGTGCTGCAGGTGGCTACGAGCTGCGTGAC

CGGACATATGGGCAGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

SEQ ID NO: 240: Anti IL-4 heavy Chain-GS-NARV

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCGCGCG

CGTCGACCAGACGCCGCGCAGCGTCACGAAGGAAACCGGCGAGTCCCTCACCATCAACTGCG

TGCTGCGGGATGCCTCCTACGCCCTGGGCAGCACATGTTGGTACAGAAAGAAGAGCGGGGAA

GGCAACGAGGAGTCCATCTCCAAGGGGGGAAGATACGTCGAGACCGTGAACAGCGGAAGCAA

GAGCTTCAGCCTGCGGATCAACGACCTCACCGTCGAGGACGGGGGCACCTACCGTTGCGGTC

TGGGCGTGGCCGGCGGCTATTGCGATTACGCCCTGTGCAGTAGCCGGTATGCTGAGTGCGGC

GACGGCACCGCTGTGACCGTGAAC

SEQ ID NO: 241 anti-IGF1R Heavy Chain-TVAAPSGS-CT01 adnectin

CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTGTC

CTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATGAACTGGGTGCGGCAGGCCCCAG

GCCAGGGACTGGAATGGATGGGCAACATCAACCCCAACAACGGCGGCACCAACTACAACCAG

AAGTTCAAGGACCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAACT

GCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCCCGGTGGATCCTGTACTACG

GCCGGTCCAAGTGGTACTTCGACGTGTGGGGCAGGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC

AGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACA

GCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTAC

AGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAA

CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACA

AGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTG

TTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT

GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCC

GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGC

CCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACC

TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCC

CGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACA

GCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATG

CACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGT

GGCCGCCCCCTCGGGATCCGAGGTGGTGGCCGCCACCCCCACCAGCCTGCTGATTTCCTGGA

GGCACCCCCACTTCCCCACACGCTACTACAGGATCACCTACGGCGAGACCGGCGGCAACAGC

CCCGTGCAGGAGTTCACCGTGCCCCTGCAGCCTCCCACTGCCACCATCAGCGGCCTCAAGCC

CGGCGTGGACTACACCATCACCGTGTACGCCGTCACCGACGGAAGGAACGGCAGGCTGCTGA

GCATCCCCATCAGCATCAACTACAGGACC

SEQ ID NO: 242: Erbitux Heavy chain-RS-CT01 adnectin

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCAC

CTGTACCGTGAGCGGCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAG

GCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGACTACAACACCCCC

TTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAA

CAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACTACGACT

ACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGA

CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA

GCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCA

AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACA

CCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGTCTGAGCCTGTCTCCTGGCAAGAGATCCGAGGTGGTGGC

CGCCACCCCCACCAGCCTGCTGATTTCCTGGAGGCACCCCCACTTCCCCACACGCTACTACA

GGATCACCTACGGCGAGACCGGCGGCAACAGCCCCGTGCAGGAGTTCACCGTGCCCCTGCAG

CCTCCCACTGCCACCATCAGCGGCCTCAAGCCCGGCGTGGACTACACCATCACCGTGTACGC

CGTCACCGACGGAAGGAACGGCAGGCTGCTGAGCATCCCCATCAGCATCAACTACAGGACC

SEQ ID NO: 243: Erbitux Light Chain

GACATCCTGCTGACCCAGAGCCCCGTGATCCTGAGCGTGAGCCCTGGCGAGAGAGTGAGCTT

CAGCTGCCGGGCCAGCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACG

GCAGCCCCAGGCTGCTGATCAAGTACGCCAGCGAGTCCATCAGCGGCATCCCCAGCCGGTTC

AGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAACAGCGTGGAGAGCGAGGATAT

CGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCACCAAGC

TGGAACTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAG

CTCAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAA

AGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGC

AGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTAC

GAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA

GAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 244: Erbitux Light chain-RS-CT01 adnectin

GACATCCTGCTGACCCAGAGCCCCGTGATCCTGAGCGTGAGCCCTGGCGAGAGAGTGAGCTT

CAGCTGCCGGGCCAGCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACG

GCAGCCCCAGGCTGCTGATCAAGTACGCCAGCGAGTCCATCAGCGGCATCCCCAGCCGGTTC

AGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAACAGCGTGGAGAGCGAGGATAT

CGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCACCAAGC

TGGAACTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAG

CTCAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAA

AGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGC

AGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTAC

GAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA

GAGCTTCAACCGAGGCGAGTGCAGATCCGAGGTGGTGGCCGCCACCCCCACCAGCCTGCTGA

TTTCCTGGAGGCACCCCCACTTCCCCACACGCTACTACAGGATCACCTACGGCGAGACCGGC

GGCAACAGCCCCGTGCAGGAGTTCACCGTGCCCCTGCAGCCTCCCACTGCCACCATCAGCGG

CCTCAAGCCCGGCGTGGACTACACCATCACCGTGTACGCCGTCACCGACGGAAGGAACGGCA

GGCTGCTGAGCATCCCCATCAGCATCAACTACAGGACC

SEQ ID NO: 245: Erbitux Heavy Chain

CAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGCCCTCTCAGAGCCTGAGCATCAC

CTGTACCGTGAGCGGCTTCAGCCTGACCAATTACGGCGTGCATTGGGTGCGGCAGTCTCCAG

GCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGGCAACACCGACTACAACACCCCC

TTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAA

CAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCCAGGGCCCTGACCTACTACGACT

ACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGCTAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCCCTGA

CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA

GCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAGAGCTGCGATAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAAGTGCACAACGCCA

AGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCGCGTGGTGTCTGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAAGTGAGCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCCCAGGTCTACA

CCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACTCCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 246: CT01 adnectin-GSTG-Erbitux Heavy Chain

GAGGTGGTGGCCGCCACCCCCACCAGCCTGCTGATTTCCTGGAGGCACCCCCACTTCCCCAC

ACGCTACTACAGGATCACCTACGGCGAGACCGGCGGCAACAGCCCCGTGCAGGAGTTCACCG

TGCCCCTGCAGCCTCCCACTGCCACCATCAGCGGCCTCAAGCCCGGCGTGGACTACACCATC

ACCGTGTACGCCGTCACCGACGGAAGGAACGGCAGGCTGCTGAGCATCCCCATCAGCATCAA

CTACAGGACCGGATCCACCGGCCAGGTGCAGCTGAAGCAGAGCGGCCCTGGCCTGGTGCAGC

CCTCTCAGAGCCTGAGCATCACCTGTACCGTGAGCGGCTTCAGCCTGACCAATTACGGCGTG

CATTGGGTGCGGCAGTCTCCAGGCAAGGGCCTGGAATGGCTGGGAGTGATCTGGTCCGGCGG

CAACACCGACTACAACACCCCCTTCACCAGCAGACTGAGCATCAACAAGGACAACAGCAAGA

GCCAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTATTGTGCC

AGGGCCCTGACCTACTACGACTACGAGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGT

GAGCGCCGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCA

GCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCTGTGACCGTG

TCCTGGAATAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAG

CGGCCTGTACTCCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCT

ACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTGGAGCCCAAG

AGCTGCGATAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGCGGACCTAG

CGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGGACCCCCGAAGTGA

CCTGCGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGAC

GGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACCG

CGTGGTGTCTGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCA

AAGTGAGCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAG

CCTAGAGAGCCCCAGGTCTACACCCTGCCTCCCTCCAGAGATGAGCTGACCAAGAACCAGGT

GTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA

ACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTC

TTCCTGTACTCCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTG

CAGCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGTCTGAGCCTGTCCCCTG

GCAAG

SEQ ID NO: 247: CT01 adnectin-STG-Erbitux Light Chain

GAGGTGGTGGCCGCCACCCCCACCAGCCTGCTGATTTCCTGGAGGCACCCCCACTTCCCCAC

ACGCTACTACAGGATCACCTACGGCGAGACCGGCGGCAACAGCCCCGTGCAGGAGTTCACCG

TGCCCCTGCAGCCTCCCACTGCCACCATCAGCGGCCTCAAGCCCGGCGTGGACTACACCATC

ACCGTGTACGCCGTCACCGACGGAAGGAACGGCAGGCTGCTGAGCATCCCCATCAGCATCAA

CTACAGGACGTCGACCGGTGACATCCTGCTGACCCAGAGCCCCGTGATCCTGAGCGTGAGCC

CTGGCGAGAGAGTGAGCTTCAGCTGCCGGGCCAGCCAGAGCATCGGCACCAACATCCACTGG

TATCAGCAGCGGACCAACGGCAGCCCCAGGCTGCTGATCAAGTACGCCAGCGAGTCCATCAG

CGGCATCCCCAGCCGGTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAACA

GCGTGGAGAGCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACC

TTCGGAGCCGGCACCAAGCTGGAACTGAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTT

CCCCCCCAGCGATGAGCAGCTCAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACT

TCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACAGC

CAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGAC

CCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCC

TGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 248 (PascoH-616 Heavy chain)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGTGCAGCTCCTGGAGAGCGGCGGA

GGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCGTGTTC

CCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCC

AGCATCGACTGGCACGGGAAGATCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACC

ATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAG

GACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGC

CAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 249 (PascoH-TVAAPS-616 Heavy chain)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGCGTGCAG

CTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCC

GCCAGCGGCTTCGTGTTCCCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAG

GGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGAAGATCACCTACTACGCCGACAGC

GTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATG

AACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCC

GGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 250: 656H-TVAAPS-210 Heavy chain

CAGGTGCAGCTCGTCCAGTCTGGGGCCGAGGTGAAGAAGCCCGGAGCTTCTGTGAAGGTGTC

CTGCAAGGCCAGCGGCTATACCTTCATCGACTACGAGATCCATTGGGTGAGGCAGGCTCCCG

GGCAGGGCCTGGAGTGGATGGGCGCCATCGACCCAGAGACCGGAGGCACGGCGTACAACCAG

AAGTTCAAGGGACGGGTCACCATGACAACCGATACCAGCACCTCCACCGCTTACATGGAGCT

GCGCAGCCTGAGAAGCGACGACACCGCGGTGTACTACTGTACGCGCATCCTGCTCTACTACT

ACCCCATGGATTACTGGGGCCAGGGCACACTAGTGACCGTGTCTAGCGCCAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGA

CCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCAcCCAGACCTACATCTgTAACgTgAACCACAa

GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCA

AGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACA

CCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTC

GGAAGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGA

GCTGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGCTGGGTCAGGCAGGCCCCC

GGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCACCGAGACCTACTACGCCGA

CAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTGCAGA

TGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCCAAGAGCCTGGGCAGGTTC

GACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 251: 656 Light chain

GAGATCGTGCTGACCCAGAGTCCAGCCACCCTCAGCCTGAGCCCTGGGGAACGCGCCACCCT

GTCCTGCCGGGCGAGTCAGAACATCTCCGACTACCTGCATTGGTACCAGCAGAAGCCCGGCC

AGGCCCCTCGCCTGCTGATCTACTACGCCTCCCAGAGCATCAGCGGAATCCCCGCCCGGTTC

TCCGGAAGTGGGTCCGGAACCGACTTTACCCTGACCATCAGCTCTCTCGAGCCAGAGGACTT

CGCGGTGTACTACTGCCAGAACGGGCATAGTTTCCCACTGACCTTCGGAGGGGGCACAAAGG

TGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAG

CTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAA

GGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGC

AGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTAC

GAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAA

GAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 252: PascoH-TVAAPS-546_Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGCGTGCAG

CTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCC

GCCAGCGGCTTCGTGTTCCCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAG

GGCCTGGAGTGGGTGTCCAGCATCGACTGGAAGGGGGGCAAGACCTACTACGCCGACAGC

GTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATG

AACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCC

GGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 253: PascoH-546_Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGTGCAGCT

CCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCA

GCGGCTTCGTGTTCCCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTG

GAGTGGGTGTCCAGCATCGACTGGAAGGGGGGCAAGACCTACTACGCCGACAGCGTGAAGGG

CAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGA

GGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTAC

TGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 254: PascoH-TVAAPS-567_Heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGCGTGCAG

CTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCC

GCCAGCGGCTTCGTGTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAG

GGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGC

GTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATG

AACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCC

GGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 255: (PascoH-567_Heavy chain)

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG

ACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGG

CAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGG

TACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTG

GTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGG

GAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGTGCAGCTCCTGGAGAGCGGCGGA

GGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCGTGTTC

GCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCC

AGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACC

ATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAG

GACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGC

CAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 256: 656 Heavy chain

CAGGTGCAGCTCGTCCAGTCTGGGGCCGAGGTGAAGAAGCCCGGAGCTTCTGTGAAGGTGTC

CTGCAAGGCCAGCGGCTATACCTTCATCGACTACGAGATCCATTGGGTGAGGCAGGCTCCCG

GGCAGGGCCTGGAGTGGATGGGCGCCATCGACCCAGAGACCGGAGGCACGGCGTACAACCAG

AAGTTCAAGGGACGGGTCACCATGACAACCGATACCAGCACCTCCACCGCTTACATGGAGCT

GCGCAGCCTGAGAAGCGACGACACCGCGGTGTACTACTGTACGCGCATCCTGCTCTACTACT

ACCCCATGGATTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGC

CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG

CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA

CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC

GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA

GCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT

GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA

CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG

CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA

AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC

CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC

AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA

CCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA

GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA

CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG

TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG

CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

SEQ ID NO: 257: Anti IL-5 Heavy Chain-G4S-dAb474-TVAAPSGS-dAb210

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACACAGACCCTCACTCTG

ACCTGCACCGTGAGCGGCTTCAGCCTGACCTCCTACAGCGTCCACTGGGTGAGGCAGCCC

CCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGCAAGCGGCGGCACCGACTACAAC

AGCGCCCTGATGAGCAGGCTCTCCATCAGCAAGGACACCAGCCGGAACCAGGTGGTGCTG

ACCATGACCAACATGGACCCCGTGGACACCGCCACCTATTACTGCGCCAGGGACCCTCCC

TCTAGCCTGCTGAGGCTGGACTACTGGGGCAGGGGAACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGC

ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGG

AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTAC

ATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG

AGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCC

AGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAG

GTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTAC

GTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGC

ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAG

TACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAG

GCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTG

ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTG

GACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAG

CAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG

AAGAGCCTGAGCCTGTCCCCTGGCAAGGGCGGCGGCGGATCTGGCGTGCAGCTCCTGGAG

AGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGC

TTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAG

TGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGC

AGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTG

AGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGAC

TACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGCACCGTGGCCGCCCCCTCGGGATCC

GAAGTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTG

AGCTGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGCTGGGTCAGGCAGGCC

CCCGGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCACCGAGACCTACTAC

GCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCCAAGAGCCTG

GGCAGGTTCGACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 258: Anti CD-20 Heavy Chain-TVAAPSGS-dAb154-TVAAPSGS-

dAb474

CAGGTGCAGCTGCAGCAGCCTGGAGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAAATG

TCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACC

CCCGGCAGGGGCCTCGAGTGGATCGGAGCTATCTACCCCGGCAACGGCGACACTAGCTAC

AACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTAC

ATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTATTACTGCGCCAGGAGCACC

TACTACGGCGGCGACTGGTACTTCAACGTCTGGGGCGCCGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGATCTGAC

ATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATT

ACCTGCAGGGCCAGCAGGCCCATCAGCGACTGGCTGCACTGGTACCAACAGAAGCCCGGC

AAGGCTCCCAAGCTGCTGATCGCCTGGGCCAGCAGCCTGCAGGGAGGCGTGCCCAGCAGG

TTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTCACCATCTCTTCCCTGCAGCCCGAG

GACTTCGCCACCTACTACTGCCTGCAGGAGGGCTGGGGGCCCCCTACTTTCGGCCAGGGC

ACCAAGGTGGAGATCAAGAGGACCGTGGCCGCCCCCTCGGGATCCGGCGTGCAGCTCCTG

GAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGC

GGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTG

GAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAG

GGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGC

CTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTAC

GACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 259: Anti CD-20 Heavy Chain-TVAAPSGS-dAb210-TVAAPSGS-

dAb474

CAGGTGCAGCTGCAGCAGCCTGGAGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAAATG

TCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGACC

CCCGGCAGGGGCCTCGAGTGGATCGGAGCTATCTACCCCGGCAACGGCGACACTAGCTAC

AACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTAC

ATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTATTACTGCGCCAGGAGCACC

TACTACGGCGGCGACTGGTACTTCAACGTCTGGGGCGCCGGCACACTAGTGACCGTGTCC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC

GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG

TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC

AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG

ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG

CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA

GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACC

CCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAAC

TGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTAC

AACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC

AAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATC

AGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGAT

GAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGAC

ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCT

GTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGA

TGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTAC

ACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGACCGTGGCCGCCCCCTCGGGATCTGAA

GTGCAGCTCCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGC

TGCGCCGCTAGCGGCTTCACCTTCAGGAACTTCGGCATGGGCTGGGTCAGGCAGGCCCCC

GGCAAGGGCCTGGAGTGGGTCAGCTGGATCATCAGCTCCGGCACCGAGACCTACTACGCC

GACAGCGTGAAGGGCAGGTTCACCATCAGCCGCGACAACAGCAAGAACACCCTGTACCTG

CAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTCTACTACTGCGCCAAGAGCCTGGGC

AGGTTCGACTACTGGGGACAGGGGACCCTGGTGACTGTGAGCAGCACCGTGGCCGCCCCC

TCGGGATCCGGCGTGCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGC

CTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGCCTGGTATGATATGGGCTGGGTG

AGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCAGCATCGACTGGCACGGGGAGGTG

ACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAAC

ACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCAGTGTACTACTGCGCC

ACCGCCGAGGACGAACCCGGCTACGACTACTGGGGCCAGGGCACCCTGGTGACTGTGAGC

AGC

SEQ ID NO: 260: anti cMET 5D5v2 Heavy Chain (hole)-GS-dAb593

GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGAG

CTGCGCCGCTAGCGGCTACACCTTCACCAGCTACTGGCTCCACTGGGTCAGGCAGGCCCCAG

GCAAGGGACTGGAGTGGGTGGGCATGATCGACCCCAGCAACAGCGACACCAGGTTCAACCCC

AACTTCAAGGACAGGTTCACCATCAGCGCCGACACTAGCAAGAACACCGCCTACCTGCAGAT

GAACAGCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGCCACCTACAGGAGCTACGTCA

CCCCCCTGGATTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGC

CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGG

CTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGA

CCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGC

GTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAA

GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCT

GCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAG

CCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAG

CCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCA

AGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTG

CTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCC

TGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACA

CCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGAGCTGCGCCGTGAAG

GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTA

CAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGGTGAGCAAGCTGACCG

TGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTG

CACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCGAGGTGCAGCT

CCTGGTCAGCGGCGGCGGCCTGGTCCAGCCCGGAGGCTCACTGAGGCTGAGCTGCGCCGCTA

GCGGCTTCACCTTCAAGGCCTACCCCATGATGTGGGTCAGGCAGGCCCCCGGCAAAGGCCTG

GAGTGGGTGTCTGAGATCAGCCCCAGCGGCAGCTACACCTACTACGCCGACAGCGTGAAGGG

CAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCTCTGA

GGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGACCCCAGGAAGCTGGACTATTGGGGC

CAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 261: anti cMET 5D5v2 Heavy Chain (knob)-GS-dAb593

TGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCC

AAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGAT

GTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAC

AATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTG

CTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAAC

AAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAG

CCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTG

TGGTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGC

CAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTC

CTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGC

TCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCT

GGCAAGGGATCCGAGGTGCAGCTCCTGGTCAGCGGCGGCGGCCTGGTCCAGCCCGGAGGC

TCACTGAGGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAAGGCCTACCCCATGATGTGG

GTCAGGCAGGCCCCCGGCAAAGGCCTGGAGTGGGTGTCTGAGATCAGCCCCAGCGGCAGC

TACACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAG

AACACCCTGTACCTGCAGATGAACTCTCTGAGGGCCGAGGACACCGCCGTGTACTACTGC

GCCAAGGACCCCAGGAAGCTGGACTATTGGGGCCAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 262: anti cMET 5D5v2 Light Chain

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCAGTGGGAGACAGGGTGACC

ATCACCTGCAAGAGCAGCCAGAGCCTCCTGTACACCAGCAGCCAGAAGAACTACCTGGCC

TGGTACCAGCAGAAACCCGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCTAGCACCAGG

GAGTCAGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACTCTGACC

ATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACTACGCCTAT

CCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGC

GTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGT

CTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG

CAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGC

CTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGT

GAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ ID NO: 263: anti cMET 5D5v2 IgG4 Heavy Chain (UNIBODY)-GS-dAb593

GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTG

AGCTGCGCCGCTAGCGGCTACACCTTCACCAGCTACTGGCTCCACTGGGTCAGGCAGGCC

CCAGGCAAGGGACTGGAGTGGGTGGGCATGATCGACCCCAGCAACAGCGACACCAGGTTC

AACCCCAACTTCAAGGACAGGTTCACCATCAGCGCCGACACTAGCAAGAACACCGCCTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGCCACCTACAGG

AGCTACGTCACCCCCCTGGATTACTGGGGCCAGGGCACACTAGTCACCGTGAGCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGC

ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGG

AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCAAGACCTAC

ACCTGCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGCCCCCGAG

TTCCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATC

AGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCCGAGGTC

CAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAG

GAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG

CTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAG

AAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCA

TCCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTAC

CCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACC

ACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGAC

AAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGAGGCCCTGCAC

AACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGGATCCGAGGTGCAGCTC

CTGGTCAGCGGCGGCGGCCTGGTCCAGCCCGGAGGCTCACTGAGGCTGAGCTGCGCCGCT

AGCGGCTTCACCTTCAAGGCCTACCCCATGATGTGGGTCAGGCAGGCCCCCGGCAAAGGC

CTGGAGTGGGTGTCTGAGATCAGCCCCAGCGGCAGCTACACCTACTACGCCGACAGCGTG

AAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAAC

TCTCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAAGGACCCCAGGAAGCTGGAC

TATTGGGGCCAGGGCACTCTGGTGACCGTGAGCAGC

SEQ ID NO: 264: anti cMET 5D5v2 Heavy Chain (hole)

GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTG

AGCTGCGCCGCTAGCGGCTACACCTTCACCAGCTACTGGCTCCACTGGGTCAGGCAGGCC

CCAGGCAAGGGACTGGAGTGGGTGGGCATGATCGACCCCAGCAACAGCGACACCAGGTTC

AACCCCAACTTCAAGGACAGGTTCACCATCAGCGCCGACACTAGCAAGAACACCGCCTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGCCACCTACAGG

AGCTACGTCACCCCCCTGGATTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGC

ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGG

AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTAC

ATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAG

AGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCC

AGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAG

GTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTAC

GTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGC

ACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAG

TACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAG

GCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTG

ACCAAGAACCAGGTGTCCCTGAGCTGCGCCGTGAAGGGCTTCTACCCCAGCGACATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTG

GACAGCGATGGCAGCTTCTTCCTGGTGAGCAAGCTGACCGTGGACAAGAGCAGATGGCAG

CAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG

AAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 265: anti cMET 5D5v2 Heavy Chain (knob)

TGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCC

AAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGAT

GTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAC

AATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTG

CTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAAC

AAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAG

CCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTG

TGGTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGC

CAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTC

CTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGC

TCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCT

GGCAAG

SEQ ID NO: 266: anti cMET 5D5v2 IgG4 Heavy Chain (UNIBODY)

GAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTG

AGCTGCGCCGCTAGCGGCTACACCTTCACCAGCTACTGGCTCCACTGGGTCAGGCAGGCC

CCAGGCAAGGGACTGGAGTGGGTGGGCATGATCGACCCCAGCAACAGCGACACCAGGTTC

AACCCCAACTTCAAGGACAGGTTCACCATCAGCGCCGACACTAGCAAGAACACCGCCTAC

CTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGCCACCTACAGG

AGCTACGTCACCCCCCTGGATTACTGGGGCCAGGGCACACTAGTCACCGTGAGCAGCGCC

AGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGC

ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGG

AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCAAGACCTAC

ACCTGCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGCCCCCGAG

TTCCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATC

AGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAAGATCCCGAGGTC

CAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAG

GAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG

CTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAG

AAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCA

TCCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTAC

CCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACC

ACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGGAC

AAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGAGGCCCTGCAC

AACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAG

SEQ ID NO: 267: PascoH IgG2-GS-474 heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTCACCGTGAGCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTGGA

CCACAAGCCCAGCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCC

CCCCCTGCCCTGCCCCTCCTGTGGCCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAG

GACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGA

GGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCA

AGCCCCGGGAGGAACAGTTCAACAGCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCAC

CAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCC

CATCGAGAAAACCATCAGCAAGACCAAGGGCCAGCCCAGGGAACCCCAGGTGTACACCCTGC

CCCCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTC

TACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGAC

CACCCCCCCCATGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACAGTGGACA

AGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC

CACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAGGGATCCGGCGTGCAGCTCCTGGA

GAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCT

TCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGG

GTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAGGTT

CACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCG

AGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGGGGC

CAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 268: PascoH IgG4-GS-474 heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTCACCGTGAGCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGA

CCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTGCC

CCAGCTGCCCTGCCCCCGAGTTCCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCC

AAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCA

GGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGA

CCAAGCCCCGGGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTG

CACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAG

CTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCC

TGCCCCCATCCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGC

TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA

GACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGG

ACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGAGGCCCTGCAC

AACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGGATCCGGCGTGCAGCTCCT

GGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCG

GCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAG

TGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAG

GTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGG

CCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGG

GGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 269: PascoH IgG4PE-GS-474 heavy chain

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTCACCGTGAGCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGA

CCACAAGCCCAGCAACACCAAGGTGGACAAGCGGGTGGAGAGCAAGTACGGCCCTCCCTGCC

CCCCCTGCCCTGCCCCCGAGTTCGAGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCC

AAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCA

GGAAGATCCCGAGGTCCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGA

CCAAGCCCCGGGAGGAACAGTTCAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTG

CACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAG

CTCCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCC

TGCCCCCATCCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGC

TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA

GACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGGCTGACCGTGG

ACAAGAGCCGGTGGCAGGAAGGCAACGTCTTTAGCTGCAGCGTGATGCACGAGGCCCTGCAC

AACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGGGATCCGGCGTGCAGCTCCT

GGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCG

GCTTCACCTTCGCCTGGTATGATATGGGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAG

TGGGTGTCCAGCATCGACTGGCACGGGGAGGTGACCTACTACGCCGACAGCGTGAAGGGCAG

GTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGG

CCGAGGACACCGCAGTGTACTACTGCGCCACCGCCGAGGACGAACCCGGCTACGACTACTGG

GGCCAGGGCACCCTGGTGACTGTGAGCAGC

SEQ ID NO: 270: Anti IL-4 Heavy Chain-GS-anti TNF-a adnectin

CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGAC

CTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGC

CACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAAC

CCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGAC

CATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCT

TCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACC

AAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGC

CCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAG

CCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTG

AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAA

CCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCC

ACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCC

CCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGA

TGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACA

ATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTG

ACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGC

CCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGG

TGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTG

GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAA

CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGC

TGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAG

GCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCGTGAG

CGACGTGCCAAGGGACCTCGAGGTGGTGGCAGCCACTCCCACCTCTCTGCTGATCAGCTGGG

ACACACACAACGCCTACAACGGCTACTACAGGATCACCTACGGAGAGACCGGCGGCAATAGC

CCCGTGAGGGAGTTCACCGTGCCCCACCCCGAGGTGACCGCCACCATTAGCGGCCTGAAGCC

CGGCGTGGACGATACCATCACCGTCTACGCCGTGACCAACCACCACATGCCCCTGAGGATCT

TCGGCCCCATCAGCATCAACCATAGGACC

Number	Date	Country
60991449	Nov 2007	US
61027858	Feb 2008	US
61046572	Apr 2008	US
61081191	Jul 2008	US
61084431	Jul 2008	US

ANTIGEN-BINDING CONSTRUCTS

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