The invention relates to multimers, methods and uses to expand antigen specificity of binding sites, as well as vaccines, methods of vaccination and assay methods and reagents.
The invention also relates to multimers such as dimers or tetramers of polypeptides; and tetramers or higher-order multimers (eg, octamers, dodecamers and hexadecamers) of epitopes or effector domains, such as antigen binding sites (eg, antibody or TCR binding sites that specifically bind to antigen or pMHC, or variable domains thereof) or peptides such as incretin, insulin or hormone peptides.
Multimers of effector domains have recognized utility in medical and non-medical applications for combining and multiplying the activity and presence of effector domains, eg, to provide for higher avidity of antigen binding (for effector domains that are antibody or TCR binding domains, for example) or for enhancing biological or binding activity, such as for providing bi- or multi-specific targeting or interaction with target ligands in vivo or in vitro.
Multimerisation domains which cause self-assembly of protein monomers into multimers are known in the art. Examples include domains found in transcription factors such as p53, p63 and p73, as well as domains found in ion channels such as TRP cation channels. The transcription factor p53 can be divided into different functional domains: an N-terminal transactivation domain, a proline-rich domain, a DNA-binding domain, a tetramerisation domain and a C-terminal regulatory region. The tetramerisation domain of human p53 extends from residues 325 to 356, and has a 4-helical bundle fold (Jeffrey et al., Science (New York, N.Y.) 1995, 267(5203): 1498-1502). The TRPM tetramerisation domain is a short anti-parallel coiled-coil tetramerisation domain of the transient receptor potential cation channel subfamily M member proteins 1-8. It is held together by extensive core packing and interstrand polar interactions (Fujiwara et al., Journal of Molecular Biology 2008, 383(4):854-870). Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Another example is the potassium channel BTB domain. This domain can be found at the N terminus of voltage-gated potassium channel proteins, where represents a cytoplasmic tetramerisation domain (T1) involved in assembly of alpha-subunits into functional tetrameric channels (Bixby et al., Nature Structural Biology 1999, 6(1):38-43). This domain can also be found in proteins that are not potassium channels, like KCTD1 (potassium channel tetramerisation domain-containing protein 1; Ding et al., DNA and Cell Biology 2008, 27(5):257-265).
Multimeric antibody fragments have been produced using a variety of multimerisation techniques, including biotin, dHLX, ZIP and BAD domains, as well as p53 (Thie et al., Nature Boitech., 2009:26, 314-321). Biotin, which is efficient in production, is a bacterial protein which induces immune reactions in humans.
Human p53 (UniProtKB - P04637 (P53_HUMAN)) acts as a tumor suppressor in many tumor types, inducing growth arrest or apoptosis depending on the physiological circumstances and cell type. It is involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. Human p53 is found in increased amounts in a wide variety of transformed cells. It is frequently mutated or inactivated in about 60% of cancers. Human p53 defects are found in Barrett metaplasia a condition in which the normally stratified squamous epithelium of the lower esophagus is replaced by a metaplastic columnar epithelium. The condition develops as a complication in approximately 10% of patients with chronic gastroesophageal reflux disease and predisposes to the development of esophageal adenocarcinoma.
Nine isoforms of p53 naturally occur and are expressed in a wide range of normal tissues but in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues but is not detected in brain, lung, prostate, muscle, fetal brain, spinal cord and fetal liver. Isoform 3 is expressed in most normal tissues but is not detected in lung, spleen, testis, fetal brain, spinal cord and fetal liver. Isoform 7 is expressed in most normal tissues but is not detected in prostate, uterus, skeletal muscle and breast. Isoform 8 is detected only in colon, bone marrow, testis, fetal brain and intestine. Isoform 9 is expressed in most normal tissues but is not detected in brain, heart, lung, fetal liver, salivary gland, breast or intestine.
The invention provides; A protein multimer comprising 4 copies of a binding site, wherein the binding site is capable of binding to a virus spike protein of a coronavirus.
There is also provided:-
The invention provides: A polypeptide comprising an antibody Fc region, wherein the Fc region comprises an antibody CH2 and an antibody CH3; and a self-associating multimerisation domain (SAM); wherein the CH2 comprises an antibody hinge sequence and is devoid of a core hinge region. Advantageously, the Fc does not directly pair with another Fc, which is useful for producing multimers by multimerization using SAM domains. For example, a benefit may be aiding desired multimer formation and/or enhancing multimer purity formed by such multimerization.
The invention also provides: A multimer of a plurality of antibody Fc regions, wherein each Fc is comprised by a respective polypeptide and is unpaired with another Fc region; optionally wherein the multimer is for medical use.
The invention also provides:-
A protein multimer of at least first, second, third and fourth copies of an effector domain (eg, a protein domain or a peptide), wherein the multimer is multimerised by first, second, third and fourth self-associating tetramerisation domains (TDs) which are associated together, wherein each tetramerisation domain is comprised by a respective engineered polypeptide comprising one or more copies of said protein domain or peptide.
An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of a TCR binding site, insulin peptide, incretin peptide or peptide hormone; or a plurality of said tetramers or octamers.
An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of an antibody binding site or an antibody variable domain (eg, a single variable domain); or a plurality of said tetramers or octamers.
In an example the tetramer or octamer is soluble in aqueous solution (eg, aqueous eukaryotic cell culture medium). In an example the tetramer or octamer is expressible in a eukaryotic cell. Exemplification is provided below.
A tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, a tetramer or octamer) of
An engineered polypeitide or monomer of a multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, a tetramer or octamer) of the invention.
An engineered (and optionally isolated) engineered polypeptide (P1) which comprises (in N- to C-terminal direction):-
A nucleic acid encoding an engineered polypeptide or monomer of the invention, optionally wherein the nucleic acid is comprised by an expression vector for expressing the polypeptide.
Use of a nucleic acid or vector of the invention in a method of manufacture of protein multimers for producing intracellularly expressed and/or secreted multimers, wherein the method comprises expressing the multimers in and/or secreting the multimers from eukaryotic cells comprising the nucleic acid or vector.
A method producing
Use of a nucleic acid or vector of the invention in a method of manufacture of protein multimers for producing glycosylated multimers in eukaryotic cells comprising the nucleic acid or vector.
Use of self-associating tetramerisation domains (TD) (eg, NHR2 TD, p53 TD, p63 TD or p73 TD or a homologue or orthologue thereof) in a method of the manufacture of a tetramer of polypeptides, for producing a higher yield of tetramers versus monomer and/or dimer polypeptides.
Use of an engineered polypeptide in a method of the manufacture of a tetramer of a polypeptide comprising multiple copies of a protein domain or peptide, for producing a higher yield of tetramers versus monomer and/or dimer polypeptides, wherein the engineered polypeptide comprises one or more copies of said protein domain or peptide and further comprises a self-associating tetramerisation domains (TD) (eg, NHR2 TD, p53 TD, p63 TD or p73 TD or a homologue or orthologue).
Use of self-associating tetramerisation domains (TD) (eg, NHR2 TD, p53 TD, p63 TD or p73 TD or a homologue or orthologue thereof) in a method of the manufacture of a tetramer of a polypeptide, for producing a plurality of tetramers that are not in mixture with monomers, dimers or trimers.
A eukaryotic host cell comprising the nucleic acid or vector for intracellular and/or secreted expression of the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, tetramer, octamer), engineered polypeptide or monomer of the invention.
Use of an engineered polypeptide in a method of the manufacture of a tetramer of a polypeptide comprising multiple copies of a protein domain or peptide, for producing a plurality of tetramers that are not in mixture with monomers, dimers or trimers, wherein the engineered polypeptide comprises one or more copies of said protein domain or peptide and further comprises a self-associating tetramerisation domains (TD) (eg, NHR2 TD, p53 TD, p63 TD or p73 TD or a homologue or orthologue).
A multivalent heterodimeric soluble T cell receptor capable of binding pMHC complex comprising:
A multimeric immunoglobulin, comprising
method for assembling a soluble, multimeric polypeptide, comprising:
A mixture comprising (i) a cell line (eg, a eukaryotic, mammalian cell line, eg, a HEK293, CHO or Cos cell line) encoding a polypeptide of the invention; and (ii) tetramers of the invention.
A method for enhancing the yield of tetramers of an protein effector domain (eg, an antibody variable domain or binding site), the method comprising expressing from a cell line (eg, a mammalian cell, CHO, HEK293 or Cos cell line) tetramers of a polypeptide, wherein the polypeptide is a polypeptide of the invention and comprises one or more effector domains; and optionally isolating said expressed tetramers.
A polypeptide comprising (in N- to C-terminal direction; or in C- to N-terminal direction)
A method of expanding the antigen binding specificity of a binding site, wherein the binding site binds a first antigen, but not a second antigen (eg, when administered to humans) when the binding site is comprised in monovalent or bivalent form by a protein that specifically binds to the first antigen, the method comprising providing a plurality of copies of a polypeptide of the invention, and multimerising at least 4 of the polypeptides to produce a multimer comprising at least 4 copies of the polypeptide, wherein the polypeptide comprises one, two or more copies of the binding site, whereby binding sites of the multimer are capable of binding the first and second antigens.
Use of a polyepeptide of the invention in a method of manufacturing a multimer for expanding the antigen binding specificity of a binding site, wherein the binding site binds a first antigen, but not a second antigen (eg, when administered to humans) when the binding site is comprised in monovalent or bivalent form by a protein that specifically binds to the first antigen, wherein the method comprises providing a plurality of copies of a polypeptide of the invention, and multimerising at least 4 of the polypeptides to produce a multimer comprising at least 4 copies of the polypeptide, wherein the polypeptide comprises one, two or more copies of the binding site, whereby binding sites of the multimer are capable of binding the first and second antigens.
In an embodiment, the polypeptide comprises aspects useful for treating or preventing a viral infection or cancer wherein the polypeptide comprises
The invention also provides:
The invention also provides a pharmaceutical composition, cosmetic, foodstuff, beverage, cleaning product, detergent comprising the multimer(s), tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, tetramer(s) or octamer(s)) of the invention.
A multimer herein is, eg, a dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer.
As demonstrated in Example 22, dodecamer and hexadecamer multimers surprisingly display a very high functional affinity for antigen binding due to the increasing avidity effect. The functional affinity for these going from 8 to 12 binding sites (compare Tables 15 and 16) or from 8 to 16 binding sites is much more than additive; a synergistic increase is seen as a result of enhanced avidity. Thus, in one embodiment, a multimer which is 12-valent for an antigen (ie, a dodecamer as described herein) is preferred; in another embodiment a multimer which is 16-valent for an antigen (ie, hexadecamer as described herein) is preferred.
Thus, a further configuration of the invention provides:
As demonstrated in Example 32, multimers comprising 4 copies of an antigen binding site of REGN10987, REGN10933 or CB6 surprisingly display much improved neutralization potency of the Quad formats over the parental IgG format (
As demonstrated in Example 33, multimers comprising 4 copies of antigen binding domain Nb-112 (a VHH domain comprising the amino acid sequence of SEQ ID NO: 288) surprisingly display much improved neutralization potency of the Quad formats over the parental VHH format (
A method of purifying a multimer of the invention from a composition comprising the multimer, the method comprising contacting the composition with an antigen (eg, a supergantigen) and binding the multimer to the antigen, and optionally isolating antigen/multimer complexes.
The multimer may be obtained from the complexes.
Preferably, the multimer comprises at least 4 copies (eg, 4, 8, 12, 16, 20, 24 or 28 copies) of a VH3 family domain and the superantigen is Protein A. Preferably, the multimer comprises at least 4 copies (eg, 4, 8, 12, 16, 20, 24 or 28 copies) of a Vκ domain and the superantigen is Protein L.
The polypeptide described herein may, for example, comprise binding domain QB-GB or a binding domain (eg, an antibody single variable domain) that competes with QB-GB for binding to SARS-CoV-2 spike in an in vitro competition assay.
The polypeptide described herein may, for example, comprise binding domain QB-GB or a binding domain (eg, an antibody single variable domain) that binds to the same SARS-CoV-2 spike epitope (or an overlapping epitope) as QB-GB.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state.
Similarly, the multimer herein may comprise copies of such a binding domain. The multimer described herein may, for example, bind to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike. The multimer described herein may, for example, bind to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state.
As explained in Example 37 Quad multimers that have such features have been found to be highly advantageous and may be more resistant to receptor-driven selection pressure associated with SARS-Cov-2 mutation.
The invention also provides polypeptide dimers, as well as tetramers of dimers.
The invention relates to multimers such as tetramers of polypeptides and tetramers, octamers, dodecamers, hexadecamers or 20-mesr (eg, tetramers and octamers) of epitopes or effector domains (such as antigen binding sites (eg, antibody or TCR binding sites that specifically bind to antigen or pMHC, or variable domains thereof)) or peptides such as incretin, insulin or hormone peptides. In embodiments, multimers of the invention are usefully producible in eurkaryotic systems and can be secreted from eukaryotic cells in soluble form, which is useful for various industrial applications, such as producing pharmaceuticals, diagnostics, as imaging agents, detergents etc. Higher order multimers, such as tetramers or octamers of effector domains or peptides are useful for enhancing antigen or pMHC binding avidity. This may be useful for producing an efficacious medicine or for enhancing the sensitivity of a diagnostic reagent comprising the multimer, such as tetramer or octamer. An additional or alternative benefit is enhanced half-life in vivo when the multimers of the invention are administered to a human or animal subject, eg, for treating or preventing a disease or condition in the subject. Usefully, the invention can also provide for multi-specific (eg, bi- or tri-specific) multivalent binding proteins. Specificity may related to specificity of antigen or pMHC binding. By using a single engineered polypeptide comprising binding domains or peptides, the invention in certain examples usefully provides a means for producing multivalent (eg, bi-specific) proteins at high purity. Use of a single species of engineered polypeptide monomer avoids the problem of mixed products seen when 2 or more different polypeptide species are used to produce multi- (eg, bi-) specific or multivalent proteins.
The invention also relates to methods and uses to expand antigen specificity of binding sites, as well as vaccines, methods of vaccination and assay methods and reagents.
The invention provides the following Clauses, Aspects, Paragraphs and Concepts (which are not intended to represent “Claims”; Claims are presented towards the end of this disclosure after the Examples and Tables). Any Clause herein can be combined with any Aspect or Concept herein. Any Aspect herein can be combined with any Concept herein.
The following Aspects are not to be interpreted as Claims. The Claims start after the Examples section.
1. A protein multimer of at least first, second, third and fourth copies of an effector domain (eg, a protein domain) or a peptide, wherein the multimer is multimerised by first, second, third and fourth self-associating tetramerisation domains (TDs) which are associated together, wherein each tetramerisation domain is comprised by a respective engineered polypeptide comprising one or more copies of said protein domain or peptide.
In an example, each TD is a TD of any one of proteins 1 to 119 listed in Table 2. In an example, each TD is a p53 TD or a homologue or orthologue thereof. In an example, each TD is a NHR2 TD or a homologue or orthologue thereof. In an example, each TD is a p63 TD or a homologue or orthologue thereof. In an example, each TD is a p73 TD or a homologue or orthologue thereof. In an example, each TD is not a NHR2 TD. In an example, each TD is not a p53 TD. In an example, each TD is not a p63 TD. In an example, each TD is not a p73 TD. In an example, each TD is not a p53, 63 or 73 TD. In an example, each TD is not a NHR2, p53, 63 or 73 TD.
By being “associated together”, the TDs in Aspect 1 multimerise first, second, third and fourth copies of the engineered polypeptide to provide a multimer protein, for example, a multimer that can be expressed intracellulary in a eukaryotic or mammalian cell (eg, a HEK293 cell) and/or which can be extracellularly secreted from a eukaryotic or mammalian cell (eg, a HEK293 cell) and/or which is soluble in an aqueous medium (eg, a eukaryotic or mammalian cell (eg, a HEK293 cell) culture medium). Examples are NHR TD, p53 TD, p63 TD and p73 TD (eg, human NHR TD, p53 TD, p63 TD and p73 TD) or an orthologue or homologue thereof.
In an example, the TD is not a p53 TD (or homologue or orthologue thereof), eg, it is not a human p53 TD (or homologue or orthologue thereof). In an example, the TD is a NHR2 TD or a homologue or orthologue thereof, but excluding a p53 TD or a homologue or orthologue thereof. In an example, the TD is a human NHR2 TD or a homologue or orthologue thereof, but excluding a human p53 TD or a homologue or orthologue thereof. In an example, the TD is human NHR2. In an example, the amino acid sequence of the TD is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to the sequence of human NHR2. In an example, the domain or peptide is not naturally comprised by a polypeptide that also comprise a NHR2 TD.
In an example, all of the domains of the polypeptide are human.
The engineered polypeptide may comprise one or more copies of said domain or peptide N-terminal to a copy of said TD. Additionally or alternatively, the engineered polypeptide may comprise one or more copies of said domain or peptide C- terminal to a copy of said TD. In an example, the engineered polypeptide comprises a first said domain or peptide and a TD, wherein the first domain or peptide is spaced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous amino acids from the TD, wherein there is no further said domain or peptide between the first domain or peptide and the TD.
In an example, the multimer (eg, tetramer of said engineered polypeptide) comprises 4 (but no more than 4) TDs (eg, identical TDs) and 4, 8, 12 or 16 (but no more than said 4, 8, 12 or 16 respectively) copies of said domain or peptide. In an example, each TD and each said domain or peptide is human.
In an example, the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, tetramer or octamer) comprises first, second, third and fourth identical copies of an engineered polypeptide, the polypeptide comprising a TD and one (but no more than one), two (but no more than two), or more copies of the said protein domain or peptide. For example, a tetramer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has 1 such epitope or effector domain. For example, an octamer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has 2 such epitope or effector domain. For example, a dodecamer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has 3 such epitope or effector domain. For example, a hexadecamer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has 4 such epitope or effector domain. For example, a 20-mer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has 5 such epitope or effector domain. Generally, for example, a X-mer of the epitope or effector domain has 4 identical copies of the polypeptide comprising a TD and each polypeptide has X/4 such epitope or effector domain, where X= any multiple of 4, eg, 4, 8, 12, 16, 20, 24, 28 or 32.
In some embodiments, by requiring just one type of engineered polypeptide to form the multimer, eg, tetramer or octamer, of the invention, the invention advantageously provides a format that can be readily isolated in pure (or highly pure, ie >90, 95, 96, 97, 98 or 99% purity) format, as well as a method for producing such a format in pure (or highly pure) form. Purity is indicated by the multimer of the invention not being in mixture in a composition with any other multimer or polypeptide monomer, or wherein the multimer of the invention comprises >90, 95, 96, 97, 98 or 99% of species in a composition comprising the multimer of the invention and other multimers and/or polypeptide monomers which comprise the engineered polyeptide. Thus, mixtures of different types of polypeptide in these embodiments are avoided or minimised. This advantageously also provides, therefore, plurality of multimers (eg, a plurality of tetramers or octamers or dodecamers or hexadecamers) that comprise only one (and no more than one) type of engineered polypeptide, wherein the multimers are monospecific (but multivalent) for antigen binding, or alternatively bi- or multi-specific for antigen binding. Thus, the invention provides a plurality of multimers (eg, a plurality of tetramers or octamers or dodecamers or hexadecamers, each polypeptide being at least tetra-valent for antigen binding and (i) bi-specific (ie, capable of specifically binding to 2 different antigens) or (ii) mono-specific and at least tetravalent for antigen binding. Herein, where antigen binding is mentioned this can instead be pMHC binding when the domain is a TCR V domain. Advantageously, the plurality is in pure form (ie, not mixed with multimers (eg, tetramers or octamers or dodecamers or hexadecamers) that comprise more than one type of polypeptide monomer. In an example, the multimer comprises at least 2 different types of antigen binding site. In an example, the multimer is bi-specific, tri-specific or tetra-specific. In an example, the multimer has an antigen binding site or pMHC binding site valency of 4, 6, 8, 10 or 12, preferably 4 or 8.
In an example, a peptide MHC (pMHC) is a class I or class II pMHC.
By the term “specifically binds,” as used herein, eg, with respect to a domain, antibody or binding site, is meant a domain, antibody or binding site which recognises a specific antigen (or pMHC) with a binding affinity of 1 mM or less as determined by SPR
Target binding ability, specificity and affinity (KD (also termed Kd), Koff and/or Kon) can be determined by any routine method in the art, eg, by surface plasmon resonance (SPR). The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular binding site/ligand, receptor/ligand or antibody/antigen interaction. In one embodiment, the surface plasmon resonance (SPR) is carried out at 25° C. In another embodiment, the SPR is carried out at 37° C. In one embodiment, the SPR is carried out at physiological pH, such as about pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (also referred to as HBS-EP)). In one embodiment, the SPR is carried out at a physiological salt level, eg, 150 mM NaCl. In one embodiment, the SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-20™) at 0.05% and EDTA at 3 mM. In one example, the SPR is carried out at 25° C. or 37° C. in a buffer at pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, the SPR is carried out at 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022). In an example, the affinity (eg, of a VH/VL binding site) is determined using SPR by using any standard SPR apparatus, such as by Biacore™ or using the ProteOn XPR36™ (Bio-Rad®). The binding data can be fitted to 1:1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36™ analysis software.
In an example, a multimer, tetramer or octamer or dodecamer or hexadecamer or 20-mer of the invention is an isolated multimer, tetramer or octamer or dodecamer or hexadecamer or 20-mer. In an example, a multimer, tetramer or octamer of the invention consists of copies of said engineered polypeptide. Optionally the multimer, tetramer or octamer or dodecamer or hexadecamer or 20-mer of the invention comprises 4 or 8 or 12 or 16 or 20 but not more than 4 or 8 or 12 or 16 or 20 copies respectively of the engineered polypeptide.
By “engineered” is meant that the polypeptide is not naturally-occurring, for example the protein domain or peptide is not naturally comprised by a polypeptide that also comprises said TD.
Each said protein domain or peptide may be a biologically active domain or peptide (eg, biologically active in humans or animals), such as a domain that specifically binds to an antigen or peptide-MHC (pMHC), or wherein the domain is comprised by an antigen or pMHC binding site. In an alternative, the domain or peptide is a carbohydrate, glucose or sugar-regulating agent, such as an incretin or an insulin peptide. In an alternative, the domain or peptide is an inhibitor or an enzyme or an inhibitor of a biological function or pathway in humans or animals. In an alternative, the domain or peptide is an iron-regulating agent. Thus, in an example, each protein domain or peptide is selected from an antigen or pMHC binding domain or peptide; a hormone; a carbohydrate, glucose or sugar-regulating agent; an iron-regulating agent; and an enzyme inhibitor.
The immunoglobulin superfamily (IgSF) is a large protein superfamily of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Molecules are categorized as members of this superfamily based on shared structural features with immunoglobulins (also known as antibodies); they all possess a domain known as an immunoglobulin domain or fold. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular muscle proteins. They are commonly associated with roles in the immune system.
T-cell receptor (TCR) domains can be Vα (eg. paired with a Vβ), Vβ (eg. paired with a Vα), Vγ (eg, paired with a V8) or Vδ (eg, paired with a Vy).
4. The multimer of Aspect 3, wherein the binding site comprises a first variable domain paired with a second variable domain.
In a first example, the first and second variable domains are comprised by the engineered polypeptide. In another example, the first domain is comprised by the engineered polypeptide and the second domain is comprised a by a further polypeptide that is different from the engineered polypeptide (and optionally comprises a TD or is devoid of a TD).
In the alternative, the domains are constant region domains. In an alternative, the domains are FcAbs. In an alternative, the domains are non-Ig antigen binding sites or comprises by a non-Ig antigen binding site, eg, an affibody.
In an example, the or each antigen binding site (or effector domain) is selected from the group consisting of an antibody variable domain (eg, a VL or a VH, an antibody single variable domain (domain antibody or dAb), a camelid VHH antibody single variable domain, a shark immunoglobulin single variable domain (NA V), a Nanobody™ or a camelised VH single variable domain); a T-cell receptor binding domain; an immunoglobulin superfamily domain; an agnathan variable lymphocyte receptor (J Immunol; 2010 Aug 1;185(3):1367-74; “Alternative adaptive immunity in jawless vertebrates; Herrin BR & Cooper M D.); a fibronectin domain (eg, an Adnectin™); an scFv; an (scFv)2; an sc-diabody; an scFab; a centyrin and an antigen binding site derived from a scaffold selected from CTLA-4 (Evibody™); a lipocalin domain; Protein A such as Z-domain of Protein A (eg, an Affibody™ or SpA); an A-domain (eg, an Avimer™ or Maxibody™); a heat shock protein (such as and epitope binding domain derived from GroEI and GroES); a transferrin domain (eg, a trans-body); ankyrin repeat protein (eg, a DARPin™); peptide aptamer; C-type lectin domain (eg, Tetranectin™); human γ- crystallin or human ubiquitin (an affilin); a PDZ domain; scorpion toxin; and a kunitz type domain of a human protease inhibitor.
Further sources of antigen binding sites are variable domains and VH/VL pairs of antibodies disclosed in WO2007024715 at page 40, line 23 to page 43, line 23. This specific disclosure is incorporated herein by reference as though explicitly written herein to provide basis for epitope binding moieties for use in the present invention and for possible inclusion in claims herein.
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” or “antibody single variable domain” refers to an antibody variable domain (VH, VHH, VL) 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 VHH immunoglobulin single variable domains. Camelid VHH sre 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 VHH domains 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 “VH includes camelid VHH domains. NA V 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. 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 β-sheet secondary structure with a numer 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), US7250297B1 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 a-helices and a β-turn. They can be engineered to bind different target antigens by randomising residues in the first a-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™ consist 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 US6818418B1. 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 KalataBI and conotoxin and knottins. The microproteins have a loop which can be engineered to include upto 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see WO2008098796. Other epitope binding moieties and 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).
In an example, the or each antigen binding site (or effector domain) comprises a non-Ig scaffoled, eg, is selected from the group consisting of Affibodies, Affilins, Anticalins, Atrimers, Avimers, Bicycle Peptides, Cys-knots, DARpins, Fibronectin type III, Fyomers, Kunitz Domain, OBodies, Aptamers, Adnectins, Armadillo Repeat Domain, Beta-Hairpin mimetics and Lipocalins.
For example, the polypeptide comprises in N-terminal direction (i) P1- TD -P2; or (ii) TD -P1-P2, wherein P1=a copy of a domain or peptide of the first type (ie, the type of domain or peptide of the multimer of Aspect 1); and P2=a copy of a domain or peptide of said second type.
For example, a said protein domain of the engineered polypeptide is a V domain (a VH or VL) of an antibody binding site of an antibody selected from said group, wherein the multimer comprises a further V domain (a VL or VH respectively) that pairs with the V domain of the engineered polypeptide to form the antigen binding site of the selected antibody. Advantageously, therefore, the invention provides tetramer, octamer, 12-mer, 16-mer or 20-mer (eg, a tetramer, octamer, 12-mer or 16-mer; or tetramer or octamer)of a binding site of said selected antibody, which beneficially may have improved affinity, avidity and/or efficacy for binding its cognate antigen or for treating or preventing a disease or condition in a human or animal wherein the multimer is administered thereto to bind the cognate antigen in vivo.
For example, the multimer, tetramer, octamer, 12-mer, 16-mer or 20-mer (eg, a tetramer, octamer, 12-mer or 16-mer; or tetramer or octamer) comprises 4 (or said X/4 as described above) copies of an antigen binding site of an antibody, wherein the antibody is adalimumab, sarilumab, dupilumab, bevacizumab (eg, AVASTIN™), cetuximab (eg, ERBITUX™), tocilizumab (eg, ACTEMRA™) or trastuzumab (HERCEPTIN™). In an alternative the antibody is an anti-CD38 antibody, an anti-TNFa antibody, an anti-TNFR antibody, an anti-IL-4Ra antibody, an anti-IL-6R antibody, an anti-IL-6 antibody, an anti-VEGF antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PCSK9 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD138 antibody, an anti-IL-1 antibody. In an alternative the antibody is selected from the antibodies disclosed in WO2007024715 at page 40, line 23 to page 43, line 23, the disclosure of which is incorporated herein by reference.
A binding site herein may, for example, be a ligand (eg, cytokine or growth factor, eg, VEGF or EGFR) binding site of a receptor (eg, KDR or Flt). A binding site herein may, for example, be a binding site of Eyelea™, Avastin™ or Lucentis™, eg, for ocular or oncological medical use in a human or animal. When the ligand or antigen is VEGF, the mutlimer, tetramer or octamer may be for treatment or prevention of a caner or ocular condition (eg, wet or dry AMD or diabetic retinopathy) or as an inhibitor of neovascularisation in a human or animal subject.
17. An isolated tetramer, octamer, dodecamer, hexadecamer or 20-mer of a TCR binding site, insulin peptide, incretin peptide or peptide hormone; or a plurality of said tetramers, octamers, dodecamers, hexadecamers or 20-mers.
Several important peptide hormones are secreted from the pituitary gland. The anterior pituitary secretes three hormones: prolactin, which acts on the mammary gland; adrenocorticotropic hormone (ACTH), which acts on the adrenal cortex to regulate the secretion of glucocorticoids; and growth hormone, which acts on bone, muscle, and the liver. The posterior pituitary gland secretes antidiuretic hormone, also called vasopressin, and oxytocin. Peptide hormones are produced by many different organs and tissues, however, including the heart (atrial-natriuretic peptide (ANP) or atrial natriuretic factor (ANF)) and pancreas (glucagon, insulin and somatostatin), the gastrointestinal tract (cholecystokinin, gastrin), and adipose tissue stores (leptin). In an example, the peptide hormone of the invention is selected from prolactin, ACTH, growth hormone (somatotropin), vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin (eg, selected from human prolactin, ACTH, growth hormone, vasopressin, oxytocin, glucagon, insulin, somatostatin, cholecystokinin, gastrin and leptin).
In an example, the incretin is a GLP-1, GIP or exendin-4 peptide.
The invention provides, in embodiments, the following engineered multimers:-
In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the domain or peptide is human. In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises a NHR2 TD (eg, a human NHR2). In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises a p53 TD (eg, a human p53 TD). In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises a p63 TD (eg, a human p63 TD). In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises a p73 TD (eg, a human p73 TD). In an example of any of these tetramer, octamer, dodecamer, hexadecamer or 20-mers, the tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises a tetramer of TDs (eg, human NHR2 TDs), whereby the domains or peptides form a multimer of 4 or 8 domains or peptides.
In an example, the plurality is pure, eg, is not in mixture with multimers of said binding site or peptide wherein the multimers comprise more than one type of polypeptide monomer.
18. The multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer of any preceding Aspect, wherein the mulitmer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is
In an example the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is secretable from a HEK293T (or other eukaryotic, mammalian, CHO or Cos) cell in stable form as indicated by a single band at the molecular weight expected for said multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer on a PAGE gel using a sample of supernatant from such cells and detected using Western Blot.
19. A tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, atetramer or octamer) of
An example of the medium is SFMII growth medium supplemented with L-glutamine (eg, complete SFMII growth medium supplemented with 4 mM L-glutamine). In an example, the medium is serum-free HEK293 cell culture medium. In an example, the medium is serum-free CHO cell culture medium.
For example, a cell herein is a human cell, eg, a HEK293 cell (such as a HEK293T cell).
For example, the glycosylation is CHO cell glycosylation. For example, the glycosylation is HEK (eg, HEK293, such as HEK293T) cell glycosylation. For example, the glycosylation is Cos cell glycosylation. For example, the glycosylation is Picchia cell glycosylation. For example, the glycosylation is Sacchaaromyces cell glycosylation.
The monomer is an engineered polypeptide as disclosed herein, comprising a said protein domain or peptide and further comprising a TD.
Optionally, the engineered polypeptide comprises (in N- to C-terminal direction) a variable domain (V1) - a constant domain (C) (eg, a CH1 or Fc) - optional linker - TD.
28. An engineered (and optionally isolated) engineered polypeptide (P1) which comprises (in N-to C-terminal direction):-
In (a) or (b), in an example, the TCR V is comprised by an single chain TCR binding site (scTCR) that specifically binds to a pMHC, wherein the binding site comprises TCR V-linker -TCRV. In an example, the engineered polypeptide comprises (in N- to C-terminal direction) (i) V1 -linker - V - optional C - optional linker - TD, or (ii) Va - linker - V1 - optional C - optional linker -TD, wherein Va is a TCR V domain and C is an antibody C domain (eg, a CH1 or CL) or a TCR C.
Preferably, the antibody C is CH1 (eg, IgG CH1).
In an example the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer has a size of no more than 155 kDa, eg, wherein said protein domain is an antibody variable domain comprising a CDR3 of at least 16, 17, 18, 19, 20, 21 or 22 amino acids, such as a Camelid CDR3 or bovine CDR3.
In an example, the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer comprises TCR binding sites and antibody binding sites. For example, each polypeptide comprises a TCR V (eg, comprised by a scTCR that specifically binds a pMHC) and an antibody V (eg, comprised by a scFv or paired with a second V domain comprised by a said second polypeptide to form a V/V paired binding site that specifically binds to an antigen). In an example, the pMHC comprises a RAS peptide. In an example the antigen is selected from the group consisting of PD-1, PD-L1 or any other antigen disclosed herein. For example, the antigen is PD-1 and the pMHC comprises a RAS peptide.
29. The polypeptide of Aspect 28, wherein the engineered polypeptide P1 is paired with a further polypeptide (P2), wherein P2 comprises (in N- to C-terminal direction):-
Optionally, V1 and V2 form a paired variable domain binding site that is capable of specifically binding to an antigen or pMHC. In an example, V1 and V2 are variable domains of an antibody, eg, selected from the group consisting of ReoPro™; Abciximab; Rituxan™; Rituximab; Zenapax™; Daclizumab; Simulect™; Basiliximab; Synagis™; Palivizumab; Remicade™; Infliximab; Herceptin™; Mylotarg™; Gemtuzumab; Campath™; Alemtuzumab; Zevalin™; Ibritumomab; Humira™; Adalimumab; Xolair™; Omalizumab; Bexxar™; Tositumomab; Raptiva™; Efalizumab; Erbitux™; Cetuximab; Avastin™; Bevacizumab; Tysabri™; Natalizumab; Actemra™; Tocilizumab; Vectibix™; Panitumumab; Lucentis™; Ranibizumab; Soliris™; Eculizumab; Cimzia™; Certolizumab; Simponi™; Golimumab, Ilaris™; Canakinumab; Stelara™; Ustekinumab; Arzerra™; Ofatumumab; Prolia™; Denosumab; Numax™; Motavizumab; ABThrax™; Raxibacumab; Benlysta™; Belimumab; Yervoy™; Ipilimumab; Adcetris™; Brentuximab; Vedotin™; Perjeta™; Pertuzumab; Kadcyla™; Ado-trastuzumab; Keytruda™, Opdivo™, Gazyva™ and Obinutuzumab. Optionally, the binding site of the polypeptide of the multimer comprises a VH of the binding site of the antibody and also the CH1 of the antibody (ie, in N- to C-terminal direction the VH-CH1 and SAM). In an embodiment, the polypeptide may be paired with a further polypeptide comprising (in N- to C-terminal direction a VL-CL, eg, wherein the CL is the CL of the antibody).
In one embodiment, the antibody is Avastin.
In one embodiment, the antibody is Actemra.
In one embodiment, the antibody is Erbitux.
In one embodiment, the antibody is Lucentis.
In one embodiment, the antibody is sarilumab.
In one embodiment, the antibody is dupilumab.
In one embodiment, the antibody is alirocumab.
In one embodiment, the antibody is bococizumab.
In one embodiment, the antibody is evolocumab.
In one embodiment, the antibody is pembrolizumab.
In one embodiment, the antibody is nivolumab.
In one embodiment, the antibody is ipilimumab.
In one embodiment, the antibody is remicade.
In one embodiment, the antibody is golimumab.
In one embodiment, the antibody is ofatumumab.
In one embodiment, the antibody is Benlysta.
In one embodiment, the antibody is Campath.
In one embodiment, the antibody is rituximab.
In one embodiment, the antibody is Herceptin.
In one embodiment, the antibody is durvalumab.
In one embodiment, the antibody is daratumumab.
In an example, any binding domain herein (eg, a dAb or scFv or Fab) or V1 is capable (itself when a single variable domain, or when paired with V2) of specifically binding to an antigen selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AWI; AIG1; AKAP1; AKAP2; AIYIH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLRl (MDR15); BlyS; BM Pl; BMP2; BMP3B (GDFIO); BMP4; BMP6; BM P8; BMPRIA; BMPRIB; BM PR2; BPAG1 (plectin); BRCA1; CI9orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (M IP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (M PIF-1); CCL24 (MPIF-2 I eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK /ILC) ; CCL28; CCL3 (MIP-la); CCL4 (M IP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA);CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CM KBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CM KBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p2IWapl/Cipl); CDKN1B (p27Kipl); CDKNIC; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDi) ; CX3CR1 (V28); CXCL1 (GRO1); CXCLIO (IP-10); CXCL11 (1-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTRISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; EN01; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FU12584; FU25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-I); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13; IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R; 1L18; IL18BP; IL18R1; IL18RAP; 1L19; ILIA; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1;IL1RL2 IL1RN; 1L2; 1L20; IL20RA; IL21R; 1L22; 1L22R; 1L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG; 1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6R; IL6ST (glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAKI; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; M IB1; midkine; M IF; M IP-2; MK167 (Ki-67); MMP2; M MP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); MTSS 1; M UC 1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NM E1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2IRac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRRIB (Spri); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-i); T]MP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a (also referred to herein as TNF alpha or TNFα); TNFAIP2 (B94); TNFAIP3; TNFRSF1 1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSF1 1 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF1 5 (VEGI); TNFSF1 8; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM 1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM 1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2.
For example in any configuration of the invention, the multimer, octamer, dodecamer, hexadecamer or 20-mer specifically binds to first and second (eg, for an octamer, dodecamer, hexadecamer or 20-mer); optionally, first, second and third (eg, for a dodecamer, hexadecamer or 20-mer); or optionally, first, second, third and fourth (eg, for a hexadecamer or 20-mer); or optionally, first, second, third, fourth and fifth (eg, for a 20-mer) epitopes or antigens, each of which is selected from the group consisting of EpCAM and CD3; CD19 and CD3; VEGF and VEGFR2; VEGF and EGFR; CD138 and CD20; CD138 and CD40; CD20 and CD3; CD38 and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD19 and CD20; CD-8 and IL-6; PDL-1 and CTLA-4; CTLA-4 and BTN02; CSPGs and RGM A; IGF1 and IGF2; IGF1 and/or 2 and Erb2B; IL-12 and IL-18; IL-12 and TWEAK; IL-13 and ADAM8; IL-13 and CL25; IL-13 and IL-lbeta; IL-13 and IL-25; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-9; IL-13 and LHR agonist; IL-13 and MDC; IL-13 and MIF; IL-13 and PED2; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and TARC; IL-13 and TGF-beta; IL-1 alpha and IL-1 beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; RGM A and RGM B; Te38 and TNF alpha; TNF alpha and IL-12; TNF alpha and IL-12p40; TNF alpha and IL-13; TNF alpha and IL-15; TNF alpha and IL-17; TNF alpha and IL-18; TNF alpha and IL-1 beta; TNF alpha and IL-23; TNF alpha and M IF; TNF alpha and PEG2; TNF alpha and PGE4; TNF alpha and VEGF; and VEGFR and EGFR; TNF alpha and RANK ligand; TNF alpha and Blys; TNF alpha and GP130; TNF alpha and CD-22; and TNF alpha and CTLA-4
For example, the first epitope or antigen is selected from the group consisting of CD3; CD16; CD32; CD64; and CD89; and the second epitope or antigen is selected from the group consisting of EGFR; VEGF; IGF-1R; Her2; c-Met (aka HGF); HER3; CEA; CD33; CD79a; CD19; PSA; EpCAM; CD66; CD30; HAS; PSMA; GD2; ANG2; IL-4; IL-13; VEGFR2; and VEGFR3.
In an example, any binding domain herein (eg, a dAb or scFv or Fab) or V1 is capable (itself when a single variable domain, or when paired with V2) of specifically binding to an antigen selected from the group consisting of human IL-1A, IL-1β, IL-1RN, IL-6, BLys, APRIL, activin A, TNF alpha, a BMP, BMP2, BMP7, BMP9, BMP10, GDF8, GDF11, RANKL, TRAIL, VEGFA, VEGFB or PGF; optionally the multimer comprises a cytokine amino acid sequence (eg, C-terminal to a TD), such as IL-2 or an IL2-peptide; and the multimer, octamer, dodecamer, hexadecamer or 20-mer is for treating or preventing a cancer in a human subject. In an example the said effector or protein domain is capable of binding to such an antigen; optionally the multimer comprises a cytokine amino acid sequence (eg, C-terminal to a TD), such as IL-2 or an IL2-peptide; and the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer is for treating or preventing a cancer in a human subject.
30. A multimer (eg, a dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer) of P1 as defined in Aspect 28; or of P1 paired with P2 as defined in Aspect 29; or a plurality of said multimers, optionally wherein the multimer is according to any one of aspects 1 to 24.
Preferably the multimer is a tetramer of the engineered polypeptide and/or effector domain. In one example, the plurality of tetramers are not in mixture with monomers, dimers or trimers of the polypeptide,
In one example the multimer, eg, tetramer, is a capable of specifically binding to two different pMHC.
31. A nucleic acid encoding an engineered polypeptide or monomer of any one of Aspects 27 to 29, optionally wherein the nucleic acid is comprised by an expression vector for expressing the polypeptide.
In an example, the nucleic acid is a DNA, optionally operably connected to or comprising a promoter for expression of the polypeptide or monomer. In another example the nucleic acid is a RNA (eg, mRNA).
Mammalian glycosylation of the invention is useful for producing medicines comprising or consisting of the multimers, tetramer, octamer, dodecamer, hexadecamer or 20-merof the invention for medical treatment or prevention of a disease or condition in a mammal, eg, a human. The invention thus provides such a method of use as well as the multimer, tetramer, octamer, dodecamer, hexadecamer or 20-merof the invention for this purpose. Similarly, intracellular and/or secreted expression in one or more host cells (or cell lines thereof) that are mammalian according to the invention is useful for producing such medicines. Particularly useful is such expression in HEK293, CHO or Cos cells as these are commonly used for production of medicaments.
In an embodiment, the invention comprises a detergent or personal healthcare product comprising a multimer, tetramer, octamer, dodecamer, hexadecamer or 20-merof the invention. In an embodiment, the invention comprises a foodstuff or beverage comprising a multimer, tetramer, octamer, dodecamer, hexadecamer or 20-merof the invention.
In an example, the multimer, monomer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer, polypeptide, composition, mixture, use or method of the present invention is for an industrial or domestic use, or is used in a method for such use. For example, it is for or used in agriculture, oil or petroleum industry, food or drink industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aeorspace industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dentistry industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry or steel industry.
The invention further provides
The invention also provides
The invention provides a claim multimer (eg, tetramer) of NHR2 or p53 (or another TD disclosed herein) fused at its N- and/or C-terminus to an amino acid sequence (eg, a peptide, protein domain or protein) that is not an NHR2 sequence. For example, sequence is selected from a TCR (eg, TCRα, TCRβ, Cα or Cβ), cytokine (eg, interleukin, eg, IL-2, IL-12, IL-12 and IFN), antibody fragments (eg, scFv, dAb or Fab) and a antibody domain (eg, V or C domain, eg, VH, VL, VK, Vλ, CH, CH1, CH2, CH3, hinge, CK or Cλ domain). Optionally, the multimer is the molecule is
The invention provides:-
Optionally, the amino acid is an amino acid sequence of a human peptide, protein domain or protein,eg, a TCR (eg, TCRα, TCRβ, Cα or Cβ), cytokine (eg, interleukin, eg, IL-2, IL-12, IL-12 and IFN), antibody fragments (eg, scFv, dAb or Fab), or an antibody domain (eg, V or C domain, eg, VH, VL, VK, Vλ, CH, CH1, CH2, CH3, hnige, CK or Cλ domain).
Optionally, the or each polypeptide comprises a polypeptide selected from the group consisting of Quad 1-46 (ie, a polypeptide as shown in
Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequence) that is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-50. Optionally, the or each polypeptide comprises a polypeptide (excluding any leader or tag sequence) that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-115. Optionally, the invention provides a multimer (eg, a dimer, trimer, tetramer, pentamer, hexamer, septamer or octamer, preferably a tetramer or octamer) of such a polypeptide, eg, for medical or diagnostic use, eg, medical use for treating or preventing a disease or condition in a human or animal (eg, a human).
In an example, the TD is a TD comprised by any one of SEQ ID NOs: 1-9. In an example, the TD is a TD comprising SEQ ID NO: 10 or 126. In an example, the TD is encoded by SEQ ID NO: 124 or 125. In an example, the amino acid sequence of each TD is SEQ ID NO: 10 or 126 or is at least 80, 85, 90, 95, 96m 97, 98 or 99% identical to SEQ ID NO: 10 or 126.
In an example, the TD is a TD comprising SEQ ID NO: 120 or 123. In an example, the TD is encoded by SEQ ID NO: 116 or 119. In an example, the amino acid sequence of each TD is SEQ ID NO: 120 or 123 or is at least 80, 85, 90, 95, 96m 97, 98 or 99% identical to the SEQ ID NO: 120 or 123.
Optionally, the domain or peptide comprised by the engineered polypeptide or monomer comprises an amino acid selected from SEQ ID NOs: 51-82.
As exemplified herein, the invention in one configuration is based on the surprising realization that tetramerisation domains (TD), eg, p53 tetramerisation domain (p53 TD), can be used to preferentially produce tetramers of effector domains over the production of lower-order structures such as dimers and monomers. This is particularly useful for secretion of tetramers is desirable yields from mammalian expression cell lines, such as CHO, HEK293 and Cos cell lines. The invention is also particularly useful for the production of tetramers no more than 200, 160, 155 or 150 kDa in size.
Thus, the invention provides the following Concepts:-
The following Concepts are not to be interpreted as Claims. The Claims start after the Examples section.
1. Use of a tetramerisation domain (TD) (eg, p53 tetramerisation domain (p53 TD) or NHR2 TD) or a homologue or orthologue thereof in a method of the manufacture of a tetramer of polypeptides, for producing a higher yield of tetramers versus monomer and/or dimer polypeptides.
The monomers and dimers comprise one or two copies of the TD, homologue or orthologue respectively
In an example, the TD, orthologue or homologue is a human domain.
Herein, the TD is a human TD or a homologue, eg, a TD selected from any of the p53 TD sequences disclosed in UniProt (www.uniprot.org), for example the p53 TD is a TD disclosed in Table 13. In an example, the homologue is a p53TD of a non-human animal species, eg, a mouse, rat, horse cat or dog p53TD. See
In an example, the yield of tetramers is higher than the yield of monomers; In an example, the yield of tetramers is higher than the yield of dimers; In an example, the yield of tetramers is higher than the yield of trimers; In an example, the yield of tetramers is higher than the yield of monomers and dimers; In an example, the yield of tetramers is higher than the yield of monomers and trimers; In an example, the yield of tetramers is higher than the yield of monomers, dimers and trimers
For example, the TD is the TD of p53 isoform 1. In an example, the TD comprises or consists of an amino acid sequence that is identical to positions 325 to 356 (or 319-360; or 321-359) of human p53 (eg, isoform 1). Optionally, the TD, orthologue or homologue comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10, 126, 11 or 12. For example the sequence is identical to said selected sequence. Optionally, the TD, orthologue or homologue comprises or consists of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 120, 121, 122 or 123. For example the sequence is identical to said selected sequence.
Optionally, the yield is at least 2× 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the yield of monomers and/or dimers. Optionally, the ratio of tetramers produced : monomers and/or dimers is at least 90:10, eg, at least 95:5; or 96:4; or 97:3; or 98:2; or 99:1. Optionally only tetramers are produced.
In an embodiment, each domain comprised by each monomer, dimer or tetramer is a human domain; and optionally the monomer, dimer or tetramer does not comprise non-human amino acid sequences or linkers.
Amounts of tetramers, monomers, dimers and trimers can be determined, for example, using Western Blot analysis of a gel described herein, eg, a native gel, ie, a gel not under denatured conditions, such as in the absence of SDS.
For example, the monomer has a size of no more than 35, 30, 25, 24, 23, 22, 21 or 20 kDa
11. The use of any preceding Concept, wherein each tetramer has a size of no more than 150 kDa.
For example, the tetramer has a size of no more than 80, 90, 100, 110, 120, 130 or 140 kDa.
12. The use of any preceding Concept, wherein the method comprises expressing the tetramers from a mammalian cell line, eg, a HEK293, CHO or Cos cell line.
For example, the cell line is a HEK293 (eg, HEK293T) cell line. In the alternative, the cell line is a yeast (eg, Saccharomyces or Pichia, eg, P pastoris) or bacterial cell line.
13. The use of any preceding Concept, wherein the method comprises secreting the tetramers from a mammalian cell line, eg, a HEK293, CHO or Cos cell line.
Thus, advantageously in an example, the use or tetramer is for expression from a mammalian cell line (eg, a HEK293, CHO or Cos cell line) or a eukaryotic cell line. This is useful for large-scale manufacture of the products, eg, tetramers, of the invention.
For example, the cell line is a HEK293 (eg, HEK293T) cell line. In the alternative, the cell line is a yeast (eg, Saccharomyces or Pichia, eg, P pastoris) or bacterial cell line.
For example, the tetramer has a size of no more than 80, 90, 100, 110, 120, 130 or 140 kDa. In an example, any multimer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-merherein has a size of at least 60 or 80 kDa; this may be useful for example to increase half -life in a human or animal subject administered with the multimer, dimer, trimer, tetramer, octamer, dodecamer, hexadecamer or 20-mer (eg, to treat or prevent a disease or condition in the subject). Sizes in these ranges may be above the renal filtration size.
In an alternative, the invention provides a monomer, dimer, octamer, dodecamer, hexadecamer or 20-merinstead of a tetramer.
In an embodiment, each polypeptide comprises only 2 (ie, only a first and a second, but not a third) effector domains or only 2 dAbs, VHH, scFvs, scTCRs, Fabs or antigen binding sites.
30. A pharmaceutical composition comprising a tetramer of any one of Concepts 22 to 29 and a pharmaceutically acceptable carrier, diluent or excipient.
Optionally the composition is comprised by a sterile medical container or device, eg, a syringe, vial, inhaler or injection device.
Optionally, the mixture is comprised by a sterile container.
The homologue, orthologue or equivalent has multimerisation or tetramerisation function.
Homologue: A gene, nucleotide or protein sequence related to a second gene, nucleotide or protein sequence by descent from a common ancestral DNA or protein sequence. The term, homologue, may apply to the relationship between genes separated by the event of or to the relationship between genes separated by the event of genetic duplication.
Orthologue: Orthologues are genes, nucleotide or protein sequences in different species that evolved from a common ancestral gene, nucleotide or protein sequence by speciation. Normally, orthologues retain the same function in the course of evolution.
In an example, the TD, orthologue or homologue is a TD of any one of proteins 1 to 119 listed in Table 2. In an example, the orthologue or homologue is an orthologue or homologue of a TD of any one of proteins 1 to 119 listed in Table 2. In an embodiment, instead of the use of a p53 tetramerisation domain (p53-TD) or a homologue or orthologue thereof, all aspects of the invention herein instead can be read to relate to the use or inclusion in a polypeptide, monomer, dimer, trimer or tetramer of aTD of any one of proteins 1 to 119 listed in Table 2 or a homologue or orthologue thereof. The TD may be a NHR2 (eg, a human NHR2) TD or an orthologue or homologue thereof. The TD may be a p63 (eg, a human p63) TD or an orthologue or homologue thereof. The TD may be a p73 (eg, a human p73) TD or an orthologue or homologue thereof. This may have one or more advantages as follows:-
In an example, the tetramer comprises 4 copies of the antigen binding site of a first antibody selected from the group consisting of ipilimumab (or YERVOY™), tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™), pidilizumab, BMS-936559, durvalumab and atezolizumab and optionally 4 copies of the antigen binding site of a second antibody selected from said group, wherein the first and second antibodies are different. For example, the first antibody is ipilimumab (or YERVOY™) and optionally the second antibody is nivolumab (or OPDIVO™) or pembrolizumab (or KEYTRUDA™). This is useful for treating or preventing a cancer in a human.
In an example, the tetramer comprises 4 copies of the antigen binding site of Avastin. In an example, the tetramer comprises 4 copies of the antigen binding site of Humira. In an example, the tetramer comprises 4 copies of the antigen binding site of Erbitux. In an example, the tetramer comprises 4 copies of the antigen binding site of Actemra™. In an example, the tetramer comprises 4 copies of the antigen binding site of sarilumab. In an example, the tetramer comprises 4 copies of the antigen binding site of dupilumab. In an example, the tetramer comprises 4 copies of the antigen binding site of alirocumab or evolocumab. In an example, the tetramer comprises 4 copies of the antigen binding site of In an example, the tetramer comprises 4 copies of the antigen binding site of Remicade. In an example, the tetramer comprises 4 copies of the antigen binding site of Lucentis. In an example, the tetramer comprises 4 copies of the antigen binding site of Eylea™. Such tetramers are useful for administering to a human to treat or prevent a cancer. Such tetramers are useful for administering to a human to treat or prevent an ocular condition (eg, wet AMD or diabetic retinopathy, eg, when the binding site is an Avastin, Lucentis or Eylea site). Such tetramers are useful for administering to a human to treat or prevent angiogenesis.
In an example, the tetramer comprises 4 copies of insulin. In an example, the tetramer comprises 4 copies of GLP-1. In an example, the tetramer comprises 4 copies of GIP. In an example, the tetramer comprises 4 copies of Exendin-4. In an example, the tetramer comprises 4 copies of insulin and 4 copies of GLP-1. In an example, the tetramer comprises 4 copies of insulin and 4 copies of GIP. In an example, the tetramer comprises 4 copies of insulin and 4 copies of Exendin-4. In an example, the tetramer comprises 4 copies of GLP-1 and 4 copies of Exendin-4. Such tetramers are useful for administering to a human to treat or prevent diabetes (eg, Type II diabetes) or obesity.
The polypeptide, multimer may bind to one or more antigens or epitopes, or each of the binding sites herein (eg, dAb or scFv binding sites) herein may bind to an antigen or epitope. In an example, an (or each) antigen herein is selected from the following list. In an example, an (or each) epitope herein is an epitope of an antigen selected from the following list.
Activin type-II receptor; Activin type-IIB receptor; ADAM11; ADAM12; ADAM15; ADAM17; ADAM18; ADAM19; ADAM1A; ADAM1B; ADAM2; ADAM20; ADAM21; ADAM22; ADAM23; ADAM24P; ADAM28; ADAM29; ADAM30; ADAM32; ADAM33; ADAM3A; ADAM3B; ADAM5; ADAM6; ADAM7; ADAM8; ADAM9; ADORA2A; AKT; ALK; alpa-4 integrin; alpha synuclein; anthrax protective antigen; BACE1; BCMA; beta amyloid; BRAF; BTLA; BTNL2; CCR4; CCR5; CD126; CD151; CD16; CD160; CD19; CD20; CD22; CD226; CD244; CD27; CD274 (PDL1); CD276; CD28; CD3; CD30; CD300A; CD300C; CD300E; CD300LB; CD300LF; CD33; CD38; CD3; CD3 epsilon; CD3 delta; CD3 gamma; CD40; CD40L; CD47; CD48; CD5; CD52; CD59; CD6; CD70; CD72; CD73; CD80; CD81; CD84; CD86; CD96; CDK4/6; CEA; CEACAM1; CEACAM3; CGRP receptor; CLEC12A; CLEC1B; CLEC4A; CLEC5A; CLEC7A; Clostridium difficile toxin ; cMET; Complement C5 factor; Complement factor D; CSF1R; CSF2RA; CTAG1B; CTLA4; CXCL12; CXCR2; CXCR4; DR4; DR5; EDA; EDA2R; EGFR; EGFRvIII; EMR1; ENTPD1; EpCAM; Factor IX; Factor X; Factor VII; FAP; FAS; FCAR; FCER1G; FCER2 ; TFR2; 4-1BB; FCGR2A; FCGR2B; FCGR3A; FCGR3B; FCRL1; FCRL3; FCRL4; FCRL6; FGRF1/2/3; FLT3; GAL; GEM; GITR; GITRL; GM-CSF; GM-CSF receptor; GP IIb IIIa; gpNMB; TIM3; HDAC1; HER-2; HER3; HFE; HHLA2; Histone H1 modulator; HLA-C; HLA-G; HMGB1; HMMR; HVEM; ICAM-1; ICOS; ICOSL; IDO1; IFNG; IL-1 beta; IL-12; IL-13; IL-2; IL-22R; IL-23; IL-23a; IL-24; IL-2R; IL-34; IL-8; IL10; IL11; IL13; IL17A; IL17D; IL22; IL2RA; IL36G; IL4; IL4a; IL5; IL6; Immunoglobulin E; Immunoglobulin E; Immunoglobulin G; INHBA; INHBB; Interferon type I receptor; INF-a-2a/2b; INF-b-1a/1b; ITGA2B; ITGB3; KIR; KIR2DL1; KIR2DL2; KIR2DL3; KIR2DL4; KIR2DL5A; KIR3DL1; KIR3DL3 ;KIR3DS1; KIT; KLRC1; KLRC2; KLRF1; KLRG1 ;KLRK1 ;KRAS; LAG3; LAIR1; LAIR2; LFA-1; LIGHT; LILRA1; LILRA2; LILRA3; LILRA4; LILRA5; LILRA6; LILRB1; LILRB2; LILRB3; LILRB4; LILRB5 ;LILRP1; LILRP2; LTA; LTBR; LY9; MadCam; MAGE-C1; MAGE-C2; MARCO; MEK-½; MIA3; MIC; MICA; MICB; MMP9; MS4A1; MS4A2; mTOR; MUC1; MUCIN-1; Nav1.7; Nav1.8; NCR1; NCR2; NCR3; NGF; NGFR; NT5E; NY-ESO-1; OX40; OX40L; p53; PARP; PCSK9; PD-1; PDCD1LG2; PDCD6; PDGF receptor alpha; PECAM1; PI3K delta; PILRA; PPP1R1B; PSMA; PTPN6; PVR; PVRL2; PVRL3; RANKL; Respiratory syncytial virus protein; SIGLEC10; SIGLEC12; SIGLEC15; SIGLEC5; SIGLEC6; SIGLEC7; SIGLEC9; SIRPA; SIRPB1; SLAMF1; SLAMF6; SLAMF7; SLAMF8; SNCA; SOD1; STAT3; STING; SURVIVIN; TARM1; Tau; TDP43; TfR1; TGF-b; TGM2; TIGIT; TIM-3; TLR-4; TLR03; TMEM30a; TMIGD2; TNFa; TNFRSF10A; TNFRSF10B; TNFRSF10C; TNFRSF10D; TNFRSF11A; TNFRSF11B; TNFRSF12A; TNFRSF13B; TNFRSF13C; TNFRSF14; TNFRSF17; TNFRSF18; TNFRSF19; TNFRSF21; TNFRSF4; TNFRSF6B; TNFRSF8; TNFRSF9; TNFSF10; TNFSF11; TNFSF12; TNFSF13; TNFSF13B; TNFSF14; TNFSF15; TNFSF18; TNFSF4; TNFSF8; TNFSF9; Transmembrane glycoprotein NMB modulator; TREML1; TREML2; TSLP; VEGF; VEGF-2R; VEGF1; VEGFA; VEGFL; VEGFR; Viral envelope glycoprotein; Viral protein haemagglutinin; VISTA; VSIG4; VSTM1; VTCN1; and WEE-1. In an example, an antigen herein is a PCSK9, eg, human PCSK9; optionally the multimer has 4, 8, 12 or 16 copies an anti- PCSK9 binding site (eg, a dAbs).
An example antigen is a toxin, such as a snake venom toxin, eg, wherein a multimer of the invention is administered (such as systemically or by IV injection) to a human or animal subject and the antigen binding sites comprised by the multimer specifically bind to the toxin in the subject. Preferably, each binding site or domain of the multimer is a dAb (eg, a Nanobody™). For example, each snake venom toxin antigen binding site of the multimer of the invention is a C33 single domain VH as disclosed in
Another example of a toxin is a blood toxin, eg, wherein a multimer of the invention is administered (such as systemically or by IV injection) to a human or animal subject and the antigen binding sites comprised by the multimer specifically bind to the toxin in the blood of the subject. These examples are useful for sequestering the toxin or for reducing the toxic effect of the toxin to the subject or to promote excretion or metabolism of the toxin.
In an example, the antigen is a viral antigen, each a capsid protein or carbohydrate (eg, a sugar). In an example, a multimer of the invention binds to a virus or virus antigen, eg, a virus selected from Table 19 wherein the virus comprises a surface antigen that is bound by the multimer; or the multimer of the invention binds to a cell or virus antigen, eg, selected from an antigen disclosed in Table 20. Binding to the virus may, for example, reduce or inhibit attachment of the virus to its host cell or infection of the cell by the virus. For example, the invention provides a method of treating or preventing (eg, reducing the risk of) a viral or cell infection in a human or animal or plant subject (eg, in a human subject), the method comprising administering a multimer of the invention to the subject wherein the multimer binds to a surface antigen of the virus, thereby inhibiting the virus from attaching to a host cell; inhibiting infection of a host cell by the virus and/or sequestering the virus in the subject (eg, to mark the bound virus for clearance from the systemic circulation or a tissue of the subject). In an alternative, For example, the invention provides a method of treating or preventing (eg, reducing the risk of) a bacterial or archaeal cell infection in a human or animal or plant subject (eg, in a human subject), the method comprising administering a multimer of the invention to the subject wherein the multimer binds to a surface antigen of the cell, thereby inhibiting infection of the subject by the cell and/or sequestering the cell in the subject (eg, to mark the bound cell for clearance from the systemic circulation or a tissue of the subject). In an alternative, For example, the invention provides a method of treating or preventing (eg, reducing the risk of) a cancer in a human or animal subject (eg, in a human subject), the method comprising administering a multimer of the invention to the subject wherein the multimer binds to a surface antigen of a tumour cell, thereby sequestering the cell in the subject (eg, to mark the bound cell for clearance from the systemic circulation or a tissue of the subject) or marking the cell for targeting by the immune sytem or another therapy (eg, immune checkpoint therapy or CAR-T therapy) administered to the subject.
In an example, the antigen is selected from CXCR2, CXCR4, GM-CSF, ICAM-1, IFN-g, IL-1, IL-10, IL-12, IL-1R1, IL-1R2, IL-1Ra, IL-1β, IL-4, IL-6, IL-8, MIF, TGF-β, TNF-α, TNFR1, TNFR2 and VCAM-1. Targeting one or more of these antigens may be useful for treating or preventing sepsis in a subject. Thus, in an example the multimer of the invention comprises one or more antigen binding sites (eg, each one provided by a dAb), wherein the multimer is for use in a method of treating or preventing sepsis in a human or animal subject, wherein the multimer is administered to the subject (eg, systemically or intravenously). Optionally, the multimer is monospecific, bispecific, trispecific or tetraspecific for antigen binding. For example, the multimer is bispecific, trispecific or tetraspecific for an antigen selected from CXCR2, CXCR4, GM-CSF, ICAM-1, IFN-g, IL-1, IL-10, IL-12, IL-1R1, IL-1R2, IL-1Ra, IL-1β, IL-4, IL-6, IL-8, MIF, TGF-β, TNF-α, TNFR1, TNFR2 and VCAM-1. There is also provided a pharmaceutical composition comprising such a multimer and a pharmaceutically acceptable diluent, carrier or excipient. There is also provided a method of treating or preventing sepsis in a human or animal subject, the method comprising administering the multimer to the subject, eg, systemically or intravenously.
The polypeptide monomer or multimer (eg, dimer, trimer, tetramer or octamer) of the invention can be used in a method for administration to a human or animal subject to treat or prevent a disease or condition in the subject.
Optionally, the disease or condition is selected from
In an example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease, geriopsychosis, Down syndrome, Parkinson’s disease, Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome, Huntington’s disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt- Jakob disease. For example, the disease is Alzheimer disease. For example, the disease is Parkinson syndrome.
In an example, wherein the method of the invention is practised on a human or animal subject for treating a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus into the brain of the subject, whereby the disease or condition (eg, Alzheimer’s disease) is treated, prevented or progression thereof is reduced. In an embodiment the method causes an increase of IFN-gamma in the CNS system (eg, in the brain and/or CSF) of the subject. In an example, the method restores nerve fibre and//or reduces the progression of nerve fibre damage. In an example, the method restores nerve myelin and//or reduces the progression of nerve myelin damage. In an example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method can be used with any method disclosed in WO2015136541 (the disclosure of this document is incorporated by reference herein in its entirety, eg, for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatement and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).
Cancers that may be treated include tumours that are not vascularized, or not substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as haematological tumours, for example, leukaemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukaemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and paediatric tumours/cancers are also included.
Haematologic cancers are cancers of the blood or bone marrow. Examples of haematological (or haematogenous) cancers include leukaemias, including acute leukaemias (such as acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myelogenous leukaemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronic leukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukaemia and myelodysplasia.
Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumours, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
The present configuration relates to a multivalent soluble TCR protein. In one aspect, the invention relates to tetravalent and octavalent soluble TCR analogues. The TCR proteins of the invention are capable of self-assembly from monomers and are entirely of human origin. The proteins are multimers which comprise an ETO NHR2 multimerisation domain. The invention also relates to methods of constructing multimeric soluble TCRs, and methods of using such proteins.
Attempts to exploit alternative soluble TCR formats as therapeutic molecules have lagged far behind compared to the plethora of antibody formats. This is largely due to TCR, a heterodimeric transmembrane protein having the intrinsic problem of solubility once the extracellular TCR α/β chains are dissociated from their transmembrane and cytoplasmic domain. Secondly the intrinsic low affinity and avidity of these molecules for their cognate ligand at the target site has to a large degree hampered their development as a therapeutic molecule.
In order to overcome these drawbacks, the present configuration of the invention provides a TCR protein which is both multivalent and soluble. Multivalency increases the avidity of the TCR for cognate pMHC, and solubility allows the TCR to be used outside of a transmembrane environment. Accordingly, in a first aspect there is provided a multivalent heterodimeric soluble T cell receptor capable of binding pMHC complex comprising:
The use of Ig constant domains provides the TCR extracellular domains with stability and solubility; multimerisation via the NHR2 domains provides multivalency and increased avidity. Advantageously, all of the domains are of human origin or conform to human protein sequences.
Using the Ig constant domain to stabilise and render soluble the TCR avoids the use of non-native disulphide bonds. Advantageously, therefore, the TCR of the invention does not comprise a non-native disulphide bond.
In one embodiment, said complex comprises a heavy chain and a light chain, and each light chain comprises a TCR Vα domain and an immunoglobulin Cα domain, and each heavy chain comprises a TCR Vβ domain and an immunoglobulin CH1 domain.
In one embodiment, each light chain additionally comprises a TCR Cα domain, and each heavy chain additionally comprises a TCR Cβ domain.
In embodiments, the TCR and immunoglobulin domains can be separated by a flexible linker.
The NHR2 multimerisation domain is advantageously attached to the C-terminus of an immunoglobulin domain. Thus, each dimer of heavy and light chains will be attached to one multimerisation domain, so that the heavy chain-light chain dimers associate into multivalent oligomers.
In embodiments, the multimerisation domain and the immunoglobulin domain are separated by a flexible linker. In certain embodiments, this allows the multimerisation domain to multimerise without hindrance from the immunoglobulin domain(s).
In embodiments, the TCR protein may further comprise an immunoglobulin hinge domain. Hinge domains allow dimerization of heavy chain-light chain dimers; this allows further multimerisation of the TCR proteins. For example, a multimerisation domain which forms polypeptide tetramers can, using an immunoglobulin hinge domain, form multimers up to polypeptide octamers. Likewise, a dimerising multimerisation domain can form tetramers in the presence of a hinge domain.
In embodiments, the TCR protein of the invention is tetravalent.
In embodiments, the TCR protein of the invention is octavalent
The present invention provides a soluble TCR where it is stably assembled in a tetravalent heterodimeric format using the nervy homology region 2 (NHR2) domain found in the ETO family protein in humans (Liu et al. 2006). The NHR2 domain is found naturally to form homotetramer, which is formed from pairing of two NHR2 homodimers. NHR2 linked operably to the extracellular TCRα or TCRβ chain will preferentially form tetravalent heterodimeric soluble TCR protein molecules sequentially self-assembled from a monomer followed by a homodimer (
TCR proteins assembling into octamers can be created using the NHR2 domain, by employing immunoglobulin hinge domains.
In a further aspect, the TCR proteins of the invention can be coupled to biologically active polypeptides/effector molecules. Examples of such polypeptides can include immunologically active moieties such as cytokines, binding proteins such as antibodies or targeted polypeptides, and the like.
The invention further relates to methods for making tetravalent and octavalent heterodimeric soluble TCR, the DNA vectors encoding the proteins used for transfecting host cells of interests and the use of these novel highly sensitive multivalent soluble TCR protein molecules. Applications for use could include but not limited to, therapeutics, diagnostics and drug discovery.
In a further aspect, the invention provides a method for constructing multivalent immunoglobulin molecules in an efficient manner, without employing non-human construct components.
Accordingly, there is provided a multimeric immunoglobulin comprising
The immunoglobulin variable domains are preferably antibody variable domains. Such domains are fused to the ETO NHR2 multimerisation domain, which provides means for forming tetramers of the immunoglobulin variable domains.
The ETO NHR2 domain is more efficient than p53 and similar multimerisation domains in the production of immunoglobulin multimers, and permits the production of multimeric immunoglobulin molecules without the use of non-human components in the construct.
Also provided is a method for producing a multimeric immunoglobulin, comprising expressing immunoglobulin variable domains in fusion with an NHR2 domain of ETO, and allowing the variable domains to assemble into multimers.
Preferably, the immunoglobulin variable domains are attached to one or more immunoglobulin constant domains.
Advantageously, the immunoglobulin domains are antibody domains. For example, the variable domains can be VH and VL antibody domains. For example, the constant domains are antibody CH1 domains.
In one embodiment, the multimeric immunoglobulin molecules according to the invention, both TCR and non-TCR immunoglobulins, are produced for screening by phage display or another display technology. For example, therefore, the multivalent immunoglobulins are produced as fusions with a phage coat protein. For each immunoglobulin produced fused to a coat protein, other immunoglobulin molecules are produced without a coat protein, such that they can assemble on the phage surface as a result of NHR2 multimerisation.
The present configuration of the invention as detailed above relates to the nucleic acid sequences and methods for producing novel multivalent, for example tetravalent and octavalent, soluble proteins. In one aspect in particular the soluble protein is a TCR assembled into a tetravalent heterodimeric format that can bind four pMHC with high sensitivity, affinity and specificity. The soluble tetravalent heterodimeric TCR is a unique protein molecule composed from either the entire or in part the extracellular TCR α/β chains. The extracellular TCR α/β chains are linked to immunoglobulin CH1 and CL (either CK or Cλ) domains. This linkage allows stable formation of heterodimeric TCR α/β. In the context of soluble tetravalent TCR the unique feature is the NHR2 homotetramer domain of the ETO family of proteins, which is operably linked to the C-terminus of CH1 or the C-terminus of CL. Linkage of the NHR2 domain to the heterodimeric α/βTCR in this manner allows it to self-assemble into a tetravalent format inside cells and be subsequently secreted into the supernatant as a soluble protein.
TCR extracellular domains are composed of variable and constant regions. These domains are present in T-cell receptors in the same way as they are present in antibodies and other immunoglobulin domains. The TCR repertoire has extensive diversity created by the same gene rearrangement mechanisms used in antibody heavy and light chain genes (Tonegawa, S. (1988) Biosci. Rep. 8:3-26). Most of the diversity is generated at the junctions of variable (V) and joining (J) (or diversity, D) regions that encode the complementarity determining region 3 (CDR3) of the α and β chains (Davis and Bjorkman (1988) Nature 334:395-402). Databases of TCR genes are available, such as the IMGT LIGM database, and methods for cloning TCRs are known in the art - for example, see Bentley and Mariuzza (1996) Ann. Rev. Immunol. 14:563-590; Moysey et al., Anal Biochem. 2004 Mar 15;326(2):284-6; Walchli, et al. (2011) A Practical Approach to T-Cell Receptor Cloning and Expression. PLoS ONE 6(11): e27930.
Antibody variable domains are known in the art and available from a wide variety of sources. Databases of sequences of antibody variable domains exist, such as IMGT and Kabat, and variable domains can be produced by cloning and expression of natural sequences, or synthesis of artificial nucleic acids according to established techniques.
Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (McCafferty et al. (1990) Nature, 348: 552; Kang et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 4363; Clackson et al. (1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991) Proc. Natl. Acad Sci USA., 88: 10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang et al. (1991) J Immunol., 147: 3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra; Barbas et al. (1992) supra; Hawkins and Winter (1992) J Immunol., 22: 867; Marks et al., 1992, J Bioi. Chem., 267: 16007; Lerner et al. (1992) Science, 258: 1313, incorporated herein by reference).
One particularly advantageous approach has been the use of scFv phage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991) Nature, 352: 624; Marks et al. (1991) J Mol. Bioi., 222: 581; Chiswell et al. (1992) Trends Biotech., 10: 80; Marks et al. (1992) J Bioi. Chem., 267). Various embodiments of scFv libraries displayed on bacteriophage coat proteins have been described. Refinements of phage display approaches are also known, for example as described in W096/06213 and W092/01047 (Medical Research Council et al.) and W097/08320 (Morphosys), which are incorporated herein by reference.
Such techniques can be adapted for the production of multimeric immunoglobulins by the fusion of NHR2 multimerisation domains to the antibody variable domains
An immunoglobulin constant domain, as referred to herein, is preferably an antibody constant domain. Constant domains do vary in sequence between antibody subtypes; preferably, the constant domains are IgG constant domains. Preferably, the constant domains are CH1 constant domains. Antibody constant domains are well known in the art and available from a number of sources and databases, including the IMGT and Kabat databases.
The fusion of antibody constant domains to immunoglobulin variable domains is also known in the art, for example in the construction of engineered Fab antibody fragments.
Flexible linkers can be used to connect TCR variable domain - Ig constant domain to the NHR2 multimerisation domain. This allows the TCR domains and the multimerisation domain to function without steric hindrance from each other or other molecules in the multimeric complex. Suitable linkers comprise, for example, glycine repeats, glycine-alanine repeats, Gly(4)Ser linkers, or flexible polypeptide linkers as set forth in Reddy Chichili et al., 2012 Protein Science 22:153-167.
The Ig Hinge domain, herein preferably an antibody hinge domain, is the domain which links antibody constant regions in a natural antibody. This domain therefore provides for natural dimerization of molecules which include an antibody constant domain. It is present, for example, in a F(ab)2 antibody fragment, as well as whole antibodies such as IgG. This region comprises two natural interchain disulphide bonds, which connect the two CH1 constant domains together.
The multimerisation domain, in one embodiment, may be attached to the Ig constant domain or to the hinge domain. If a hinge domain is present, the multimerisation domain will form a TRC octamer, comprising four dimers of TCR variable-Ig Constant domains joined at a hinge region. Without the hinge region, the multimerisation domain will lead to the formation of a tetramer. Preferably, the multimerisation domain is attached to the C-terminal end of the constant domain or the hinge region.
One or more biologically active molecules or effector molecules (EM) can be attached to the multimer, eg, multimeric TCR proteins, of the present invention. Such molecules may be, for example, antibodies, especially antibodies which may assist in immune recognition and functioning of the TCR, such as anti-CD3 antibodies or antibody fragments.
In some aspects, the biologically active molecule can be a cytotoxic drug, toxin or a biologically active molecule such as a cytokine, as described in more detail below. Examples of biologically active molecules include chemokines such as MIP-1b, cytokines such as IL-2, growth factors such as GM-CSF or G-CSF, toxins such as ricin, cytotoxic agents, such as doxorubicin or taxanes, labels including radioactive and fluorescent labels, and the like. For examples of biologically active molecules conjugatable to TCRs, see US20110071919.
In other aspects, the biologically active molecule is, for example, selected from the group consisting of: a group capable of binding to a molecule which extends the half-life of the polypeptide ligand in vivo, and a molecule which extends the half-life of the polypeptide ligand in vivo. Such a molecule can be, for instance, HSA or a cell matrix protein, and the group capable of binding to a molecule which extends the half-life of the TCR molecule in vivo is an antibody or antibody fragment specific for HSA or a cell matrix protein.
In one embodiment, the biologically active molecule is a binding molecule, for example an antibody fragment. 2, 3, 4, 5 or more antibody fragments may be joined together using suitable linkers. The specificities of any two or more of these antibody fragments may be the same or different; if they are the same, a multivalent binding structure will be formed, which has increased avidity for the target compared to univalent antibody fragments.
The biologically active molecule can moreover be an effector group, for example an antibody Fc region.
Attachments to the N or C terminus may be made prior to assembly of the TCR molecule or engineered polypeptide into multimers, or afterwards. Thus, the TCR fusion with an Ig Constant domain may be produced (synthetically, or by expression of nucleic acid) with an N or C terminal biologically active molecule already in place. In certain aspects, however, the addition to the N or C terminus takes place after the TCR fusion has been produced. For example, Fluorenylmethyloxycarbonyl chloride can be used to introduce the Fmoc protective group at the N-terminus of the TCR fusion. Fmoc binds to serum albumins including HSA with high affinity, and Fmoc-Trp or FMOC-Lys bind with an increased affinity. The peptide can be synthesised with the Fmoc protecting group left on, and then coupled with the scaffold through the cysteines. An alternative is the palmitoyl moiety which also binds HSA and has, for example been used in Liraglutide to extend the half-life of this GLP-1 analogue.
Alternatively, the TCR fusinon can be modified at the N-terminus, for example with the amine- and sulfhydryl-reactive linker N-e-maleimidocaproyloxy)succinimide ester (EMCS). Via this linker the TCR can be linked to other polypeptides, for example an antibody Fc fragment.
AML1/ETO is the fusion protein resulting from the t(8;21) found in acute myeloid leukemia (AML) of the M2 subtype. AML1/ETO contains the N-terminal 177 amino acids of RUNX1 fused in frame with most (575 aa) of ETO. The nervy homology domain 2 of ETO is responsible for many of the biological activities associated with AML1/ETO, including oligomerisation and protein-protein interactions. This domain is characterised in detail in Liu et al (2006). See Genbank accession number NG_023272.2.
In one aspect of the present invention, the protein assembled into a soluble multivalent format is a TCR composed of either in part or all of the extracellular domains of the TCR α and β chains. The TCR α and β chains are stabilized by immunoglobulin CH1 and CL domains and could be arranged in the following configurations:
In one aspect of this invention, the extracellular TCR domains are linked to immunoglobulin CH1 and CL domains via an optional peptide linker (L) to promote protein flexibility and facilitate optimal protein folding.
In another aspect of this invention, a tetramerisation domain (TD) such as NHR2 homotetramer domain is linked to the C-terminus of either the immunoglobulin CH1 or CL domain, which is linked to the extracellular TCR α and β chain. The NHR2 domain could be optionally linked to CH1 or CL domain via a peptide linker. The resulting tetravalent heterodimeric TCR protein could be arranged in the following configurations where (L) is an optional peptide linker:
The sensitivity of the soluble TCR for its cognate pMHC can be enhanced by increasing the avidity effect. This is achieved by increasing the number of antigen binding sites, facilitated by the tetramerisation domain. This in turn also increases the molecular weight of the protein molecule compared to a monovalent soluble TCR and thus extends serum retention in circulation. Increasing the serum half-life also enhances the likelihood of these molecules interacting with their cognate target antigens.
The tetravalent heterodimeric soluble TCR protein molecule is capable of binding simultaneously to one, two, three or four pMHC displayed on a single cell or bind simultaneously to one, two, three or four different cells displaying its cognate pMHC.
TCR α and β chain sequences used in this invention could be from a known TCR specific for a particular pMHC or identified de novo by screening using techniques known in the art, such as phage display. Furthermore, TCR sequences are not limited to α and β chain in this invention but can also incorporate TCRδ and γ or ε chain and sequence variations thereof either directly cloned from human T cells or identified by directed evolution using recombinant DNA technology.
In another aspect to this invention, the tetravalent heterodimeric soluble TCR protein molecules are preferentially produced in mammalian cells for optimal production of soluble, stable and correctly folded protein molecules.
Multimer (eg, tetramer or octamer), or multivalent TCR according to the present invention may be expressed in cells, such as mammalian cells, using any suitable vector system. The pTT5 expression vector is one example of an expression system is used to express multivalent soluble TCR. The pTT5 expression system allows for high-level transient production of recombinant proteins in suspension-adapted HEK293 EBNA cells (Zhang et al. 2009). It contains origin of replication (oriP) that is recognized by the viral protein Epstein-Barr Nuclear Antigen 1 (EBNA-1), which together with the host cell replication factor mediates episomal replication of the DNA plasmid allowing enhanced expression of recombinant protein. Other suitable vector system for mammalian cell expression known in the art and commercially available can be used with this invention.
The tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced by transiently expressing genes from an expression vector.
In another embodiment, tetravalent heterodimeric soluble TCR protein molecules or other multimers can be produced from an engineered stable cell line. Cell lines can be engineered to produce the protein molecule using genome-engineering techniques known in the art where the gene(s) encoding for the protein molecule is integrated into the genome of the host cells either as a single copy or multiple copies. The site of DNA integration can be a defined location within the host genome or randomly integrated to yield maximum expression of the desired protein molecule. Genome engineering techniques could include but not limited to, homologous recombination, transposon mediated gene transfer such as PiggyBac transposon system, site specific recombinases including recombinase-mediated cassette exchange, endonuclease mediated gene targeting such as CRISPR/Cas9, TALENs, Zinc-finger nuclease, meganuclease and virus mediated gene transfer such as Lentivirus.
Also, in another aspect to the invention, the tetravalent heterodimeric soluble TCR protein molecule or other multimer is produced by overexpression in the cytoplasm of E. coli as inclusion bodies and refolded in vitro after purification by affinity chromatography to produce functional protein molecules capable of correctly binding to its cognate pMHC or antigen.
In another aspect to the invention, expression of the tetravalent heterodimeric soluble TCR protein molecule or other multimer is not limited to mammalian or bacterial cells but can also be expressed and produced in insect cells, plant cells and lower eukaryotic cells such as yeast cells.
In another aspect to this invention, the heterodimeric soluble TCR molecule or other multimer is produced as an octavalent protein complex, eg, having up to eight binding sites for its cognate pMHC (
The heterodimeric soluble TCR portion of the molecule is made into a bivalent molecule by fusing the immunoglobulin hinge domain to the C-terminus of either the CH1 or CL domain, which is linked itself either to TCR α or β chain. The hinge domain allows for the connection of two heavy chains giving a structure similar to IgG. To the C-terminus of the hinge domain, a tetramerisation domain such as NHR2 is linked via an optional peptide linker. By joining immunoglobulin hinge to C- and N-terminus of Ig CH1 or CL domain and NHR2 domain respectively, it allows for the assembly of two NHR2 monomers referred to as monomer2. In this conformation we predict the two NHR2 domains will most likely not form a homodimer by an antiparallel association due to structural constraints unless a long flexible linker is provided between the hinge and NHR2 domain. Linkage of the tetramerisation and the hinge domain to the to the heterodimeric soluble TCR via immunoglobulin CH1 or CL domain allows for the stepwise self-assembly of an octavalent soluble TCR formed through a NHR2 homotetramer2. The self-assembly of the octavalent soluble TCR is via NHR2 monomer2 and homodimer2 intermediate protein complexes (
In another aspect to this invention, the self-assembled multivalent protein preferentially tetravalent and octavalent heterodimeric soluble TCR are fused or conjugated to biologically active agent/effector molecule thus allowing these molecules to be guided to the desired cell population such as cancers cells and exert their therapeutic effect specifically. The tumour targeting ability of monoclonal antibodies to guide an effector molecule such as a cytotoxic drug, toxins or a biologically active molecule such as cytokines is well established (Perez et al. 2014; Young et al. 2014). In a similar manner the multivalent soluble TCR molecules outlined in this invention can also be fused with effector proteins and polypeptide or conjugated to cytotoxic agents. Examples of effector protein molecules suitable for use as a fusion protein with the multivalent protein complexes outlined in this invention include but are not limited to, IFNα, IFNβ, IFNγ, IL-2, IL-11, IL-13, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor [TNF]α, IL-7, IL-10, IL-12, IL-15, IL-21, CD40L, and TRAIL, the costimulatory ligand is B7.1 or B7.2, the chemokines DC-CK1, SDF-1, fractalkine, lyphotactin, IP-10, Mig, MCAF, M1P-1α, MIP-⅓, IL-8, NAP-2, PF-4, and RANTES or an active fragment thereof. Examples of toxic agent suitable for use as a fusion protein or conjugated to the multivalent protein complexes described in this invention include but not limited to, toxins such as diphtheria toxin, ricin, Pseudomonas exotoxin, cytotoxic drugs such as auristatin, maytansines, calicheamicin, anthracyclines, duocarmycins, pyrrolobenzodiazepines. The cytotoxic drug can be conjugated by a select linker, which is either non-cleavable or cleavable by protease or is acid-labile.
To eliminate heterogeneity and improve conjugate stability the cytotoxic drug can be conjugated in a site-specific manner. By engineering specific cysteine residues or using enzymatic conjugation through glycotransferases and transglutaminases can achieve this (Panowski et al. 2014).
In another aspect of the invention, the multivalent protein complex is covalently linked to molecules allowing detection, such as fluorescent, radioactive or electron transfer agents.
In another aspect of the invention, an effector molecule (EM) is fused to the multivalent protein complex via the C-terminus of the tetramerisation domain such as NHR2 via an optional peptide linker. Fusion via the NHR2 domain can be arranged to produce multivalent protein complexes in a number of different configurations. Examples of some of the protein configurations that can be produced using the tetravalent heterodimeric soluble TCR is shown below:
In another aspect of the invention, the effector molecule (EM) is fused to the multivalent protein complex at the C-terminus of either the immunoglobulin CH1 or CL1 domain via an optional peptide linker. Fusion of the EM via the immunoglobulin domain can be arranged to produce multivalent protein complexes in a number of different configurations. Examples of some of the protein configurations that can be produced using the tetravalent heterodimeric soluble TCR is shown below:
In another aspect of the invention, effector molecules (EM) are fused to the multivalent protein complex at the C-terminus of either the immunoglobulin CH1 or CL1 domain and also the C-terminus of the tetramerisation domain (e.g. NHR2) via an optional peptide linkers. This approach allows for the fusion of two effector molecules to be fused per TCR heterodimer complex. Fusion of the EM via the immunoglobulin domain and the tetramerisation domain can be arranged to produce multivalent protein complexes in a number of different configurations. Examples of some of the protein configurations that can be produced using the tetravalent heterodimeric soluble TCR is shown below:
In another aspect of the invention, the multivalent protein complex is fused to a protein tag to facilitate purification. Purification tags are known in the art and they include, without being limited to, the following tags: His, GST, TEV, MBP, Strep, FLAG.
The present invention provides a unique method for assembling proteins in a soluble multivalent format with potential to bind multiple interacting domains or antigens. The protein can be a monomer, homodimer, heterodimer or oligomer preferentially involved either directly or indirectly in the immune system, or having the potential to regulate immune responses. Examples include, but not limited to, TCR, peptide MHC class I and class II, antibodies or antigen-binding portions thereof and binding proteins having alternative non-antibody protein scaffolds.
In another aspect of the invention, the interacting domains or antigens could be any cell surface expressed or secreted proteins, peptide-associated with MHC Class I or II or any proteins associated with pathogens including viral and bacterial proteins.
Non-TCR multimers may be multimers of antibodies or antibody fragments, such as dAbs of Fabs. Examples of dAbs and Fabs in accordance with the invention include the following:
Examples of multivalent dAbs
Examples of multivalent Fabs
In the examples above, (L) denotes an optional peptide linker, whilst EM denotes a biologically active agent or effector molecule such as toxins, drugs or cytokines, and including binding molecules such as antibodies, Fabs and ScFv.
The variable light chain can be either Vλ or Vκ.
In one aspect of the invention, the assembled tetramerized protein molecule in one example could be a human pMHC for the application in drug discovery using animal drug discovery platforms (e.g. mice, rats, rabbits, chicken). In such a context, the tetramerisation domain is preferentially expressed and produced from genes originating from the animal species it is intended for. One example of such drug discovery applications would be the use of the tetramerized human pMHC as an antigen for immunization in rats for example. Once rats are immunized with pMHC the immune response is directed specifically towards the human pMHC and not the tetramerisation domain of the protein complex.
Multivalent antibodies can be produced, for example using single domain antibody sequences, fused to the NHR2 multimerisation domain.
In a related aspect to the invention, the tetravalent protein can be a peptide used as a probe for molecular imaging of tumour antigens. The multivalent binding of such a probe will have distinctive advantage over monovalent molecular probes as it will have enhanced affinity, avidity and retention time in vivo and this in turn will enhance in vivo tumour targeting.
The multimerisation domain is the NHR2 domain set forth above. Preferably, polypeptides are stabilized and/or rendered soluble by the use of Ig constant domains fused to the polypeptides, such that the fusions provide tetramers of polypeptides. Ig hinge domains can be used to provide octamers.
Multimeric TCR proteins according to the invention are useful in any application in which soluble TCR proteins are indicated. Particular advantages of the TCR proteins of the invention include increased avidity for the selected target, and/or the ability to bind a plurality of targets.
Thus, in one aspect, the multivalent heterodimeric soluble TCR protein molecules of the invention can be used for selectively inhibiting immune responses, for example suppression of an autoimmune response. The multivalent, for example tetravalent, nature of these soluble protein molecules gives it exquisite sensitivity and binding affinity to compete antigen-specific interactions between T cells and antigen presenting cells. This kind of neutralization effect can be therapeutically beneficial in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, inflammatory bowel diseases, graves disease, vasculitis and type 1 diabetes.
Similarly, the tetravalent heterodimeric soluble TCR protein molecules can be used to prevent tissue transplant rejection by selectively suppressing T cell recognition of specific transplantation antigen and self antigens binding to target molecule and thus inhibiting cell-to-cell interaction.
In another aspect of the invention, the tetravalent heterodimeric soluble TCR protein molecules can be used in clinical studies such as toxicity, infectious disease studies, neurological studies, behavior and cognition studies, reproduction, genetics and xenotransplantation studies.
The tetravalent heterodimeric soluble TCR protein molecules with enhanced sensitivity for cognate pMHC can be used for the purpose of diagnostics using biological samples obtained directly from human patients. The enhanced sensitivity of the tetravalent heterodimeric soluble TCRs allows detection of potential disease-associated peptides displayed on MHC, which are naturally found to be expressed at low density. These molecules can also be used for patient stratification for enrolling patient onto relevant clinical trials.
In another aspect of the invention, octavalent heterodimeric soluble TCR protein molecules can be used in pharmaceutical preparations for the treatment of various diseases.
In another related aspect to this invention, octavalent heterodimeric soluble TCR protein molecules can be used as a probe for tumour molecular imaging or prepared as a therapeutic protein.
Optionally, the polypeptide (first polypeptide) comprises or consists of a polypeptide disclosed in Table 8. In an example the invention provides a multimer (eg, a dimer, trimer or tetramer, preferably a tetramer) of such a polypeptide. In an example, in Table 8, the multimerization domain (SAM) is a p53 domain (eg, a human p53 domain). In an example, in Table 8, the multimerization domain (SAM) is an orthologue or homologue of a p53 domain (eg, a human p53 domain).
Optionally, the invention provides a polypeptide (eg, said polypeptide or said first polypeptide), wherein the polypeptide comprises or consists of (in N- to C-terminal direction*);
*In an alternative the components are written in the C- to N-terminal direction.
In an embodiment, polypeptide H, L, O or Q is associated with a second polypeptide, wherein the second polypeptide comprises (in N- to C-terminal direction) VL and CL, wherein the CL is associated with the CH1 of the first polypeptide.
In an embodiment, polypeptide I, M, P or R is associated with a second polypeptide, wherein the second polypeptide comprises (in N- to C-terminal direction) VH and CH1, wherein the CH1 is associated with the CL of the first polypeptide.
In an example, the polypeptide is encoded by a nucleotide sequence disclosed in Table 9. In an example, the polypeptide comprises or consists of an amino acid sequence disclosed in Table 10.
In an example (i) the polypeptide comprises (in N- to C-terminal direction);
Optionally, the SAM is a tetramerisation domain, eg, a p53 TD.
In an example the first, second, third (when present) and fourth (when present) dAbs have the same antigen binding specificity. In an example the first, second, third (when present) and fourth (when present) dAbs have the same different binding specificity.
In an example the first and second scFvs have the same antigen binding specificity. In an example the first and second scFvs have the same different antigen binding specificity.
In an example the first dAb, second dAb and first scFv have the same antigen binding specificity. In an example the first dAb, second dAb and first scFv have the same different antigen binding specificity.
Herein, where a dAb is provided in the polypeptide, in an alternative there may be provided instead any different type of antigen binding domain, such as a scFv or Fab or non-Ig binding domain (eg, an affibody, avimer or fibronectin domain).
Herein, where a scFv is provided in the polypeptide, in an alternative there may be provided instead any different type of antigen binding domain, such as a dAb or Fab or non-Ig binding domain (eg, an affibody, avimer or fibronectin domain).
Herein, where a Fab is provided in the polypeptide, in an alternative there may be provided instead any different type of antigen binding domain, such as a scFv or dAb or non-Ig binding domain (eg, an affibody, avimer or fibronectin domain).
Each antigen may be any antigen disclosed herein.
In an example, the CH1 (when present), CH2 and CH3 are a human Ig CH1, a CH2 a CH3, eg, a IgG1 CH1, CH2 and CH3. In an example, the CH2 comprises a CH2 domain, the CH3 comprises a CH3 domain and the CH2 comprises a hinge amino acid sequence. In this example, the CH2 comprises (in N- to C-terminal direction) the hinge amino acid sequence and the CH2 domain. In an example the hinge amino acid sequence (i) is a complete hinge; (ii) is a hinge amino acid sequence that is non-functional to dimerise the polypeptide with another such polypeptide; (iii) a hinge amino acid sequence devoid of a hinge core comprising the amino acid motif CXXC (and optionally also devoid of an upper hinge amino acid sequence); or (iv) an upper hinge fused to a lower hinge, but devoid of a hinge core comprising the amino acid motif CXXC; or (v) a lower hinge, but devoid of a hinge core comprising the amino acid motif CXXC (and optionally also devoid of an upper hinge amino acid sequence). Examples of upper, core and lower hinge sequences are disclosed in Table 12. In an example, the CH2 is devoid of a functional hinge region, ie, wherein the hinge region is non-functional to dimerise the polypeptide with another such polypeptide. In an example, the CH2 is devoid of a hinge region. In an example, the CH2 is devoid of a complete hinge region sequence. In an example, the CH2 is devoid of a core hinge region sequence.
In an example, the CH2 comprises (in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and wherein the CH2 (and the polypeptide) is devoid of a core hinge region that is functional to dimerise the polypeptide with another said polypeptide.
In an example, the CH2 comprises in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S).
In an example, the CH2 comprises in N- to C- terminal direction) an amino acid selected from SEQ ID NOs: 163-178 and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S).
In an embodiment, the core hinge region amino acid sequence is selected from SEQ ID Nos: 180-182. In an embodiment, the CH2 (an the polyeptide) is devoid of amino acid sequences SEQ ID NOs: 183-187.
In an embodiment, the CH2 domain is a human IgG1 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CPPC (SEQ ID NO: 180). Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180).
In an embodiment, the CH2 domain is a human IgG2 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ERKCCVE (SEQ ID NO: 184) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180).
In an embodiment, the CH2 domain is a human IgG3 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181).
In an embodiment, the CH2 domain is a human IgG4 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ESKYGPP (SEQ ID NO: 187) and core hinge region amino acid sequence CPSC (SEQ ID NO: 182).
In an example, the CH2 of a polypeptide herein is devoid of a core hinge (and optionally also an upper hinge) amino acid sequence. In an example, the CH2 of a polypeptide herein is devoid of a core hinge CXXC amino acid sequence, wherein X is any amino acid, preferably P, R or S, most preferably P. In an example, the CH2 comprises an APELLGGPSV amino acid sequence, or an PAPELLGGPSV amino acid sequence. In an example, the CH2 comprises an APPVAGPSV amino acid sequence, or an PAPPVAGPSV amino acid sequence. In an example, the CH2 comprises an APEFLGGPSV amino acid sequence, or an PAPEFLGGPSV amino acid sequence.
In an example, the CH2 and CH3 of a polypeptide herein are human IgG1 CH2 and CH3 domains, wherein the CH2 is devoid of a core hinge (and optionally also an upper hinge) amino acid sequence, eg, wherein the CH2 is devoid of a CPPC sequence. In an example, the CH2 comprises an APELLGGPSV amino acid sequence, or an EPKSCDKTHT[P]APELLGGPSV amino acid sequence, wherein the bracketed P is optional.
In an example, the CH2 and CH3 of a polypeptide herein are human IgG2 CH2 and CH3 domains, wherein the CH2 is devoid of a core hinge (and optionally also an upper hinge) amino acid sequence, eg, wherein the CH2 is devoid of a CPPC sequence. In an example, the CH2 comprises an APPVAGPSV amino acid sequence, or an ERKCCVE[P]APPVAGPSV amino acid sequence, wherein the bracketed P is optional.
In an example, the CH2 and CH3 of a polypeptide herein are human IgG3 CH2 and CH3 domains, wherein the CH2 is devoid of a core hinge (and optionally also an upper hinge) amino acid sequence, eg, wherein the CH2 is devoid of a CPRC sequence. In an example, the CH2 comprises an APELLGGPSV amino acid sequence, or an ELKTPLGDTTHT[P]APELLGGPSV amino acid sequence, wherein the bracketed P is optional. In an example, the CH2 comprises an EPKSCDTPPP[P]APELLGGPSV amino acid sequence, wherein the bracketed P is optional.
In an example, the CH2 and CH3 of a polypeptide herein are human IgG4 CH2 and CH3 domains, wherein the CH2 is devoid of a core hinge (and optionally also an upper hinge) amino acid sequence, eg, wherein the CH2 is devoid of a CPSC sequence. In an example, the CH2 comprises an APEFLGGPSV amino acid sequence, or an ESKYGPP[P]APEFLGGPSV amino acid sequence, wherein the bracketed P is optional.
When the polypeptide comprises a V-CH1, a CH2 may also be present, but in this case optionally lacking the core hinge region (or at least a sequence selected from CXXC as disclosed herein and SEQ ID Nos: 180-182) and optionally lacking the upper and/or the lower hinge region to prevent F(ab′)2 formation.
By way of example the invention provides the following Aspects, some of which have been exemplified herein. The following Aspects are not to be interpreted as Claims. The Claims start after the Examples section.
1. A polypeptide comprising (in N- to C-terminal direction; or in C- to N-terminal direction)
In an example, each linker is a peptide linker comprising (or comprising up to, or consisting of) 40, 30, 25, 20, 19, 18, 17, 16, 15 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or amino acids.
In an alternative, the domain of (a) is a non-Ig domain or comprises a non-Ig scaffold.
In an alternative herein, instead of using a TD or copies of a TD, in an embodiment any other self-associating multimerization domain (SAM) may be used. In an example, the SAM (eg, TD) is a human, dog, cat, horse, monkey (eg, cynomolgus monkey), rodent (eg, mouse or rat), rabbit, bird (eg, chicken) or fish SAM (or TD).
Optionally, the domain of (a) is capable of specifically binding to an antigen selected from PD-L1, PD-1, 4-1BB, CTLA-4, 4-1BB, CD28, TNF alpha, IL17 (eg, IL17A), CD38, VEGF-A, EGFR, IL-6, IL-4, IL-6R, IL-4R (eg, IL-4Ra), OX40, OX40L, TIM-3, CD20, GITR, VISTA, ICOS, Death Receptor 5 (DR5), LAG-3, CD40, CD40L, CD27, HVEM, KRAS, haemagglutinin, transferrin receptor 1, amyloid beta, BACE1, Tau, TDP43, SOD1, Alpha Synculein and CD3.
In an example, the antigen is a peptide-MHC.
In some embodiments (eg, some embodiments of Aspect 16 below or 17 below), the polypeptide comprises at least two binding moieties, eg, two dAbs, two scFvs, or a dAb and a scFv. In an example, these binding moieties bind to the same antigen (eg, an antigen disclosed herein or in the immediately preceding paragraph herein). In another example, the moieties bind to different antigens (eg, an antigen disclosed herein or in the immediately preceding paragraph herein). For example, in Aspect 16B, D, E or F the variable domains or scFvs are capable of specifically binding to the same or different antigens selected from TNF alpha, CD38, IL17a, CD20, PD-1, PD-L1, CTLA-4 and 4-1BB. For example, one of the moieties binds to TNF alpha and the other binds to IL17a; one of the moieties binds to PD-1 and the other binds to 4-1BB; or one of the moieties binds to PD-L1 and the other binds to 4-1BB; one of the moieties binds to PD-1 and the other binds to CTLA-4; or one of the moieties binds to PD-L1 and the other binds to CTLA-4.
2. The polypeptide of any preceding Aspect, wherein the domain of (a) is an antibody variable domain.
For example, a variable domain herein is a VH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a VHH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a humanised VH, humanised VHH or a human VH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a VL (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a Vκ. In another example, it is a Vλ.
In another example, the domain of (a) is a TCR variable domain (eg, a TCRα, TCRβ, TCRγ or TCRδ).
In an example the immunoglobulin superfamily domain is an antibody single variable domain (dAb).
3. The polypeptide of any preceding Aspect, wherein the domain of (a) is selected from an antibody single variable domain, a VH and a VL; or wherein the domain is comprised by an scFv.
In an example, a single variable domain herein is a human or humanised dAb or nanobody; or is a camelid VHH domain.
In an example, the domain of (a) is comprised by a single-chain TCR (scTCR).
In an example, the single variable domain is a human or humanised dAb or nanobody; or is a camelid VHH domain.
Optionally, the TD herein is a TD of a protein disclosed in Table 2.
Optionally, each variable domain is a VH or a VL (eg, a Vκ or a Vλ).
Optionally, each domain of the polypeptide herein is a human domain. Optionally, each domain of the polypeptide herein is a human or humanised domain.
17. The polypeptide of any of Aspects 1 to 15, wherein
Thus, in some embodiments the polypeptide comprises an antibody Fc region, wherein the Fc comprises the CH2 and CH3 domains.
This further variable domain may be different from the first single variable domain or may have a target binding specificity that is different from the target binding specificity of the first single variable domain or scFv.
In an example, the CH2 is a CH2′ disclosed herein. Optionally, the CH2 comprises (in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and wherein the CH2 (and the polypeptide) is devoid of a core hinge region that is functional to dimerise the polypeptide with another said polypeptide. Optionally, the CH2 comprises in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S). Optionally, the CH2 comprises in N- to C- terminal direction) an amino acid selected from SEQ ID NOs: 163-178 and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S). In an embodiment, the core hinge region amino acid sequence is selected from SEQ ID Nos: 180-182. In an embodiment, the CH2 (an the polyeptide) is devoid of amino acid sequences SEQ ID NOs: 183-187. In an embodiment, the CH2 domain is a human IgG1 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CPPC (SEQ ID NO: 180). Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180). In an embodiment, the CH2 domain is a human IgG2 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ERKCCVE (SEQ ID NO: 184) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180). In an embodiment, the CH2 domain is a human IgG3 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181). In an embodiment, the CH2 domain is a human IgG4 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ESKYGPP (SEQ ID NO: 187) and core hinge region amino acid sequence CPSC (SEQ ID NO: 182).
26. The polypeptide of any preceding Aspect, wherein the first or each linker is a (G4S)n linker, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In an example, n is 3. In an example, n is 4. In an example, n is 5.
In an example, the binding antagonises the antigen. In another example, the binding agonises the antigen.
29. The polypeptide of any preceding Aspect, wherein the polypeptide comprises binding specificity for more than one antigen, optionally 2, 3 or 4 different antigens.
For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (eg, dAb or scFv) and at least one anti-4-1BB binding domain. For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (eg, dAb or scFv) and at least one anti-PD-L1 binding domain. For example, the polypeptide comprises at least one anti-CTLA-4 binding domain (eg, dAb or scFv) and at least one anti-PD-1 binding domain. For example, the polypeptide comprises at least one anti-TNF alpha binding domain (eg, dAb or scFv) and at least one anti-IL-17A binding domain.
For example, the polypeptide comprises a first antigen binding domain (eg, a said VH, VL, VHH, dAb, scFv or Fab variable region) that is N-terminal of the SAM and a second antigen binding domain (eg, a said VH, VL, VHH, dAb, scFv or Fab variable region) that is C-terminal of the SAM. In an example, the polypeptide comprises a third antigen binding domain (eg, a said VH, VL, VHH, dAb, scFv or Fab variable region) that is N-terminal of the SAM (eg, and also N-terminal of the first domain; or between the first domain and the SAM); and optionally the polypeptide a fourth antigen binding domain (eg, a said VH, VL, VHH, dAb, scFv or Fab variable region) that is C-terminal of the SAM (eg, and also C-terminal of the second domain; or between the second domain and the SAM).
In an embodiment, the first domain is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3) and the second binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an example, the domains have the same antigen binding specificity. In an example, the domains have the same epitope binding specificity. In an example, the domains have different antigen binding specificity. In an example, the domains have different epitope binding specificity on the same antigen. In an example, the domains bind TNF alpha. In an example, the domains bind CD20. In an example, the domains bind PD-1. In an example, the domains bind PD-L1. In an example, the domains bind CTLA-4.
In an embodiment, the first domain is capable of specifically binding to 4-1BB, PD-1 or PD-L1 and the second binding site is capable of specifically binding to CTLA-4. In an embodiment, the second domain is capable of specifically binding to 4-1BB, PD-1 or PD-L1 and the first binding site is capable of specifically binding to CTLA-4. In an example, the first domain is capable of specifically binding to 4-1BB, the second binding site is capable of specifically binding to CTLA-4, the third binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3) and the fourth binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an example, the first domain is capable of specifically binding to PD-1, the second binding site is capable of specifically binding to CTLA-4, the third binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3) and the fourth binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an example, the first domain is capable of specifically binding to PD-L1, the second binding site is capable of specifically binding to CTLA-4, the third binding site is capable of specifically binding to CTLA-4, PD-L1, CD3 or CD28 and the fourth binding site is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3).
In an embodiment, the first domain is capable of specifically binding to TNF alpha and the second binding site is capable of specifically binding to IL-17 (eg, IL-17A). In an embodiment, the second domain is capable of specifically binding to TNF alpha and the first binding site is capable of specifically binding to IL-17 (eg, IL-17A).
In an example, the polypeptide comprises a cytokine, eg, an IL-2, IL-15 or IL-21. In an example, the cytokine is a truncated cytokine, eg, a truncated IL-2, IL-15 or IL-21. In an example, the cytokine is C-terminal of the SAM (eg, C-terminal of the C-terminal most antigen binding domain). In an example, the cytokine is N-terminal of the SAM (eg, N-terminal of the N-terminal most antigen binding domain). In an embodiment of these examples, the first domain is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an embodiment of these examples, the first domain is capable of specifically binding to 4-1BB, PD-1, PD-L1 or CTLA-4. In an embodiment of these examples, the second domain is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an embodiment of these examples, the second domain is capable of specifically binding to 4-1BB, PD-1, PD-L1 or CTLA-4. In an embodiment of these examples, the third domain is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an embodiment of these examples, the third domain is capable of specifically binding to 4-1BB, PD-1, PD-L1 or CTLA-4. In an embodiment of these examples, the fourth domain is capable of specifically binding to an immune checkpoint or T-cell co-stimulatory antigen (eg, selected from OX40, GITR, VISTA, CD40, CD28, LAG3 and TIM-3). In an embodiment of these examples, the fourth domain is capable of specifically binding to 4-1BB, PD-1, PD-L1 or CTLA-4.
30. A multimer (optionally a tetramer) of a polypeptide according to any preceding Aspect.
In an example, the multimer is a polypeptide dimer.
In an example, the multimer is a polypeptide trimer.
In an example, the multimer is a polypeptide tetramer.
In an example the bodily fluid is a blood, saliva, semen or urine sample.
In an example, the method is for pregnancy testing or diagnosing a disease or condition in a subject from which the sample has been previously obtained.
In another embodiment, the binding agonises the antigen.
Optionally, at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% of the polypeptides are comprised by tetramers of said polypeptides.
The invention also provides polypeptides and multimers comprising antibody Fc region(s). This is useful, for example, to harness FcRn recycling when administered to a subject, such as a human or animal, which may contribute to a desirable half-life in vivo. Fc regions are also useful for providing Fc effector functions. For example, an IgG1 Fc may be useful when the multimer is used to treat a cancer or where cell killing is desired, eg, by ADCC.
To this end, the invention provides the following:
A polypeptide comprising an antibody Fc region, wherein the Fc region comprises an antibody CH2 and an antibody CH3; and a self-associating multimerisation domain (SAM); wherein the CH2 comprises an antibody hinge sequence and is devoid of a core hinge region.
In an embodiment, the polypeptide comprises an epitope binding site, eg, an antibody VH single variable domain or an antibody VH/VL pair that binds to an epitope. Additionally or alternatively, the polypeptide comprises an epitope which is cognate to an antibody. This is useful, for example, as the polypeptide can form a multimer that binds copies of the antibodies, such as when the multimer is contacted with a sample comprising the antibodies (eg, for medical use as disclosed herein). In this way, for example, the multimer can be used in a method of diagnosis or testing to determine the presence and/or quantity (or relative amount) of the antibody in the sample. As the multimer provides multiple copies of the epitope (at least one for each polypeptide comprised by the multimer), this can be useful to bind many copies of the antibody, which may be present in relatively small amounts in the sample, thereby having the effect of enhancing the chances of detecting (or amplifying) a positive signal denoting presence of the antibody. Thus, assay sensitivity may be enhanced so that relatively rare antibodies in samples can be detected.
Optionally, the CH2 is devoid of (i) a core hinge CXXC amino acid sequence, wherein X is any amino acid or wherein each amino acid X is selected from a P, R and S; and/or (ii) an upper hinge amino acid sequence. For the CH2 is devoid of (i) a core hinge CXXC amino acid sequence, wherein X is any amino acid or wherein each amino acid X is selected from a P, R and S and the Fc does not directly pair with another Fc.
Optionally, the CXXC sequence is selected from SEQ ID NOs: 180-182; or the CH2 is devoid of amino acid sequences SEQ ID NOs: 183-187.
Optionally, the CH2 comprises
Optionally, the CH2 comprises (in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and wherein the CH2 (and the polypeptide) is devoid of a core hinge region that is functional to dimerise the polypeptide with another said polypeptide. Optionally, the CH2 comprises in N- to C- terminal direction) an optional upper hinge region, a lower hinge region and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S). Optionally, the CH2 comprises in N- to C- terminal direction) an amino acid selected from SEQ ID NOs: 163-178 and a CH2 domain and the wherein the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CXXC, wherein X is any amino acid (optionally wherein each amino acid X is selected from a P, R and S). In an embodiment, the core hinge region amino acid sequence is selected from SEQ ID Nos: 180-182. In an embodiment, the CH2 (an the polyeptide) is devoid of amino acid sequences SEQ ID NOs: 183-187. In an embodiment, the CH2 domain is a human IgG1 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG1 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of a core hinge region amino acid sequence CPPC (SEQ ID NO: 180). Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDKTHT (SEQ ID NO: 183) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180). In an embodiment, the CH2 domain is a human IgG2 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 180. Optionally, any CH1 and CH3 present in the polypeptide are human IgG2 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ERKCCVE (SEQ ID NO: 184) and core hinge region amino acid sequence CPPC (SEQ ID NO: 180). In an embodiment, the CH2 domain is a human IgG3 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 181. Optionally, any CH1 and CH3 present in the polypeptide are human IgG3 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 185) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181). Optionally, alternatively the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence EPKSCDTPPP (SEQ ID NO: 186) and core hinge region amino acid sequence CPRC (SEQ ID NO: 181). In an embodiment, the CH2 domain is a human IgG4 CH2 domain and the core hinge region amino acid sequence is SEQ ID NO: 182. Optionally, any CH1 and CH3 present in the polypeptide are human IgG4 CH1 and CH3 respectively. Optionally, the CH2 (and the polypeptide) is devoid of upper hinge region amino acid sequence ESKYGPP (SEQ ID NO: 187) and core hinge region amino acid sequence CPSC (SEQ ID NO: 182).
Optionally, the polypeptide comprises an antibody CH1-hinge sequence devoid of core region-CH2-CH3.
Optionally, the CH2 and CH3 comprise
Optionally, the CH2 and CH3 comprise
Optionally,
Optionally, the polypeptide comprises (in N- to C-terminal direction) the Fc region and the SAM, the Fc region comprising (in N- to C-terminal direction) the hinge sequence, a CH2 domain and a CH3 domain.
Optionally, the polypeptide comprises one or more epitope binding sites, eg, an antibody variable domain that is capable of specifically binding to a first epitope. Optionally, the first epitope is comprised by an antigen (eg, a human antigen) selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AWI; AIG1; AKAP1; AKAP2; AIYIH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BM Pl; BMP2; BMP3B (GDFIO); BMP4; BMP6; BM P8; BMPRIA; BMPRIB; BM PR2; BPAG1 (plectin); BRCA1; CI9orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (M IP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (M PIF-1); CCL24 (MPIF-2 I eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK /ILC) ; CCL28; CCL3 (MIP-la); CCL4 (M IP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA);CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CM KBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CM KBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p2IWapl/Cipl); CDKN1B (p27Kipl); CDKNIC; CDKN2A (pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDi) ; CX3CR1 (V28); CXCL1 (GROl); CXCLIO (IP-10); CXCL11 (1-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; EN01; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILl (EPSILON); FILl (ZETA); FU12584; FU25530; FLRTl (fibronectin); FLTl; FOS; FOSLl (FRA-I); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRHl; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPl; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13; IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R; 1L18; IL18BP; IL18R1; IL18RAP; 1L19; ILIA; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1;IL1RL2 IL1RN; 1L2; 1L20; IL20RA; IL21R; 1L22; 1L22R; 1L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG; 1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6R; IL6ST (glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAKI; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; M IB1; midkine; M IF; M IP-2; MK167 (Ki-67); MMP2; M MP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); MTSS 1; M UC 1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB 1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NM E1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2IRac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPl; SPRRIB (Spri); ST6GAL1; STABl; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-i); T]MP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF1 1A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSF1 1 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF1 5 (VEGI); TNFSF1 8; TNFSF4 (0X40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53; TPM 1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM 1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2. Optionally, the second epitope (as discussed below) is comprised by the same antigen as the first epitope (eg, comprised by the same antigen molecule). In another example, the second antigen is comprised by said group. In an example, the first and second epitopes are comprised by different antigens selected from said group.
For example, the polypeptide has 1, 2, 3, 4 or 5 epitope binding sites (optionally wherein the polypeptide comprises 2 or more binding sites (eg, single variable domains) that bind to different epitopes, or wherein the polypeptide binding sites are identical). In an embodiment, the SAM is a TD (eg, a p53 TD, such as a human p53 TD) and the polypeptide has 2, 3 or 4 binding sites, such as 3 sites or such as 4 sites. Preferably, the polypeptide has 3 binding sites. Preferably, the polypeptide has 4 binding sites. For example, the binding sites each binds TNF alpha (eg, wherein the binding sites are identical, eg, identical antibody single variable domains).
In an example, the multimer is an octavalent bispecific multimer comprising 4 copies of an anti-PD-L1binding site (eg, dAb) and 4 copies of an anti-4-1BB binding site (eg, dAb).
In an example, the multimer is a tetravalent multimer comprising copies of an anti-PD-L1 binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-PD-L1 binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-PD-L1 binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-PD-L1 binding site (eg, dAb). In an example, the anti-PD-L1 binding site comprises an avelumab or atezolizumab binding site that specifically binds to PD-L1. Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-PD-1 binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-PD-1 binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-PD-1 binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-PD-1 binding site (eg, dAb). In an example, the anti- PD-1 binding site comprises a nivolumab or pembrolizumab binding site that specifically binds to PD-1. Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-DR5 (Death Receptor 5) binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-DR5 binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-DR5 binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-DR5 binding site (eg, dAb). Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-OX40 or OX40L binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-OX40 or OX40L binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti- OX40 or OX40L binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti- OX40 or OX40L binding site (eg, dAb). Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-glucocorticoid-induced tumor necrosis factor receptor (GITR) binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-GITR binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-GITR binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-GITR binding site (eg, dAb). Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-antibody kappa light chain (KLC) binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-KLC binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-KLC binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-KLC binding site (eg, dAb). Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-VEGF binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-VEGF binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-VEGF binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-VEGF binding site (eg, dAb). In an example, the anti-VEGF binding site comprises a VEGF receptor domain that specifically binds to VEGF (eg, a VEGF binding site of human flt (eg, flt-1) or KDR, eg, Ig domain 2 from VEGFR1 or Ig domain 3 from VEGFR2)). In an example, the anti-VEGF binding site comprises an aflibercept, bevacizumab or ranibizumab binding site that specifically binds to VEGF. Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
In an example, the multimer is a tetravalent multimer comprising copies of an anti-TNF alpha binding site (eg, dAb). In an example, the multimer is a tetravalent multimer comprising copies of an anti-TNF alpha binding site (eg, dAb). In an example, the multimer is an octavalent multimer comprising copies of an anti-TNF alpha binding site (eg, dAb). In an example, the multimer is a 12-valent multimer comprising copies of an anti-TNF alpha binding site (eg, dAb). In an example, the multimer is a 16-valent multimer comprising copies of an anti-TNF alpha binding site (eg, dAb). Preferably, in these examples the SAM domain is a TD, eg, a p53 TD, such as a human p53 TD. For the tetramer, each polypeptide comprises one copy of the binding site. For the octamer each polypeptide comprises 2 copies of the binding site. For the 12-mer each polypeptide comprises 3 copies of the binding site. For the 16-mer each polypeptide comprises 4 copies of the binding site.
Optionally, the variable domain is selected from an antibody single variable domain, a VH and a VL; or wherein the domain is comprised by an scFv. Optionally, the domain is comprised by an antibody VH/VL pair that binds to said first epitope. In an example, epitope binding herein is specific binding as herein defined.
Optionally, the polypeptide comprises (in N- to C-terminal direction)
Optionally, the polypepide comprises a second antibody variable domain N- or C-terminal to the SAM, wherein the second variable domain is capable of specifically binding to a second epitope, wherein the first and second epitopes are identical or different.
Optionally, the SAM is a self-associating tetramerisation domain (TD); optionally wherein the TD is a p53, p63 or p73 TD or a homologue or orthologue thereof; or wherein the TD is a NHR2 TD or a homologue or orthologue thereof; or wherein the TD comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 10 or 126.
Optionally,
Optionally, any single variable domain or dAb herein is a Nanobody™ or a Camelid VHH (eg, a humanised Camelid VHH). For example, a variable domain, such as a dAb (AKA antibody single variable domain) herein is a VH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a VHH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a humanised VH, humanised VHH or a human VH (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a VL (eg, comprised by a scFv or a Fab polypeptide chain). In another example, it is a Vκ. In another example, it is a Vλ.
In an embodiment, each polypeptide of the multimer is paired with a copy of a further polypeptide, wherein the further polypeptide comprises an antibody light chain constant region (eg, a Cκ or a Cλ) that pairs with the Fc of the first polypeptide. In an example, the first polypeptide comprises an antibody VH domain, the further polypeptide comprises an antibody VL domain (eg, a Vκ or a Vλ), wherein the VH and VL form an epitope binding site. In this way, the multimer may be a multimer of Fab-like structures, such as comprising multiple copies of an adalimumab (Humira) or avelumab (Bavencio) binding site as exemplified in the Examples below. All of the antibody domains in such a multimer may, for example, be human, and optionally the SAM is a human domain. When the SAM is a TD (eg, a p53 TD), the multimer comprises a tetramer of the VH/VL epitope binding sites. The first polypeptide or the further polypetide may comprise a second epitope binding site, for example, wherein the multimer is octavalent. When the 8 binding sites may bind the same antigen; alternatively the 4 VH/VL binding sites bind a first antigen and the other 4 binding sites bind to a second antigen, wherein the antigens are different. Thus, the multimer may be octavalent and bispecific. If the first or further polypetide comprises yet another antigen binding site, the multimer may be 12-valent (and, eg, monospecific, bispecific or trispecific for antigen binding). If the first or further polypetide comprises yet another antigen binding site, the multimer may be 16-valent (and, eg, monospecific, bispecific, trispecific or tetraspecific for antigen binding).
The invention further provides:-
The invention provides the following Paragraphs. The following Paragraphs are not to be interpreted as Claims. The Claims start after the Examples section.
In certain embodiments, the invention is useful for providing multimers for treating cancer in humans or animals. In this respect, it may be useful to use the multimers to target tumours by binding to tumour-associated antigen and/or to bind to T-cells to modulate their activity. For example the multimers may bind to an antigen on T regulatory cells (Tregs) to downregulate their activity. Additionally or alternatively, the multimers may bind to T effector (Teff) cells to upregulate their activity. The provision of an antibody Fc region in the polypeptides of multimers may be advantageous for providing Fc effector functions and/or cytotoxicity for killing tumour cells. In one advantageous configuration, the invention exploits the ability to provide multiple identical antigen or epitope binding sites that can be used to bind to several copies of the same antigen or epitope on tumour cells, thereby providing for an avidity affect wherein the multimers bind preferentially to tumour cells over any non-target or normal cells, since the former surface-express more copies of the antigen than normal cells. In one configuration, when the multimer also comprises binding sites for an immune checkpoint regulator. In one example the regulator is an immune checkpoint inhibitor and the binding sites antagonise the inhibitor. This is useful, for example when the inhibitor is expressed by Teff cells, for upregulating Teff activity in the vicinity of tumour cells that are targeted by the multimer (eg, by binding TAA on the tumour cells). In another example the regulator is an immune checkpoint stimulator and the binding sites agonise the inhibitor. This is useful, for example when the inhibitor is expressed by Teff cells, for upregulating Teff activity in the vicinity of tumour cells that are targeted by the multimer (eg, by binding TAA on the tumour cells). Thus, upregulation of T-cell activity may be stimulated in the vicinity of tumour cells, rather in the vicinity of non-target (eg, normal or non-cancerous) cells. To this end, the invention provides the following Concepts. The following Concepts are not to be interpreted as Claims. The Claims start after the Examples section.
In an example, said first dAb or first scFv of the polypeptide herein is the first antigen binding site of these Concepts; and optionally when a further dAb or scFv binding site is present this is the second antigen binding site of the Concepts.
In an example, said first dAb or first scFv of the polypeptide herein is the second antigen binding site of these Concepts; and optionally when a further dAb or scFv binding site is present this is the first antigen binding site of the Concepts.
In a configuration, the invention provides the following Clauses. The following Clauses are not to be interpreted as Claims. The Claims start after the Examples section.
In any disclosure herein, the or each constant region or domain, the CH2, the CH3, the CH2 and CH3 or the Fc is respectively a constant region or domain, the CH2, the CH3, the CH2 and CH3 or the Fc of a human constant region. For example, the constant region is selected from the group IGHA1*01, IGHA1*02, IGHA1*03, IGHA2*01, IGHA2*02, IGHA2*03, IGHD*01, IGHD*02, IGHE*01, IGHE*02, IGHE*03, IGHE*04, IGHEP1*01, IGHEP1*02, IGHEP1*03, IGHEP1*04, IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, IGHG1*05, IGHG1*06, IGHG1*07, IGHG1*08, IGHG1*09, IGHG1*10, IGHG1*11, IGHG1*12, IGHG1*13, IGHG1*14, IGHG2*01, IGHG2*02, IGHG2*03, IGHG2*04, IGHG2*05, IGHG2*06, IGHG2*07, IGHG2*08, IGHG2*09, IGHG2* 10, IGHG2* 11, IGHG2* 12, IGHG2* 13, IGHG2* 14, IGHG2* 15, IGHG2* 16, IGHG2* 17, IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3* 10, IGHG3* 11, IGHG3* 12, IGHG3* 13, IGHG3* 14, IGHG3* 15, IGHG3* 16, IGHG3 * 17, IGHG3* 18, IGHG3* 19, IGHG3*20, IGHG3*21, IGHG3*22, IGHG3*23, IGHG3*24, IGHG3*25, IGHG3*26, IGHG3*27, IGHG3*28, IGHG3*29, IGHG4*01, IGHG4*02, IGHG4*03, IGHG4*04, IGHG4*05, IGHG4*06, IGHG4*07, IGHG4*08, IGHGP*01, IGHGP*02, IGHGP*03, IGHM*01, IGHM*02, IGHM*03 and IGHM*04 (eg, the constant region is a *01 allele listed in said group, preferably the constant region is a human IGHG1*01 or IGHM*01 constant region). In an alternative, the constant region is a non-human (eg, mammal, rodent, mouse, rat, dog, cat or horse) constant region, such as a homologue of a human constant region listed in said group.
The polypeptide, in one embodiment, comprises (in N- to C-terminal direction) a first antigen binding site (eg, a dAb), an antibody CH1 (eg, human IgGl CH1), a hinge sequence comprising a lower hinge and devoid of a core hinge region (and optionally devoid of an upper hinge region), an antibody Fc region and a SAM (eg, a TD, such as a p53 TD). For example, the core hinge region sequence is a CXXC amino acid sequence. The polypeptide may comprise another antigen binding site (eg a dAb or scFv) between the first binding site and the CH1, between the Fc and SAM and/or C-terminal to the SAM. Optionally, the multimer comprises a plurality (eg, 4 copies) of such polypeptide, for example wherein each polypeptide is paired with a further polypeptide comprising (in N- to C-terminal direction) a second antigen binding site (eg, a dAb), an antibody CL (eg, a human Cκ) and optionally a third antigen binding site. Optionally the binding sites have the same antigen specificity (eg, all bind TNF alpha). In another option, the first and second (and optionally said another binding site) bind to different antigens. The or each binding site can bind any antigen disclosed herein, eg, each binding site binds TNF alpha (as shown in Example 17). In another example, the first antigen binding site is a VH of an antigen binding site of a predetermined antibody that specifically binds to the antigen (and the CH1 is optionally the CH1 of the antibody), and the second binding site of the further polypeptide is a VL of the antigen binding site of the predetermined antibody (and the CL is optionally the CL of the antibody), wherein the VH and VL pair to form a VH/VL binding site which has binding specificity for the antigen. The predetermined antibody may be a marketed antibody, for example, as shown in Example 19. For example, the VH/VL binding site specifically binds to CTLA-4, eg, wherein the predetermined antibody is ipilimumab (or Yervoy™). For example, the VH/VL binding site specifically binds to TNF alpha, eg, wherein the predetermined antibody is adalimumab, golimumab, infliximab (or Humira™, Simponi™ or Remicade™). For example, the VH/VL binding site specifically binds to PD-L1, eg, wherein the predetermined antibody is avelumab (or Bavencio™) or atezolizumab (or Tecentriq™). For example, the VH/VL binding site specifically binds to PD-1, eg, wherein the predetermined antibody is nivolumab (or Opdivo™) or pembrolizumab (or Keytruda™). For example, the VH/VL binding site specifically binds to VEGF, eg, wherein the predetermined antibody is bevacizumab (or Avastin™) or ranibizumab (or Lucentis™). In another example, the polypeptide comprises (in N- to C-terminal direction) a first VEGF binding site, an optional second VEGF binding site, an antibody CH1 (eg, human IgGl CH1), a hinge sequence comprising a lower hinge and devoid of a core hinge region (and optionally devoid of an upper hinge region), an antibody Fc region and a SAM (eg, a TD, such as a p53 TD). In an example, the first binding site is a Ig domain 2 from VEGFR1 and the second binding site is Ig domain 3 from VEGFR2 (as shown in Example 20). In another example, the first binding site is a Ig domain 3 from VEGFR2 and the second binding site is Ig domain 2 from VEGFR2. In an example, the first and second binding domains are (in N- to C-terminal direction) the first and second VEGF binding sites of aflibercept (or Eylea™).
Suitable predetermined antibodies are ReoPro™; Abciximab; Rituxanh™; Rituximab; Zenapaxh™; Daclizumab; Simulecth™; Basiliximab; Synagis™; Palivizumab; Remicadeh™; Infliximab; Herceptinh™; Trastuzumab; Mylotargh™; Gemtuzumab; Campathh™; Alemtuzumab; Zevalinh™; Ibritumomab; Humirah™; Adalimumab; Xolair™; Omalizumab; Bexxarh™; Tositumomab; Raptivah™; Efalizumab; Erbituxh™; Cetuximab; Avastinh™; Bevacizumab; Tysabrih™; Natalizumab; Actemrah™; Tocilizumab; Vectibixh™; Panitumumab; Lucentish™; Ranibizumab; Solirish™; Eculizumab; Cimziah™; Certolizumab; Simponih™; Golimumab, Ilaris™; Canakinumab; Stelara™; Ustekinumab; Arzerrah™; Ofatumumab; Prolie™; Denosumab; Numaxh™; Motavizumab; ABThraxh™; Raxibacumab; Benlystah™; Belimumab; Yervoyh™; Ipilimumab; Adcetrish™; Brentuximab; Vedotin™; Perjeta™; Pertuzumab; Kadcyla™; Ado-trastuzumab; Gazyva™ and Obinutuzumab. Also disclosed are the generic versions of these and the corresponding INN names - each of which is a suitable predetermined antibody for use as a source of antigen binding sites for use in the present invention. Suitable sequences of VH and VL domains of predetermined antibodies are disclosed in Table 4. Thus, for example, the multimer of the invention comprises a plurality (eg, 4, 8, 12, 16 or 20) copies of the VH/VL antigen binding site of any of these antibodies, eg, wherein the VH of the binding site is comprised by a polypeptide of the invention that comprises a SAM (eg, a TD) and each polypeptide is paired with a further polypeptide comprising the VL that pairs with the VH, thus forming an antigen binding site. In an example, the polypeptide comprising the SAM also comprises a CH1 which pairs with a CL of the further polypeptide. Optionally, the binding site of the polypeptide of the multimer comprises a VH of the binding site of the antibody and also the CH1 of the antibody (ie, in N- to C-terminal direction the VH-CH1 and SAM). In an embodiment, the polypeptide may be paired with a further polypeptide comprising (in N- to C-terminal direction a VL-CL, eg, wherein the CL is the CL of the antibody).
In one embodiment, the predetermined antibody is Avastin.
In one embodiment, the predetermined antibody is Actemra.
In one embodiment, the predetermined antibody is Erbitux.
In one embodiment, the predetermined antibody is Lucentis.
In one embodiment, the predetermined antibody is sarilumab.
In one embodiment, the predetermined antibody is dupilumab.
In one embodiment, the predetermined antibody is alirocumab.
In one embodiment, the predetermined antibody is evolocumab.
In one embodiment, the predetermined antibody is pembrolizumab.
In one embodiment, the predetermined antibody is nivolumab.
In one embodiment, the predetermined antibody is ipilimumab.
In one embodiment, the predetermined antibody is remicade.
In one embodiment, the predetermined antibody is golimumab.
In one embodiment, the predetermined antibody is ofatumumab.
In one embodiment, the predetermined antibody is Benlysta.
In one embodiment, the predetermined antibody is Campath.
In one embodiment, the predetermined antibody is rituximab.
In one embodiment, the predetermined antibody is Herceptin.
In one embodiment, the predetermined antibody is durvalumab.
In one embodiment, the predetermined antibody is daratumumab.
In another embodiment, the polypeptide comprises (in N- to C-terminal direction) a first antigen binding site, an optional linker (eg, a G4Sn, wherein n=1, 2, 3, 4, 5, 6, 7, ot 8, preferably 3), a second antigen binding site, a hinge sequence comprising a lower hinge and devoid of a core hinge region (and optionally devoid of an upper hinge region), an antibody Fc (eg, an IgGl Fc) and a SAM (eg, a TD, such as a p53 TD). For example, the core hinge region sequence is a CXXC amino acid sequence. The polypeptide may comprise another antigen binding site (eg a dAb or scFv) between the Fc and SAM and/or C-terminal to the SAM. Optionally, the multimer comprises a plurality (eg, 4 copies) of such polypeptide. Optionally the binding sites have the same antigen specificity (eg, all bind TNF alpha). In another option, the first and second (and optionally said another binding site) bind to different antigens. The or each binding site can bind any antigen disclosed herein, eg, each binding site binds PD-L1, or the first binding site binds PD-L1 and the second binding site binds 41-BB, or the first binding site binds 4-1BB and the second binding site binds PD-L1 (as shown in Example 18).
The polypeptide, in one embodiment, comprises (in N- to C-terminal direction) a first antigen binding site (eg, a dAb), an optional linker (eg, a G4Sn, wherein n=1, 2, 3, 4, 5, 6, 7, ot 8, preferably 3), a second antigen binding site (eg, a dAb), an antibody CH1 (eg, human IgGl CH1) and a SAM (eg, a TD, such as a p53 TD). The polypeptide may comprise another antigen binding site (eg a dAb or scFv) C-terminal to the SAM. Optionally, the multimer comprises a plurality (eg, 4 copies) of such polypeptide, for example wherein each polypeptide is paired with a further polypeptide comprising (in N- to C-terminal direction) a third antigen binding site (eg, a dAb), an optionaly fourth antigen binding site (eg, a dAb), an antibody CL (eg, a human Cκ or Cλ) and optionally a furhter antigen binding site. For example, the fourth and further binding sites are omitted. In another example, the third and fourth binding sites, but not the further binding site, are present. In another example, the third and further (but not the fourth) binding sites are present. Optionally the binding sites have the same antigen specificity (eg, all bind TNF alpha). In another option, the first and second (and optionally said another said binding site) bind to different antigens. The or each binding site can bind any antigen disclosed herein, eg, each binding site binds TNF alpha (as shown in Examples 21 and 22). In an example, the first and third, or the second and third binding sites pair to form a VH/VL pair that is identical to the VH/VL binding site of an anti-TNF alpha antibody, such as adalimumab, golimumab, infliximab (or Humira™, Simponi™ or Remicade™). In an example, the first and third, or the second and third binding sites pair to form a VH/VL pair that is identical to the VH/VL binding site of an anti-PD-L1 antibody, such as avelumab (or Bavencio™) or atezolizumab (or Tecentriq™). In an example, the first and third, or the second and third binding sites pair to form a VH/VL pair that is identical to the VH/VL binding site of an anti-PD-1 antibody, such as nivolumab (or Opdivo™) or pembrolizumab (or Keytruda™). In an example, the first and third, or the second and third binding sites pair to form a VH/VL pair that is identical to the VH/VL binding site of an anti-VEGF antibody, such as bevacizumab (or Avastin™) or ranibizumab (or Lucentis™). Predetermined antibodies as discussed above can be used as the source of the VH/VL pairs.
In an example, the polypeptide of the invention is any Quad polypeptide disclosed herein, eg, comprising the Quad amino acid shown in any of the Tables herein (eg, any one of SEQ ID Nos: 81-115, 151-162, 190, 191, 209-224 and 179) or encoded by any of the Quad nucleotide sequences in any of the Tables herein (eg, Table 9, 14 or 17), or having the structure of a polypeptide shown in Table 8. The SAM may be any SAM disclosed herein, eg, any p53 or homologue TD disclosed in any Table herein (eg, as shown in Table 7 or comprised by a protein in Table 13).
Where amino acid sequences are shown with plural histidines at their C-terminus (eg, “HHHHHH” optionally followed by “..AAA”), such histidines and the optional ..AAA are in one embodiment omitted and the corresponding nucleotides encoding this are omitted from the nucleic acid encoding the amino acid sequence. Where amino acid sequences are shown with a DYKDDDDK motif (eg, a DYKDDDDKHHHHHH or DYKDDDDKHHHHHH..AAA), such a motif is in one embodiment omitted and the corresponding nucleotides encoding this are omitted from the nucleic acid encoding the amino acid sequence.
As discussed herein, the invention provides configurations in which the polypeptide a self-associating multimerisation domain (SAM, eg, a TD) and a peptide, domain or an epitope or antigen binding site (eg, a dAb or an antibody variable domain). In an embodiment, the SAM is a TD, such a p53 TD as disclosed herein.
In an example, the polypeptide comprises (eg, in N- to C-terminal direction) at least an extracellular domain (ECD) of a cell-surface protein that is a receptor for a virus or required for virus activation. For example, the protein poteolytically cleaves and activates a spike glycoprotein of the virus (eg, Coronoavirus or any other virus disclosed herein, such as in Table 19). Optionally, the entire cell-surface portion of the receptor is comprised by the polypeptide of the invention. In an example, the virus is capable of infecting human cells and the receptor is a cell-surface protein found on human cells (such as lung cells). In an example, the virus is capable of infecting non-human animal cells and the receptor is a cell-surface protein found on cells of such animal (such as lung cells). In an example, the virus is capable of infecting plant cells and the receptor is a cell-surface protein found on cells of such plant (such as a crop, wheat, corn, barley, tobacco, grass, fruiting plant or tree). By forming multimers using copies of the polypeptide according to the invention, multimers of the invention comprising copies of all or portions of such cell-surface proteins may act as sinks for binding several virus particles per copy of multimer. Advantageously, this will prevent the bound viruses from infecting cells of the human, animal, plant or other subject or environment in which the cells are present. So, for example, the invention provides a method of treating a viral infection in a human or animal subject, the method comprising administering a composition comprising a plurality of the multimers to a human or animal subject (eg, intravenously or by inhalation), wherein the subject is suffering from a virus infection and copies of the multimer bind to copies of the virus, thereby reducing the severity of the infection and/or reducing progression of the infection and/or reducing one or more symptoms of the infection (such as a inflammatory response). In another example, the composition can be used prophylactically; thus the invention provides a method of preventing or reducing the risk of a viral infection or a symptom thereof in a human or animal subject, the method comprising administering a composition comprising a plurality of the multimers to a human or animal subject (eg, intravenously or by inhalation), wherein the subject is at risk of suffering from a virus infection, thereby preventing or reducing the risk of the viral infection and/or preventing or reducing one or more symptoms of the infection (such as a inflammatory response). In an example, the virus is a Coronavirus.
In an example, the virus is a virus selected from Table 19.
For example, the virus is a Coronavirus, a MERS-Cov, a SARS-Cov, SARS-Cov-1 or preferably SARS-Cov-2. In this example, the receptor may be ACE2. In an alternative, the cell-surface protein is a TMPRSS protein, preferably a TMPRSS2 protein. Optionally, the polypeptide of the invention in this example comprises an ACE2 extracellular domain and a TMPRSS protein extracellular domain, optionally wherein the domains are human domains and the polypeptides (or multimers according to the invention comprising copies of such a polypeptide) are for treating or preventing a Coronavirus infection in a human. In an embodiment in this example, the SAM is a TD, such a p53 TD as disclosed herein.
An example of a protein required for virus activation is TMPRSS2 protein, eg, human TMPRSS2 protein (UniProtKB - 015393 (TMPS2 _HUMAN), the sequence with of which with identifier 015393-1 is explicitly incorporated herein for use in the invention and possible inclusion in one or more claims herein). In an example, the polypeptide of the invention comprises amino acids of human TMPRSS2 protein from amino acid 106 to 492.
In an alternative, the virus is selected from Coronavirus 229E (HCoV-229E), Coronavirus EMC (HCoV-EMC), Sendai virus (SeV), human metapneumovirus (HMPV), human parainfluenza 1, 2, 3, 4a and 4b viruses (HPIV), and influenza A virus (eg, strains H1N1, H3N2 and H7N9).
In an embodiment, the polypeptide comprises an angiotensin converting enzyme 2 (ACE2) protein as disclosed in any of US9,561,263; US 8,586,319; or EP2089715, EP2047867, EP2108695, EP2543724, EP2155871, EP2274005, EP3375872, EP2222330, EP2943216, EP2332582 or any US counterpart patent application or patent of any of these that shares a common priority. The disclosures of such proteins and their sequences as disclosed in these European and US patents and applications are explicitly incorporated herein for possible use in the invention, such as in polypepides or multimers of the invention. For example, the peptide or domain of the polypeptide of the invention comprises an ECD of entire ACE2 protein as disclosed in any one of these European and US patents and applications. Each such protein and sequence is also individually and explicitly incorporated herein such that any one of such proteins or sequences can be included in any claim herein as a component of a polypeptide or multimer of the invention. Also explicitly incorporated herein are the uses and medical diseases and conditions disclosed in any of such European and US patents and applications and the polypeptide or multimer or method or use of the present invention may be for treating, preventing or reducing the risk of any of such diseases or conditions and may be included in any claim herein.
As discussed, a polypeptide of the invention may comprises amino acid sequence from an ACE2 protein. In an embodiment, there is provided a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 229-231. In an embodiment, there is provided a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 229-231 with the exception that the polypeptide comprises an alternative SAM other than the p53 TD disclosed in such sequence. For example, the SAM is a p63, p73 or homologue TD as disclosed herein. In an embodiment, there is provided a polypeptide comprising an ACE2 amino acid sequence as comprised by any one of SEQ ID NOs: 229-231. The invention also provides a tetramer of the invention comprising 4 copies of such a polypeptide, as well as a composition of the invention comprising such a tetramer. Such a multimer or composition may preferably be for use in a method of treating, preventing or reducing the risk of a viral infection (eg, a Coronavirus, or preferably SARS-Cov-2 infection), hypertension or a lung condition (eg, an acute lung injury or inflammation) in a human.
For example, a polypeptide of the invention (eg, for treating or preventing a viral infection, preferably a Coronavirus, a MERS-Cov, a SARS-Cov, SARS-Cov-1 or SARS-Cov-2 infection) comprises the amino acid sequence from amino acid 18 to 615; or from 18 to 656 of SEQ ID NO: 1 disclosed in US9,561,263, which sequences are explicitly incorporated herein by reference for use in the present invention and for possible inclusion in one or more claims herein. Optionally, the polypeptide comprises the amino acid sequence from amino acid 18 to 615; or from 18 to 656; or from 18 to 740 of SEQ ID NO: 1 disclosed in US9,561,263, but does not comprise any other amino acids from such SEQ ID NO: 1. Optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 disclosed in EP2332582, or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% homologous thereto, which sequences are explicitly and individually incorporated herein by reference for use in the invention and possible inclusion in one or more claims herein. Optionally, N-glycosylation sites of Asn53, Asn90, Asn103, Asn322, Asn432, Asn546 and Asn690 of SEQ ID NO: 1 are sialyzed, eg, as disclosed in US8,568,319, which disclosure of sialyzation is explicitly incorporated herein for use in the invention.
In an alternative to treating, preventing or reducing the risk of a viral infection as discussed above, the invention instead provides polypeptides, multimers, methods and uses for treating, preventing or reducing the risk of inflammation in the subject (eg, in a human suffering from lung inflammation or at risk of such).
In an alternative to treating, preventing or reducing the risk of a viral infection as discussed above, the invention instead provides polypeptides, multimers, methods and uses for treating, preventing or reducing the risk of hypertension, heart failure (eg, congestive heart failure or chronic heart failure or acute heart failure), myocardial infarction, atherosclerosis, renal failure or insufficiency, polycystic kidney disease (PKD), or a pulmonary disease. Examples are disclosed in EP2543724, the disclosure of which are explicitly incorporated herein for use in the invention.
In an alternative to treating, preventing or reducing the risk of a viral infection as discussed above, the invention instead provides polypeptides, multimers, methods and uses for treating, preventing or reducing the risk of an acute lung injury (ALI), eg, ARDS (Adult Respiratory Distress Syndrome), SARS (Severe Acute Respiratory Syndrome) or MERS (Middle East Respiratory Syndrome).
Optionally, the polypeptide of the invention comprises eukaryotic cell, mammalian or human cell glycosylation eg, CHO or HEK293 or Cos cell glycosylation.
Optionally, the invention provides a composition (eg, for medical use as described herein, such as for treating, preventing or reducing the risk of a viral infection) comprising a plurality of polypeptides of the invention, wherein less than 15, 10, 5, 4, 3, 2, or 1% of all the polypeptides are comprised by the group consisting of polypeptide monomers, dimers and trimers. Additionally or alternatively, at least 80, 85, 90, 95, 96, 97, 98 or 99% of all of the polypeptides are comprised by multimers comprising 4 copies of the SAM (eg, p53 TD).
In an alternative to treating, preventing or reducing the risk of a viral infection as discussed above, the invention instead provides polypeptides, multimers, methods and uses for treating, preventing or reducing the risk of hypertension in the subject (eg, in a human suffering from a lung or cardiovascular condition or at risk of such).
In a configuration, the polypeptide of the invention comprises one or more binding sites for an antigen comprised by the extracellular part of a cell-surface protein that is a receptor (eg, ACE2 or a homologue or orthologue) for a virus or a protein (eg, TMPRSS2 protein) required for virus activation. In an embodiment, 1, 2, 3, 4 or 5 such binding sites are comprised by the polypeptide or by a multimer of the invention comprising copies of the polypeptide. In an example, each binding site is comprised by a dAb, Fv or scFv. In an example, the multimer comprises a plurality (eg, 4 and no more and no less than 4) copies of a polypepide of the invention comprising a SAM (eg, a TD) and each polypeptide as paired with a respective copy of a further polypeptide, wherein each polypeptide pair comprises a VH/VL antigen binding site. In an example, the binding site specifically binds to a spike glycoprotein of the virus, eg, any virus disclosed herein, preferably a Coronavirus, more preferably SARS-Cov-2. In an example, the binding site binds to 2 or more different Coronaviruses, eg, SARS-Cov-1 and 2. Advantageously, multimers comprising 4 (and no more or less than 4) copies of a heavy chain polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 225-227, wherein each copy is paired with a copy of a polypeptide comprising the amino acid of SEQ ID NO: 228 will bind to a virus, such as a Coronavirus (eg, SAR-Cov-1 and/or SARS-Cov-2) and preferably SAR-Cov-1 and SARS-Cov-2. In an example, the binding site is a VH/VL antigen binding site of a SAR-Cov antibody, such as antibody CR3022, CR3006, CR3013 or CR3014 disclosed in PLoS Med. 2006 Jul;3(7):e237; “Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants”, ter Meulen J et al and J Virol. 2005 Feb; 79(3): 1635-1644; doi: 10.1128/JVI.79.3.1635-1644.2005; PMCID: PMC544131; PMID: 15650189, “Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus”, Edward N. van den Brink et al, the sequences of these antibodies and all anti-virus antibodies in these papers and individually of their VH and VL domains are incorporated herein by reference for use in the invention and possible inclusion in one or more claims herein. In an example, the polypeptide of the invention comprises a first SAR-Cov antigen binding site and a second SAR-Cov antigen binding site wherein the first site comprises a VH/VL binding site of CR3022 and the second site compriss a VH/VL binding site of an antibody selected from CR3006, CR3013 and CR3014 (eg, CR3022/3014; CR3022/3006; CR3022/3013 or CR3022/3014). In an example, the multimer of the invention comprises a first SAR-Cov antigen binding site and a second SAR-Cov antigen binding site wherein the first site comprises a VH/VL binding site of CR3022 and the second site compriss a VH/VL binding site of an antibody selected from CR3006, CR3013 and CR3014 (eg, CR3022/3014; CR3022/3006; CR3022/3013 or CR3022/3014).
In a configuration, the polypeptide of the invention comprises one or more binding sites for human TMPRSS2 protein, for example, the polypeptide comprises a binding site for TMPRSS2 protein as disclosed in US20190300625, eg, the VH/VL pair of any anti-TMPRSS2 antibody disclosed in US20190300625, eg wherein the binding site comprises SEQ ID NOs: 17 and 18 disclosed in US20190300625; all of these sequences and binding site disclosures are incorporated herein by reference for use in the present invention and for possible inclusion in one or more claims herein. In a configuration, the polypeptide of the invention comprises one or more binding sites for human IL-6R, for example, the polypeptide comprises the VH/VL pair of sarilumab. In a configuration, the polypeptide of the invention comprises one or more binding sites for human IL-4R, for example, the polypeptide comprises the VH/VL pair of dupilumab. In a configuration, the polypeptide of the invention comprises one or more binding sites for human OX40L or OX40, eg, the VH/VL pair of oxelumab.
In a configuration, the multimer of the invention comprises binding sites for human TMPRSS2 protein, for example, the multimer comprises a plurality of copies of a binding site for TMPRSS2 protein as disclosed in US20190300625, eg, the VH/VL pair of any anti-TMPRSS2 antibody disclosed in US20190300625, eg wherein the binding site comprises SEQ ID NOs: 17 and 18 disclosed in US20190300625; all of these sequences and binding site disclosures are incorporated herein by reference for use in the present invention and for possible inclusion in one or more claims herein. In a configuration, the multimer of the invention comprises a plurality of copies of a binding sites for human IL-6R, for example, the VH/VL pair of sarilumab. In a configuration, the multimer of the invention comprises a plurality of copies of a binding sites for human IL-4R, for example, the VH/VL pair of dupilumab. In a configuration, the multimer of the invention comprises binding sites for human OX40L or OX40, eg, a plurality of clpies of the VH/VL pair of oxelumab.
Thus, generally multimers of the invention may advantageously be cross-reactive to more than one antigen (ie, bind to more than one antigen, such as first and second antigens which are different from each other); or may be capable of binding to an antigen using binding sites of the multimer, wherein the binding site as a monomer or dimer is not capable of binding to the antigen. For example, the binding by the multimer and by the monomer or dimer form are tested under identical conditions (eg, of temperature, pH, time and antigen concentration). For example, the binding site as a monomer or dimer means that one or two copies (but no more than one or two respectively) of the binding site when comprised by a protein are not capable of binding to the antigen (first antigen). Thus, in that example the protein is monovalent or bivalent for the antigen. For example, the binding site is a VH/VL binding site of a 4-chain antibody having 2 copies (but no more than 2) of the antigen binding site, wherein the antibody is not capable of binding to the antigen (eg, TACI); optionally the antigen is an antigen that is cognate to a receptor or ligand (eg, APRIL when the antigen is TACI; eg, the antigen is a ligand is cognate to a receptor (or another, second ligand); or the antigen is a receptor and is cognate to a ligand) wherein the receptor or first ligand is capable of binding to a second antigen and the antibody is capable of binding to the second antigen (eg, BCMA when the first antigen is TACI), and wherein a multimer of the invention is capable of binding to the first and second antigens. Thus, in an example, the first antigen is TACI and the second antigen is BCMA, and the multimer of the invention is capable of binding to TACI and BCMA. In another example, the first antigen is a SARS-Cov-2 antigen (eg, spike protein antigen) and the second antigen is a SARS-Cov-1 antigen (eg, spike protein antigen), and optionally the multimer of the invention is capable of binding to the first and second antigens. In another example, the first antigen is an antigen (eg, spike protein) of a first virus and the second antigen is an antigen (eg, spike protein) of a second virus, and optionally the multimer of the invention is capable of binding to the first and second antigens. The viruses are different, eg, the viruses are Coronaviruses; eg, the viruses are different strains of influenza viruses. For example, the first and second antigens are HIV antigens (eg, for the treatment or prevention of HIV infection or a symptom thereof); or P. falciparum antigens, such first and second CSP epitopes (eg, for the treatment or prevention of malaria or a symptom thereof); or Salmonella typhimurium antigens (eg, for the treatment or prevention of Salmonella infection or a symptom thereof. In an example, a multimer of the invention specifically binds to human BCMA and human TACI, and optionally the multimer comprises a plurality (eg, 4 and no more or less than 4) of a polypeptide of the invention wherein the polypeptide comprises a BCMA binding site as disclosed herein, such as in the next paragraph. Multimers that bind in these ways can be used in any method or use disclosed herein.
The invention, thus, provides:
A “4-chain antibody”, as the skilled addressee will understand, is a conventional antibody format having 2 copies of a heavy chain and 2 copies of a light chain, wherein each heavy chain is paired with a respective light chain and the heavy chain Fc regions pair to form heavy chain dimers.
The invention, by providing the ability to create multimers with broadened antigen specificity, provides useful multimers, compositions, methods and uses to target viruses whose antigens evolve through mutation during the natural history of a viral infection. In this respect, the invention may provide broadly-antigen-neutralising multimers, which can be useful for treatment or prevention of HIV infections, CoV (eg Cov-1 or Cov-2) infections or malaria.
Thus, the invention may find application to shift antigen-binding specificity of a predetermined binding site against a first antigen so that the multimer additionally or alternatively binds to a second antigen. For example, this can be demonstrated where the predetermined binding site specifically binds to BCMA, wherein the multimer of the invention binds to BCMA and TACI. For example, the predetermined binding site is the BCMA binding site of JNJ64007957 (Johnson & Johnson), AMG420 (Amgen), AMG701 (Amgen), CC-93269 (Cellgene), RGN5458, (Regeneron), PF-06863135 (Pfizer), SEA-BCMA (Seattle Genetics), MEDI2228 (AstraZeneca), belantamab (GlaxoSmithKline), idecabtagene vicleucel (Celgene), JNJ-4528 (Johnson & Johnson, Nanjing Legend Biotech), P-BCMA-01 (Poseidon Therapeutics), bb21217 (Bluebird Bio), JCARH125 (Celgene, Juno) or ALLO-715 (Allogene). Preferably, the binding site is the BCMA binding site of JNJ64007957. Preferably, the binding site is the BCMA binding site of JNJ-4528. Preferably, the binding site is the BCMA binding site of RGN5458. In an example, the multimer in this paragraph is for treating a cancer, eg, multiple myeloma.
In any aspect of the invention herein, the polypeptide of the invention optionally comprises
In one embodiment, the multimer is useful for binding to a first epitope and a second epitope which is a mutant of the first epitope, ie, wherein the second epitope differs from the first epitope by one or more amino acids or one or more sugar residues. For example, the epitopes differ by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids or sugar residues. As shown in Example 25, multimerization of polypeptides described herein may produce multimers that can use the same binding site to bind more than one (eg, 2) different epitopes or antigens. Thus, in an example, the first epitope is comprised by a first antigen and the second epitope is comprised by a second antigen, eg, the antigens are different receptors (such as cell-surface receptors), or different ligands (such as different forms of spike protein of a virus). Optionally, the virus is a SARS virus (eg, SARS-Cov, SARS-Cov-2 or MERS-Cov), HIV or influenza virus. Optionally, the virus is HIV and the epitope is an Env epitope, gp41 epitope or gp120 epitope. Optionally, the virus is influenza virus and the epitope is an epitope of haemagglutinin or matrix protein 2. By providing a multimer that is capable of binding to different forms of a spike protein of such a virus, the multimer is useful for treating or preventing or reducing a seasonal viral infection in humans or animals. For example, the multimer is useful for treating, preventing or reducing infection by a virus comprising a first form of spike protein, and the multimer is useful for treating, preventing or reducing infection by a virus comprising a second form of the spike protein. Thus, in this way the multimer is useful for treating or preventing viral infection in a first and second season wherein humans are infected in the first season by the virus comprising the first spike form and humans are infected in the second season by the virus comprising the second spike form. Instead of a spike protein, the epitope may be a different virus antigen, such as a capsid or tail protein. The multimer, therefore, is capable of binding to different strains of a virus and preferably neutralises the virus (eg, renders it non-infective and/or reduces proliferation of the virus). Thus, in an embodiment the invention provides a seasonal virus treatment or prophylaxis medicament for administration to a human or animal subject, wherein the medicament comprises a plurality of multimers of the invention, such as multimers according to this paragraph, wherein the medicament comprises a pharmaceutically acceptable diluent, carrier or excipient. Such diluents, carriers and excipients are well known to the skilled person. Administration is, eg, intravenous, inhaled, oral or intranasal administration. The invention therefore provides in an embodiment: a multi-seasonal (eg, 2-seasonal or 3-seasonal) anti-viral medicament comprising a plurality of multimers of the invention which are capable of binding to first and second strains of the virus, wherein the strains differ in a surface-exposed antigen to which the multimers can bind. In an example, the seasons are a first year and a second year (eg, two consecutive years or two consecutive winters thereof, or two consecutive summers thereof, or two consecutive springs thereof, or two consecutive falls/autumns thereof). Optionally in any embodiment herein, the virus is a SARS virus (eg, SARS-Cov, SARS-Cov-2, MERS-Cov), HIV, ebola virus, zika virus, norovirus, rotovirus, respiratory synctial virus (RSV), an exanthematous virus, papilloma virus, hepatitis (eg, A, B, C, D or E) virus, Lassa fever virus, dengue fever virus, yellow fever virus, Marburg fever virus, Crimean-Congo fever virus, polio virus, viral meningitis virus, viral encephalitis virus, rabies virus, smallpox virus, hantavirus or influenza virus. When the multimer is useful or used in a method for reducing the virus in an animal, this is advantageous for reducing a zoonotic population of viruses that are transmissible to humans, wherein the viruses are capable of causing a disease or condition (or death) in humans. In this respect, the animal may be a livestock animal, such as a pig, poultry (eg, chicken, duck or turkey), sheep, cow, goat, fish or shellfish. In an example, the animal is a bat, racoon dog, dog, cat, palm civet or camelid (eg, a camel or dromedary). In an example, the animal is a bird.
By presenting multiple copies of the epitope(s) multimers of the invention are believed to provide useful means for vaccination and for stimulating strong immune responses in a human or animal. Thus, in an alternative, the multimers comprise a plurality (eg, 4, 8, 12, 16 or 20) of copies of a peptide, wherein the peptide comprises an epitope of a pathogen, such as a surface-exposed epitope of a virus or bacterium. For example, the peptide comprises a first and/or second epitope as described in the immediately preceding paragraph. In this way, there is provided a vaccine composition for administration to a human or animal for preventing or reducing an infection by the virus or bacterium. In an example, each polypeptide of the multimer comprises a first peptide comprising a first said epitope of the pathogen and a second peptide comprising a second said epitope of the pathogen. Optionally, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 (eg, 2 or 3) said epitopes of the pathogen. Optionally, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 (eg, 2 or 3) different epitopes of the pathogen.
For example, the virus is Coronavirus, eg, SARS-Cov, SARS-Cov-2, a SARS-related coronavirus (a SARSr-Cov), HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E. In an example, the virus is SARS-CoV ZXC21, ZC45, RaTG13, CUHK-W1, Urbani, GZ02, A031, A022, WIV16, WIV1, Rp3, Rs672 or HKU4. For example, the virus is Coronavirus is a group 1, group 2 or group 3 Coronavirus. For example, the multimer is a vaccine antigen composition comprising copies of a polypeptide of the invention. In an aspect, the polypeptide comprises one or more S epitopes of said virus. In an aspect, the polypeptide of the invention comprises a S1 and/or S2 epitope of said virus; or a SA and/or SB epitope of said virus (eg, a SARS-Cov-2 SA and/or SB epitope, preferably SARS-Cov-2 SB epitope). In an aspect, the polypeptide comprises a peptide which comprises all or part of the SA domain and/or all or part of the SB domain. In an aspect, the polypeptide comprises a peptide which comprises all or part of the SB domain and all or part of the S2 subunit, and optionally also the S 1/S2 boundary. Additionally or alternatively, the polypeptide comprises a peptide which comprises the virus spike protein S1 subunit/ S2 subunit boundary. Additionally or alternatively, the polypeptide comprises a peptide which comprises the virus spike protein furin cleavage site. In an alternative, the multimer comprises a plurality of binding sites for one or more of the epitopes, wherein the multimer comprises copies of a polypeptide of the invention wherein the polypeptide comprises one or more epitope binding sites, each epitope being an epitope as described in this paragraph.
In an example, the SARS-Cov epitope comprises one or more N-linked glycans, eg, where each N is an N selected from the following table or is a corresponding N in the virus.
Italic font indicates the absence of a glycosylation sequon and deletions are indicated with periods. Glycans observed in the SARS-CoV-2 S cryo-EM map are underlined. (see Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein [published online ahead of print, 2020 Mar 6]. Cell. 2020;S0092-8674(20)30262-2. doi: 10.1016/j.cell.2020.02.058, the disclosure of which is incorporated herein by reference.
Position numbers are with reference to SARS-CoV-2 sequence with Accession Number: YP_009724390.1; and SARC-CoV Urbani sequence with Accession Number: AAP13441.1.
In an example, the virus is selected from SARS-CoV-2 (YP_009724390.1), SARSr-CoV RaTG13 (QHR63300.2), SARS-CoV Urbani (AAP13441.1), SARS-CoV CUHK-W1 (AAP13567.1), SARS-CoV GZ02 (AAS00003.1), SARS-CoV A031 (AAV97988.1), SARS-CoV A022 (AAV91631.1), WIV-16 (ALK02457.1), WIV-1 (AGZ48828.1), SARSr-CoV ZXC21 (AVP78042.1), SARSr-CoV ZC45 (AVP78031.1), SARSr-CoV Rp3 (Q3I5J5.1), SARSr-CoV Rs672 (ACU31032.1). Accession numbers are shown in brackets; the sequences thereof are explicitly incorporated herein by reference for use in the present invention, eg, for providing epitope or antigen sequence.
For example, the pathogen is HIV and the polypeptide comprises a gp120 epitope and a gp41 epitope, eg, the polypeptide comprises a SOSIP peptide.
For example, the pathogen is a Coronavirus (eg, SARS-Cov, SARS-Cov-2 or MERS-Cov) and the polypeptide comprises a first spike epitope and a second spike epitope, wherein the epitopes are different from each other. For example, the first epitope and/or second epitope comprises a sugar residue. For example, the first epitope comprises a viral contact residue for ACE2 and/or the second epitope comprises a viral contact reside for TMPRSS2 and optionally the virus is SARS-Cov or SARS-Cov-2. For example, the first epitope comprises a viral contact residue for DPP4 and/or the second epitope comprises a viral contact reside for TMPRSS2 and optionally the virus is MERS-Cov.
For example, the pathogen is influenza and the polypeptide comprises one or more haemagglutinin epitopes. Optionally, any influenza herein is influenza A, B or H1N1.
For example, the pathogen is HIV and the polypeptide comprises a plurality of Env epitopes.
For example, the pathogen is a virus (eg, a Coronavirus) and the polypeptide comprises a plurality of spike epitopes.
For example, the pathogen is a virus and the polypeptide comprises a plurality of capsid or tail epitopes.
In an example, the virus is a bacteriophage that is capable of infecting a host bacterial cell; or the virus is a virus that is capable of infecting an archaeal cell.
In an example, the pathogen is a bacterium selected from
The invention provides a protein comprising 4, 12, 16, 20, 24, 28 or 32 copies of an epitope disclosed herein, optionally also comprising 4, 12, 16, 20, 24, 28 or 32 copies of a second second epitope disclosed herein. Optionally, the protein is useful as a vaccine for treating or preventing an infection of a virus or bacterium in a human or animal subject, wherein the virus or bacterium comprises the epitope(s). Optionally, the protein is a multimer as disclosed herein, the multimer comprising four copies of a polypeptide, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, or 8 copies of the first epitope (and optionally comprises 1, 2, 3, 4, 5, 6, 7, or 8 copies of the second epitope). In an alternative, the invention provides a protein comprising 4, 12, 16, 20, 24, 28 or 32 copies of a binding site that is capable of binding to an epitope disclosed herein, optionally also comprising 4, 12, 16, 20, 24, 28 or 32 copies of a second binding site that is capable of binding to a second epitope disclosed herein. Optionally, the protein is useful as a therapy for treating or preventing an infection of a virus or bacterium in a human or animal subject, wherein the virus or bacterium comprises the epitope(s). Optionally, the protein is a multimer as disclosed herein, the multimer comprising four copies of a polypeptide, wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, or 8 copies of the first epitope (and optionally comprises 1, 2, 3, 4, 5, 6, 7, or 8 copies of the second epitope). In an example, the protein is useful as an assay reagent for detecting a virus of bacterium comprising the epitope(s). To this end there is provided a first method of detecting the virus or bacterium in a sample (eg, in vitro), the method comprising contacting the sample with the protein to allow the protein to bind to one or more copies of the virus or bacterium in the sample, and detecting the binding, eg, using a detection reagent that binds to virus or bacteria that have bound to the protein. There is also provided a method of detecting antibodies that are capable of binding (and optionally neutralising) the virus or bacterium in a sample (eg, in vitro), the method comprising contacting the sample with the protein to allow the protein to bind to such antibodies in the sample, and detecting the binding, eg, using a detection reagent that binds to the antibodies that have bound to the protein. The detection reagent may be an anti-virus or bacterium agent (such as a labelled antibody) in the first method; or an anti-antibody (eg, anti-IgG or anti-IgM) agent (such as a labelled antibody) in the second method. The label may, for example, be a fluorescence label, eg, GFP. The sample may be a blood, spit, sputum or cell sample, eg, a patient sample, such as a patient that is suffering from, is suspected of suffering from or has suffered from an infection by the virus or bacterium. In an example, the protein or multimer of the invention is immobilised on a solid surface, eg, a petri dish or test tube surface, or a flow chamber surface. For example, the surface is a particle surface, eg, a bead surface, such a magenetic bead, magnetisable bead, metal or ferrous bead. In an example, the protein or multimer of the invention is comprised by a fluid, eg, a liquid, eg, a liquid in a droplet, such as an emulsion droplet. Thus, the protein or multimer is useful in a microfluidics method of detecting the virus, bacterium or antibody (eg, IgG or IgM that binds the virus or bacterium).
In an example, the polypeptide comprises one or more (eg, 1, 2, 3 or 4) protein G peptides each of which is capable of binding to IgG, or the protein or multimer comprises a plurality of such polypeptides. Such a multimer or protein is useful to capture IgG when the protein or sample is contacted with a sample (eg, blood, sputum, saliva, semen or cell sample), such as wherein the contacting is carried out in vitro, such as in an in vitro assay. The avidity effect of the multimer’s plurality of protein G peptides is useful to enhance IgG detection sensitivity . The invention, therefore, provides such an assay method and a kit comprising the protein or multimer (optionally immobilised on a solid surface, such as on the surface of a container) and a detection reagent. In an example, the reagent comprises an antigen or epitope that is bound (eg, specifically bound) by the captured IgG. Optionally, the epitope is a viral or bacterial epitope, eg, a viral spike, capsid or tail fibre epitope; or eg, a bacterial cell surface epitope. Optionally, the epitope is a virus spike epitope, eg, a Coronavirus spike epitope, such as a SARS-CoV or SARS-Cov-2 or MERS-CoV spike epitope. Optionally, the reagent comprises a label that is detectable, such as a fluorescence marker, eg, GFP or an Alexa fluor marker. Instead of a protein G peptide, additionally or alternatively the polypeptide, multimer or protein comprises one or more protein A peptides that are each capable of binding to an antibody Fc, such as a Fc of an anti-viral or anti-bacterial antibody from a patient sample. Instead of a protein G peptide, additionally or alternatively the polypeptide, multimer or protein comprises one or more protein L peptides that are each capable of binding to an antibody light chain, such as a light chain of an anti-viral or anti-bacterial antibody from a patient sample (eg, an IgG, IgM, IgA, IgE or IgD antibody).
See
Optionally, where the protein or multimer comprises binding sites for ACE2, the protein or multimer may be used for treating or preventing hypertension in a human or animal subject. Optionally, the protein or multimer comprises one or more ACE2 epitopes, wherein the protein or multimer may be used for treating or preventing hypertension in a human or animal subject, such as by administration of the protein or multimer to the subject to raise antibodies against ACE2 in the subject. Alternatively, the treatment or prevention is an inflammatory condition (eg, lung inflammation), pneumonia, COPD, asthma or any treatment or prevention of a condition disclosed in US20110020315A1, the disclosure of which is incorporated herein by reference.
Optionally, where the protein or multimer comprises binding sites for TMPRSS2, the protein or multimer may be used for treating or preventing a cancer (eg, prostate cancer) or viral infection (eg, influenza infection) in a human or animal subject. Optionally, the protein or multimer comprises one or more TMPRSS2 epitopes, wherein the protein or multimer may be used for treating or preventing a cancer (eg, prostate cancer) or viral infection (eg, influenza infection) in a human or animal subject, such as by administration of the protein or multimer to the subject to raise antibodies against TMPRSS2in the subject. Alternatively, the treatment or prevention is any treatment or prevention of a condition disclosed in US 9,498,529, the disclosure of which is incorporated herein by reference. For example, the inflammation is local inflammation of a tissue or an organ and/or a systemic inflammation. For example, the inflammation comprises sepsis. For example, the inflammation comprises an autoimmune disease.
Optionally, where the protein or multimer comprises copies of a binding site for a virus spike, the binding site may be the binding site of antibody 80R, CR3014, CR3006, CR3013 or CR3022. The VH and VL domain sequences of these antibodies are incorporated herein by reference for possible inclusion in a protein or multimer of the invention. Optionally, where the protein or multimer comprises copies of a binding site for a virus spike, the binding site is capable of binding to amino acid residues 426-492, 318-510, or 318-510 of S1 subunit of SARS-CoV, and wherein optionally the protein or multimer binds SARS-CoV and SARS-CoV-2.
Optionally, where the protein or multimer herein is for treating or preventing viral pneumonia in a human or animal subject, eg, wherein the subject is suffering from or is at risk of suffering from a Coronavirus infection. Optionally, where the protein or multimer herein is for treating or preventing Coronavirus viral pneumonia in a human or animal subject. Optionally, where the protein or multimer herein is for treating or preventing Coronavirus viral pneumonia in a human or animal subject, wherein the binding sites are capable of binding to a Pseudomonoas aeruginosa epitope, or wherein the protein or multimer comprises Pseudomonoas aeruginosa epitopes.
Optionally, where the protein or multimer herein is for treating or preventing a viral infection or symptom thereof in a human or animal subject, wherein the binding sites are capable of binding to a Cathepsin L epitope, or wherein the protein or multimer comprises Cathepsin L epitopes. In an example, the virus is Ebola virus or a SARS virus or a Coronavirus (eg, SARS-CoV or SARS-CoV-2).
Optionally, where the protein or multimer herein is for treating or preventing a Coronavirus infection or symptom thereof in a human or animal subject, wherein the binding sites are capable of binding SARS-CoV S1 RBD or RBDR, or wherein the protein or multimer comprises SARS-CoV S1 RBD or RBDR. The receptor-binding determining region (RBDR) that recognizes ACE2. For example, the binding sites are capable of binding the peptide S471-503 of the RBD; or the protein or multimer comprises copies of S471-503 of the RBD.
Optionally, where the protein or multimer herein is for treating or preventing a Coronavirus infection or symptom thereof in a human or animal subject, wherein the binding sites are capable of binding SARS-CoV-2 S1 RBD or RBDR, or wherein the protein or multimer comprises SARS-CoV-2 S1 RBD or RBDR. The receptor-binding determining region (RBDR) that recognizes ACE2. For example, the binding sites are capable of binding the peptide S471-503 (ALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFEL) (SEQ ID NO: 500) of the RBD; or the protein or multimer comprises copies of S471-503 of the RBD. In an example, the protein or multimer comprises copies of a peptide comprises by the amino acid sequence from position 318 to 536 of SARS-CoV or the equivalent amino acid sequence of SARS-CoV-2, wherein the peptide comprises the amino acid sequence from position 424 to position 494. Optionally, the peptide is RBD219-N1 (see For example, Chen, W.; Hotez, P.J.; Bottazzi, M.E. Potential for Developing a SARS-CoV Receptor Binding Domain (RBD) Recombinant Protein as a Heterologous Human Vaccine against Coronavirus Infectious Disease (COVID)-19. Preprints 2020, 2020020449, the sequences of (i) RBD219-N1, (ii) the sequence in the blue box (the epitope consisting S343-367, 373-390 and 411-428 (reported by Bian et al)) and (iii) the sequences in the green box from the first N to QPY for each of RBD219-N1 and SARS-CoV-2 spike shown in
Optionally, the protein or multimer of the invention is for treating or preventing HIV and comprises one or more copies of the antigen binding site of an antibody shown in Table 1 or
In an example, the protein or multimer comprises a plurality of (eg, 4, 8, 12, 12, 16, 20, 24, 28 or 32) copies of a peptide disclosed herein, eg, a peptide disclosed in the immediately preceding paragraph.
Optionally, where the protein or multimer herein is for treating or preventing a RSV infection or symptom thereof in a human or animal subject, wherein the binding sites are palivizumab binding sites.
Optionally, the binding site of the protein, multimer or polypeptide of the invention disclosed herein comprises a binding site for a peptide or epitope disclosed herein, or for a peptide or epitope whose amino acid sequence is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the amino acid sequence of a peptide or epitope disclosed herein. Alternatively, the protein, multimer or polypeptide binding site competes (eg, in SPR) with a binding site disclosed in the first sentence of this paragraph.
In one configuration, the invention provides a RNA (eg, mRNA or self-amplifying mRNA, or saRNA) that encodes a polypeptide or protein of the invention. In an example, there is provided a medicament (eg, a vaccine) comprising the RNA, wherein the RNA is for administration to a human or animal subject for treating or preventing a disease or condition in the subject, wherein the RNA is expressed in the subject to produce polypeptides, proteins or multimers of the invention. Optionally, the medicament is a vaccine and the condition is a virial or bacterial infection, such as when the encoded polypeptide comprises an epitope of the virus or bacterium or a binding site that is capable of binding to such an epitope.
The polypeptide, protein or multimer can comprise multiple (i.e. 2, 3 or 4) different peptides of the target virus or bacterium (eg, peptides of cell-surface proteins) for use as vaccine. In another embodiment, there is provided a composition comprising first and a second multimer or protein of the invention wherein the multimers/proteins comprise peptides of the target virus or bacterium (eg, peptides of cell-surface proteins) for use as vaccine, wherein the peptides of the first multimer or protein differ from the peptides of the second multimer or protein. For example, the proteins or multimers do not comprise a common such peptide. For example, the second multimer or protein comprises a such peptide that is not comprised by the first protein or multimer. Optionally, the composition comprises a third protein or multimer which is different from the first and second proteins/multimers, wherein the third protein or multimer comprises a said peptide that is not comprised by the first and second proteins/multimers.
For a vaccine herein that targets a virus, the peptide or epitope is not limited to a spike epitope (eg, S1 or S2 subunit epitope); the virus epitope could be from any region of the virus, preferably a region that is exposed on the cell surface of the viral host.
Multimerizing virus or bacterial peptides or epitopes according to the invention may advantageously enhance immunogenicity in the subject and thus promote generation of anti-viral/bacterial antibodies that are desirably affinity matured and may give rise to antibodies with a broad epitope coverage (ie, more recognising more than one epitope) of the virus/bacterium.
Through multimerization made possible by the invention, certain aspects advantageously enable provision of new antigen specificities for binding sites; or new cross-reactivity of binding sites (eg, wherein a binding site in a control binds, such as an IgG, a first but not second antigen, but when present as a multimer of the invention binds both antigens - as exemplified herein). The multimerization also or alternatively can greatly enhance binding strength for an antigen, such as a viral antigen, thereby providing multimer format that are useful for human or animal therapy and for highly sensitive assays, eg, to detect antigen or virus in a sample, such as a serum sample of a subject. Again, such highly sensitive assaying is exemplified herein. Thus, the invention may render therapeutically- or prophylactically-useful a binding site that has hitherto been useless for therapy of prophylaxis of a disease or condition (eg, infection by a certain virus) in humans or animals. As exemplified herein, the multimerization of the invention converts binding based on anti-SARS-CoV-2 binding sites from therapeutically- or prophylactically-useless to therapeutically- or prophylactically-useful for administration of the multimer of the invention to a human or animal subject for treating (eg, reducing) or preventing a SARS-CoV-2 infection. Thus, the invention enables re-purposing of pre-existing antigen binding sites to provide for possible new applications for treatement, prevention or detection of a disease, condition or infection.
In a first aspect, the invention provides:
A protein multimer (first multimer) comprising more than 2 copies of a binding site, wherein the binding site is capable of binding to a first antigen, optionally wherein the multimer is capable of binding to the first antigen and a second antigen, wherein the antigens are different.
For example, the multimer comprises from 4 to 32 (eg, from 4 to 24, or from 4 to 20, or from 4 to 16) copies of the binding site, ie, this means that the multimer does not comprise any more or less than said number. In an embodiment, the multimer comprises, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 copies of the binding site.
For example, the multimer contains from 4 to 32 (eg, from 4 to 24, or from 4 to 20, or from 4 to 16) copies of the binding site, ie, this means that the multimer does not have any more or less than said number. In an embodiment, the multimer contains, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 copies of the binding site. For example, the binding site is any binding site mentioned herein, for example, any VH, VL, VHH, dAb, nanobody, VH/VL pair, sybody or scFv.
In an embodiment, a control protein multimer comprising 1 or 2 (but no more than 1 or 2 respectively) of said binding sites is not capable of binding to the first antigen; or is capable of binding to the first antigen, but not to the second antigen. This is exemplified herein in Example 25. Binding may be determined by an ELISA assay, such as by determining OD450, for example in an ELISA assay described herein. In an example, the first antigen is BCMA and the second antigen is TACI. Optionally, the antigens are human antigens. Optionally, the antigens are bacterial, archaeal or fungal antigens. Alternatively, the antigens are different viral antigens, or antigens of first and second viruses which viruses are different from each other, eg, SARS-CoV and SARS-CoV-2. Optionally, the virus antigens are spike proteins. Alternatively, the virus antigens are nucleocapsid (N) proteins. Alternatively, the virus antigens are envelope (E) proteins. Alternatively, the virus antigens are membrane (M) proteins. Optionally, the antigens of first and second viruses are different and the viruses are different strains of the same type of virus (eg, SARS-CoV strains; or SARS-CoV-2 strains; or influenza strains). Optionally, the antigens of first and second viruses are different and the viruses are different types of virus, eg, SARS-CoV and SARS-CoV-2.
The invention also provides multimers of binding sites that bind to virus antigens, as expemplified herein. Thus, the invention provides:
A protein multimer (first multimer) comprising more than 2 copies of a binding site, wherein the binding site is capable of binding to a virus protein (eg, a virus spike, E, M or N protein) of a first virus, optionally wherein the multimer is capable of binding to the first and a second virus, wherein the viruses are different. This is exemplified herein for 2 different viruses. For example, the multimer comprises 4 copies of the binding site, wherein the binding site is capable of binding to the virus protein (first virus protein) and a second virus protein which is a mutated version of the first virus protein, wherein the second virus protein is found in a second virus that is infectious to humans. For example, the first and second viruses are SARS viruses or coronaviruses, eg, SARS-CoV-2 viruses. For example, the first protein is a SARS-CoV-2 spike protein comprising the amino acid N501 (ie, asparagine at position 501) and the second protein is a a SARS-CoV-2 spike protein comprising the amino acid Y501 (ie, tyrosine at position 501) or T501. Additionally or alternatively, the first protein comprises E484 and the second protein comprises K484; and/or the first protein comprises K417 and the second protein comprises N417 or T417. Additionally or alternatively, compared to the first protein, the second protein comprises deletion ΔHV69-70, ΔY144 or ΔLLA242-244. Additionally or alternatively, the first protein comprises A222 and the second protein comprises V222; and/or the first protein comprises N439 and the second protein comprises K439; and/or the first protein comprises S477 and the second protein comprises N477; and/or the first protein comprises Y453 and the second protein comprises F453; and/or the first protein comprises F486 and the second protein comprises L486; and/or the first protein comprises G261 and the second protein comprises D261; and/or the first protein comprises V367 and the second protein comprises F367. Thus, we have discovered that multimers, as per the invention, comprising at least 4 copies of a binding site that binds to SARS-CoV-2 spike protein, such as the RBD (and preferably the inner face of the RBD) are particularly useful as medicaments (or diagnostic agents to idenfity the presence of the virus). As exemplified in Example 37, a multimer that recognises the epitope recognised by QB-GB binding site is capable of binding to several such mutant forms of SARS-CoV-2 spike and is well suited for administration to patients (or a human population) for treating or preventing SARS-CoV-2 infection, such as where some degree of resistance to mutants occurring during the life history of the virus is desired.
Optionally, each virus is a coronavirus. Optionally, one of the viruses is SARS-CoV and the other virus is SARS-Cov-2. For example, the first virus is SARS-CoV and the second virus is SARS-Cov-2. In an alternative, the first virus is SARS-CoV-2 and the second virus is SARS-Cov.
Optionally, the multimer comprises 4 copies of the binding site. Optionally, the multimer comprises 4 (but no more than 4) copies of the binding site. Optionally, the multimer comprises 8 (but no more than 8) copies of the binding site. Optionally, the multimer comprises 12 (but no more than 12) copies of the binding site. Optionally, the multimer comprises 16 (but no more than 16) copies of the binding site. Optionally, the multimer comprises 20 (but no more than 20) copies of the binding site. Optionally, the multimer comprises 24 (but no more than 24) copies of the binding site. Optionally, the multimer comprises 4, 8, 12, 16, 20 or 24 copies of the binding site.
Optionally,
Optionally,
Optionally,
Optionally,
Optionally,
Preferably, binding or binding strength is determined by ELISA, eg, by determining OD450. An ELISA herein may be carried out at room temperature and pressure (rtp), or preferably at 20 or 25 centigrade and 1 atmosphere.
Optionally,
In an example, the multimer binds according to (a). In an example, the multimer binds according to (b). In an example, the multimer binds according to (c). In an example, the multimer binds according to (a) and (b). In an example, the multimer binds according to (a) and (c). In an example, the multimer binds according to (a), (b) and (c). In an example, the multimer binds according to (b) and (c). These are exemplified herein.
Binding of the the first multimer with said OD450 indicates that the first multimer (ie, multimer of the invention) is useful for medical use, ie, therapy or prophylaxis of a disease or condition in a human or animal subject wherein the disease or condition is mediated by the first antigen (or a pathogen comprising the first antigen). Binding of the the second multimer (eg, IgG having only 2 of said binding sites) with said OD450 indicates that the second multimer is not useful for medical use or said therapy or prophylaxis.
Binding of the the first multimer with said OD450 indicates that the first multimer (ie, multimer of the invention) is useful for assaying for detecting the presence of the first antigen or antibodies against the first antigen in a bodily fluid sample of a human or animal, eg, a serum, saliva or cell sample obtained from a human or animal, wherein the human or animal (i) is suffering from, has suffered from or is suspected of suffering from a disease or conditionthat is mediated by the first antigen, or (ii) is suffering from, has suffered from or is suspected of suffering from an infection by a pathogen that comprises the first antigen, such as a virus, bacterium or fungus (eg, a yeast). Binding of the the second multimer (eg, IgG having only 2 of said binding sites) with said OD450 indicates that the second multimer is not useful for such assaying or detection.
Generally, an Ig (eg, IgG) that binds to its cognate antigen with an affinity (Kd) higher than 1, 10, 100 or 1000 mM are not useful as medicaments. In an example, the binding site of the multimer of the invention is an antigen binding site of an Ig (eg, IgG) Fab fragment that binds to the antigen with an affinity (Kd) higher than 0.1, 1, 10, 100 or 1000 mM (eg, higher than 1 or 10 mM). Optionally, the Ig is said second multimer. In an example, the multimer of the invention binds to the antigen with an apparent affinity (avidity) of lower than 0.1 mM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM or 100 fM. These affinities are amenable to medical use. Affinities are may be determined by any standard method, for example by surface plasmon resonance (SPR) or ELISA, or bilayer interferometry (eg, as per the example below). The method may be carried out at rtp, or optionally at 20 or 25° C.entrigrade and 1 atm and optionally at a pH from 6.5 to 7.5 (eg, at pH 7). Reference is made to Science. 2020 May 8;368(6491):630-633. doi: 10.1 126/science.abb7269. Epub 2020 Apr 3, “A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV”, Yuan M et al, which determined binding affinity using a Fab version of CR3022, it can be seen CR3022 binds spike RBD of CoV-2 with at least 100x less affinity than CoV-1. The Kd for CR3022 for CoV-2 is around 115 nM. A multimer of the invention comprising 4 copies of the CR3022 binding site will have a Kd in the low pM range, thereby greatly improving on the apparent affinity and rendering the multimer useful as a medicament. We expect also a large improvement in affinity (expected to be in the double of single digit pM range or less) for the multimer binding to RBD of the CoV-1 strain. We would expect binding to such RBD with higher affinity again in the low pM range.
Binding assays may performed by biolayer interferometry (BLI) using an Octet Red® instrument (ForteBio). Briefly, His6-tagged antigen (eg, S or RBD protein) at 20 to 100 µg/mL in 1x kinetics buffer (1x PBS, pH 7.4, 0.01% BSA and 83 0.002% Tween 20) are loaded onto Anti-Penta-HIS™ (HIS1K) biosensors and incubated with the indicated concentrations of Fab or IgG (eg, CR3022 Fab or IgG) or multimer. The assay comprises fivesteps: 1) baseline: 60 s with 1x kinetics buffer; 2) loading: 300 s with his6-tagged proteins; 3) baseline: 60 s with 1x kinetics buffer; 4) association: 120 s with samples (Fab or IgG or multimer); and 5) dissociation: 120 s with 1x kinetics buffer. For estimating the exact Kd, a 1:1 binding model is used.
ELISAs are performed in duplicates to compare the binding affinities of the different product formats. Recombinant antigen is diluted to 1 ug/ml in ELISA coating buffer (50 mM carbonate/bicarbonate). One hundred ul of 1 ug/ml antigen is added to each well of an ELISA plate and the plates are incubated overnight at 4° C. The plates are washed three times with PBS containing 0.05% Tween-20 before being blocked with 200 ul 1% bovine serum albumin in PBS for 4 hrs at room temperature. The plates are washed three times as before. Products (multimers or other protein to be tested) are serially diluted in PBS containing 0.05% Tween-20. One hundred ul of sample is added to each well and the plates are incubated overnight at 4° C. The plates are washed four times with PBS containing 0.05% Tween-20. One hundred ul of detection antibody (anti-His-HRP, A7058, Sigma; or antiHuman-IgG HRP, 31410, Thermo Fisher Scientific; or Protein L HRP, M00098, Genscript) diluted in blocking buffer (according to the manufacturers’ recommendations) is added to each well and the plates are incubated at room temperature for 2 h. Following four plate washes, 25 ul of TMB substrate solution (Thermo Fisher Scientific) is added to each well. The reaction is terminated after ~ 15 min by the addition of 25 ul 3 M HCl. The absorbance at 450 nm is read using a CLARIOstar™ microplate reader (BMG Labtech).
The SPR is carried out at a detergent level of no greater than 0.05% by volume, eg, in the presence of P20 (polysorbate 20; eg, Tween-20™) at 0.05% and EDTA at 3 mM. In one example, the SPR is carried out at 25° C. or 37° C. in a buffer at pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. The buffer can contain 10 mM Hepes. In one example, the SPR is carried out at 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc (California; catalogue number H8022). In an example, the affinity (eg, of a VH/VL binding site) is determined using SPR by using any standard SPR apparatus, such as by Biacore™ or using the ProteOn XPR36™ (Bio-Rad®). The binding data can be fitted to 1:1 model inherent using standard techniques, eg, using a model inherent to the ProteOn XPR36™ analysis software.
Optionally,
In an example, the multimer binds according to (a). In an example, the multimer binds according to (b). In an example, the multimer binds according to (c). In an example, the multimer binds according to (a) and (b). In an example, the multimer binds according to (a) and (c). In an example, the multimer binds according to (a), (b) and (c). In an example, the multimer binds according to (b) and (c). These are exemplified herein.
Optionally, binding of the first multimer to the first antigen or protein is saturated as determined by OD450 in an ELISA assay in which the antigen or protein is at a concentration between 10 and 100 nM in the assay (and optionally the second multimer binds to the first antigen or protein with an OD450 less than 2.5 (eg, from 2 to 2.5) in an ELISA assay in which the antigen or protein is at a concentration between 10 and 100 nM in the assay).
Optionally,
Optionally, binding of the first multimer to the second antigen or protein is saturated as determined by OD450 in an ELISA assay in which the antigen or protein is at a concentration between 10 and 100 nM in the assay (and optionally the second multimer binds to the second antigen or protein with an OD450 less than 1.5 (eg, from 1 to 1.5) in an ELISA assay in which the antigen or protein is at a concentration between 10 and 100 nM in the assay).
Optionally, the multimer is capable of detectably binding to antibodies that bind to the first antigen or the second antigen or virus protein (eg, anti-virus protein antibodies, such as anti-SARS-Cov spike antibodies or anti-SARS-Cov-2 spike antibodies or anti-influenza haemagglutinin antibodies) in an ELISA assay, wherein detection of the multimer binding is measured by OD450 and the assay comprises
ELISA herein may be a sandwich ELISA.
Optionally, the dilution is from 10 to 104, 105 or 106-fold. Optionally, the dilution is from 100 to 104, 105 or 106-fold. Optionally, the dilution is from 1000 to 104, 105 or 106-fold. Preferably, the dilution is 1000 to 1,000,000-fold (such as 1000 to 100,000-fold or 1000 to 10,000-fold). Optionally, dilution is dilution with water or an aqueous solution, eg, PBS, such as PBS containing from 0.1 to 0.05% (eg, either 0.1% or 0.05%) Tween-20. This is exemplified herein, demonstrating the possibility of extremely sensitive assaying using multimers of the invention comprising more than 2 (eg, at least 4) binding site copies.
Optionally, the spike protein is a trimer of polypeptides.
Optionally, the binding site is an antibody VH/VL pair or an antibody single variable domain (such as a nanobody, VHH or a dAb).
Optionally, the binding site is
Optionally, the binding site comprises or consists of an ACE2 extracellular protein. Optionally, the ACE2 protein is human ACE2 protein. For example, an extracellular protein of ACE2 having UNIPROT number Q9BYF1, the sequence of such ACE2 and the extracellular domain thereof being incorporated herein by reference, along with the nucleotide sequence encoding such. In an example, ACE2 extracellular protein comprises or consists of positions 18 to 615 or 18 to 740 of ACE2 having UNIPROT number Q9BYF1, the sequence comprising or consisting of positions 18 to 740 being incorporated herein by reference, along with the nucleotide sequence encoding such.
Optionally, the binding site comprises or consists of an TMPRSS2 extracellular protein. Optionally, the TMPRSS2 protein is human TMPRSS2 protein. For example, an extracellular protein of TMPRSS2 having UNIPROT number 015393, the sequence of such TMPRSS2 and the extracellular domain thereof being incorporated herein by reference, along with the nucleotide sequence encoding such. In an example, TMPRSS2 extracellular protein comprises or consists of positions 106 to 492 of TMPRSS2 having UNIPROT number 015393, the sequence comprising or consisting of positions 106 to 492 being incorporated herein by reference, along with the nucleotide sequence encoding such.
Optionally, the binding site is an antibody VH/VL pair, wherein the VH comprises an amino acid sequence of a VH disclosed in Table 23 and the VL comprises the amino acid sequence of the cognate VL disclosed in Table 23. Optionally, the binding site comprises an scFv disclosed in Table 23. Optionally, the binding site comprises an antibody single variable domain (eg, a VHH, nanobody, dAb, VH or VL) disclosed in Table 23, Table 32 or elsewhere herein.
Optionally, the binding site is an antibody VH/VL pair, wherein the VH comprises an amino acid sequence of a VH disclosed in Table 32 and the VL comprises the amino acid sequence of the cognate VL disclosed in Table 32. Optionally, the binding site comprises an scFv disclosed in Table 32.
Optionally, the multimer comprises a multimer of a polypeptide disclosed in Table 23, optionally wherein the polypeptide is a polypeptide in the Table that comprises a TD.
Optionally, the multimer comprises a multimer of a polypeptide disclosed in Table 32, optionally wherein the polypeptide is a polypeptide in the Table that comprises a TD.
Any amino acid sequence in Table 23, Table 32 or elsewhere herein that ends at its C-terminus in TVS may in the alternative be provided as the indentical sequence except that the alternative ends in TVSS. Any amino acid sequence in Table 23, Table 32 or elsewhere herein that ends at its C-terminus in TVSS may in the alternative be provided as the indentical sequence except that the alternative ends in TVS.
The multimer may be a multimer of any format disclosed herein. The multimer may be a multimer of any polypeptide dislosed herein.
Optionally, the multimer comprises more than 2 (eg, comprises 4) copies of a heavy/light chain pair, wherein each heavy chain comprises (in N- to C-terminal direction) a VH and an antibody constant region (eg, an Fc) and wherein each light chain comprises (in N- to C-terminal direction) a VL and an antibody constant region (eg, a CL), wherein the binding site of the multimer comprises the VH paired with the VL; optionally wherein each heavy chain comprises a self-assembly multimerization domain (such as a tetramerization domain, such as a p53 TD). Optionally, each heavy chain comprises a hinge region as disclosed herein.
Optionally, the multimer comprises more than 2 (eg, comprises 4) copies of a polypeptide, wherein the polypeptide comprises (in N- to C-terminal direction) a single variable domain and a multimerization domain (eg, a tetramerization domain, such as a p53 TD), and optionally an antibody constant region (eg, an Fc or CL) between the single variable domain and the multimerization domain, or the multimersiation domain is between the single variabl domain and the constant region.
In an aspect, the invention provides assays and methods:-
Optionally, the method is an ELISA method, eg, a sandwich ELISA. The method is carried out in vitro.
Suitable assay example formats are shown
For example, the invention provides an assay comprising a format shown in any of
In an embodiment, there is provided:-
A method of detecting the presence of anti-SARS-Cov-2 protein (eg, spike, M, E or N) antibodies in a serum sample, the method comprising carrying out an ELISA assay (eg, an assay disclosed herein), and the assay comprises
The method is carried out in vitro. In an alternative, the antibodies are antibodies that bind to a protein of SARS-CoV or a different coronavirus. Optionally, the antibodies are antibodies that bind to a N, M or E proteins of a coronavirus, eg SARS-CoV or SARS-Cov-2.
Optionally, the presence of anti-antigen or protein antibodies (eg, anti-virus protein antibodies, such as anti-SARS-Cov-2 spike antibodies) in the sample is detected when the optical density (eg, OD450) is greater than 0.1 or 0.5 (optionally, greater than 1, 1.5 or 2) in the assay.
Optionally wherein the spike protein is immobilised on a solid surface. Alternatively, the multimers are immobilised on a solid surface.
Optionally, the dilution is 1000 to 1,000,000-fold (such as 1000 to 100,000-fold or 1000 to 10,000-fold) or any other fold dilution disclosed herein. Optionally, the dilution is from 10 to 104, 105 or 106-fold. Optionally, the dilution is from 100 to 104, 105 or 106-fold. Optionally, the dilution is from 1000 to 104, 105 or 106-fold. Optionally, dilution is dilution with water or an aqueous solution, eg, PBS, such as PBS containing from 0.1 to 0.05% (eg, either 0.1% or 0.05%) Tween-20.
Optionally, the spike protein is a trimer of polypeptides. For example, the spike protein is a monomer of either S1 or S2 spike ectodomain, a trimer of the spike, monomer of the spike receptor binding domain (RBD domain); or a RBD multimer, such as a dimer, trimer, tetramer or octamer of the RBD.
In an alternative to binding spike, the multimer of may bind a Nucleocapsid (N protein), membrane protein (M protein) or envelope protein (E protein) and the disclsoures herein referring to spike protein binding can apply mutatis mutandis to those alternatives.
Before carrying the method herein the serum sample may have been obtained by taking a blood sample or other bodily fluid sample from a mammal (eg, a human or animal, such as any animal disclosed herein). In an example, the human is a human suspected of having previously been infected or currently infected by a pathogen, eg a virus, bacterium or fungus comprising the antigen (first antigen), eg, SARS-CoV or SARS-Cov-2. For example, the human is a male, female, adult, teenager, child, baby or a human of at least 10, 20, 30, 40, 50, 60, 70 or 80 years’ of age (preferably over 50).
In embodiments, the binding site of the multimer is
Optionally, the multimer is a multimer of a polypeptide disclosed in Table 24, optionally wherein the polypeptide is a polypeptide in the Table that comprise a TD.
Optionally, the multimer comprises a plurality of copies of an Ig binding domain disclosed in Table 25, optionally wherein the multimer further comprises a plurality of copies of a further (ie, different) Ig binding domain disclosed in Table 25,
Optionally, the binding site of the multimer is alternatively capable of binding to an antibody (eg, an antibody that is capable of binding a human antigen, viral antigen, bacterial antigen or fungal antigen, such as an anti-SARS-Cov2 antibody, optionally wherein the binding site is comprised by
Optionally, step (c) is carried out before step (b), wherein the protein A, G, L or fragment, scFv or variable domain binding sites of the multimers bind a plurality of copies of the antibody (eg, anti-SARS-Cov2 antibody). For this option, the multimers may be immobilised on a solid support.
Optionally, the multimers are immobilised on a solid surface. Optionally, in any method or assay herein, the step of determining optical density (eg, OD450) comprises labelling complexes comprising first antigen or protein (eg, spike protein) and multimers with horseradish peroxidase (HRP) and detecting the label (optionally at a wavelength of 450 nm). For example, the HRP is contacted with tetramethyl benzidine and abosorbance is read at 450 nm, whereby OD450 is determined.
The invention also provides:-
A pharmaceutical composition or assay reagent comprising a plurality of multimers of the invention, optionally wherein the reagent comprises said multimers immobilised on a solid support.
In an example the following provide the solid support: Beads, petri dish, a laboratory apparatus, flow cell or a swab or dipstick. The support may be sterile or suitable for medical use.
For example, the pharmaceutical composition comprises a pharmaceutically-acceptable carrier, diluent or excipient.
The invention also provides:-
A multimer of the invention for administration to a human or animal subject for medical use.
A multimer of the invention for administration to a human or animal subject for treatment or prevention of an infection by a pathogen (eg, a virus, bacterium or fungus) that comprises the first antigen or protein, or a symptom of such an infection (eg, an unwanted inflammatory response).
A multimer of the invention for administration to a human or animal subject for treatment or prevention of an infection by the first and/or second virus, or a symptom of such an infection (eg, an unwanted inflammatory response).
A method of treating a disease, condition or symptom thereof in a human or animal subject, the method comprising administering to the subject a plurality of multimers of the invention. For example, the disease, condition or symptom is caused by the first antigen or protein (or by a pathogen that comprises the first antigen or protein, such as a virus that comprises the antigen or protein).
A method of treating a viral infection or symptom thereof in a human or animal subject, the method comprising administering to the subject a plurality of multimers of the invention.
The composition or multimers of the invention may be admistered in said use or method to the subject by any means, such as intravenously, orally, by inhalation or any other route disclosed herein.
The invention also provides:-
An assay kit comprising a reagent of the invention and an amount of the first antigen or protein (eg, viral spike protein), optionally wherein the reagent and protein are comprised by different containers.
Examples 23 -26 demonstrate how advantageously multimerization of the invention can repurpose a binding site which otherwise would not be useful or much less useful, such as for medical use (eg, for treatment or prophylaxis of a disease or condition mediated by or associated with an antigen to which the binding site binds), or for assay use (eg, detecting a pathogen or antigen that mediates, causes or is adversely associated with a a disease or condition in a subject). Through multimerization of the invention, very high-order multimers (eg, containing 8-24 copies of a binding site) can easily be achieved in a stable multimer that can be readily expressed, such as in eukaryotic expression systems and host cells (as demonstrated in the exemplification herein). The high-order multimers usefully can repurpose binding sites that individually have relatively low binding strength for an antigen, wherein in the multimers an avidity effect is produced rendering the combined binding strength of copies of the binding site well suited to medical applications or very sensitive assay detection of low levels of antigens in samples. Usefully, for example, we demonstrate this even for very diluted samples where the antigen is at very low concentration. This is advantageous, for example where the antigen is an antigen of a pathogen (eg, a virus, bacterium or fungus that causes disease, such as in humans, animals or plants); or where the antigen is comprised by antibodies produced by a human or animal subject in response to immunisation, such as in response to a pathogen or a human protein in the subject.
Thus, in an embodiment, the invention provides:-
A method of expanding a utility of an antigen (eg, a protein) binding site, the method comprising producing a multimer of the invention, wherein the multimer comprises a plurality of copies (eg, at least 4 or 8 copies) of the binding site.
In an example the utility is a medical utility, such as treating or preventing a disesase or condition mediated by the antigen in a human or animal subject (eg, an infection caused by a pathogen comprising the antigen). In an example the utility is an assay or detection method for determining the presence or relative amount of the antigen (or a pathogen comprising the antigen) or antibodies that bind the antigen in a sample (eg, an environmental sample or any sample of a human or animal subject disclosed herein). In an example, the method increases the sensitivity of assaying for the antigen or antibodies in a sample. For example, the sample is a blood or serum or saliva sample which has been diluted, such as diluted with fold dilution disclosed herein. For example, the utlity is a reduced propensity for producing false positive results in assaying for the presence of the antigen or antibodies that bind the antigen in a sample.
In examples, the invention provides a polypeptide comprising one or more copies of an antigen binding domain (BD), the polypeptide comprising or consisting of, in N- to C-terminal direction
Preferably in these examples, TD is a p53 TD, eg, a human p53TD.
Optionally, the BD is a single variable domain (also referred to as a domain antibody or dAb, eg, a nanobody or VHH, eg, a single variable domain comprising SEQ ID NO: 288, preferably Nb-112). Preferably, BD comprises the amino acid of QB-GB (SEQ ID NO: 307). Preferably, BD comprises the amino acid of QB-BG. Preferably, the BD comprises the amino acid of QB-FE. Preferably, BD comprises the amino acid of SEQ ID NO: 288. For example, the BD comprises an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 288. Preferably, BD comprises the amino acid sequence of a VH or VL disclosed in Table 32 (optionally wherein in the multimer each said VH is paired with the cognate VL shown in Table 32; or optionally wherein in the multimer each said VL is paired with the cognate VH shown in Table 32, eg, the pair comprises the VH and VL of REGN10987, REGN10933 or CB6). Preferably, BD comprises the VH or VL of REGN10987 (optionally wherein in the multimer each said VH is paired with the VL of REGN10987; or optionally wherein in the multimer each said VL is paired with the VH of REGN10987). Preferably, BD comprises the VH or VL of REGN10933 (optionally wherein in the multimer each said VH is paired with the VL of REGN10933; or optionally wherein in the multimer each said VL is paired with the VH of REGN10933). Preferably, BD comprises the VH or VL of CB6 (optionally wherein in the multimer each said VH is paired with the VL of CB6; or optionally wherein in the multimer each said VL is paired with the VH of CB6). Antibody CB6 is also known as LY-CoV555.
Optionally, example (a) is BD-CH1-TD, where BD= an antibody VH domain and CH1 is an antibody CH1 domain. In an embodiment, this polypeptide is paired with a second polypeptide comprising or consisting of, in N- to C-terminal direction BD2-CL, wherein BD2=an antibody VL domain, wherein the VH and VL form an antigen binding site and the CH1 pairs with the CL. An optional peptide linker may be between the TD and a domain (eg, the CH1) that is immediately N-terminal to the TD in the polypeptide. Multimerisation of 4 copies of the polypeptide using TDs produces a multimer (ie, tetramer) comprising 4 identical antigen binding sites, see, eg,
For example, in the immediately preceding paragraph BD and BD2 respectively comprise the VH and VL of an antibody selected from REGN10987, REGN10933 and CB6 (see Table 32 for sequences). For example, the multimer comprises the monomer (middle schematic) shown in any of
For example, the effector domain or binding domain or binding site of a polypeptide herein comprises the VH and/or VL of an antibody selected from REGN10987, REGN10933, CB6, rRBD-15 (ABLINK Biotech Co., Ltd / Chengdu Medical College), B38, H4 (Capital Medical University, Beijing), FYB-207 (Formycon AG), ABP300 (Abpro Corporation), BRII-198 (Brii Biosciences, TSB Therapeutics (Beijing) CO.LTD), BRII-196 (Brii Biosciences, TSB Therapeutics (Beijing) CO.LTD), CT-P59 (Celltrion), HFB-3013, or HFB30132A (HiFiBiO Therapeutics), MW33 (Mabwell), SAB-185 (SAB Biotherapeutics), Etesevimab (Junshi Biosciences), SCTA01 or H014 (University of Chinese Academy of Sciences), STI-1499 or COVI-GUARD (Sorrento Therapeutics), TY027 (Tychan), COVI-AMG™ or STI-2020 (Sorrento Therapeutics), HLX70 (Hengenix Biotech Inc), ADM03820 (Ology Bioservices), an antibody comprised by XAV-19 (Nantes University Hospital), BGB DXP-593 or DXP-604 (BeiGene), VIR-7831 or GSK4182136 (Vir Biotechnology, GSK), AZD8895 or AZD1061 (AstraZeneca), HBM9022 or 47D11 (AbbVie, Harbour BioMed, Utrecht University and Erasmus Medical Center), Ab8 (University of Pittsburgh), MAbCo19 (AchilleS Vaccines Srl), AR-701 or AR-711 (Aridis Pharmaceuticals), DXP-604 (BeiGene), Centi-B9 (Centivax), GIGA-2050 (GigaGen), TATX-03 or TATX-06 or TATX-09 or TATX-13 or TATX-16 (ImmunoPrecise Antibodies), MTX-COVAB (Memo Therapeutics), NOVOAB-20 (NovoAb), COVI-SHIELD (Sorrento Therapeutics), STI-4920 or ACE-MAB or CMAB020 (MabPharm), IDB003 (IDBiologics) and VIR-7832 (Vir Biotechnology, GSK).
For example, the multimer herein comprises at least 4 copies (eg, 4, 8, 12, 16, 20, 24 or 28 copies) of the VH and/or VL of an antibody selected from REGN10987, REGN10933, CB6, rRBD-15 (ABLINK Biotech Co., Ltd / Chengdu Medical College), B38, H4 (Capital Medical University, Beijing), FYB-207 (Formycon AG), ABP300 (Abpro Corporation), BRII-198 (Brii Biosciences, TSB Therapeutics (Beijing) CO.LTD), BRII-196 (Brii Biosciences, TSB Therapeutics (Beijing) CO.LTD), CT-P59 (Celltrion), HFB-3013, or HFB30132A (HiFiBiO Therapeutics), MW33 (Mabwell), SAB-185 (SAB Biotherapeutics), Etesevimab (Junshi Biosciences), SCTA01 or H014 (University of Chinese Academy of Sciences), STI-1499 or COVI-GUARD (Sorrento Therapeutics), TY027 (Tychan), COVI-AMG™ or STI-2020 (Sorrento Therapeutics), HLX70 (Hengenix Biotech Inc), ADM03820 (Ology Bioservices), an antibody comprised by XAV-19 (Nantes University Hospital), BGB DXP-593 or DXP-604 (BeiGene), VIR-7831 or GSK4182136 (Vir Biotechnology, GSK), AZD8895 or AZD1061 (AstraZeneca), HBM9022 or 47D11 (AbbVie, Harbour BioMed, Utrecht University and Erasmus Medical Center), Ab8 (University of Pittsburgh), MAbCo19 (AchilleS Vaccines Srl), AR-701 or AR-711 (Aridis Pharmaceuticals), DXP-604 (BeiGene), Centi-B9 (Centivax), GIGA-2050 (GigaGen), TATX-03 or TATX-06 or TATX-09 or TATX-13 or TATX-16 (ImmunoPrecise Antibodies), MTX-COVAB (Memo Therapeutics), NOVOAB-20 (NovoAb), COVI-SHIELD (Sorrento Therapeutics), STI-4920 or ACE-MAB or CMAB020 (MabPharm), IDB003 (IDBiologics) and VIR-7832 (Vir Biotechnology, GSK). The multimer may comprise no more than said number of copies.
In an example, there is a provided a tetramer of an antibody (or a fragment of an antibody, eg, a Fab of an antibody), wherein the tetramer is tetramersised using tetramerization domains (TDs). For example, the tetramer has the configuration shown in the right-hand-side schematic of any one of
A multimer or tetramer herein may have the configuration shown in the any one of the Figures herein (and there may further be other moieties, such as one or more additional peptides, domains or proteins comprised by the multimer or tetramer that are not shown in said Figure). A multimer or tetramer herein may have the configuration shown in the right-hand-side schematic of any one of
There is provided a mixture of at least 2 (eg, 2 or 3) different multimers, wherein each multimer is according to the invention. For example, a first of said multimers comprises 4 copies of an antigen binding site that is capable of binding to a first antigen; and a second of said multimers comprises 4 copies of an antigen binding site that is capable of binding to a second antigen, optionally the antigens are identical and the binding sites bind different epitopes comprised by the antigen, or the antigens are different. For example, the or each antigen is an antigen of a virus, eg, SARS-CoV or SARS-Cov-2 antigen, such as spike antigen, or the virus is influenza virus or any other virus disclosed herein. For example, wherein a first of said multimers comprises 4 copies of the SARS-CoV-2 antigen binding site of REGN10987, and a second of said multimers comprises 4 copies of the SARS-CoV-2 antigen binding site of REGN10933.
For example, the binding site (eg, BD or BD2) of a polypeptide or multimer herein comprises the variable domain of Nb11-59 (Shanghai Novamab Biopharmaceuticals Co., Ltd.), MERS VHH-55, SARS VHH-72 or VHH303 72-Fc. For example, the binding site (eg, BD or BD2) of a polypeptide or multimer herein comprises a Darpin of MP0420 or MP0423.
Optionally, example (a) is BD-CH1-hinge-Fc-TD, where BD= an antibody VH domain and CH1 is an antibody CH1 domain, optionally the hinge is devoid of a core hinge region or is any other hinge disclosed herein and Fc is an antibody Fc region (ie, CH2-CH3). In an embodiment, this polypeptide is paired with a second polypeptide comprising or consisting of, in N- to C-terminal direction BD2-CL, wherein BD2=an antibody VL domain, wherein the VH and VL form an antigen binding site and the CH1 pairs with the CL. An optional peptide linker may be between the TD and a domain (eg, the CH3) that is immediately N-terminal to the TD in the polypeptide. Multimerisation of 4 copies of the polypeptide using TDs produces a multimer (ie, tetramer) comprising 4 identical antigen binding sites, see, eg,
In an example, the invention provides a polypeptide comprising an antigen binding domain (BD), the polypeptide comprising or consisting of, in N- to C-terminal direction BD-Fc-Td, wherein Fc is an antibody Fc region. In an embodiment, there is provided a dimer of first and second copies of such a polypeptide, wherein the Fc of the first polypeptide is associated with the Fc of the second polypeptide. In an embodiment there is a dimer of such a dimer, eg, as shown in
In an example, the invention provides a provides polypeptide comprising an antigen binding domain (BD), the polypeptide comprising or consisting of, in N- to C-terminal direction BD-CH1-Fc-Td, wherein Fc is an antibody Fc region. In an embodiment, there is provided a dimer of first and second copies of such a polypeptide, wherein the Fc of the first polypeptide is associated with the Fc of the second polypeptide. In an embodiment there is a dimer of such a dimer, eg, as shown in
In an example, the invention provides a provides polypeptide comprising an antigen binding domain (BD), the polypeptide comprising or consisting of, in N- to C-terminal direction BD-CH1-Td, wherein CH1 is an antibody CH1. In an embodiment, there is provided a dimer of first and second copies of such a polypeptide, eg, wherein the TDs of the polypeptides are associated together. In an embodiment there is a dimer of such a dimer, eg, as shown in
BD and BD2 may be a VH/VL pair of an antigen binding site of an antibody selected from the group consisting of REGN10987, REGN10933 and CB6.
There is also provided:
Advantageously, as shown in Example 33, a VH or VHH herein may be a VH3 family VH or VHH. As shown in the example, multimerization of such a variable domain can surprisingly produce a multimer of the invention that can be readily purified by binding to protein A. Thus, preferably, the multimer can be devoid of an affinity tag, such as a His tag.
Herein, any Fc may be a human antibody Fc. Herein, an Fc may be a gamma antibody Fc, mu antibody Fc, delta antibody Fc, epsilon antibody Fc or alpha antibody Fc, preferably a gamma (eg, gamma-1, gamma-2, gamma-3 or gamma-4) antibody Fc (preferably a gamma-1 antibody Fc).
Optionally, the invention provides a protein multimer comprising the configuration of ACE2-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of ACE2 monomeric Ig-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of ACE2 dimer-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of ACE2- Ig-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-Heavy Chain Only -TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-Ig-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-Fab-like -TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-Fab-like monomeric Ig-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of Dimeric-TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-Fab′-like -TD shown in
Optionally, the invention provides a protein multimer comprising the configuration of BD-monomeric Ig-TD shown in
Optionally, BD-TD denotes the binding domain directly linked N-terminal to the TD.
Optionally, TD-BD denotes the binding domain directly linked C-terminal to the TD.
Optionally, BD-BD denotes the binding domains directly linked to each other.
Optionally, 2 copies of the polypeptide are associated or joined together so that the N- and C-termini of each polypeptide is not directly joined to the other polypeptide to form a polypeptide dimer (eg, see
The BD or binding domain herein may be a binding domain disclosed in Table 23, eg, QB-GB, QB-BG or QB-FE (see Table 23 for sequences), or as disclosed in Table 32. The VH/VL pair may be a VH/VL pair of antibody CR3022: CR3022 VH (ie, QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMGIIYPGDSETR YSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAGGSGISTPMDVWGQGTTVTV) paired with CR3022 VL (ie, DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIK).
In an alternative, instead of BD, the polypeptide comprises or contains a peptide (eg, an insulin peptide or a superantigen peptide or domain) or a receptor (eg, ACE2 ECD). See, eg,
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 copies of a binding domain or a peptide. For example, the binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 8 copies of a binding domain or a peptide. For example, the binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 16 copies of a binding domain or a peptide. For example, the binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 20 copies of a binding domain or a peptide. For example, the binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 24 copies of a binding domain or a peptide. For example, the binding domain may be any binding domain disclosed herein.
Herein, by “comprising” said number of copies of the binding domain or peptide, the multimer may, for example, comprise no more than said number of the domain or peptide. For example, the multimer may contain exactly said number of copies of the binding domain or peptide.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 copies of a first binding domain or a peptide; and 4 copies of a second binding domain or a peptide, wherein the first and second binding domains are different (eg, they bind to different antigens of a virus, eg, a coronavirus or HIV or influenza, or immune checkpoint antigens, or cytokine antigens, or growth factor antigens, or venom (eg, snake venom) antigens). For example, the first binding domain may be any binding domain disclosed herein. For example, the second binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 or 8 copies of a first binding domain or a peptide; and 4 copies of a second binding domain or a peptide, wherein the first and second binding domains are different (eg, they bind to different antigens of a virus, eg, a coronavirus or HIV or influenza, or immune checkpoint antigens, or cytokine antigens, or growth factor antigens, or venom (eg, snake venom) antigens). For example, the first binding domain may be any binding domain disclosed herein. For example, the second binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 or 8 copies of a first binding domain or a peptide; and 8 copies of a second binding domain or a peptide, wherein the first and second binding domains are different (eg, they bind to different antigens of a virus, eg, a coronavirus or HIV or influenza, or immune checkpoint antigens, or cytokine antigens, or growth factor antigens, or venom (eg, snake venom) antigens). For example, the first binding domain may be any binding domain disclosed herein. For example, the second binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 copies of a first binding domain or a peptide; 4 copies of a second binding domain or a peptide; and 4 copies of a third binding domain or a peptide, wherein the first, seond and third binding domains are different from each other (eg, they bind to different antigens of a virus, eg, a coronavirus or HIV or influenza, or immune checkpoint antigens, or cytokine antigens, or growth factor antigens, or venom (eg, snake venom) antigens). For example, the first binding domain may be any binding domain disclosed herein. For example, the second binding domain may be any binding domain disclosed herein. For example, the third binding domain may be any binding domain disclosed herein.
The invention comprises a multimer (eg, a multimer described elsewhere herein) comprising 4 copies of a first binding domain or a peptide; 4 copies of a second binding domain or a peptide; 4 copies of a third binding domain or a peptide; and 4 copies of a fourth binding domain or a peptide, wherein the first, second, third and fourth binding domains are different from each other (eg, they bind to different antigens of a virus, eg, a coronavirus or HIV or influenza, or immune checkpoint antigens, or cytokine antigens, or growth factor antigens, or venom (eg, snake venom) antigens). For example, the first binding domain may be any binding domain disclosed herein. For example, the second binding domain may be any binding domain disclosed herein. For example, the third binding domain may be any binding domain disclosed herein. For example, the fourth binding domain may be any binding domain disclosed herein.
Optionally, the multimer comprises said number of first and second binding domains. Optionally, the multimer comprises said number of first and second peptides.
Optionally, the multimer comprises mammalian cell (eg, human cell) glycosylation.
Optionally, the multimer binds to the antigen with an affinity of less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15 or 10 pM (preferably less than 40 or 20 pM) in an ELISA assay, such as an ELISA assy disclosed herein. Optionally, the multimer binds to the antigen with an affinity of less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15 or 10 pM (preferably less than 40 or 20 pM) in an SPR assay, such as an SPR assy disclosed herein. In an embodiment of these options, the multimer comprises or contains 16 copies of a binding domain or peptide.
Optionally, the multimer neutralises the antigen with an IC50 of less than 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 nM (preferably from 0.06 to 0.01 nM) in an ELISA assay, such as an ELISA assay disclosed herein. Optionally, the multimer neutralises the antigen with an IC50 of less than 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 nM (preferably from 0.06 to 0.01 nM) in an SPR assay, such as an SPR assay disclosed herein. In an embodiment of these options, the multimer comprises or contains 16 copies of a binding domain or peptide.
In an embodiment, the multimer comprises anti-coronavirus (eg, SARS-Cov-2) spike protein binding sites or receptor peptides, wherein the multimer binds to spike trimer or spike RBD. For example, the binding is with an affinity of less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15 or 10 pM (preferably less than 40 or 20 pM) in an SPR assay, such as an SPR assay disclosed herein; or in an ELISA assay, such as an ELISA assay disclosed herein (eg, an assay as disclosed in Example 28). See, eg, Example 28.
An antigen binding domain or site comprised by a polypeptide or multimer of the invention may be any binding domain or binding site selected from those disclosed herein (eg, any VH, VL, dAb, VHH or scFv) or may be a binding domain or binding site that comprises an amino acid sequence that is at least 70, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the amino acid sequence of said selected domain or site.
An antigen binding domain or site comprised by a polypeptide or multimer of the invention may be any binding domain or binding site selected from those disclosed herein (eg, any VH, VL, dAb, VHH or scFv) or may be a binding domain or binding site that comprises an amino acid sequence that is identical to the amino acid sequence of said selected domain or site except for 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid differences (eg, conseravative amino acid changes).
An antigen binding domain or site comprised by a polypeptide or multimer of the invention may be a domain or site that competes with any binding domain or binding site selected from those disclosed herein (eg, any VH, VL, dAb, VHH or scFv) for binding to the antigen. Competition may be determined by a standard competition assay, such as an SPR competition assay or an ELISA assay.
A polypeptide of the invention may have a configuration shown for a polypeptide in any of the figures herein. A multimer (eg, polyeptide dimer or tetramer) of the invention may have a configuration shown for a multimer in any of the figures herein.
In a configuration, the invention provides:
In an alternative, the multimer comprises or contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 copies of the peptide or binding site. Preferably, the multimer comprises or contains 4, 8, 12, 16, 20, 24, 28, 32 or 36 copies of the peptide or binding site. For example, the binding site is a VH/VL pair comprising VH-ICC paired with VL-ICC or VH-IHG paired with VL-IHG. For example, the binding site comprises QB-GB, QB-DD, QB-BG or QB-FE.
Optionally, the multimer comprises or contains 16 copies of the peptide or binding site.
Optionally, the multimer comprises (i) 4 copies of a polypeptide, wherein each polypeptide copy comprises a tetramerization domain and 2 or more (eg, 2, 3 or 4) copies of the peptide or binding site; or (ii) 4 copies of a dimer of a polypeptide, wherein each polypeptide copy comprises a tetramerization domain and 1 or more (eg, 2) copies of the peptide or binding site.
Optionally, the polypeptide comprises a self-assembly multimerization domain (SAM) (preferably a tetramerization domain (TD)) and one or more copies of an antigen binding site (BD), the polypeptide comprising or consisting of, in N- to C-terminal direction
Optionally, the BD is a single variable domain.
Optionally, the BD comprises the amino acid sequence of QB-GB (SEQ ID NO: 307), QB-DD, QB-BG or QB-FE.
Optionally, the polypeptide comprises, in N- to C-terminal direction, BD-CH1-TD, BD-CL-TD, BD-CH1-Fc-TD or BD-Fc-TD, where BD is an antibody V domain (eg, a VH), Fc is an antibody Fc region, and CH1 is an antibody CH1 domain; and optionally each BD-CH1-TD or BD-CH1-Fc-TD polypeptide of the multimer is paired with a respective second polypeptide, wherein the second polypeptide comprises, in N- to C-terminal direction BD2-CL, wherein BD2 is an antibody V domain (eg, a VL or single variable domain), wherein the CH1 pairs with the CL.
In an example, tandam dAbs are provided N-termial to the TD, preferably at the N-terminus of the polypeptide. For example the polypeptide comprises, in N- to C-terminal direction, BD′-optionaly linker-BD-CH1-TD, BD′-optionaly linker-BD -CL-TD, BD′-optionaly linker-BD -CH1-Fc-TD or BD′-optionaly linker-BD -Fc-TD, wherein each of BD and BD′ is an antibody single variable domain (eg, a nanobody), Fc is an antibody Fc region, and CH1 is an antibody CH1 domain. BD′ and BD may be the same or different (eg, comprising different antigen specificities).
In an example, the dimer comprises a first polypeptide comprising, in N- to C-terminal direction, BD-hinge-TD; BD′-optional linker-BD-Hinge-TD; or BD-optional linker-CH1-Hinge-TD. Optionally the first polypeptide is associated with a second polypeptide. In an embodiment, the second polypeptide comprises, in N- to C-terminal direction, BD″-optional Linker 1-BD-optional linker 2-CL (kappa or lambda) (eg, BD″- Linker 1-BD-optional linker 2-CL; BD″-BD-optional linker-CL; or BD″-BD-CL), wherein the first polyeptide comprises a CH1 domain that is paired with the CL, and wherein each of BD and BD′ is an antibody single variable domain (eg, a nanobody).
Optionally, (i) each of BD and BD2 is an antibody single variable domain; or (ii) BD1 is an antibody VH domain and BD2 is an antibody VL domain, wherein the VH and VL form a VH/VL pair comprising an antigen binding site.
Optionally, the polypeptide comprises, in N- to C-terminal direction, BD-CH1-Fc-TD or BD-CH1-Linker-Fc-TD (optionally wherein the Linker is an antibody hinge, wherein the hinge is devoid of a core hinge region).
In configuration, the invention further provides:-
Optionally, the (or the first) polypeptide comprises, in N- to C-terminal direction, BD-TD.
Optionally, the 2 copies of the polypeptide are disulphide bonded together in the dimer.
The invention also provides:
A protein dimer containing 2 copies of a polypeptide recited herein, wherein the polypeptide comprises BD-CH1-Fc-TD, wherein the Fc regions of the polypeptides associate with each other to form the dimer.
The invention also provides:
A multimer comprising or containing 4 copies of the dimer of the invention.
The invention also provides:
A polypeptide as recited for the multimer or dimer of the invention.
Optionally, the polypeptide is isolated or recombinant.
The multimer, dimer or polypeptide may be comprised by a medical or sterile container, eg, a syringe, vial, IV bag, container connected to a needle or a subcutaneous injection administration device.
Optionally, the antigen is a viral antigen, bacterial antigen, fungal antigen, toxin antigen, venom antigen, immune checkpoint protein antigen, cytokine antigen, growth factor antigen, hormone antigen (eg, chorionic gonadotropin), sugar antigen, lipid antigen or protein antigen.
Optionally, BD and BD2 are different from each other and each comprises a binding site for an antigen of a virus, an antigen of a bacterium, an antigen of a fungus, an antigen of a toxin, an antigen of a venom, an antigen of an immune checkpoint protein, an antigen of a cytokine, an antigen of a growth factor antigen or an antigen of a hormone; optionally wherein both BD and B2 comprises a binding site for a virus.
Optionally, the multimer binds to the antigen with an affinity of less than 200 pM in an ELISA assay; and/or the multimer neutralises the antigen with an IC50 of less than 0.2 nM in an ELISA assay.
Optionally, the multimer is capable of detectably binding to anti-first antigen antibodies (optionally anti-SARS-Cov-2 spike antibodies) in an ELISA assay, wherein detection of the multimer binding is measured by OD450 and the assay comprises
Optionally, the dilution is 1000 to 1,000,000, 100,000 or 10000-fold (preferably 10,000 to 100,000-fold).
The invention provides:
A method of detecting the presence of anti-first antigen antibodies (eg, anti-SARS-Cov-2 spike antibodies) in a bodily fluid sample of a human or animal, the method comprising carrying out an ELISA assay, and the assay comprises
Optionally, the presence of anti-first antigen antibodies in the sample is detected when the optical density (eg, OD450) is greater than 0.1 or 0.5 (optionally, greater than 1, 1.5 or 2) in the assay. Optionally, the dilution is 1000 to 1,000,000, 100,000 or 10000-fold (preferably 10,000 to 100,000-fold).
Optionally, the binding site is
Optionally, the multimers are immobilised on a solid surface; or the first antigen is immobilised on a solid surface.
Optionally, determining optical density (eg, OD450) comprises labelling complexes comprising spike protein and multimers with horseradish peroxidase (HRP) and detecting the label (eg, at a wavelength of 450 nm).
For the multimer, dimer, polypeptide or method, each multimer may comprise a polypeptide; or variable domain or binding site amino acid disclosed herein.
The invention provides:
A multimer comprising 4 copies of a binding site for an antigen, wherein the multimer comprises a dimer of an antibody or a dimer of an antigen binding fragment (eg, Fab) of an antibody, optionally wherein the multimer is according to any preceding claim. The antibody can be any antibody disclosed herein, eg, an antibody selected from REGN10987, REGN10933, CB6, rRBD-15, B38, H4, FYB-207, ABP300, BRII-198, BRII-196, CT-P59, HFB-3013, HFB30132A, MW33, SAB-185, Etesevimab, SCTA01, H014, STI-1499, COVI-GUARD™, TY027, COVI-AMG™, STI-2020, HLX70, ADM03820, an XAV-19 antibody, BGB DXP-593, DXP-604, VIR-7831, GSK4182136, AZD8895, AZD1061, HBM9022, 47D11, Ab8, MAbCo19, AR-701, AR-711, DXP-604, Centi-B9, GIGA-2050, TATX-03, TATX-06, TATX-09, TATX-13, TATX-16, NOVOAB-20, COVI-SHIELD™, STI-4920, ACE-MAB, CMAB020, IDB003 and VIR-7832.
The multimer may be a tetramer having a configuration shown in the right-hand-side schematic of any one of
Optionally,
The invention provides:
A pharmaceutical composition or assay reagent comprising a plurality of multimers of the invention, optionally wherein the reagent comprises said multimers immobilised on a solid support.
A multimer of the invention (or a combination of at least 2 or 3 multimers of the invention claim) for administration to a human or animal subject for medical use.
There is provided a composition comprising a multimer of the invention, eg, for medical use or for use in vitro.
A VH herein may be a VH encoded by a VH DNA sequence shown in Table 21(b) and a VL herein may be a VL encoded by the cognate VL DNA sequence shown in Table 21(b), wherein the VH and VL form an antigen binding VH/VL pair (eg, that is capable of binding to a SARS-CoV-2 antigen, such as spike antigen). Alternatively, the VH sequence is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VH DNA sequence; and/or the VL sequence is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VL DNA sequence. In an example, the VH DNA sequence is VH-ICC (see Table 21(b)) or a VH sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VH-ICC DNA sequence; and the VL DNA sequence is VL-ICC (see Table 21(b)) or a VL sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VL-ICC DNA sequence. In an example, the VH DNA sequence is VH-IHG (see Table 21(b)) or a VH sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VH-IHG DNA sequence; and the VL DNA sequence is VL-IHG (see Table 21(b)) or a VL sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said VL-IHG DNA sequence.
A VH herein may comprise the amino acid sequence of SEQ ID NO: 288 or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 288. Preferably, such a VH is unpaired with a second variable domain (eg, a VL), since the VH in this instance is a single variable domain, it is able to bind to a SARS-Cov-2 antigen (eg, spike) without requirement for pairing.
A VH herein may comprise antibody single variable domain Nb11-59 (Novamab Biopharmaceuticals Co. Ltd) or an antibody single variable domain of ALX-0171 (Ablynx). A VH herein may comprise antibody single variable domain comprising SEQ ID NO: 203, or an antibody single variable domain comprising amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to the amino acid sequence of Nb11-59 or SEQ ID NO: 293.
Optionally, the combination comprises first and second multimers of the invention, wherein the first multimer comprises a binding site comprising a first VH/VL pair and the second multimer comprises a second VH/VL pair which is different from the first VH/VL pair. In an example, the VH of the first VH/VL pair is encoded by the DNA sequence of VH-ICC and the VL is encoded by the DNA sequence of VL-ICC. In an example, the VH of the first VH/VL pair is encoded by the DNA sequence of VH-IHG and the VL is encoded by the DNA sequence of VL-IHG.
In an example, the VH of the first VH/VL pair is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VH-ICC and the VL is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VL-ICC. In an example, the VH of the first VH/VL pair is encoded by the DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VH-IHG and the VL is encoded by the DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VL-IHG.
Optionally, the multimer comprises first and second antigen binding sites which are different from each other. For example, the first binding site comprises a first VH/VL pair and the second binding site comprises a second VH/VL pair. In an example, the VH of the first VH/VL pair is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to a first VH DNA sequence disclosed in Table 21(b) and the VL is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the cognate VL DNA sequence disclosed in Table 21(b); and the VH of the second VH/VL pair is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to a second VH DNA sequence disclosed in Table 21(b) and the VL is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the cognate VL DNA sequence disclosed in Table 21(b), wherein the first and second VH DNA sequences are different. For example, the first VH DNA sequence is the sequence of VH-IHI (see Table 21(b)), the cognate VH DNA sequence is VL-IHI; and the second VH DNA sequence is the sequence of VH-IHG, VH-ICC, VH-ICD, VH-IGG, VH-IFD, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IHG (see Table 21(b)), the cognate VH DNA sequence is VL-IHG; and the second VH DNA sequence is the sequence of VH-IHI, VH-ICC, VH-ICD, VH-IGG, VH-IFD, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-ICC (see Table 21(b)), the cognate VH DNA sequence is VL-ICC; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHQ, VH-ICD, VH-IGG, VH-IFD, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-ICD (see Table 21(b)), the cognate VH DNA sequence is VL-ICD; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHI, VH-ICC, VH-IGG, VH-IFD, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IGG (see Table 21(b)), the cognate VH DNA sequence is VL-IGG; and the second VH DNA sequence is the sequence of VH-ICC, VH-ICD, VH-IHI, VH-IFD, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IFD (see Table 21(b)), the cognate VH DNA sequence is VL-IFD; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHI, VH-ICC, VH-ICD, VH-GG, VH-IED, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IED (see Table 21(b)), the cognate VH DNA sequence is VL-IED; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHI, VH-ICC, VH-ICD, VH-IGG, VH-IFD, VH-IHD or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IHD (see Table 21(b)), the cognate VH DNA sequence is VL-IHD; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHI, VH-ICC, VH-ICD, VH-GG, VH-IFD, VH-IED, or VH-IHF. For example, the first VH DNA sequence is the sequence of VH-IHF (see Table 21(b)), the cognate VH DNA sequence is VL-IHF; and the second VH DNA sequence is the sequence of VH-IHG, VH-IHI, VH-ICC, VH-ICD, VH-GG, VH-IFD, VH-IED or VH-IHD. Preferably, the second VH DNA sequence is VH-ICC or VH-IHG.
Optionally, the multimer comprises first and second antigen binding sites which are different from each other. For example, the first binding site comprises a first VH/VL pair and the second binding site comprises a second VH/VL pair. In an example, the VH of the first VH/VL pair is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VH-ICC and the VL is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VL-ICC; and the VH of the second VH/VL pair is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VH-IHG and the VL is encoded by a DNA sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical (or 100% identical) to the DNA sequence of VL-IHG. For example, the multimer comprises (i) at least 4, 8, 12, 16, 20, 24 or 28 (optionally no more than 4, 8, 12, 16, 20, 24 or 28 respectively) copies of a further antibody variable domain that is different from the domain of (i). For example, each domain is capable of specifically binding to a SARS-CoV-2 antigen.
In an embodiment, the multimer comprises a tetramer of a polyeptide dimer, wherein the dimer comprises a first polypeptide associated with a second polypeptide, wherein the first polypeptide comprises at least one copy of a first peptide or a first antigen binding site and a teramerisation domain (TD), the second polypeptide comprises at least one copy of a second peptide or a second antigen binding site and a teramerisation domain (TD).
Preferably, the TDs of the first and second polypeptides are identical. Optionally, the TDs are p53 TDs, such as human p53 TDs. In one embodiment, each of the first and second polypeptides comprises a said peptide and the first and second peptides are different. In one embodiment, each of the first and second polypeptides comprises a said peptide and the first and second peptides are the same. In one embodiment, each of the first and second polypeptides comprises a said binding site and the first and second binding sites are different.
In one embodiment, each of the first and second polypeptides comprises a said binding site and the first and second binding sites are the same. In an example, in each dimer the first and second polypeptides are disulphide bonded together.
In an example, in each dimer the first polypeptide comprises an antibody CH3 domain (eg, a CH2-CH3) and the second polypeptide comprises an antibody CH3 domain (eg, a CH2-CH3), wherein the CH3 domains associate together to form said dimer. For example, each of the first and second polypeptides comprises, in N- to C-terminal direction, a peptide or antigen binding site -TD -optional CH2 domain -CH3 domain, wherein the CH3 domains are associated together. For example, each of the first and second polypeptides comprises, in N- to C-terminal direction, a peptide or antigen binding site - optional CH2 domain - CH3 domain - TD, wherein the CH3 domains are associated together.
“Knobs into holes” technology for making bispecific antibodies was described in [1] and in US5,731,168, both incorporated herein by reference. The principle is to engineer paired CH3 domains of heterodimeric heavy chains so that one CH3 domain contains a “knob” and the other CH3 domains contains a “hole” at a sterically opposite position. Knobs are created by replacing small amino acid side chain at the interface between the CH3 domains, while holes are created by replacing large side chains with smaller ones. The knob is designed to insert into the hole, to favour heterodimerisation of the different CH3 domains while destabilising homodimer formation. In in a mixture of antibody heavy and light chains that assemble to form a bispecific antibody, the proportion of IgG molecules having paired heterodimeric heavy chains is thus increased, raising yield and recovery of the active molecule
Mutations Y349C and/or T366W may be included to form “knobs” in an IgG CH3 domain. Mutations E356C, T366S, L368A and/or Y407V may be included to form “holes” in an IgG CH3 domain. Knobs and holes may be introduced into any human IgG CH3 domain, e.g., an IgG1, IgG2, IgG3 or IgG4 CH3 domain. A preferred example is IgG4. The IgG4 may include further modifications such as the “P” and/or “E” mutations. A “P” substitution at position 228 in the hinge (S228P) stabilises the hinge region of the heavy chain. An “E” substitution in the CH2 region at position 235 (L235S) abolishes binding to FcyR. A bispecific antibody of the present invention may contain an IgG4 PE human heavy chain constant region, optionally comprising two such paired constant regions, optionally wherein one has “knobs” mutations and one has “holes” mutations.
While knobs-into-holes technology involves engineering amino acid side chains to create complementary molecular shapes at the interface of the paired CH3 domains in the bispecific heterodimer, another way to promote heterodimer formation and hinder homodimer formation is to engineer the amino acid side chains to have opposite charges. Association of CH3 domains in the heavy chain heterodimers is favoured by the pairing of oppositely charged residues, while paired positive charges or paired negative charges would make homodimer formation less energetically favourable. WO2006/106905 described a method for producing a heteromultimer composed of more than one type of polypeptide (such a heterodimer of two different antibody heavy chains) comprising a substitution in an amino acid residue forming an interface between said polypeptides such that heteromultimer association will be regulated, the method comprising:
An example of this is to suppress association between heavy chains by introducing electrostatic repulsion at the interface of the heavy chain homodimers, for example by modifying amino acid residues that contact each other at the interface of the CH3 domains, including:
By modifying one or more of these pairs of residues to have like charges (both positive or both negative) in the CH3 domain of a first heavy chain, the pairing of heavy chain homodimers is inhibited by electrostatic repulsion. By engineering the same pair or pairs of residues in the CH3 domain of a second (different) heavy chain to have an opposite charge compared with the corresponding residues in the first heavy chain, the heterodimeric pairing of the first and second heavy chains is promoted by electrostatic attraction.
In one example, amino acids at the heavy chain constant region CH3 interface of the dimer of the invention are modified to introduce charge pairs, the mutations being listed in Table 1 of WO2006/106905. It was reported that modifying the amino acids at heavy chain positions 356, 357, 370, 399, 409 and 439 to introduce charge-induced molecular repulsion at the CH3 interface had the effect of increasing efficiency of formation of the intended bispecific antibody. WO2006/106905 also exemplified bispecific IgG antibodies in which the CH3 domains of IgG4 were engineered with knobs-into-holes mutations.
Further examples of charge pairs are disclosed in WO2013/157954, which described a method for producing a heterodimeric CH3 domain-comprising molecule from a single cell, the molecule comprising two CH3 domains capable of forming an interface. The method comprised providing in the cell
Further methods of engineering electrostatic interactions in polypeptide chains to promote heterodimer formation over homodimer formation were described in WO2011/143545.
Another example of engineering at the CH3-CH3 interface that can be used in the dimer of the invention is strand-exchange engineered domain (SEED) CH3 heterodimers. The CH3 domains are composed of alternating segments of human IgA and IgG CH3 sequences, which form pairs of complementary SEED heterodimers referred to as “SEED-bodies” [2; WO2007/110205].
Bispecifics have also been produced with heterodimerised heavy chains that are differentially modified in the CH3 domain to alter their affinity for binding to a purification reagent such as Protein A. WO2010/151792 described a heterodimeric bispecific antigen-binding protein comprising
Thus, in the dimer of the present invention, the CH3 of one (but not the other) of the first and second polypeptides comprises a modification that reduces or eliminates binding of the respective CH3 domain to Protein A.
Dimers and antibodies of the present invention may employ any of these techniques and molecular formats as desired.
Optionally, each of the first and second polypeptides comprises an antigen binding site, wherein each binding site is an antibody single variable domain (eg, a VHH or nanobody).
Optionally, the dimer comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide is associated with the first polypeptide, and the fourth polypeptide is associated with the second polypeptide, wherein each polyeptide comprises an antibody variable domain, wherein (i) the variable domain of the first polypeptide is paired with the variable domain of the third polypepeptide to form a first VH/VL binding site for binding a first antigen; (ii) and the variable domain of the second polypeptide is paired with the variable domain of the fourth polypepeptide to form a second VH/VL binding site for binding a second antigen. Preferably, the first antigen is different from the second antigen. In an alternative, the first and second antigens are the same. In an example, the variable domain of the first polypeptide is a VH and the variable domain of the third polypeptide is a VL. In an example, the variable domain of the first polypeptide is a VL and the variable domain of the third polypeptide is a VH. In an example, the variable domain of the second polypeptide is a VH and the variable domain of the fourth polypeptide is a VL. In an example, the variable domain of the second polypeptide is a VL and the variable domain of the fourthe polypeptide is a VH.
For example, the first polypeptide comprises a CH1 domain that associates with a CL domain that is comprised by the third polypeptide and/or the second polypeptide comprises a CH1 domain that associates with a CL domain that is comprised by the fourth polypeptide.
For example, (i) the first polypeptide comprises, in N- to C-terminal direction, a peptide or a variable domain of a first VH/VL antigen binding site - CH1 domain - optional hinge region -TD - [a Fc region comprising a CH2 domain and aCH3 domain]; and (ii) the second polypeptide comprises, in N- to C-terminal direction, a peptide or a variable domain of a second VH/VL antigen binding site - CH1 domain - optional hinge region -TD - [a Fc region comprising a CH2 domain and a CH3 domain], wherein the CH3 domains of the first and second polypeptides are associated together. In this example, (i) the third polypeptide comprises, in N- to C-terminal direction, a peptide or a variable region of the first antigen binding site - CL; (ii) the fourth polypeptide comprises, in N- to C-terminal direction, a peptide or a variable region of the second antigen binding site - CL, wherein (iii) said variable domains or the first and third polypeptides form the first VH/VL binding site (eg, wherein the variable domain of the first polypeptide is a VH and the variable domain of the third polypeptide is a cognate VL), (iv) said variable domains of the second and fourth polypeptides form the second VH/VL binding site (eg, wherein the variable domain of the second polypeptide is a VH and the variable domain of the fourth polyeptide is a cognate VL), (v) the CH1 of the first polypeptide is associated with the CL of the third polypeptide, (vi) the CH1 of the second polypeptide is associated with the CL of the fourth polypeptide, and (vii) the Fc of the first polyeptide is associated with the Fc of the seond polypeptide (eg, the CH3 domains are associated together).
Optionally, the Fc regions (or CH3 domains) of the first and second polypeptides are associated together using knob-in-hole technology or charge pairing.
The multimer of the invention may be a multimer for administration to a human or animal subject for treatment or prevention of a disease or condition (eg, an infection by the first and/or second virus, or a symptom of such an infection (eg, an unwanted inflammatory response)) in the subject.
The invention provides:
A method for the treatment or prevention of a disease or condition (eg, an infection by the first and/or second virus, or a symptom of such an infection (eg, an unwanted inflammatory response)) in a human or animal subject, the method comprising administering to the subject a plurality of multimers of the invention.
An assay kit comprising an assay reagent as mentioned above and an amount of the first antigen (eg, viral spike protein), optionally wherein the reagent and protein are comprised by different containers.
A method for detecting the presence of an antigen in a sample, the method comprising combining the sample with a multimer of the invention, allowing antigen in the sample to bind multimers to form antigen/multimer complexes and detecting antigen/multimer complexes.
A method of expanding a utility of an antigen (eg, a protein) binding site, the method comprising producing a multimer of the invention, wherein the multimer comprises a plurality of copies (eg, at least 8 or 16 copies) of the binding site.
Optionally for the multimer, dimer, polypeptide, method, kit or composition, the multimer comprises a tetramer of a polyeptide dimer, wherein the dimer comprises a first polypeptide associated with a second polypeptide, wherein the first polypeptide comprises at least one copy of a first peptide or a first antigen binding site and a teramerisation domain (TD), the second polypeptide comprises at least one copy of a second peptide or a second antigen binding site and a teramerisation domain (TD).
Optionally, each of the first and second polypeptides comprises a said binding site and the first and second binding sites are different.
Optionally, in each dimer the first polypeptide comprises an antibody CH3 domain (eg, a CH2-CH3) and the second polypeptide comprises an antibody CH3 domain (eg, a CH2-CH3), wherein the CH3 domains associate together to form said dimer.
Optionally, each of the first and second polypeptides comprises, in N- to C-terminal direction, (i) a peptide or antigen binding site -TD - optional CH2 domain -CH3 domain, wherein the CH3 domains of the first and second polypeptides are associated together; or (ii) a peptide or antigen binding site - optional CH2 domain - CH3 domain - TD, wherein the CH3 domains of the first and second polypeptide are associated together.
Optionally, each of the first and second polypeptides comprises an antigen binding site, wherein each binding site is an antibody single variable domain (eg, a VHH or nanobody).
Optionally, the dimer is associated with a third polypeptide and a fourth polypeptide, wherein the third polypeptide is associated with the first polypeptide, and the fourth polypeptide is associated with the second polypeptide, wherein each polyeptide comprises an antibody variable domain, wherein (i) the variable domain of the first polypeptide is paired with the variable domain of the third polypepeptide to form a first VH/VL binding site for binding a first antigen; and (ii) the variable domain of the second polypeptide is paired with the variable domain of the fourth polypepeptide to form a second VH/VL binding site for binding a second antigen.
Optionally, the first and second antigens are different.
Optionally, the variable domain of the first polypeptide is a VH and the variable domain of the third polypeptide is a VL and/or the variable domain of the second polypeptide is a VH and the variable domain of the fourth polypeptide is a VL.
Optionally, (A) (i) the first polypeptide comprises a CH1 domain that associates with a CL domain that is comprised by the third polypeptide and (ii) the second polypeptide comprises a CH1 domain that associates with a CL domain that is comprised by the fourth polypeptide; or (B) (i) the first polypeptide comprises a CH1 domain that associates with a CL domain that is comprised by the third polypeptide and (ii) the second polypeptide comprises a CL domain that associates with a CH1 domain that is comprised by the fourth polypeptide.
In an embodiment of option (A), the third and fourth polypeptides are identical. Thus, a common chain or polypeptide is used. Thus, the common polypeptide associates with each of the first and second polypeptides. This may simplify production by requiring only 3, instead of 4 different polypeptides to be expressed together.
Option (B) is useful to reduce chances of undesirable light chain pairing, ie, the fourth polypeptide pairing with the first polypeptide and/or the third polypeptide pairing with the second polypeptide. Thus, having the CH1 in the first polypeptide and the CL in the third polypeptide, this avoids the risk of the third polypeptide pairing with the second polypeptide, since these two polypeptides comprise CL domains that do not pair with each other. Similarly, the CH domains of the first and fourth polypeptides do not pair with each other. This is advantageous for favouring production of multimers of the invention where there is a first/third polypeptide pair and a second/fourth polypeptide pair comprised by each dimer of the multimer. For example (i) the first/third polypeptide pair comprises the following configuration wherin the first polypeptide comprises in N- to C-terminal direction [VH-CL-Hinge-CH2-CH3-TD] paired with the third polypeptide wherein the third polypeptide comprises in N- to C-terminal direction [VL-CH1]; and (ii) the second/fourth polypeptide pair comprises the following configuration wherein the second polyeptide comprises in N- to C-terminal direction [VH-CH1-Hinge-CH2-CH3-TD] paired with the fourth polypeptide wherein the fourth polypeptide comprises in N- to C-terminal direction [VL-CL].
Optionally:
Optionally, the Fc regions of the first and second polypeptides are associated by knob-in-hole or charge pairing technology.
Optionally:
Optionally, (i) the first/third polypeptide pair comprises a configuration wherein the first polypeptide comprises in N- to C-terminal direction [VH-CL-Hinge-CH2-CH3-TD] paired with the third polypeptide comprising in N- to C-terminal direction [VL-CH1]; and (ii) the second/fourth polypeptide pair comprises a wherein the second polyeptide comprises in N- to C-terminal direction [VH-CH1-Hinge-CH2-CH3-TD] paired with the fourth polypeptide wherein the fourth polypeptide comprises in N- to C-terminal direction [VL-CL].
Optionally, the Fc regions of the first and second polypeptides are associated using knob-in-hole technology, wherein (i) the Fc of the first polypeptide comprises a CH3 domain having a knob that associates with a hole of a CH3 domain of the Fc of the second polypeptide; or (ii) the Fc of the first polypeptide comprises a CH3 domain having a hole that associates with a knob of a CH3 domain of the Fc of the second polypeptide.
Optionally, the Fc regions of the first and second polypeptides are associated using charge pairing technology, wherein (i) the Fc of the first polypeptide comprises a first amino acid positive charge that associates with a second amino acid negative charge of the Fc of the second polypeptide; or (ii) the Fc of the first polypeptide comprises a first amino acid negative charge that associates with a second amino acid positive charge of the Fc of the second polypeptide.
In an alternative, the first, but not the second, polypeptide comprises a TD. In another alternative each of the first and second polypeptides are devoid of a TD.
In an alternative, the dimer of the invention may be devoid of a TD, wherein the Fc regions of the first and second polypeptides are associated together in the dimer. For such an embodiment where the dimer is devoid of a TD, all other features of the dimer disclosed herein (in respect of dimers comprsing a TD) are otherwise applicable mutatis mutandis and combinable with the alternative emobidment that is devoid of a TD. For example, the Fc regions may be associated using any technology described herein, such as using knob-in-hole or charge pairing technology. In an embodiment, the first polyeptide comprises a first antigen binding site (eg, a single variable domain or a variable domain that is paired with a variable domain of the third polypeptide (when present) to form a first VH/VL binding site) and/or the second polyeptide comprises a second antigen binding site (eg, a single variable domain or a variable domain that is paired with a variable domain of the fourth polypeptide (when present) to form a second VH/VL binding site).
Optionally, the dimer is an antibody and the first polypeptide is a first heavy chain, the second polypeptide is a second heavy chain, the third polypeptide is a first light chain and the fourth polypeptide is a second light chain. For example, the first and second heavy chains are identical. In an example, they are different (eg, they comprise different peptides or they comprise different antigen binding sites or V domains). For example, the first and second light chains are identical. In an example, they are different (eg, they comprise different different peptides or they comprise different antigen binding sites or V domains). In an embodiment the dimer is a bispecific antibody wherein the first and second heavy chains comprise different binding sites capable of binding a respective antigen and the light chains are identical; alternatively, the light chains are different (eg, V′-CL and V″-CL wherein V′=a first variable domain (eg, a VL, VH or dAb) that is capable (alone or paired with a V domain of the associated heavy chain) of binding a first antigen; and V″= a second variable domain (eg, a VL, VH or dAb) that is capable (alone or paired with a V domain of the associated heavy chain) of binding a second antigen). In an embodiment, a V domain of the first heavy chain is paired with a V domain of the first light chain and is comprised by a first VH/VL binding site that is capable of binding to a first antigen; and/or a V domain of the second heavy chain is paired with a V domain of the second light chain and is comprised by a first VH/VL binding site that is capable of binding to a second antigen. The antigens in this case are different, eg, different antigens of a virus, bacterium or cell.
Optionally each single variable domain is selected from a variable domain disclosed herein. Optionally, each VH/VL binding site is a VH/VL binding site comprised by an antibody disclosed herein (or encoded by VH and VL DNA sequences disclosed herein). Optionally, the antigen is a viral antigen (eg, a coronavirus or SARS-CoV or SARS-Cov-2 antigen, such as RBD or spike antigen) and each single variable domain is an anti-viral antigen variable domain (eg, nanobody or VH or VHH) disclosed herein, such as a variable domain that comprises the amino acid sequence of QB-GB (SEQ ID NO: 307), QB-DD, QB-BG or QB-FE). Optionally, the first and second antigen binding sites are different. Optionally, they are the same. Optionally, the first and second VH/VL sites are different. Optionally, they are the same. Optionally, the first VH/VL site is (i) a VH/VL binding site comprising a VH and a VL encoded by a VH DNA sequence and the cognate VL DNA sequence shown in Table 21(b), or (ii) a VH/VL binding site of any antibody disclosed in Table 21(a). For example, the VH and VL are encoded by VH-ICC and VL-ICC DNA sequences. Optionally, the second VH/VL site is (i) a VH/VL binding site comprising a VH and a VL encoded by a VH DNA sequence and the cognate VL DNA sequence shown in Table 21(b), or (ii) a VH/VL binding site of any antibody disclosed in Table 21(a). For example, the VH and VL are encoded by VH-IHG and VL-IHG DNA sequences. In one embodiment, the first and second VH/VL sites are the same. In another embodiment, they are different.
Optionally, a polypeptide herein (eg, a first; second; third; fourth; first and second; third and fourth; or first, second, third and fourth polypeptide) comprises in N-to C-terminal direction (i) a first antibody single variable domain, a second antibody single variable domain and TD; or a first antibody single variable domain, a second antibody single variable domain and Fc; or (iii) a first antibody single variable domain, a second antibody single variable domain and CH1. Each single variable domain is capable of binding to a respective antigen. Preferably, the antigens are different, although they may be the same. Optionally, the single variable domains are connected by a peptide linker (eg, a (G4S)n linker, wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2 or 3). Optionally, the single variable domains are directly connected together.
Optionally, when the first and second polypeptides of the dimer are associated with the third and fourth polypeptides respectively, the first and third polypeptides comprise a respective variable domain, wherein the variable domains are comprised by a VH/VL pair that is capable of binding a first antigen; and wherein the second polypeptide comprises a first antibody single variable domain that is capable of binding a second antigen; and the fourth polypeptide comprises a second antibody variable domain that is capable of binding a third antigen. In an example, the first antigen is different from the second and third antigens. In an example, the second and third antigens are the same, or they may be different. Each single variable domain (dAb) may, for example, be a nanobody. Each single variable domain (dAb) may, for example, be a human dAb. In an embodiment, (i) the first polypeptide comprises a configuration of, in N- to C-terminal direction, V1-CH1-Hinge-CH2-CH3(with optional knob or first charged amino acid)-TD which is paired with the third polypeptide, wherein the third polypeptide comprises a configuration of, in N- to C-terminal direction, V2-CL, wherein V1 and V2 comprise a first VH/VL pair that is capable of binding to the first antigen (eg, wherein V1=VH and V2=VL; or V1=VL and V2=VH); and (ii) the second polypeptide comprises a configuration of, in N- to C-terminal direction, V3-CH1-Hinge-CH2-CH3(with optional hole that pairs with the knob; or optionally a second charged amino acid that pairs with the first charged amino acid)-TD, wherein the fourth polypeptide comprises a configuration of, in N- to C-terminal direction, V4-CL, wherein each of V3 and V4 is an antibody single variable domain that is capable of binding to a second and third antigen respectively. In an alternatiave, hinge is absent from the first and second polypeptides. Optionally, the first charge is a positive charge and the second charge is a negative charge. Optionally, the second charge is a positive charge and the first charge is a negative charge.
Optionally, wherein the dimer comprises the first and second polypeptides and a third (but not fourth) polypeptide, wherein the first polypeptide is associated with the third polypeptide, the first and third polypeptides comprise a respective variable domain, wherein the variable domains are comprised by a VH/VL pair that is capable of binding a first antigen; and wherein the second polypeptide comprises a first antibody single variable domain that is capable of binding a second antigen. In an example, the first antigen is different from the second antigen. Preferably, the second polypeptide is devoid of a CH1 domain. In an example, the first and second antigens are the same, or they may be different. The single variable domain (dAb) may, for example, be a nanobody. The single variable domain (dAb) may, for example, be a human dAb. In an embodiment, (i) the first polypeptide comprises a configuration of, in N- to C-terminal direction, V1-CH1-Hinge-CH2-CH3(with optional knob or first charged amino acid)-TD which is paired with the third polypeptide, wherein the third polypeptide comprises a configuration of, in N- to C-terminal direction, V2-CL, wherein V1 and V2 comprise a first VH/VL pair that is capable of binding to the first antigen (eg, wherein V1=VH and V2=VL; or V1=VL and V2=VH); and (ii) the second polypeptide comprises a configuration of, in N- to C-terminal direction, V3-Hinge-CH2-CH3 (with optional hole that pairs with the knob; or optionally a second charged amino acid that pairs with the first charged amino acid)-TD, wherein V3 is an antibody single variable domain that is capable of binding to the second antigen. In an alternatiave, hinge is absent from the first and second polypeptides. Optionally, the first charge is a positive charge and the second charge is a negative charge. Optionally, the second charge is a positive charge and the first charge is a negative charge. This arrangement, wherein the second polypeptide is devoid of a CH1, avoids the need for a polypeptide (such as a fourth polypeptide) that pairs with the second polypeptide. Thus, the third polypeptide will pair with the first polypeptide and undesirable pairing of the third polypeptide with the second polypeptide is avoided. This is useful for providing favourable yields of the desired dimer (comprising the first, second and third polypeptides with the third polypeptide paired with the first, but not the second polypeptide) and reduce contamination by the undesired configuration (comprising the first, second and third polypeptides with the third polypeptide paired with the first and the second polypeptides).
A inhalable pharmaceutical composition (or dose of said composition) is provided which comprises particles of any multimer disclosed herein in the size range from 0.5 to 5.0 µm. For example, at least 60% (eg, 60-80% or 60-90%) of particles are in said size range. Optionally, at least 20% (eg, 20-40% or 20-35% or 20-30%) of particles are in the size range >4.7 µm (coarse particles), and optionally in the size range >4.7 µm but no more than 5.0 µm. Optionally, at least 50% (eg, at least 60%, 50-80%, 50-75%, 50-70% 50-65%) of particles are in the size range <4.7 µm (fine particles). Optionally, at least 15% (eg, at least 10%, at least 5%, at least 4, 3, 2 or 1%) of particles are in the size range <1.0 µm (ultra-fine particles). Preferably, the particles are nebulised particles. For example, the composition or dose is comprised by a nebuliser. For example, the composition or dose is comprised by an inhaler. For example, the composition or dose is obtainable by nebulising the multimer, eg, using an Aeroneb Solo™ nebuliser. The composition or dose comprises the multimer and a pharmaceutically acceptable carrier; such carriers for inhalable formulations will be familiar to the skilled addressee.
In a preferred example,
In a preferred example,
In a preferred example,
The composition may comprise particles of the multimer and the median mass aerodynamic particle diameter (MMAD) is 2 to 4.5, eg, 2.5 to 4 or 3 to 3.5 µm.
In an example, the multimer comprises at least 4 copies of an antibody single variable domain, eg, a that specifically binds to a virus antigen, for example a RSV or SARS-CoV-2 antigen, eg, RBD or NTD. For example, the variable domain is CoVnb-112 (also called Nb-112 herein (SEQ ID NO: 288), and the virus antigen is a SARS-CoV-2 antigen), Nb11-59 (Novamab Biopharmaceuticals Co. Ltd, and the virus antigen is a SARS-CoV-2 antigen) or a variable domain of ALX-0171 (Ablynx BV, and the virus antigen is an RSV antigen).
Preferably, the polypeptide herein comprises the amino acid sequence of any one of SEQ ID NOs: 289 to 292. Preferably, the multimer herein comprises 4 copies of a polypeptide that comprises the amino acid sequence of any one of SEQ ID NOs: 289 to 292, or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 289 to 292. The pharmaceutical composition herein may comprise such a multimer, eg, for administration to a human or animal subject for treating or preventing a lung condition. The lung condition may be a lung infection, such as a viral infection, or symptom thereof.
The polypeptide described herein may, for example, comprise a SARS-CoV-2 antigen binding domain disclosed herein or a binding domain (eg, an antibody single variable domain) that competes with a SARS-CoV-2 antigen binding domain disclosed herein for binding to SARS-CoV-2 spike in an in vitro competition assay. In vitro competition may be determined by standard SPR or ELISA, for example. Any SPR herein is, for example, surface plasmon resonance (SPR) at 37° C. and pH 7.6.
The polypeptide described herein may, for example, comprise a SARS-CoV-2 antigen binding domain disclosed herein or a binding domain (eg, an antibody single variable domain) that binds to the same SARS-CoV-2 spike epitope (or an overlapping epitope) as a SARS-CoV-2 antigen binding domain disclosed herein.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state. Similarly, the multimer herein may comprise copies of such a binding domain. The multimer described herein may, for example, bind to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike. The multimer described herein may, for example, bind to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state.
The multimer described herein may, for example, comprise copies of a SARS-CoV-2 antigen binding domain, wherein the multimer competes with a SARS-CoV-2 antigen binding domain-containing multimer (eg, Q185B see right-hand-side schematic in
The multimer described herein may, for example, comprise copies of a SARS-CoV-2 antigen binding domain, wherein the multimer binds to the same SARS-CoV-2 spike epitope (or an overlapping epitope) as a SARS-CoV-2 antigen binding domain-containing multimer (eg, Q185B see right-hand-side schematic in
The polypeptide described herein may, for example, comprise binding domain QB-GB or a binding domain (eg, an antibody single variable domain) that competes with QB-GB for binding to SARS-CoV-2 spike in an in vitro competition assay. In vitro competition may be determined by standard SPR or ELISA, for example. Any SPR herein is, for example, surface plasmon resonance (SPR) at 37° C. and pH 7.6.
The polypeptide described herein may, for example, comprise binding domain QB-GB or a binding domain (eg, an antibody single variable domain) that binds to the same SARS-CoV-2 spike epitope (or an overlapping epitope) as QB-GB.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike.
The polypeptide described herein may, for example, comprise a binding domain that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state.
Similarly, the multimer herein may comprise copies of such a binding domain.
As explained in Example 37 Quad multimers that have such features have been found to be highly advantageous and may be more resistant to receptor-driven selection pressure associated with SARS-Cov-2 mutation.
Furthermore, there is provided a method of producing a polypeptide multimer, the method comprising multimerising first, second, third and fourth copies of a polypeptide (eg, any polypeptide disclosed herein) that comprises at least one copy of an SARS-CoV-2 antigen binding domain (eg, QB-GB or a binding domain (eg, an antibody single variable domain) that competes with QB-GB for binding to SARS-CoV-2 spike in an in vitro competition assay, and optionally formulating the multimer in a pharmaceutical composition for administration (eg, injected or pulmonary administration) to a human or animal subject to treat or prevent a coronavirus (preferably, SARS-CoV-2) infection.
Furthermore, there is provided a method of producing a polypeptide multimer, the method comprising multimerising first, second, third and fourth copies of a polypeptide (eg, any polypeptide disclosed herein) that comprises at least one copy of an SARS-CoV-2 antigen binding domain (such as an antibody variable domain) that binds to the same SARS-CoV-2 spike epitope (or an overlapping epitope) as QB-GB, and optionally formulating the multimer in a pharmaceutical composition for administration (eg, injected or pulmonary administration) to a human or animal subject to treat or prevent a coronavirus (preferably, SARS-CoV-2) infection.
Furthermore, there is provided a method of producing a polypeptide multimer, the method comprising multimerising first, second, third and fourth copies of a polypeptide (eg, any polypeptide disclosed herein) that comprises at least one copy of an SARS-CoV-2 antigen binding domain (such as an antibody variable domain) that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike, and optionally formulating the multimer in a pharmaceutical composition for administration (eg, injected or pulmonary administration) to a human or animal subject to treat or prevent a coronavirus (preferably, SARS-CoV-2) infection.
Furthermore, there is provided a method of producing a polypeptide multimer, the method comprising multimerising first, second, third and fourth copies of a polypeptide (eg, any polypeptide disclosed herein) that comprises at least one copy of an SARS-CoV-2 antigen binding domain (such as an antibody variable domain) that binds to the inner face of the RBD (receptor-binding domain) of SARS-CoV-2 spike that is in the up state, and optionally formulating the multimer in a pharmaceutical composition for administration (eg, injected or pulmonary administration) to a human or animal subject to treat or prevent a coronavirus (preferably, SARS-CoV-2) infection.
The binding domain may be an immunoglobulin domain (eg, an antibody variable domain or single variable domain (dAb or nanboby), or any other type of binding site or domain disclosed herein.
Any example, configuration, Aspect, Concept, Clause or Paragraph or disclosure herein is combinable with any feature of any further example, configuration, Aspect, Concept, Clause or Paragraph or disclosure herein.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognise, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications (including US equivalents of all mentioned patent applications and patents) are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This example demonstrates a method for generating tetravalent and octavalent soluble heterodimeric TCR molecules referred to as ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR respectively. These formats overcome the problems associated with solubility and avidity for cognate ligand at the target site.
To exemplify ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR as stable and soluble molecules, TCR αβ variable sequences with high affinity for NY-ESO-1 together with immunoglobulin constant domains and the NHR2 tetramerisation domain are used in this example.
The high-affinity NY-ESO TCR αβ chains (composing of TCR Vα-Cα and Vβ-Cβ respectively) specific for SLLMWITQC-HLA-A∗0201 used in this example is as reported in WO 2005/113595 A2 with the inclusion of a signal peptide sequence (MGWSCIILFLVATATGVHS). To aid protein purification, a histidine tag was incorporated to the C-terminus of NHR2 domain.
DNA constructs encoding components of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR are synthetically constructed as a two-vector system to allow for their soluble expression and functional assembly in mammalian cells. A schematic representation of the two assembled TCR chains (α and β chains) whose DNA sequences are synthesized for cloning into the expression vector are shown in
The pTT5 vector system allows for high-level transient production of recombinant proteins in suspension-adapted HEK293 EBNA cells (Zhang et al., 2009). It contains origin of replication (oriP) that is recognized by the viral protein Epstein-Barr Nuclear Antigen 1 (EBNA-1), which together with the host cell replication factor mediates episomal replication of the DNA plasmid allowing enhanced expression of recombinant protein. Therefore the pTT5 expression vector is selected for cloning the components for the ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR molecules.
Synthesized DNA fragments containing the TCR αβ chains are digested with restriction enzymes at the restriction sites (RS) (FastDigest, Fermantas) and the DNA separated out on a 1% agarose gel. The correct size DNA fragments is excised and the DNA purified using Qiagen gel extraction kit. The pTT5 vector was also digested with the same restriction enzymes and the linearized plasmid DNA is purified from excised agarose gel. The digested TCR αβ chains is cloned into the digested pTT5 vector to give four expression vectors (pTT5-ts-NY-ESO-1-TCRα, pTT5-ts-ESO-1-TCRβ, pTT5-os-NY-ESO-1-TCRα and pTT5-os-ESO-1-TCRβ).
Functional expression of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR is carried out in suspension-adapted HEK293 EBNA cells. HEK293-EBNA cells are cultured in serum-free Dulbecco’s Modified Eagle Medium (DMEM, high glucose (4.5 g/L) with 2 mM L-glutamine) at 37° C., 5% CO2 and 95% humidity.
For each transfection, HEK293-EBNA cells (3 × 107 cells) are freshly seeded into 250 mL Erlenmeyer shaker Flask (Corning) from ~60% confluent cells. Transfections are carried out using FreeStyle MAX cationic lipid base reagent (Life Technologies) according to the supplier’s guidelines. For expression of ts-NY-ESO-1 TCR, 37.5 µg of total plasmid DNA (18.75 µg plasmid DNA each of pTT5-ts-NY-ESO-1-TCRα and pTT5-ts-ESO-1-TCRβ vectors are used or varying amounts of the two expression plasmids) are used per transfection. Similarly for expression of os-NY-ESO-1 TCR, 18.75 µg plasmid DNA each of pTT5-os-NY-ESO-1-TCRα and pTT5-os-ESO-1-TCRβ isare used for transfection. Following transfection, cells were recovered in fresh medium and cultivated at 37° C. with 5% CO2 in an orbital shaker at 110 rpm for between 4-8 days. Smaller scale transfections are done similarly in 6 well or 24 well plates.
The ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR protein molecules secreted into the supernatant are analyzed either directly by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or after protein purification. Protein samples and standards are prepared under both reducing and non-reducing conditions. SDS-PAGE was performed using cast mini gels for protein electrophoresis in a Mini-PROTEAN Tetra cell electrophoresis system (Bio-Rad). Coomassie blue dye was used to stain proteins in SDS-PAGE gel.
Soluble ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from cell supernatant are purified in two steps. In the first step immobilized metal affinity chromatography (IMAC) are used with nitrilotriacetic acid (NTA) agarose resin loaded with nickel (HisPur Ni-NTA Superflow agarose -Thermo fisher). The binding and washing buffer consists of Tris-buffer saline (TBS) at pH7.2 containing low concentration of imidazole (10-25 mM). Elution and recovery of the His-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR from the IMAC column are achieved by washing with high concentration of imidazole (>200 mM). The eluted protein fractions are analysed by SDS-PAGE and the fractions containing the protein of interest are pooled. The pooled protein fraction is used directly in binding assays or further purified in a second step involving size-exclusion chromatography (SEC). Superdex 200 increase prepacked column (Gelifesciences) are used to separate out monomer, oligomer and any aggregated forms of the target protein.
The specific binding and affinity analysis of ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR to its pMHC is performed using BIAcore. Briefly, the purified Histidine-tagged ts-NY-ESO-1 TCR and os-NY-ESO-1 TCR proteins are captured onto sensor surface via Ni2+ chelation of nitrilotriacetic acid (NTA). Varying concentration of the analyte solution containing NY-ESO pep(SLLMWITQV)-MHC (ProImmune)is injected and the binding signals were monitored
The DNA encoding the domains required for expressing ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 protein complexes are synthesized and cloned into the expression vector pTT5 as described above. A schematic representation of the domains within the TCR αβ chains for ts-NY-ESO-1 TCR-IL2 and os-NY-ESO-1 TCR-IL2 are shown in
Briefly, using conventional genetic engineering techniques, a HEK293-T cell line was made that encodes Quad 16 (
Protein expression took place and protein was secreted from the cell lines. Samples of the medium in which the cells were incubated were subjected to PAGE under denaturing conditions (SDS-PAGE) or under native conditions (no SDS). The former was further under reduced conditions (using mercaptoethanol), whereas the latter was not.
The reduced gel showed a distinct banding (
For Quad 16, the tetramer peak from SEC was run on SDS-PAGE and the obtained band was cut out for mass spectrometry. The data were obtained with trypsin digests and p53 was detected in 100% of the protein. This was conclusive evidence that the secreted Quad 16 was multimerised
All DNA fragments were synthesized and cloned into the expression vector, pEF/myc/cyto (Invitrogen) by Twist Bioscience (California). Schematics and sequences of the synthesized DNA fragments and Quad polypeptides are shown in
Lyophilised plasmid DNA synthesized by Twist Bioscience, were resuspended with MQ water to a concentration of 50 ng/µl. 50 ng of DNA was transformed into 50 µl of competent DH5α cells using a conventional heat shock method. The cells were plated on LB agar plates containing 100 µg/mL ampicillin and grown overnight at 37° C. Individual colonies were picked and grown overnight at 37° C., 220 rpm. The DNA was purified from the cells using the QIAprep Spin Miniprep Kit, according to the manufacturers instructions (Qiagen).
Briefly, HEK293T cells were maintained in high glucose DMEM supplemented with 10% FBS and Pen/Strep. Cells were seeded at 6 × 105 cells per well of a 6-well plate in 2 ml media and were allowed to adhere overnight at 37° C., 5% CO2. 7.5 µl of Lipofectamine 2000 was diluted in 150 µl of OptiMem and incubated at room temperature for 5 mins. Plasmid DNA (2.5 µg) was diluted in 150 µl of OptiMem. Diluted DNA was combined with the diluted Lipofectamine 2000, mixed gently and incubated at R.T. for 20 mins. The 300 µl of complexes were added to one well of the 6-well plates. When analysis required the media to be serum free, the media was aspirated and replaced with CD293 media 6 hours post-transfection. The cells were incubated for 48 hours at 37° C., 5% CO2 prior to analysis.
Accordingly, different formats of TCR-linked NHR2 tetramerisation domain (TD) constructs (Quads) were transfected into HEK293T cells. Quads 3 & 4 resembling a TCR tetravalent format (structure schematically represented in
BCA Protein Assay Kit™, according to the manufacturer’s instructions. 100 µg was diluted with MQ water to give a volume of 80 µl. 20 µl of 5× SDS loading buffer was then added giving samples of 1 mg/ml. Samples were incubated at 95° C. for 5 min prior to SDS-PAGE and Western blot analysis.
Protein samples were separated on SDS-PAGE under denaturing condition. Typically, 25 µg of whole cell lysate (25 µl) were loaded on to the gel for Western blot analysis. 5 µl of PageRule Prestained 10-180 kDa Protein Ladder was loaded into the gel alongside the protein samples. The gels were run in Tris-Glycine buffer containing 0.1% SDS. A constant voltage of 150 volts was used and the gels were run for ~70 mins until the dye front has migrated fµlly.
SDS-PAGE (15% Bis-Tris) gels were prepared using the following resolving and stacking gels.
Resolving Gel:
Stacking Gel:
Western blotting was performed for the specific and sensitive detection of protein expression of TCR-NHR2 TD fusion proteins from Quads 3, 4, 12 and 13. Proteins separated out on SDS-PAGE were transferred onto Amersham Hybond™ 0.45 µM PVDF membrane as follows. Briefly, Amersham Hybond 0.45 µM PVDF membrane was activated with MeOH for ~1 min and rinsed with transfer buffer (25 mM Tris, 190 mM Glycine, 20% MeOH) before use. The sponge, filter paper, gel, membrane, filter paper, sponge stack was prepared and placed in the cassette for transfer. Transfer was carried out on ice at 280 mA for 75 mins. The membrane was incubated for ~2 hours in blocking buffer (TBST, 5% milk powder). The membrane was washed briefly with TBST before being incubated at 4° C. overnight with anti-human IgG HRP (Thermo, 31410) diluted 1/2500 in TBST, 1% milk powder. The membrane was washed thoroughly (three washes of TBST, 15 mins each) before being developed using the Pierce ECL Western Blotting Substrate.
Using anti-human IgG detection antibody to probe Western blots, specific protein band at the expected molecular weight can be detected from samples prepared from Quads 3 (46.1 kDa), 4 (46.4 kDa), 12 (47.8 kDa) and 13 (48.1 kDa) (
For all of the Quads analysed, a clear single band can be detected indicating TRVβ-TRCβ-IgG1-CH1 (+/- IgG hinge domain) fusions with the NHR2 TD are stable. These expression data also confirm the possibility of assembling tetravalent (Quads 3 & 4) and octavalent (Quads 12 & 13) molecules as exemplified in this example.
The difference between Quads 3 and 4 is the presence of a small peptide linker (G4S) located between the IgG1 CH1 domain and NHR2 TD. This is also true for Quads 12 and 13 where Q13 contains a peptide linker between the IgG1 CH1 domain and NHR2 TD. From the expression data, it can be seen the peptide linker does not effect protein expression. However, it may be desirable to include a peptide linker to aid antigen binding and or stabilizing the multimerisation complex in these TCR-NHR2 TD formats.
TCR-NHR2 TD fusion proteins were shown in Example 4 to be expressed intracellularly in HEK293T cells. Here again Quads 3, 4, 12 and 13 were used to demonstrate soluble expression of these fusion proteins. As described above, Quads 3, 4, 12 and 13 were transfected into HEK293T cells and soluble proteins from the cell supernatant were concentrated. Briefly, the media was harvested 48 hours post-transfection and centrifuged at 2,000 rpm for 5 mins to remove any cells or debris. Typically, 500 µl of media was concentrated to 100 µl using Amicon™ Ultra 0.5 Centrifugal Units with a MWCO of 10 kDa. 25 µl of 5× SDS loading buffer was added to the sample, which was then incubated at 95° C. for 5 mins prior to gel/Western blot analysis. Concentrated protein samples were separated out on SDS-PAGE gel and transferred onto Amersham Hybond 0.45 µM PVDF membrane. Western blotting and protein detection was done using anti-human IgG HRP using the methods described above.
Protein samples concentrated and prepared from cell supernatants show specific protein band at the expected molecular weight on Western blots corresponding to Quads 3 (46.1 kDa), 4 (46.4 kDa), 12 (47.8 kDa) and 13 (48.1 kDa) (
Detection of soluble protein expression from both tetrameric (Quads 3 & 4) and octameric (Quads 12 & 13) TCR-NHR2 TD formats highlights the potential applicability of NHR2 TD in a broad setting. Use of NHR2 TD fusion molecules could be used for the preparation of therapeutic molecules and protein molecules for use in diagnostics and imaging.
To further exemplify the versatility of NHR2 TD, several different antibody fragment formats fused to NHR2 TD were constructed for testing their expression in HEK293T cells.
Quads 14 and 15 contain an antibody VH domain fused to NHR2 TD either with or without a peptide linker located between the VH and NHR2 TD as schematically depicted in
Quads 38 and 44 were further developed to include an additional binding arm with the inclusion of a second VH domain specific for EGFR and CD138 respectively yielding Quads 42 and 46. Quads 42 and 46 represent bispecific molecules with the capability to multimerise via the NHR2 TD domain to form bispecific tetramers.
In another example, an effector molecule (human IL2) was linked to the C-terminus of Quads 14 & 15 resulting in Quads 18 and 19, whereby the VH-NHR2-IL2 molecule is tetravalent and bifunctional.
In another example, antibody Fab fragment (VH-CH1) was linked to NHR2 TD (Quads 23 and 24) and as schematically depicted in
In yet another example, a human IgG1 hinge domain was included to Quads 23 and 24, which is referred to as Quads 26 and 27 and as schematically depicted in
The following Quad vectors, Quads 14, 15, 18, 19, 23, 24, 26, 27, 34, 38, 40, 42, 44 and 46 all of which are His-tagged were transfected in HEK293T cells. Protein samples were prepared from whole-cell extracts as described above, separated out on SDS-PAGE and transferred onto Amersham Hybond 0.45 µM PVDF membrane. Specific protein expression were probed using anti-His HRP (Sigma, A7058) diluted 1/2500 in TBST, 1% milk powder.
Specific protein expression in whole cell extracts could be detected for all the different antibody-NHR2 TD fusion proteins tested using Quads 14, 15, 18, 19, 23, 24, 26, 27, 34, 38, 40, 42, 44 and 46 (
Expression of Quads 23 and 24 polypeptides highlights the potential to use NHR2 TD to form tetravalent antibody Fab molecules when co-expressed or mixed in-vitro with a partner soluble Quad molecule (e.g. Quad 25). Similarly expression of Quads 26 and 27, which include human IgG1 hinge domain highlight the potential to use NHR2 TD to form octavalent antibody Fab molecules when co-expressed or mixed in-vitro with a partner soluble second partner chain (e.g. Quad 25).
Quads 42 and 46 bispecific molecules containing an additional VH domain fused to the C-terminus of NHR2 TD domain also showed good protein expression. These data highlights the versatility of the NHR2 TD domain and its ability to be fused to different binding and effector molecules for developing a vast array of protein formats. The data also suggest it is possible to fuse protein molecules to both the N-terminus and C-terminus of NHR2 TD, which allows for the development of bispecific multivalent protein molecules.
NHR2 TD is responsible for the oligomerisation of ETO into a tetrameric complex. Using the NHR2 TD domain, it is possible to fuse binding domains and effector molecules to the N-terminus or C-terminus or both N- and C-terminus without effecting expression as shown in examples 4-6. Binding domains could be TCR variable and constant domains, antibody and antibody fragments or effector molecules such as IL2. It is also possible to express proteins in a soluble format when fused to NHR2 TD (
To demonstrate whether NHR2 TD retains its potential to oligomerise once it is fused to a binding domain, Quads 14 and 15 were expressed in HEK293T cells and protein samples were prepared from whole cell extracts as described above. Protein samples were separated out on PAGE gel under denaturing and non-denaturing (native) conditions. Native gels were prepared using the protocol described above, but without SDS. Proteins from PAGE gels were transferred onto Amersham Hybond 0.45 µM PVDF membrane. Specific protein expression was probed with anti-human IgG HRP detection antibody.
As expected under denaturing conditions, expression of VH-NHR2 TD from Quads 14 and 15 can be seen as a monomer where a specific protein band can be detected at the expected molecular weight (22 and 22.3 kDa) (
Together with the data in examples 4-7, there is conclusive evidence NHR2 TD is highly versatile allowing fusion of various protein binding domains and effector molecules. NHR2 TD allows soluble expression of proteins from eukaryotic cells such as HEK293T cells and they form highly stable and pure tetrameric molecules.
All DNA fragments were synthesized by Twist Bioscience (California) and cloned into the expression vector, pEF/myc/cyto (Invitrogen). Schematics and sequences of the synthesized DNA fragments are shown in
Lyophilised plasmid DNA synthesized by Twist Bioscience, were resuspended with MQ water to a concentration of 50 ng/µl. Competent E. coli DH5α cells were transformed with 50 ng of DNA using a conventional heat shock method. Transformed cells were plated on LB agar plates containing 100 µg/mL ampicillin and grown overnight at 37° C. Individual colonies were picked and grown in LB broth overnight at 37° C., 220 rpm. Plasmid DNA were purified from the cells using Qiagen plasmid extraction kits, according to the manufacturer’s instructions (Qiagen).
Expi293F™ cells (Thermo Fisher Scientific) were cultured in Expi293™ Expression Medium (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The only exception was that 5% CO2 was added directly to the flasks when the cells were split and non-vented caps were used.
Two methods involving different transfection reagents were utilised for protein expression. The methods for 30 ml cultures are described below and the protocol was adapted to either scaled up or down according to the experimental requirements.
For PEI transfections the cells were counted one day prior to transfection using a NC-3000™ (ChemoMetec) and were diluted to 1.5 × 106 cells/ml using Expi293™ Expression Medium. The cells were cultured in 5% CO2 at 37° C., 125 rpm overnight. The following day the cells were counted, spun down for 5 minutes at 1000 rpm and resuspended at 2 × 106 cells/ml in 30 ml of fresh media. 33 ug of plasmid DNA was added to 900 ul media and 90 ul of PEI Max (Polysciences Inc.) was added to 900 ul media. The DNA and transfection reagent samples were mixed and incubated at room temperature for 15 minutes. The DNA/transfection reagent mixture was added to the cells, which were cultured as before and incubated for a further 72 hrs.
For transfections with Expifectamine™ 293 Reagent (Thermo Fisher Scientific) the cells were also diluted to 1.5 × 106 cells/ml in Expi293™ Expression Medium one day prior to transfection. On the day of transfection the cells were centrifuged and resuspended at 2.5 × 106 cells/ml in 30 ml of fresh media. Two tubes containing 1.5 ml of Gibco™ Opti-MEM™ (Thermo Fisher Scientific) were prepared. 30 ug of plasmid DNA was added to one tube and 80 ul of Expifectamine was added to the other. The solutions were mixed and incubated at room temperature for 30 minutes. The DNA-transfection reagent complex was added to the cells, which were cultured in 5% CO2 at 37° C., 125 rpm. Following 16-18 hrs incubation, transfection enhancers 1 and 2 were added to the cells according to the manufacturers protocol. The cells were incubated for a further 96 hours.
The cells were harvested by centrifugation for 10 minutes at 4000 rpm. The ~30 ml supernatant was filtered through a 0.22 µm filter and diluted to 50 ml with binding buffer (50 mM HEPES, pH 7,4, 250 mM NaCl, 20 mM imidazole) containing Complete™ EDTA-free protease inhibitors (Roche) to facilitate binding to the column. A 1 ml HisTrap™ HP column (GE Healthcare) was connected to an AKTA Start (GE Healthcare) and pre-equilibrated with binding buffer. The protein-containing media was loaded onto the column using a flow rate of 1 ml/min. The column was washed with >10 CV of binding buffer before the protein was eluted using a 20-300 mM imidazole gradient over 12 ml. 0.5 ml fractions were collected and analysed by SDS-PAGE. Protein containing fractions were pooled and concentrated using Amicon® Ultra centrifugal filter units (Millipore).
Following affinity chromatography the proteins were either snap frozen and stored at -80° C., dialysed into an alternative buffer for a specific application of gel filtrated to assess the molecular weight of the various Quad formats. For the latter analyses, protein samples were concentrated to 1.5-2 ml and gel filtrated on a Superdex 75 16/600 column (GE Healthcare) using 10 mM HEPES, pH 7.4, 250 mM NaCl.
Purified proteins were analysed by separating out on SDS-PAGE under denaturing condition. Typically, 1-2 µg of purified protein were loaded per lane on SDS-PAGE gel. The gels were run in Tris-Glycine buffer containing 0.1% SDS. A constant voltage of 150 volts was used and the gels were run for ~70 mins until the dye front has migrated fully.
SDS-PAGE (15% Bis-Tris) gels were prepared using the following resolving and stacking gels.
Resolving Gel:
Stacking Gel:
The potential of the purified Quad proteins to bind its target protein was confirmed by directing binding ELISA. Briefly, high binding 96 well plates (Corning) were used for coating recombinant target protein (1-5 ug/ml diluted in PBS or as indicated), which were typically stored at 4° C. overnight. Plates are then washed 3 times with 200 ul wash buffer (PBS + 0.1% Tween) and blocked using 200 ul blocking buffer (PBS + 1% BSA) for 1 hour at room temperature. Purified protein samples are typically serially diluted in dilution buffer (PBS + 0.1% BSA) and 100ul/well is added. Samples are incubated at room temperature for 1 hour after which the plate is washed again 3 times using 200 ul wash buffer. Anti-His HRP (Abcam) Detection antibody diluted according to the manufacturer recommendation is added and incubated at room temperature for 1 hour. The plate is washed for the final time using 3×200 ul wash buffer and 50 ul pre-warmed detection reagent (TMB -Sigma) is added per well and the plate incubated in the dark for 10-30 mins. The reaction is stopped by adding 25 ul/well of 1 M sulfuric acid. The absorbance at 450 nm was read using a CLARIOstar microplate reader (BMG Labtech).
A multimer format (“Quad” format) where a single domain of an antibody variable fragment (dAb) (VH or VL either Vκ or Vλ) fused to p53 tetramerisation domain is exemplified in this example. The dAb VH sequence for anti-IL17A (sequence adapted from WO 2010/142551 A2) was engineered into a tetravalent dAb Quad format (Quad 57) (Seq ID: 146). Expression vector for Quad 57 was synthesized by Twist Bioscience and expressed in HEK293 cells as described above. Secreted Quad 57 protein was collected from cell supernatant and purified using HisTrap HP column. To demonstrate soluble expression and purity of Quad 57, a small portion of the purified protein (1.5 ug) was separated out on SDS-PAGE gel (
A bispecific tetravalent dAb Quad is exemplified in this example where two different dAb binding domains are linked to p53 tetramerisation domain via the N- and C-terminus and as schematically represented in
In a specific example of a bispecific tetravalent dAb Quad, an anti-TNFa dAb VH binding domain from Ozoralizumab was linked to the N-terminus of p53 tetramerisation domain and an anti-IL17A dAb VH binding domain (sequence adapted from WO2010/142551 A2) was linked to the C-terminus of the p53 tetramerisation domain (Quad 54) (Seq ID: 143). Although in this specific example both the dAb binding domains were VH’s, the dAb binding domains could be Vκ or Vλ or a combination of the different dAb formats.
To demonstrate whether such a bispecific tetravalent dAb Quad format could be expressed as a soluble protein, expression construct containing dual anti-TNFa and anti-IL17A dAb binding domains were synthesized as a Quad format and expressed in HEK293 cells. Following protein purification from culture supernatant using HisTrap HP column, ~1.5 ug of the purified protein was separated out on SDS-PAGE gel (
To further exemplify the functionality of such bispecific tetravalent anti-TNFa x anti-IL17A dAb Quad, direct binding ELISA assay was performed. High protein binding 96 well plates (coming) were coated with 1 ug/ml recombinant human TNFa protein (Abcam) and direct binding ELISA assay was performed with serially diluted Quad 54 protein using the method described above. A typical sigmoidal dose response curve was yielded from Quad 54 direct binding ELISA with recombinant human TNFa protein with a half-maximal binding at the low pM range (~10 pM) (
In this example, scFv binding domains for two different targets were selected and linked separately to the N-terminus of p53 tetramerisation domain to generate monospecific tetravalent scFv Quads as schematically represented in
In one example the scFv binding domain was an anti-TNFa where the VH and Vκ sequence was adapted from Humira into a scFv Quad format. To analyse whether the presence of a peptide linker effected the expression and binding of the Quad molecule to its target protein, in this example the anti-TNFa scFv binding domain was linked to the N-terminus of p53 tetramerisation domain either via a (G4S)3 peptide linker (Quad 63) (Seq ID: 147) or without a peptide linker (Quad 51) (Seq ID: 139).
In another example, the scFv binding domain was an anti-CD20 adapted from Wu et al., (Wu et al. 2001). The anti-CD20 scFv binding domain was engineered into a tetravalent Quad format by linking the binding domain directly to the N-terminus of p53 tetramerisation domain without a peptide linker (Quad 53 Tet) (Seq ID: 141).
All tetravalent scFv Quads were transfected into HEK293 cells and soluble protein from the culture supernatant were purified using HisTrap™ HP column as described above. A small amount of the purified protein (~1.5 ug) was separated out on SDS-PAGE gels to confirm expression and purity.
Anti-TNFa scFv Quads (Quads 51 & 63) were found to express well as soluble protein and the presence or absence of the peptide linker joining the scFv to the N-terminus of p53 tetramerisation domain did not appear to effect expression (
To further functionally characterize the activity of anti-TNFa Quad 51 molecule to neutralize TNFa, a cell-based assay was performed using WEHI-13VAR cells (ATCC), which is highly sensitive to TNFa. The bioassay using WEHI cells was set-up as described below. A monovalent anti-TNFa (W51ScFv) was included in the assay as a control (Seq ID: 150) and Humira was used as a positive control. W51ScFv was generated by modifying Quad 51 where the p53 tetramerisation domain was removed. Expression of W51ScFV was confirmed by SDS-PAGE (
Briefly, WEHI-13VAR cells were seeded at 1 × 104 cells per well in a 96-well plate in RPMI-1640, 10% FBS and incubated overnight at 37° C., 5% CO2. The media was aspirated from the cells and replaced with media containing 2 ug/ml actinomycin D, 0.1 ng/ml recombinant human TNFα (ab9649, Abcam) and 0-2400 pM Q51, Q35, W51ScFV and Humira. The samples were set up in quadruplicate with no TNFα and no antibody controls. The cells were incubated under standard culture conditions for a further 20-22 hours.
To assess cell viability, ATP generated by metabolically active cells was quantified using the CellTiterGlo Luminescent Cell Viability Assay (Promega) according to the manufacturers’ instructions. Luminescent signals were measured using a CLARIOstar microplate reader (BMG Labtech). The luminescence signals obtained from the compound treated cells were normalised against the media on controls. The effective dose (ED) at which 50% of the WEHI cells retained viability was calculated and the ED50 for Quad 51, Humira and W51ScFV is summarised in Table 11.
As expected, Quad 51 having four anti-TNFa binding domains was found to be most effective at neutralising the cytotoxic effect of recombinant human TNFa protein on WEHI cells compared to Humira, which has two binding domains for TNFa. The W51ScFv control having only a single binding domain for TNFa had the highest ED50. The increasing valency of the anti-TNFa molecules for TNFa correlated inversely with a decrease in ED50 values. These data highlights the enhanced functional potency of Quad molecules with increasing valency and this aligns with the general concept of avidity verses potency as reported by numerous studies (Alam et al. 2018; Rudnick & Adams 2009; Adams et al. 2006; Bru nker et al. 2016). Furthermore, the increase in avidity of Quad molecules can be used to drive selectively of tumour associated antigens that are highly expressed on tumour cells and thus limit the on-target off-tumour effect on healthy cells as reported in an example outlined by Slaga et al (Slaga et al. 2018).
To further demonstrate the ability of anti-TNFa Quad 51 to block TNFa induced activation of Caspase 3, Western blot analysis were performed alongside Humira and W51ScFv as controls. Briefly, WEHI-13VAR cells were seeded at 2.5 × 106 cells per well in a 6-well plate in culture media (RPMI-1640, 10% FBS) and incubated overnight. The media was replaced with media containing 2 ug/ml actinomycin D, 1 ng/ml recombinant human TNFα and 500 pM of Quad 51, W51ScFV and Humira. Culture media only and culture media containing 2 ug/ml actinomycin D were included as controls. The cells were incubated with TNFα and +/- anti-TNFa molecules for 10 hours.
The cells were lysed using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 mM DTT and Complete™ EDTA-free protease inhibitor (Roche). Cell lysates were sonicated using a Bioruptor® Pico Sonication System (Diagenode) and the protein concentration of each sample was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 10 ug of protein was electrophoresed on a 12% Bis-Tris gel and bands were subsequently transferred to Amersham™ Hybond® P 0.45 µm PVDF membranes (GE Healthcare). The membranes were blocked with 10%-BSA-TBST or 5%-milk-TBST before being incubated overnight at 4° C. with appropriate antibodies (anti Caspase-3 (1/1000, CST, 9665) and anti-Cleaved Caspase-3 (1/100, CST, 9664). The membranes were washed with TBST and incubated with anti-rabbit IgG HRP-linked (1/2500, CST, 7074S) secondary antibody for 2 hours at room temperature. Following thorough washing, the membranes were developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and CL-XPosure™ films (Thermo Fisher Scientific).
From the Western blots, it can be seen that Quad 51 can effectively neutralise TNFa mediated activation of Caspase-3 with no detectable cleaved Caspase-3, similar to Humira (
In this example, the valency of the scFv binding domain was increased from four (tetravalent) to eight (octavalent). This was achieved by linking an scFv to both N- and C-terminus of the p53 tetramerisation domain as schematically represented in
As described in example 10, the anti-CD20 scFv binding domain was used in this example to construct a monospecific octavalent anti-CD20 scFv Quad (Quad 53 Oct) (Seq ID: 142). In this specific example, scFv’s were linked to the N- and C-terminus of the p53 tetramerisation domain without peptide linkers, however, in other examples of this format, peptide linkers could be introduced at either or both ends to aid flexibility of the binding domain.
Following expression and purification of Quad 53 Oct protein from culture supernatant, protein expression analysis and ELISA binding assays were performed. Protein separated out on SDS-PAGE gel confirmed soluble expression of Quad 53 Oct with high purity (>99%) (
To analyse the effect of increasing valency on target protein binding, scFv anti-CD20 without the tetramerisation domain was constructed to act as a monovalent control (Quad 53 Mon) (Seq ID: 140). Following protein expression and purification, all three Quad 53 anti-CD20 scFv molecules (monovalent, tetravalent and octavalent) were separated out side-by-side on a SDS-PAGE gel (
In this example, two different scFv binding domains with specificity for two different target proteins were linked to the p53 tetramerisation domain via the N- and C-terminus to give a bispecific tetravalent scFv Quad as exemplified schematically in
The specific scFv binding domains used in this example has specificity for anti-TNFa and anti-IL17A. The anti-TNFa scFv sequence was adapted from Humira and the anti-IL17A scFv sequence was adapted from Ixekizumab (Eli Lilly) into a bispecific Quad format (Quad 55) (Seq ID: 144). To confirm soluble expression, Quad 55 was expressed in HEK293 cells and the secreted protein was purified from culture supernatant followed by protein analysis. (
In this example, two different binding domain formats with specificity for two different target proteins were linked to the N- and C-terminus of p53 tetramerisation domain respectively, as exemplified schematically in
The first binding domain was an anti-TNFa scFv as detailed in Example 12, which was linked to the p53 tetramerisation domain via the N-terminus. The second binding domain was an anti-IL17A dAb sequenced, which was linked to the p53 tetramerisation domain via the C-terminus as detailed in Example 9 (Quad 56) (Seq ID: 145).
Soluble expression of this bispecific tetravalent scFv x dAb Quad format was confirmed by analysing the purified protein on SDS-PAGE gel (
In the examples above (Examples 10, 12 & 13) different bispecific Quad formats (Quads 54-56) with specificity for anti-TNFa x anti-IL17A were produced and analysed for expression and functionality by ELISA binding assay for the anti-TNFa binding arm. From the data presented thus far, it is clear that the p53 tetramerisation domain is highly versatile and amenable to fusion with different binding domains at either or both N- and C-terminus. To compare the functional binding strengths of Quads 54-56, the ELISA binding assay data for the anti-TNFa binding arm of the different bispecific Quads were plotted side-by-side (
In this example, an scFv binding domain was linked to the lower hinge/CH2 domain of IgG1 Fc without the core and upper hinge region to generate a scFv monomeric Ig Fc (scFv-mFc), ie wherein the Fc does not pair with another Fc when a multimer is formed using the polypeptide monomer. Typically a CXXC motif comprising cysteine residues present in the core hinge region is responsible for forming inter-chain disulfide bonds. Thus, by excluding the core hinge region, the Fc region is restrained from forming a tightly packed homodimer structure typically found in native IgG antibodies. The lower hinge/CH2 domain was kept intact to allow proper interaction with Fcγ-receptors required for effector function.
The scFv-mFc was engineered into a Quad format by linking it to the p53 tetramerisation domain via the N-terminus to generate a tetravalent monospecific Ig scFv Quad termed version 1 as schematically represented in
In this example the upper hinge was also removed, but in other examples the upper hinge region can be optionally retained completely or only partially kept intact to generate dAb monomeric Ig Quads (exemplified schematically in
The scFv binding domain used in this specific example was that of an anti-CD20 described in Example 10. The anti-CD20 scFv was linked to the lower hinge/CH2 domainof the Fc via a peptide (G4S)3 linker. The p53 tetramerisation domain was linked directly to the C-terminus of the CH3 domain without any peptide linkers (Quad 64) (Seq ID: 148). An optional linker could be included at this junction between the CH3 domain and the multimerisation domain to provide flexibility.
Following Quad 64 expression and purification, protein was quantified and analysed by SDS-PAGE (
In this example, a different version of the tetravalent monomeric Ig scFv Quad described in Example 14 was constructed where the anti-CD20 scFv binding domain was linked directly to the N-terminus of the p53 tetramerisation domain. The mFc containing the lower hinge, CH2 and CH3 domains was directly linked to the C-terminus of the p53 tetramerisation domain. This version of tetravalent monomeric Ig scFv Quad is termed version 2 and is schematically represented in
Although in the specific example described below, the upper hinge was not included, in other examples the upper hinge region can be optionally retained completely or only partially kept intact.
In other examples, the version 2 configuration could contain dAb binding domains as exemplified schematically in
The expression construct for the tetravalent monomeric Ig scFv Quad version 2 specific for CD20 (Quad 65) (Seq ID: 149) was expressed in HEK293 cells and the soluble secreted protein was analysed by SDS-PAGE. Protein quantification using Nanospec confirmed high protein yield after HisTrap HP column purification with protein yield equivalent to 160 mg/L. In addition, a single protein band at the expected size (57.2 kDa) as seen on the SDS-PAGE gel confirmed expression with high purity (>99%) (
The data outlined above and in Example 14, represents the first examples of soluble and functional expression of tetravalent monomeric Ig molecules. Such multivalent monomeric Fc formats would have several advantages over scaffold and antibody fragment based molecules lacking Fc. Firstly, the presence of an Fc region in a monomeric Quad format would allow neonatal Fc receptor (FcRn) binding providing an extended half-life of these molecules in-vivo. Secondly, the presence of an Fc region would have the ability to bind multiple Fc receptors to induce effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) as reported for monovalent monomeric Ig Fc molecules (Ying et al. 2017; Ying et al. 2012). Thirdly, the proof-of-concept data outlined in this example suggest that any given monoclonal IgG antibody could be rapidly formatted into a multivalent monomeric Ig Quad format as schematically represented in
In a specific example of an octavalent bispecific Quad, dAb binding domains for PD-L1 and 4-1BB were used for the dual targeting of an immune checkpoint inhibitor and an immune co-stimulatory molecule for the treatment of cancers. In other examples, dAb binding domains could have specificity for any immune checkpoint inhibitor and any co-stimulatory molecule or a mixture thereof to provide either a dual checkpoint inhibitor bispecific multimer (e.g. PD-L1 x CTLA-4) or a dual checkpoint inhibitor and immune co-stimulatory bispecific multimer (e.g. PD-L1 x 4-1BB) or a dual immune co-stimulatory bispecific multimer (e.g. 4-1BB x OX40).
To exemplify octavalent bispecific Quads, two different versions were constructed to demonstrate soluble Quad protein expression. In one format, the PD-L1 and 4-1BB dAb binding domains were linked to a p53 multimerisation domain as a tandem dAb at either the N- or C-terminus as schematically represented in
In a second example of an octavalent bispecific multimer, the dAb binding domains for PD-L1 and 4-1BB were linked to the p53 tetramerisation domain at opposite ends respectively as schematically represented in
The dAb binding domain sequence for PD-L1 and 4-1BB were adapted from WO2017/123650A2. In the first version of an octavalent bispecific Quad, a tandem dAb containing anti-PD-L1 and anti-4-1BB in a N-C terminus orientation was linked to the N-terminus of p53 tetramerisation domain without any peptide linkers. This version is referred to as Quad 68 (SEQ ID NO: 190). However, in other examples, the tandem dAb can be linked to the tetramerisation domain via an optional peptide linker and as further described in Table 8-P.
In a second version of octavalent bispecific Quad multimer, the anti-PD-L1 dAb was linked to the N-terminus of p53 tetramerisation domain and the anti-4-1BB dAb was linked to the C-terminus. This version is refered to as Quad 69 (SEQ ID NO: 191). In this specific example the dAbs were linked to the tetramerisation domain without any peptide linkers. However, in other examples, dAbs can be linked to the tetramerisation domain via an optional linkers and as further described in Table 8-C.
The expression construct for Quads 68 and 69 were expressed in HEK293 and the secreted soluble proteins were purified from cultured supernatant. The purified Quad proteins were analysed by SDS-PAGE (
The configuration of the dAbs either in tandem (as in Quad 68) or in opposite orientation (as in Quad 69) are anticipated to engage with cancer cells via PD-L1 with higher potency and selectively than to T cells expressing 4-1BB. Therefore, T cells will preferentially be recruited to cancer cells only once the cancer cells are engaged with the Quad molecule allowing selective T cell activation with improved safety profile.
A dAb VH with specificity for TNFα as detailed in Example 9 was also used in this example to generate two new versions of monomeric Ig dAb Quads. In the first version, anti-TNFα dAb VH was linked directly to IgG CH1. The CH1 region was linked to the IgG1 Fc where the hinge region was modified so that the core hinge was removed (SEQ ID NO: 168 hinge sequence was used). The Fc region was linked to the N-terminus of the p53 TD domain yielding Quad 92. The presence of CH1 region in Q92 allowed for the generation of a second version of monomeric Ig Quad where Q92 when co-expressed with anti-TNFα dAb linked to Kappa light chain constant domain yielded an octavalent anti-TNFα Fab-like dAb monomeric Ig Quad (Q93) as schematically represented in
Q92 alone or Q92 plus Q93 were expressed in HEK293 cells and the Quad proteins were purified directly from the culture supernatant. The purified proteins were analyzed by SDS-PAGE where pure products at the expected molecular weight (Q92 (tetravalent) - 55 kDa and Q92+Q93 (octavalent) - 79 kDa) could be seen confirming soluble expression of these Quad formats (
In this example, two different versions of monomeric Ig Quads were generated similar to that exemplified in Example 14. However, instead of using scFv as binding domains, in this specific example antibody single domains (VH) were linked in tandem with specificity for either PDL1 alone or PDL1 plus 4-1BB. In the first of these examples, a bispecific octavalent anti-PDL1/4-1BB monomeric Ig dAb Quad (Q113) was generated by linking in tandem an anti-PDL1 dAb with an anti-4-1BB dAb separated by a flexible linker. This tandem bispecific binding module was linked to the lower hinge/CH2 region of IgG1 Fc without the core hinge region to generate a tandem dAb monomeric Ig Fc (ie, the resulting polypeptide comprised (in N- to C-terminal direction) an anti-PDL1 dAb, an anti-4-1BB dAb, lower hinge/CH2 region of IgG1 Fc without the core hinge region and IgG1 CH3) . The Fc region was linked to the N-terminus of p53-TD domain to generate a bispecific tandem dAb monomeric Ig Fc Quad as schematically represented in
Following Quad 113 and Q114 expression in HEK293 cells and purification of proteins from culture supernatant, the recovered proteins were analysed by SDS- PAGE (
In this specific example, monomeric Ig Quads were produced from IgG monoclonal antibodies where the native pairing of the variable heavy chain (VH) and variable light chain (VL) was kept intact. This was achieved by taking the Fab fragment of IgG monoclonal antibody and converting it into a monomeric Ig Quad as schematically represented in
Fab monomeric Ig Quad proteins were purified from the culture supernatant and analysed initially by SDS-PAGE (
In the above examples, antibody fragments were used to generate Quads. In this specific example the versatility of p53 TD domain was demonstrated further whereby extracellular domains of cell surface receptors were multimerised into a Quad format without affecting its ability to bind its natural ligand. To exemplify this, the soluble extracellular domains used in the VEGF trap aflibercept (Eylea™) was used as an example. The Ig domain 2 from VEGFR1 and Ig domain 3 from VEGFR2 similar to Eylea was linked to monomeric Ig Quad similarly to that exemplified in Examples 17-19 and as schematically represented in
Q96 was expressed in HEK293 cells and soluble protein was purified directly from the culture supernatant. SDS-PAGE analysis confirmed expression of Q96 as a highly pure Quad with a single protein band at the expected molecular weight (
Similar to Example 8, antibody single domains were used to generate monospecific tetravalent and octavalent Quads without IgG Fc (referred to herein by shorhand “non-Ig”). Anti-TNFα dAb VH was linked directly to p53 TD at either the N-terminus to generate tetravalent Quad (Q88 tetravalent) or at both N- and C-terminus to generate octavalent anti-TNFα dAb Quad (Q88 octavalent) as schematically represented in
Following expression in HEK293 cells and Quad protein purification directly from culture supernatant, initial protein analysis was carried out by SDS-PAGE. The multivalent anti-TNFα dAb Quads expressed as highly pure proteins as judged by a single band on the SDS-PAGE gels corresponding to the expected molecular weight (
Increase in TNFα binding domain valency was further investigated in WEHI bioassay where the potency of the anti-TNFα Quad molecules to neutralize TNFα-mediated cytotoxicity of WEHI cells were compared. WEHI bioassay was performed as described in Example 10 using both non-Ig and Ig-like anti-TNFα dAb Quads (
To extend the concept of multivalency further and beyond tetra- and octa-valency, two further formats of anti-TNFα dAb Quads were generated with either 12 (AKA dodeca, 12-valent or 12-mer herein) or 16 (hexadeca, 16-valent or 16-mer herein) anti-TNFα dAb binding domains in a non-Ig format. The modular design and structural arrangements of these Quads can be seen in the schematic illustration in
In the dodeca version, the first chain contained tandem anti-TNFα dAbs linked to IgG CH1, which in turn is linked to the TD domain (Q142). The second chain contained a single anti-TNFα dAb linked to either kappa (Q135) or lambda (Q136) light chain constant region. Dodeca-valent Quads were generated by co-expressing the two chains in HEK293 cells where heterodimerisation of the two chains occurred through interaction between CH1 and C-kappa or C-Lambda constant region. The TD domain allowed tetramerisation of these two assembled chains into a tetramer.
For the hexadeca-valent anti-TNFα dAb Quad, the first chain was exactly the same as the dodeca valent format, however, the second chain contained a tandem anti-TNFα dAb linked to either kappa (Q145) or lambda (Q144) light chain constant region. Co-expression of these two chains allowed generation of hexadeca-valent anti-TNFα Quads.
The dodeca- and hexadeca anti-TNFα dAb Quads were expressed in HEK293 cells and the Quad proteins were purified directly from culture supernatant. The purified proteins were analysed by SDS-PAGE (
WEHI bioassay was performed using the purified dodeca- and hexadeca anti-TNFα dAb Quads and the TNFα neutralization potency was compared to the monovalent anti-TNFα dAb control (
Thus, it has been demonstrated that constructs of the invention can surprisingly achieve highly significant increases in antigen binding potency (762-fold in Table 16, for example) and advantageously this can be achieved using different types of binding site (eg, dAb or Fab-like), with or without antibody Fc presence, with possibility of repurposing clinically approved antibodies to retain their tested binding sites and at very high purity levels (almost 100% purity).
In example 19 production of tetravalent Humira Fab monomeric Ig Quad was exemplified. In this specific example, Humira Fab with intact light chain (LC) and heavy chain (HC) but without Fc region (non-Ig version) was generated. The p53 TD domain was linked at the C-terminus of Humira HC CH1 domain where the hinge region was modified to be devoid of core hinge (ie, upper hinge sequence without core or lower hinge sequence; SEQ ID NO: 183 was used). Co-expression of this modified HC with the native Humira LC, allowed generation of tetravalent Humira Fab-TD with significantly reduced molecular size compared to the Humira Fab monomeric Ig-TD version. A schematic structural representation of Humira Fab-TD is shown in
The Humira Fab-TD Quad was expressed in HEK293 cells and the Quad protein was purified directly from culture supernatant. The purified protein was analysed by SDS-PAGE (
To further characterize this Quad protein, TNFα binding assay using ELISA and TNFα neutralization potential using WEHI bioassay was performed. As a control Humira Fab was used as a monovalent control. From the ELISA binding assay, it can be seen Humira Fab-TD could bind TNFa with higher binding strength than the Humira Fab monovalent control (
In the examples above, Fabs from antibodies were used to generate tetravalent Quads either as Ig-like (Example 19) or non Ig-like (Example 23). Further iterations of Fab Quads can be made to generate Fabs with octavalent valences either as Ig-like or non Ig-like.
To generate octavalent non Ig-like Fab Quads, a Fab with an intact hinge region will be used where p53 TD domain is linked directly to the hinge region optionally via a flexible peptide linker. The intact core hinge region will allow homodimerization of the Fab-TD when co-expressed with its native LC to generate F(ab′)2 and as such the monomeric building block will be bivalent. In turn, the TD domain will allow tetramerization of the F(ab′)2 to generate an octavalent Fab Quad as schematically shown in
Similarly, to generate octavalent Ig-like Fab Quads, a TD, eg, a p53 TD domain, will be linked directly to the C-terminus of CH3 domain of an unmodified HC of a predetermined antibody via an optional peptide linker. When this is co-expressed with its native LC from the antibody, the monomeric building block will effectively resemble a fully assembled Ig antibody with p53 TD domains linked to it at the C-terminus of the HC of the assembled antibody. The TD domain in turn will allow tetramerization of the monomeric building blocks to generate an octavalent Fab Ig-like Quad as schematically shown in
Thus, an embodiment provides an antibody comprising a heavy chain, wherein the heavy chain comprises a SAM, eg, a TD (such as a p53 or homologue TD as disclosed herein). The TD may be at the C-terminus of the heavy chain. For example, the heavy chain comprises (in N-to C-terminal direction) a VH, an antibody CH1, a hinge, a CH2, a CH3 and a SAM (eg, a TD). In an example, the heavy chain is paired with a light chain (eg, wherein the light chain comprises (in N-to C-terminal direction) a VL and an antibody CL), wherein a VH and VL comprised by the heavy and light chain pair form an antigen binding site. In an example, the antibody is a 4-chain antibody, such as comprising first and second copies of the heavy and light chain pairs (ie, a first heavy chain paired with a first light chain, a second heavy chain paired with a second light chain, wherein each pair comprises a VH/VL antigen binding site, and wherein the heavy chains are paired together (such as via disulphide bonding in the constant region)). In an example, there is provided a tetramer of such an antibody, wherein the heavy chain of each antibody comprises a TD at its C-terminus and 4 copies of the antibody are tetramerised by the TDs. See, eg,
In an example, there is provided an antibody light chain comprising in N-to C-terminal direction) an antibody V domain (eg, a VL, such as a Vκ or a Vλ; or a VH), an antibody CL and a SAM, eg, a TD (such as a p53 or homologue TD as disclosed herein). For example, there is provided a multimer (eg, a tetramer) of such a light chain, optionally wherein the light chain (or each light chain in the multimer) is paired with a second antibody chain comprising (in N- to C-terminal direction) another V domain and a CH1, wherein the V domain and CL of the light chain are paired respectively with the other V domain and CH1.
Reference is made to
Summary:
This example demonstrates how advantageously multimerization of the invention can repurpose a binding site which otherwise would not be useful or much less useful for medical use (eg, for treatment or prophylaxis of a disease or condition mediated by or associated with an antigen to which the binding site binds).
A multimer was produced by multimerising copies of a polypeptide. The polypeptide had the sequence of CR3022 VH Fab-TD shown in Table 23, and comprised (in N- to C-terminal direction) a single copy of the VH domain of CR3022, and a human p53TD. Each polypeptide was paired with a light chain, wherein the light chain comprised (in N- to C-terminal direction) a single copy of the VL domain of CR3022 and a human Ck domain.
ELISA was carried out using the example assay method given above. Briefly, for
CR3022 is a derivative of a human antibody isolated from a patient who recovered from SARS-CoV-1 infection. This antibody is known to bind the receptor binding domain (RBD) of CoV-1 strongly. It also cross-reacts with the RBD of CoV-2 strain but with significantly lower binding affinity as reflected in the above data. However the multimer version of CR3022 according to the invention massively improves the cross-reactive binding to RBD of CoV-2 and well as improving binding to the RBD domain CoV-1 strain (
This example demonstrates how advantageously multimerization of the invention can repurpose a binding site which otherwise would not be useful or much less useful for assay use (eg, detecting a pathogen or antigen that mediates, causes or is adversely associated with a disease or condition in a subject). Through multimerization of the invention, very high-order multimers (eg, containing 8-24 copies of a binding site) can easily be achieved in a stable multimer that can be readily expressed, such as in eukaryotic expression systems and host cells (as demonstrated in the exemplification above). The high-order multimers usefully can repurpose binding sites that individually have relatively low binding strength for an antigen, wherein in the multimers an avidity effect is produced rendering the combined binding strength of copies of the binding site well suited to very sensitive assay detection of low levels of antigens in samples. Usefully, for example, we demonstrate this even for very diluted samples where the antigen is at very low concentration. This is advantageous, for example where the antigen is an antigen of a pathogen (eg, a virus, bacterium or fungus that causes disease, such as in humans, animals or plants); or where the antigen is comprised by antibodies produced by a human or animal subject in response to immunisation, such as in response to a pathogen or a human protein in the subject.
A multimer was produced by multimerising copies of a polypeptide. The polypeptide had the sequence of ACE2(18-615) Ig-TD shown in Table 24, and comprised (in N- to C-terminal direction) a single copy of the ACE2 amino acids 18-615, a human hinge region, a human CH2, a human CH3 and a human p53TD.
ELISA was carried out using the example assay method given above. Briefly, for
ELISA was carried out using the example assay method given above. Briefly, for
Spike glycoprotein is naturally assembled into a trimer. It can be produced recombinantly as a trimer or only the RBD domain can be produced as a monomer for use in in vitro assays. The spike protein binds ACE2 receptor as the initial step in the infection process. Thus, ACE2 can be potentially used as a decoy receptor to neutralize coronavirus. In this experiment, a tetravalent ACE2 Ig-TD multimer Quad (ie, an example of a multimer comprising more than 2 binding sites; this example had 4 ACE2 extracellular proteins) was generated and used to capture the two recombinant forms of the spike protein for comparison. Strikingly, ACE2 Ig-TD Quad binds the trimeric form, which is more analogous to the native form with significantly enhanced binding strength than the RBD domain alone suggesting ACE2 Ig-TD is a good candidate for developing as a super neutralizer of SARS-CoV-2. See
As shown in the previous
All DNA fragments were synthesized by Twist Bioscience (California) and cloned into the expression vector. Lyophilised plasmid DNA synthesized by Twist Bioscience, were resuspended with MQ water to a concentration of 50 ng/µl. Competent E. coli DH5α cells were transformed with 50 ng of DNA using a conventional heat shock method. Transformed cells were plated on LB agar plates containing 100 µg/mL ampicillin and grown overnight at 37° C. Individual colonies were picked and grown in LB broth overnight at 37° C., 220 rpm. Plasmid DNA were purified from the cells using Qiagen plasmid extraction kits, according to the manufacturers instructions (Qiagen).
Expi293F™ cells (Thermo Fisher Scientific) were cultured in Expi293™ Expression Medium (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The only exception was that 5% CO2 was added directly to the flasks when the cells were split and non-vented caps were used.
Two methods involving different transfection reagents were utilised for protein expression. The methods for 30 ml cultures are described below and the protocol was adapted to either scale up or down according to the experimental requirements.
For PEI transfections the cells were counted one day prior to transfection using a NC-3000™ (ChemoMetec) and were diluted to 1.5 × 106 cells/ml using Expi293™ Expression Medium. The cells were cultured in 5% CO2 at 37° C., 125 rpm overnight. The following day the cells were counted, spun down for 5 minutes at 1000 rpm and resuspended at 2 × 106 cells/ml in 30 ml of fresh media. 33 ug of plasmid DNA was added to 900 ul media and 90 ul of PEI Max (Polysciences Inc.) was added to 900 ul media. The DNA and transfection reagent samples were mixed and incubated at room temperature for 15 minutes. The DNA/transfection reagent mixture was added to the cells, which were cultured as before and incubated for a further 72 hrs.
For transfections with Expifectamine™ 293 Reagent (Thermo Fisher Scientific) the cells were also diluted to 1.5 × 106 cells/ml in Expi293™ Expression Medium one day prior to transfection. On the day of transfection the cells were centrifuged and resuspended at 2.5 × 106 cells/ml in 30 ml of fresh media. Two tubes containing 1.5 ml of Gibco™ Opti-MEM™ (Thermo Fisher Scientific) were prepared. 30 ug of plasmid DNA was added to one tube and 80 ul of Expifectamine was added to the other. The solutions were mixed and incubated at room temperature for 30 minutes. The DNA-transfection reagent complex was added to the cells, which were cultured in 5% CO2 at 37° C., 125 rpm. Following 16-18 hrs incubation, transfection enhancers 1 and 2 were added to the cells according to the manufacturers protocol. The cells were incubated for a further 96 hours.
The cells were harvested by centrifugation for 10 minutes at 4000 rpm. The ~30 ml supernatant was filtered through a 0.22 µm filter and diluted to 50 ml with binding buffer (50 mM HEPES, pH 7,4, 250 mM NaCl, 20 mM imidazole) containing Complete™ EDTA-free protease inhibitors (Roche) to facilitate binding to the column. A 1 ml HisTrap™ HP column (GE Healthcare) was connected to an AKTA Start (GE Healthcare) and pre-equilibrated with binding buffer. The protein-containing media was loaded onto the column using a flow rate of 1 ml/min. The column was washed with >10 CV of binding buffer before the protein was eluted using a 20-300 mM imidazole gradient over 12 ml. 0.5 ml fractions were collected and analysed by SDS-PAGE. Protein containing fractions were pooled and concentrated using Amicon® Ultra centrifugal filter units (Millipore).
Purified proteins were analysed by separating out on SDS-PAGE under denaturing condition. Typically, 1-2 µg of purified protein were loaded per lane on SDS-PAGE gel. The gels were run in Tris-Glycine buffer containing 0.1% SDS. A constant voltage of 150 volts was used and the gels were run for ~70 mins until the dye front has migrated fully.
SDS-PAGE (15% Bis-Tris) gels were prepared using the following resolving and stacking gels.
Resolving Gel:
Stacking Gel:
The potential of the purified Quad proteins to bind its target protein was confirmed by indirect binding ELISA. Briefly, high binding 96 well plates (Corning) were used for coating recombinant target protein (1-2.5 ug/ml diluted in PBS or as indicated), which were typically stored at 4° C. overnight. Plates are then washed 3 times with 200 ul wash buffer (PBS + 0.1% Tween) and blocked using 200 ul blocking buffer (PBS + 1% BSA) for 1 hour at room temperature. Purified protein samples are typically serially diluted in dilution buffer (PBS + 0.1% BSA) and 100ul/well is added. Samples are incubated at room temperature for 1 hour after which the plate is washed again 3 times using 200 ul wash buffer. Detection antibodies as indicated in the text was diluted according to the manufacturer recommendation is added and incubated at room temperature for 1 hour. The plate is washed for the final time using 3x 200 ul wash buffer and 50 ul pre-warmed detection reagent (TMB - Sigma) is added per well and the plate incubated in the dark for 10-30 mins. The reaction is stopped by adding 25 ul/well of 1 M sulfuric acid. The absorbance at 450 nm was read using a CLARIOstar microplate reader (BMG Labtech). For competitive ELISA, a fixed amount of biotinylated spike protein or CoV-2 RBD protein (0.5 nM - 7.5 nM or as indicated) is mixed with the purified protein samples and pre-incubated at room temperature for 30 minutes prior to adding to protein coated ELISA plates. Europium labeled streptavidin is used as a detection reagent (Perkin Elmer).
CR3022 is an antibody to SARS-CoV-1. It binds the receptor-binding domain (RBD) of CoV-1 more strongly than SARS-CoV-2 receptor binding domain (RBD). To enhance the CR3022 cross-reactive binding strength to CoV-2 RBD, the variable regions of CR3022 were used to generate different Fab Quad formats either with or without Fc region in order to increase the Fab binding domain valency from bivalent to tetravalent schematically shown in
Human ACE2 is a key receptor that SARS-CoV-2 virus uses to gain entry into cells to cause infection. The receptor-binding motif of SARS-CoV-2 is the main attachment point for ACE2. Thus, to generate a decoy ACE2 Quad to neutralize virus binding ACE2 expressed on host cell surface, an ACE2 Fc fusion Quad protein was generated. The extracellular domain of ACE2 was fused to IgG Fc directly at the hinge region, which was then linked to the p53 tetramerization domain as schematically shown in
CR3022, CR3022-based Quads and ACE2 Ig-TD proteins were produced in Expi293 cells as soluble secreted proteins and their binding to CoV-1 and CoV-2 RBD was analysed by indirect ELISA. For CR3022 based Quads, high-binding ELISA plates were coated with either 100 ng of CoV-1 or 100 ng CoV-2 RBD and serially diluted antibody was added from 50 nM. Bound antibody was detected using Pro L HRP. As expected, CR3022 showed stronger binding to CoV-1 RBD than CoV-2 RDB (
The different CR3022 Quad formats were also tested for their ability to neutralize ACE2: Spike interaction in a competitive ELISA. Recombinant ACE2-IgG (200 ng) was used to coat ELISA plates and then serially diluted antibody starting from 60 nM with a fixed amount of full-length spike trimer (7 nM) was preincubated at room temperature for 30 minutes before adding to each well. Wells with only spike protein added without any inhibiting antibody was used as 100% binding. Europium labelled streptavidin was used to detect the amount of spike protein that was bound to ACE2. CR3022 was unable to neutralize ACE2:spike interaction even at the maximum concentration used in this assay (
Similarly for the ACE2 Ig-TD tetravalent Quad, it was tested for it’s ability to bind both full-length spike protein trimer or CoV-2 RBD by indirect ELISA. ACE2 Ig-TD Quad (100 ng) was used to coat ELISA plates and serially diluted full-length spike protein or CoV-2 RBD from 100 nM was added to the coated ELISA plate. Bound spike protein or CoV-2 RBD to ACE2 was detected using anti-His HRP. ACE2 Ig-TD Quad bound both spike protein and CoV-2 RBD in a dose-dependent manner (
To examine whether ACE2 Ig-TD Quad was able to neutralize the interaction of either ACE2:CoV-1 RBD or ACE2:CoV-2 RBD, ELISA plates were coated with 200 ng of recombinant ACE2-Ig protein. Serially diluted ACE2 Ig-TD Quad from 100 nM with a fixed amount of CoV-1 or CoV-2 RBD (7 nM) was added to each well. Wells with only CoV-1 or CoV-2 RBD protein added without ACE2 Ig-TD was used as 100% binding. Bound CoV-1 RBD or CoV-2 RBD to ACE2 was detected using anti-His HRP (
To compare the neutralization potency of the CR3022 based Quads and ACE2 Ig-TD, the competition ELISA as described above was set up using 250 ng of recombinant ACE2-Ig protein to coat the ELISA plate and 7.5 nM fixed amount of CoV-2 RBD together with serially diluted CoV-2 inhibitor protein from 30 nM was added to each well. Wells with only CoV-2 RBD protein added without any inhibitor antibody was used as 100% binding. Europium labelled streptavidin was used to detect the amount of CoV-2 RBD protein that was bound to ACE2 (
To extend the utility, SARS CoV-2 cross-reactive nanobody termed ‘GB’ (or ‘QB-GB’) was used to format different versions of Quads with varying valency, size and structure and either with or without an Fc region. The valency of the different formats varied from monovalent to octavalent as shown schematically in
Next the GB Quad proteins were analysed for their ability to bind CoV-2 RBD in an indirect ELISA binding assay. ELISA plates were coated with 200 ng CoV-2 RBD protein and serially diluted GB Quad proteins from 100 nM was added to the wells. Binding of GB Quad proteins to CoV-2 RBD was detected using anti-FLAG HRP. With the exception of GB VHH (monovalent), which only showed very weak binding at the highest concentration (100 nM), all other GB-based Quad proteins showed a dose dependent binding to CoV-2 RBD (
To tease out the difference in binding strength and to demonstrate the difference in the neutralization potencies between the different GB Quad molecules, a competitive ELISA was performed. ELISA plates were coated with 250 ng of recombinant ACE2 Ig fusion protein and serially diluted GB proteins from 15 nM with a fixed amount of CoV-2 RBD (2 nM) was added to the coated ELISA plate. Wells with only CoV-2 RBD protein added without GB protein was used as 100% binding. Europium labelled streptavidin was used to detect the amount of CoV-2 RBD protein that was bound to ACE2. The data were plotted as percentage inhibition of ACE2:CoV-RBD interaction in three groups (
As part of this experiment, two additional nanobodies termed ‘BG’ and ‘FE’ (also known as ‘QB-BD’ and ‘QB-FE’ respectively) were selected for testing as Quad formats to bind and neutralize SARS-CoV-2. Nanobody BG and FE were produced as monovalent VHH and also reformatted into octavalent monomeric Ig-TD format as schematically represented in
A competitive ELISA was performed only with the monomeric Ig-TD formats for BG and FE as described above and the non-Quad formats of GB VHH were included for comparison (
All the IC50 values from the competitive ELISA are summarized in Table 8.
The exemplification of nanobody based Quad reformatting into multiple different formats highlighted the power of increasing binding domain valency has on the neutralization potency. In the competitive ELISA described above, the limited assay window together with the detection limitations prevented accurate measurements of some the most potent Quads such as the octavalent Quads causing rapid signal saturation. As a means to improve the assay signal and tease out the enhanced potency of two of the most potent GB-based Quads further, a competitive sandwich ELISA was performed with substantially reduced amounts of CoV-2 RBD similar to that reported by Hansen et al. In their sandwich ELISA a capture antibody was used to capture his-tagged ACE2 protein followed by the addition of a reduced amount of CoV-2 RBD (10-15 pM) plus serially diluted CoV-2 inhibitor antibody. To replicate a similar assay, ELISA plates were coated with 100 ng anti-human IgG to capture 200 ng of recombinant ACE2 IgG. Then to each well a fixed amount of CoV-2 RBD (0.5 nM) together with serially diluted CoV-2 inhibitor antibody (Q185 or Q186/Q182) was added. Europium labelled streptavidin was used to detect the amount of CoV-2 RBD protein that was bound to ACE2. In this assay set-up, the assay window was improved and the IC50 value for Q185 and Q186/Q182 (
In example 29, SARS CoV-2 cross-reactive nanobody ‘GB’ was formatted into different multivalent formats with varying size, shape and valency. In this example, the same SARS CoV-2 cross-reactive ‘GB’ nanobody was used to generate multivalent Quads where the VHH was linked in tandem via a flexible peptide linker as shown schematically in
Additional tandem VHH Quad formats were generated either as monospecific containing only ‘GB’ linked in tandem (
The tandem VHH Quad formats generated in this example were expressed in Expi293F cells and the resulting proteins were affinity purified directly from the culture supernatant as described in example 29. The purified proteins were analysed by SAS-PAGE as denatured non-reduced protein and reduced protein (
To further analyse these tandem VHH Quad formats, their enhanced potential to neutralize the interaction of SARS-CoV-2 spike protein with the ACE2 receptor, a competitive ELISA assay was performed as detailed in example 29 with the following conditions. High binding ELISA plates were coated with 100 ng of anti-human Ig, which was used to capture 100 ng of ACE2-Fc. A fixed amount of SARS-CoV-2 RBD biotinylated protein at 15 pM was pre-mixed for 30 mins with serially diluted 1 in 3 folds of anti-SARS-CoV-2 tandem VHH Quads from 30 nM before adding to the ELISA plate with the captured ACE2-Fc. Any SARS-CoV-2 RBD not competed by the Quad molecules were detected using anti-Strep-HRP detection antibody. The signal obtained from the wells with only RBD added without any competitor was used to determined 100% binding and wells with no added RBD or competitor molecule was used to determine zero binding. From this, the percentage inhibition of SARS-CoV-2 RBD interaction with ACE2 at different competitor concentration was plotted (
All of the tandem VHH Quads were found to be significantly more potent at neutralizing SARS-CoV-2 RBD interaction with ACE2 than the three clinical stage anti-SARS-CoV-2 antibodies. The most potent of the tandem VHH Quad (Q185B) with IC50 value of 0.03 pM was found to be between 23,000 - 63,000 times more potent than the three clinical stage antibodies in this competitive ELISA assay setting. The massive improvement in neutralization potency using tandem VHH to generate Quads clearly confirms the multivalent strategy is a good approach for improving antibody functionality. This opens up the scope to further developing Quad formats with even higher neutralization potencies.
To extend the multivalent Quad formats beyond octavalency using tandem VHH, in this example twelve additional Quad formats were generated utilizing either the tandem ‘GB’ VHH or ‘GB’ and ‘EE’ VHH linked in tandem to generate monospecific and bispecific Quad formats respectively. The different Quad formats varied in shape, size and valency as schematically represented in
The multivalent tandem VHH Quads were expressed in Expi293T cells as described in example 29. Surprisingly and like all of the previously described Quads formats, these tandem VHH formats all expressed well with high purity as soluble secreted protein with average protein titres >150 mg/L. Following affinity purification with Ni-NTA, the proteins were analyzed by SDS-PAGE with either non-reduced or reduced protein. All of the Quad proteins separated out on the non-reduced SDS-PAGE (
Given the further increase in binding domain valency in the Quad formats in this example compared to those in example 30, the expectation is that the neutralization potency would also be further improve. To demonstrate the potency enhancement, competitive ELISA was performed using a selection of Quad proteins with increased stringent condition. The competitive ELISA was performed similarly to that described in example 30 with the exception that 2 nM fixed amount of biotinylated SARS-CoV-2 RBD was used instead of 15 pM. The most potent Quad molecule described in example 30 (Q185B) along with the clinical stage mAb ‘REGN10987’ was also run alongside these Quads for comparison (
Furthermore and as expected, all of the new Quads tested in this example (Q203/Q205, Q177D, Q185D, Q185E, Q209/Q182 and Q209/Q205) showed improved neutralization potency compared to Q185B, which was found to be the most potent Quad in example 30. However, the extent of the improvement in potency between the new Quad formats highlighted in this example and Q185B was not observed as substantial given the binding domain valency difference between for example Q209/Q208 and Q185B being 24 and 8, respectively. The most likely explanation for this is probably due to the limited assay sensitivity and therefore any significantly gain in potency could not be differentiated due to the rapid saturation of the assay signal. To determine the true potency of these multivalent Quad formats, which are expected to be ultra-potent would warrant further investigation of these Quads in an in vivo setting where a measure of a biological effect rather than a assay signal would be measured and thus making it possible to differentiate and observe their true potency enhancement.
The first-in-class clinical stage anti-SARS-CoV-2 mAbs REGN10987, REGN10933 (Hansen et al., 2020) and CB6 (Shi et al., 2020) are currently being trialed for their efficacy in reducing viral load and viral infections in COVID-19 patients. Given the results highlighted in the above examples whereby increasing the binding domain valency, a consistent improvement in neutralization potency could be observed in the in vitro neutralization assays. Further as shown in example 31, REGN10987 could not neutralize SARS-CoV-2 RBD binding to ACE2 using the stringent assay conditions compared to the multivalent Quads. In this example, the binding domains of REGN10987, REGN10933 and CB6 were used to reformat them into three different multivalent Quad formats to show 1) How the different formats can neutralize the SARS-CoV-2 binding to ACE2 and 2) Whether the different formats effect the neutralization potential due to their size and structural configuration.
In the first format termed Ig-TD, the p53 tetramerization domain was directly linked to the C-terminus of the mAb with the CH3 domain as schematically shown in
The three different Quad formats of REGN10987, REGN10933 and CB6 were expressed in Expi293T cells. Like normal IgG antibodies, the Quad formats of these mAbs also expressed as soluble secreted proteins where the assembled tetrameric Quad proteins were harvested from the culture supernatant and affinity purified. The purified proteins were separated out on SDS-PAGE gels using either non-reduced or reduced proteins (
To check for improved neutralization potency of the Quad formats over the parental IgG format, competitive ELISA was performed as described in example 29 with some changes in the assay conditions. Instead of SARS-CoV-2 RBD, biotinylated SARS-CoV-2 Spike trimer was used at a fixed concentration of 75 pM. For each of the clinical stage mAb, the three different Quad formats were compared for their neutralization potential compared to the parental IgG mAb and against Q185B as highlighted in
As described in previous examples, VHH nanobodies can be rapidly designed into different multivalent formats starting from simple monomeric building blocks. The VHH sequence Nb-112 against SARS-CoV-2 spike protein described previously (Esparza et al., 2020) was used to design different multivalent formats with varying size, shape, flexibility and valency.
One of the advantages of these multivalent Quads, apart from them being highly soluble, stable and amenable to large-scale production, is that they are relatively small in size, particularly the tetravalent versions without Fc compared to standard mAbs. The relative small size would be particularly advantageous when formulating these molecules for delivery through inhalation. Owing to their high stability properties, Quad based nanobodies is likely to maintain the structural integrity and thus the functional properties to neutralize SARS-CoV-2 after nebulization. Further, given the size of these Quads being above the renal clearance threshold, Quads delivered through inhalation is likely to persist for a longer period and provide a molecule with significantly enhancement efficacy than the native VHH Nb-112 nanobody.
The tetravalent (also called Q232) and octavalent (also called Q233) versions of Nb-112 were produced as schematically represented in
To investigate the functionality of the multivalent Nb-112 Quad proteins, competitive ELISA was performed for their ability to neutralize the interaction of SARS-CoV-2 spike RBD with the ACE2 receptor. The monovalent Nb-112 was reported to be a potent neutralizer of spike RBD interaction with ACE2 (Esparza et al., 2020). Therefore, two different amounts of spike RBD were used in competition ELISA. Briefly, high binding ELISA plates were coated with 100 ng/well of anti-human IgG and incubated at 4° C. overnight. After blocking with 1% BSA for 1 hour at room temperature, 100 ng/well ACE2-Fc was added and the plate incubated for a further 1 hour. The plates were washed 3 times with sample buffer (PBS containing 0.1% Tween) and 100 ul/well serially diluted anti-SARS-CoV-2 molecules (Q231-Q233 and REGN10933 as positive control) starting from 30 nM premixed at room temperature for 30 minutes with a fixed about of biotinylated SARS-CoV-2 RBD either at 2 nM (
From the data, a nice dose-dependent inhibition of SARS-CoV-2 interaction with ACE2 can be seen in the competitive ELISA at both the high (2 nM -
Following on from Example 33 where tetravalent and octavalent versions of Nb-112 were generated, in this example a modified tetravalent and octavalent version of Nb-112 were designed and produced where an intact hinge region was included in the construct. The hinge region allows for the dimerization of the monomeric building blocks to form dimers upon which these dimers are further dimerized in an anti-parallel manner through the p53 tetramerization domain to form tetramers. A schematic structural arrangement of these new tetravalent (also called Q246 or Nb-112-S-S-TD) and octavalent (also called Q247 or [Nb-112]2-S-S-TD) Quads can be seen in
Expression vectors for producing Q246 and Q247 were transfected into Expi293F cells and the proteins were produced as described in Example 28. As with Q232 and Q233 described in Example 33, the intrinsic binding properties of the VH3 subfamily for Protein A resin was exploited to purify both Q246 and Q247. The protein yields after Protein A affinity purification was measured using Nanospec and it can be seen the tetravalent (Q246) and octavalent (Q247) versions of Nb-112 yielded excellent protein titers equivalent to 350 mg/L and 265 mg/L respectively. The protein yields were similar to the tetravalent and octavalent versions described in Example 33 indicating that the present of the hinge region did not hamper protein production. To analyse the produced protein, a small aliquot (2 ug/well) were separated out on denaturing SDS-PAGE under non-reduced conditions alongside Nb-112 proteins produced in Example 33. For Q246 and Q247, the protein was also separated out under reduced conditions to visualize the monomeric building block polypeptide (
To investigate the functionality of the two new tetravalent and octavalent Nb-112 Quads (Q246 and Q247), a competitive ELISA was performed for their ability to neutralize the interaction of SARS-CoV-2 spike RBD with the ACE2 receptor as described in Example 33 using the same conditions. As a comparison, Q231-Q233 was also tested alongside Q246 and Q247 including the benchmark anti-SARS-CoV-2 antibody REGN10933. The data was plotted as percentage neutralization over a range of anti-SARS-CoV-2 inhibitor concentration (
From the in vitro neutralization assay data, a nice dose-dependent inhibition of SARS-CoV-2 interaction with ACE2 can be seen where the new tetravalent and octavalent versions (Q246 and Q247 respectively) were found to be significantly more potent than the monovalent Nb-112 VHH as seen from the IC50 values (
For the reasons discussed in Example 33 in terms of the high affinity of Nb-112 VHH and the limited assay window, the real differences in neutralization potences might not be possible to be differentiated in this in vitro artificial assay setting. A cell-based assay such as a pseudovirus neutralization assay or even better an in vivo animal model would be required to fully characterize and tease out potency differences between these multivalent Quad formats. However, on the whole a good correlation with increase binding domain valency with increase neutralization potency can be seen when comparing the neutralization potences between the tetravalent and octavalent versions of Nb-112 to the monovalent version confirming that these new multivalent formats are functionally active and are capable of neutralizing SARS-CoV-2 through blocking its interaction with ACE2 receptor.
Importantly, these Quad therapeutics may be delivered via inhalation to a human or animal subject. Inhalation has major advantages over other routes of administration and could be one of the most important potential uses for a therapeutic for SARS-CoV-2. For example, nebulisation may be performed using a commercially available human-use Aeroneb Solo™ system (https://www.aerogen.com/aerogen-solo-3/), interfaced with a nose mask. The Aeroneb Solo nebuliser can, thus, be used to produce ~ 3 micron particles of Quads for lung delivery. This is suitable for deep lung delivery.
In an experiment, we will test once daily administration to lambs of a nebulised Quad. We expect that this will result in concentrations of the Quad in lung epithelial lining fluid that are at least 10 times higher than the in vitro EC50 of the Quad for the cognate virus (eg, SARS-CoV-2). We will assess whether there is any detectable infectious virus in lung epithelial lining fluid, with no or low detectable virus supporting that the proposed route of administration could reduce infectivity. After administration of nebulised Quad, we expect blood concentrations will be lower (eg, 1000 fold lower or less) than lung epithelial lining fluid concentrations, which is important because low blood concentrations likely reduce systemic toxicity and risk of host antibody formation. Alternative routes of administration of Quads include intravenous, intramuscular or subcutaneous administration
Using the commercially available Aerogen Solo™ vibrating mesh nebuliser, we will nebulise Pichia pastoris-expressed Quad into an in line custom bead condenser. Analysis by size-exclusion chromatography on a Superdex™ 75 column will be carried out to establish no evidence of degradation or aggregation relative to the pre-nebulisation sample. From this, one can conclude that the Quad is resilient to degradation or aggregation during nebulisation.
Fourt different Quads (
Stability following nebulisation of Pichia pastoris expressed Quad (eg, a Quad comprising copies of NIH-CoVnb-112) will be performed using an Aerogen Solo High-Performance Vibrating Mesh™ nebuliser placed in line with a custom glass bead condenser. A plastic culture tube will be fitted with a glass-pore frit and filled with sterilized 5 mm borosilicate glass beads. A three-way stopcock will be positioned distal to the frit to prevent pressurization during nebulisation. A 2 mg/mL SEC polished Quad solution will be prepared in 0.9% normal saline to model potential patient delivery. The Quad will be nebulised and the resulting condensate incubated at 37° C. for 24 hr to mimic exposure to body temperature. The nebulised, 37° C. treated Quad will be then collected for stability assessments and protein concentration measurements by BCA assay. Equal masses of pre and post-nebulisation samples will be denatured in LDS sample buffer (Invitrogen) and run on a NuPAGE 12% Bis-Tris precast polyacrylamide gel with SeeBlue Plue 2™ protein standards. Additional pre-and post-nebulisation samples will be injected onto a Superdex 75 Increase 10/300 GL size exclusion column operating on an AKTA Pure 25 M™ system.#
To further assess the stability of the Quad (eg, a Quad comprising copies of NIH-CoVnb-112), we will perform incubation of the Quad in pooled normal human plasma and recombinant human albumin followed by affinity measurement to assess preservation of antigen (eg, SARS-CoV-2 RBD) binding potential.
A Quad (eg, a Quad comprising copies of NIH-CoVnb-112) expressed in Pichiapastoris will be diluted from a concentrated stock solution into apheresis derived pooled human plasma (#IPLA-N, Innovative Research) to a final concentration of 5 µM and incubated at 37° C. for either 24 hr or 48 hr with gentle rotation. An identical sample set will be prepared at 5 µM in a solution containing 35 mg/mL recombinant human albumin (#A9731, Sigma-Aldrich) and incubated at 37° C. for either 24 hr or 48 hr with gentle rotation. A no-incubation control for each the plasma and recombinant human albumin conditions will be prepared at 5 µM. The samples will be prepared in a manner providing all conditions complete at the same time. Quad binding to antigen (eg, SARS-CoV-2 RBD) following treatment in pooled human plasma at time zero, 24 hr, and 48 hr will, we expect, have negligible impact on binding. Similarly, Quad binding to antigen following treatment in recombinant human albumin at all time points will, we expect, have no apparent effect on binding. Such data will support the interpretation that the Quad is satisfactorily stable in the presence of plasma.
The samples will be diluted 1:10 with IxPBS to yield a final Quad concentration of 500 nM and Bio-layer Interferometry will be performed using immobilized biotinylated antigen (eg, SARS-CoV-2 S protein RBD) to determine retention of binding potential.
Circular Dichroism (CD) will be performed using a Jasco J-815 Spectropolarimeter™. For thermal stability measurements the Quad will be diluted to 10 µg/mL in deionized water and placed in a quartz cuvette with 1 cm path length and CD measured at an ultraviolet wavelength of 205 nm. Quad will be heated from 25° C. to 85° C. at a rate of 2.5° C./min while stirring and then cooled back to 25° C. at the same rate. We expect that measurements using circular dichroism (CD) during heating will reveal that the Quad structure resists unfolding at elevated temperature (eg, until 74° C.) and upon cooling most (eg, at least 50, 60, 70 or 80%) of the structure will return to the baseline CD value. These data will support an extremely stable, robust, high affinity format.
Using Aerogen Solo™ with an 8 stage cascade impactor running at a continuous flow rate of 28.3 LPM - see Table 37.
Quads (separately, Quads 1-4) will be produced using a HEK203, CHO or Pichia pastoris X-33 expression system. Formulation buffer will contain NaCl as osmolality agent and phosphate as buffer component.
An Aeroneb Solo™ System (Aerogen Ltd, Galway, Ireland), containing the Aeroneb Solo mesh nebulizer and the Aeroneb Pro-X™ controller will be used in accordance with the instruction manual as provided by the manufacturer. The estimated particle sizes obtained with these meshes will be around 3 to 3.5 µm (MMAD, Median mass aerodynamic diameter). The assembly and operation of the Aeroneb Solo System will be performed according to the nebulizer instruction manual. The nebulizer and the T-piece will be inserted into the breathing circuit. Air will be supplied to the system at an airflow speed of 2 L/min using a compressed air canister that is attached directly to the nebulizer T-piece.
Testing will be performed in lambs. Prior to dosing, the nebulizer reservoir will be filled with following volumes of different concentrations of the Quad formulation: either 4 mL (3 mg/kg target inhaled dose), 1.3 mL (1 mg/kg target inhaled dose) 0.4 mL (0.3 mg/kg target inhaled dose), 0.2 mL (0.08 mg/kg target inhaled dose) or 0.1 mL (0.04 mg/kg target inhaled dose). A cone mask (Cat # 05305, A.M. Bickford, Inc, US) will be attached to the nebulizer T-piece and placed over each lamb’s nose, mandible and maxilla. The nebulizer will be turned on at a constant nebulization mode and the cone mask firmly held in place during the duration of the nebulization. Once the dose has been nebulized (i.e., when the nebulizer reservoir is empty), the face mask will be removed and the nebulizer switched off. The lamb will then be returned to its cage and general health (alertness, responsiveness, ability to stand and move) will be monitored for 10 minutes.
Four independent studies will be performed in SARS-CoV-2-infected neonatal lambs. In all of these studies, blood samples will be taken at selected time points followingthe first dose and all the subsequent doses for pharmacokinetic (PK) purposes. On Day 6, bronchoalveolar lavage fluid (BALF) sampling will be performed post-mortem for PK analysis in the lung compartment. Once daily administration of Quad via inhalation for 5 or 3 consecutive days is expected to result in high concentrations of Quad in lung epithelial lining fluid (ELF). A dose-dependent increase in ELF concentrations is expected to be seen on day 6.
To assess the therapeutic efficacy of Quad when administered by inhalation, thirteen lambs (twelve lambs for analysis) will be inoculated with SARS-CoV-2 virus on day 0. The day after infection (day 1), the lambs will be randomized to either the placebo group or to Quad dose groups (0.3, 3 or 1 mg/kg) and treated daily by inhalation for 5 consecutive days. Lambs will undergo daily physical examinations and body weights, heart rates, rectal temperatures, respiratory distress and viral infection-related symptoms will be recorded. On day 6, the animals will be euthanized and lung lavage samples and lung tissues will be obtained for analysis of viral load in lung, histopathology and immunohistochemical analysis. Viral inoculation by inhalation will be confirmed for robust infection of all the analyzed lambs by reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed on BALF and lung tissue. In the placebo-treated lambs, we expect gross and microscopic lung lesions induced by viral infection. We expect that treatment of lambs with Quad at all three doses will result in significant reductions in viral RNA copy numbers (eg, that range from 1.4 to 1.8 Log10 viral RNA copies/mL in BALF and between 0.8 to 1.9 Log10 viral RNA copies/mg in lung tissue). We expect that treated lambs will have much lower or undetectable infectious virus.
Since SARS-CoV-2 first emerged, recurrent mutations in spike have occurred during both human-to-human transmission (and spillover/spillback events between humans and animals. Among mutations associated with enhancement of human-to-human transmission, N501Y occurred in three distinct emerging human variants: B.1.1.7 lineage (or 20IB/501Y.V1), B.1.351 lineage (or 20H/501Y.V2), and P.1 lineage (or 20J/501Y.V3) that were originally identified in the United Kingdom, South Africa and Japan/Brazil, respectively. The key involvement of residue 501 in ACE2 binding may contribute to increased prevalence of mutations at this site in multiple distinct SARS-CoV-2 strains. The B.1.351 and P.1 lineages also carry the RBM mutations E484K and K417N/T. Meanwhile, the N-terminal domain (NTD) deletion AHV69-70 arose in the mink-associated Cluster V strain and in B.1.1.7. The B.1.1.7 also has ΔY144, and B.1.351 has a ΔLLA242-244 deletion. Other mutations that may facilitate immune escape (e.g., A222V, N439K and S477N) are frequently observed in patient samples. Three mutations in the receptor-binding motif (RBM) (Y453F, F486L and N501T), associated with cross-species transmission between minks and humans, emerged independently in distinct clusters, suggesting they could be vital points for new host adaptation. Additional selection pressures, not directly related to receptor adaptation may also exist, as evidenced by mutations outside the RBM in human-animal transmission (e.g., the N-terminal domain deletion AHV69-70, G261D and RBD point mutations V367F).
Mutations in the spike protein can have various impacts on antibody binding footprints and affinity, ranging from no effect to substantial impairment of recognition and binding. Amino acid substitutions or deletions that appeared once in spike might appear elsewhere, new variants having a different assortment of mutations may emerge, and current variants may acquire new single mutations. Thus, work was carried out to dissect the effects of key mutations individually to understand they affect binding molecule (antibody or Quad) activities. First, the binding affinity for full-length G614 HexaPro spike ectodomain and to the monomeric receptor-binding domain (RBD) was determined. Using high-throughput surface plasmon resonance analysis, those binding molecules that react with the RBD were sorted into different “communities”. Communities were defined by shared competition profiles in a matrix, in which each antibody was evaluated for its ability to either pair with or compete with other antibodies for binding to spike. Some additional communities of binding moleucles were identified against the NTD (N-terminal domain) by mapping of antigenic sites using electron microscopy. In total, 14 different communities that react with the spike S1 domain were identified. The antigenic landscape of spike determined by these 14 communities can be divided into binding footprints that: (a) overlap with the RBM (receptor-binding motif), (b) approach the RBD from the outer edge, (c) involve the inner face of the RBD and are accessible only in the “up” state, or (d) include the NTD.
To understand if certain binding molecule communities are more susceptible to particular emerging spike mutations than others, single-cycle VSV-based pseudoparticles bearing point mutations found in human and mink variants were generated and the susceptibility of each mutation to neutralization by the molecules was assessed. Notably, a Quad multimer comprising copies of variable domain QB-GB, which binds outside of the core RBM, was found to be resistant to all S1 mutations analyzed and retained neutralization comparable to the parent virus. This Quad was found to bind to the inner face of the RBD. Strong binding was observed (IC50 of 0.0017 µg/mL). Near 100% ACE2 blocking was surprisingly observed (99.84%), and yet the multimer does not contact core RBM residues. This Quad multimer and other multimers binding spike protein in the same region, therefore, may advantageously be more resistant to receptor-driven selection pressure.
Table 8: Description of monomeric building block of Quad Formats A - AC and as outlined in
In the schematics, components of polypeptide chains are N- to C-terminally from top to the bottom of each chain as shown.
Herein the terms “SAM”, “self-associating multimerization domain” and “multimerisation domain” are used interchangeably. A SAM may, for example be called a TD herein. A TD may be a tetradimerisation domain as disclosed herein, eg, a p53, p63, p73 or NHR2 domain or homologue or orthologue thereof.
Valency indicates binding site number in the tetramer form when using a TD as a SAM.
SEQ ID NO: 188 (Quad 68 nucleotide sequence)
SEQ ID NO: 189 (Quad 69 nucleotide sequence)
SEQ ID NO: 190 (Quad 68 amino acid sequence)
SEQ ID NO: 191 (Quad 69 amino acid sequence)
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGK GLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSL KASDTAIYYCAGGSGISTPMDVWGQGTTVTVASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTKKKPLDGEYFTLQIRGRERFEMFRELNEALELK DAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGK GLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSL KASDTAIYYCAGGSGISTPMDVWGQGTTVTVASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALE LKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGK GLEWMGIIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSL KASDTAIYYCAGGSGISTPMDVWGQGTTVTVASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFREL NEALELKDAQAGKEPG
DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQA EDVAVYYCQQYYSTPYTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQN GSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEP GLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEE IKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFW TNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSV GLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMC TKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAV GEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIV GTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA
KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD
KKKP LDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQN GSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEP GLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEE IKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFW TNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSV GLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMC TKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAV GEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIV GTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADEPKS CDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDA QAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQN GSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEP GLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE MARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEE IKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFW TNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSV GLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMC TKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAV GEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIV
GTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEP VPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAA KHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGA KNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRE RFEMFRELNEALELKDAQAGKEPG
(a) Taken from Trends Immunol. 2020 Apr 24, doi: 10.1016/j.it.2020.04.008, “Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses”, Trends in Immunology 41, 355-359; 2020, Shibo Jiang, Christopher Hillyer, and Lanying Du, which is incorporated herein in its entirety by reference (including the references cited therein (to which the numbers in the last column of this table refers) and antibody and V domain sequences therein).
(b) DNA sequences encoding the VH and VL of VH/VL binding sites for Anti-SARS-CoV-2 antigen (eg, spike); VH-IHI pairs with VL-IHI; VH-IHG pairs with VL-IHG, etc. VH-IHI (SEQ ID NO: 295):
VL-IHI (SEQ ID NO: 296):
VH-IHG (SEQ ID NO: 297):
VL-IHG (SEQ ID NO: 298):
VH-ICC (SEQ ID NO: 299):
VL-ICC (SEQ ID NO: 300):
VH-ICD (SEQ ID NO: 301):
VL-ICD (SEQ ID NO: 302):
VH-IGG (SEQ ID NO: 303):
VL-IGG (SEQ ID NO: 304):
VH-IFD (SEQ ID NO: 305):
VL-IFD (SEQ ID NO: 306):
VH-IED (SEQ ID NO: 308):
VL-IED (SEQ ID NO: 309):
VH-IHD (SEQ ID NO: 310):
VL-IHD (SEQ ID NO: 311):
VH-IHF (SEQ ID NO: 312):
VL-IHF (SEQ ID NO: 313):
Table 23. Amino acid sequences of anti-SARS-CoV/anti-SARS-CoV-2 in various formats for capturing and/or neutralizing SARS-CoV, SARS-CoV-2 and other related coronavirus strains. Each of the sequences outlined in this table can be further generated with or without a N- or C-terminus affinity tag as outlined in Table 22. The tetramerization domain (TD) is preferably TD from p53 but not limited by it. CR3006, CR3013, CR3014 and CR3022 are example antibody sequences for anti-SARS-CoV and CoV-2 strains. Other anti-SARS-CoV/anti-SARS-CoV-2 could be composed of single domain antibodies (dAb) such as those from camelid. Examples of anti-SARS-CoV/anti-SARS-CoV-2 dAb include QB-DD and QB-GB formatted into different Quad formats as shown in
The sequences of binding domains (eg, VH and VL) for use in the present invention are underlined and are intended to be read as written (ie, as part of the full sequence including non-underlined part) or alternatively without the futher underlined sequence.
In an embodiment, the polypeptide of the invention comprises a sequence that is shown in bold in this Table 23. In an embodiment, the polypeptide of the invention comprises, in N- to C-terminal direction, a binding domain or peptide; and a sequence that is shown in bold in this Table 23. The binding domain may be any binding domain disclosed herein, eg, a VH, VL, VHH, dAb or scFv.
The present disclosure further comprises each underlined sequence without the following bold sequence, eg, to provide a binding site or domain for use in any polypeptide or mutimer of the invention herein. The present disclosure further comprises each bold sequence without the immediately preceding underlined sequence, eg, to for use in any polypeptide or mutimer of the invention herein (such as as part of a polypeptide comprising a binding domain or peptide immediately preceding the bold sequence).
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG
DIQMTQSPHSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIY AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVGVYYCQQRFRTPVTF GQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRER FEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTVGGGGSGGGGSGGGGSDIQMTQSPHSLSAS VGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS
GSGSGTDFTLTISSLQPEDVGVYYCQQRFRTPVTFGQGTKLEIK
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGSPRTPSFDYWGQGTLVTVGGGGSGGGGSGGGGSDIQMTQSPHSLSAS VGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS GSGSGTDFTLTISSLQPEDVGVYYCQQRFRTPVTFGQGTKLEIK
KKKPL DGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYPMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGSPRTPSFDYWGQGTLVTV
GGGGSGGGGSGGGGSDIQMTQSPHSLSAS VGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFS GSGSGTDFTLTISSLQPEDVGVYYCQQRFRTPVTFGQGTKLEIKEPKSC DKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMF RELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG
ELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQ GTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRER
FEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKKKKPLDG EYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC AKGLTPLYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG
DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK
EPKSCDK THTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRE LNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG
ELTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQ GTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRER FEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK
KKKPLDG EYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVG RTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC ARGISPFYFDYWGQGTLVTVGGGGSGGGGSGGGGSELTQSPSSLSASVG DRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK
EPKSCDK THTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRE LNEALELKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG
DIQLTQSPDSLAVSLGERATINCKSSQSVLYSSINKNYLAWYQQKPGQP PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYY STPYTFGQGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRERF EMFRELNEALELKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTV
ASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTVGGGGSGGGGSGGGGSDIQLTQSPDSLAVSL GERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSTPYTFGQGTKVEIK
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTVGGGGSGGGGSGGGGSDIQLTQSPDSLAVSL GERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKVEIK
KKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QMQLVQSGTEVKKPGESLKISCKGSGYGFITYWIGWVRQMPGKGLEWMG IIYPGDSETRYSPSFQGQVTISADKSINTAYLQWSSLKASDTAIYYCAG GSGISTPMDVWGQGTTVTVGGGGSGGGGSGGGGSDIQLTQSPDSLAVSL GERATINCKSSQSVLYSSINKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSTPYTFGQGTKVEIK
E PKSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRER FEMFRELNEALELKDAQAGKEPG
QVQLQESGGGLVQTGGSLRLSCAASGSDFSSYAMAWFRQAPG
KEREFVASISRRSTNTYYRNSVKGRFTISRDNAKNTAWLQMN
SLKPEDTAVYYCAADRARYGSSWYESLAYLEVWGQGTQVTVSS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPG
KEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMN
SLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS
SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATI SWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLG TVVSEWDYDYDYWGQGTQVTVSS
GGGGSGGGGSKKKPLDGEYFTLQIRGRE RFEMFRELNEALELKDAQAGKEPG
SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QVQLVESGGGLVQAGGSLRLSCAASGFPVRKANMHWYRQAPGKEREW VAAIMSKGEQTVYADSVEGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCRVFVGWHYFGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCATSGFPVYQANMHWYRQAPGKEREW VAAIQSYGDGTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCRAVYVGMHYFGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVNYKTMWWYRQAPGKEREW VAAIWSYGHTTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCWWVGHNYEGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYAQNMHWYRQAPGKEREW VAAIYSHGYWTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCEVQVGAWYTGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVFSGHMHWYRQAPGKEREW VAAILSNGDSTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCRVHVGAHYFGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVEQGRMYWYRQAPGKEREW VAAIISHGTVTVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVYVGAQYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVLFTYMHWYRQAPGKEREW VAAIWSSGNSTWYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCFVKVGNWYAGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVNAGNMHWYRQAPGKEREW VAAIQSYGRTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCRVFVGMHYFGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVSSSTMTWYRQAPGKEREW VAAINSYGWETHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVYVGGSYIGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVQSHYMRWYRQAPGKEREW VAAIESTGHHTAYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY
YCTVYVGYEYHGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVETENMHWYRQAPGKEREW VAAIYSHGMWTAYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVY YCEVEVGKWYFGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVKASRMYWYRQAPGKEREW VAAIQSFGEVTWYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVWVGQEYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYASNMHWYRQAPGKEREW VAAIESQGYMTAYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCWVIVGEYYVGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVQAREMEWYRQAPGKEREW VAAIKSTGTYTAYAYSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVYVGSSYIGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVKNFEMEWYRKAPGKEREW VAAIQSGGVETYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCFVYVGRSYIGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVAYKTMWWYRQAPGKEREW VAAIESYGIKWTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCIVWVGAQYHGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVAGRNMWWYRQAPGKEREW VAAIYSSGTYTEYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCHVWVGSLYKGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVKHARMWWYRQAPGKEREW VAAIDSHGDTTWYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVYVGASYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVNSHEMTWYRQAPGKEREW VAAIQSTGTVTEYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCYVYVGSSYLGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVEQREMEWYRQAPGKEREW VAAIDSNGNYTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY
YCYVYVGKSYIGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVKHHWMFWYRQAPGKEREW VAAIKSYGYGTEYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCFVGVGTHYAGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYAAEMEWYRQAPGKEREW VAAISSQGTITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCFVYVGKSYIGQGTQVSVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVHAWEMAWYRQAPGKEREW VAAIRSFGSSTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDFGTHHYAYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVNTWWMHWYRQAPGKEREW VAAITSWGFRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDKGMAVQWYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYHSTMFWYRQAPGKEREW VAAIYSSGQHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDSGQWRQEYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVEHEMAWYRQAPGKEREWV AAIRSMGRKTIYADSVKGRFTISRDNAKNTVYIQMNSIKPEDTAVYY CNVKDFGYTWHEYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVTMAWMWWYRQAPGKEREW VAAIRSEGVRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDYGQAHAYYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVNSHFMEWYRQAPGKEREW VAAIQHSSGFHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCNVKDTGTTEDYDYWGQGTQVTVS
QVQLDESGGGLVQAGGSLRLSCAASGFPVYHAWMEWYRQAPGKEREW VAAITSSGRHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDAGRVYNSYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVAHAWMEWYRQAPGKEREW VAAITSYGYKTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDTGTYRFYYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVWNQTMVWYRQAPGKEREW VAAIWSMGHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CNVKDAGVYNRYYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVEHYWMEWYRQAPGKEREW VAAITSFGYRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDWGFASHAYDYWGQGIQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPEIAWEMAWYRQAPGKEREW VAAIRSFGERTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDFGWQHQEYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYHAYMEWYRQAPGKEREW VAAIYSNGEHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDSGSFNQAYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVEWSHMHWYRQAPGKEREW VAAIVSKGGYTLYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDYGVHFKRYDYWGQGTQVTVI
QVQLVESGGGLVQAGGSLRLSCAASGFPVFHVWMEWYRQAPGKEREW VAAIDSAGWHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDAGNTTSAYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYYNWMEWYRQAPGKEREW VAAIHSNGDETFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDIDAEAYAYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYHVWMEWYRQAPGKEREW VAAITSSGSHTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDSGQWRVQYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYWHHMHWYRQAPGKEREW VAAIISWGWYTTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDHGAQNQMYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYRDRMAWYRQAPGKEREW VAAIYSAGQQTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDVGHHYEYYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVDNGYMHWYRQAPGKEREW
VAAIDSYGWHTIYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDKGQMRAAYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVSWHSMYWYRQAPGKEREW VAAIFSEGDWTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDYGSSYYKYDYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVSQSVMAWYRQAPGKEREW VAAIYSKGQYTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDAGSSYWDYDYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSIGQIEYLGWFRQAPGKEREG VAALNTWTGRTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAARWGRTKPLNTYYYSYWGQGTPVTVS
QVQLVESGGGSVQAGGSLRLSCAASGYIDKIVYLGWFRQAPGKEREG VAALYTLSGHTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAATEGHAHALYRLHYYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYQGEMHWYRQAPGKEREW VAAIRSTGVQTWYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCRVWVGTHYFGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGNIQRIYYLGWFRQAPGKEREG VAALMTYTGHTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAAYVGAENPLPYSMYGYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGQISHIKYLGWFRQAPGKEREG VAALITRWGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAADYGASDPLWFIHYLYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGKIWTIKYLGWFRQAPGKEREG VAALMTRWGYTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAANYGSNFPLAEEDYWYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGNISQIHYLGWFRQAPGKEREG VAALNTDYGYTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAAYYFGDDIPLWWEAYSYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGNISTIEYLGWFRQAPGKEREG VAALYTWHGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY
YCAAARWGRHMPLSATEYSYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGNIESIYYLGWFRQAPGKEREG VAALWTGDGETYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAAAWGNSAPLTTYRYYYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGFIYGITYLGWFRQAPGKEREG VAALVTWNGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAADWGYDWPLWDEWYWYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGTIADIKYLGWFRQAPGKEREG VAALMTRWGSTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAANYGANYPLYSQQYSYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSISSIKYLGWFRQAPGKEREG VAALMTRWGMTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAANYGANEPLQYTHYNYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGEIESIFYLGWFRQAPGKEREG VAALYTYVGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAASYGAAHPLSIMRYYYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSIQAITYLGWFRQAPGKEREG VAALVTWNGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAADWGYDWPLWDEWYWYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREG VAALVTYSGNTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAATWGHSWPLYNDEYWYWGQGSQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREG VAALITVNGHTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAAAWGYAWPLHQDDYWYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREG VAALNTFNGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAATWGYSWPLIAEYNWYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREG VAALKTQAGFTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAANWGYSWPLYEADDWYWGQGTQVTVS
QVQLVESGGGLVQAGGSLRLSCAASGFPVYNTWMEWYRQAPGKEREW VAAITSHGYKTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVY YCNVKDEGDMFTAYDYWGQGTQVTVS
QVQLVESGGGSVQAGGSLRLSCAASGTIAHIKYLGWFRQAPGKEREG VAALMTKWGQTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALY YCAAASYGANFPLKASDYSYWGQGTQVTVS
IRGRERFEMFRELNEALELKDAQAGKEPG
Table 24. Amino acid sequences of ACE2 in different formats for capturing or neutralizing SARS-CoV or SARS-CoV-2 or other related coronavirus strains. Each of the sequences outlined in this table can be further generated with or without a N- or C-terminus affinity tag as outlined in Table 22 and shown schematically in
The ACE2 sequences for use in the present invention are underlined and are intended to be read as written (ie, as part of the full sequence including non-underlined part) or alternatively without the futher underlined sequence.
In an embodiment, the polypeptide of the invention comprises a sequence that is shown in bold in this Table 24. In an embodiment, the polypeptide of the invention comprises, in N- to C-terminal direction, a binding domain or peptide; and a sequence that is shown in bold in this Table 24. The binding domain may be any binding domain disclosed herein, eg, a VH, VL, VHH, dAb or scFv.
The present disclosure further comprises each underlined sequence without the following bold sequence, eg, to provide a binding site or domain for use in any polypeptide or mutimer of the invention herein. The present disclosure further comprises each bold sequence without the immediately preceding underlined sequence, eg, to for use in any polypeptide or mutimer of the invention herein (such as as part of a polypeptide comprising a binding domain or peptide immediately preceding the bold sequence).
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYAD
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYADKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGK EPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS
NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYAD
EPKSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKKKPWGE YFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYAD
EPKSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFE MFRELNEALELKDAQAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN
MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQY FLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKA IRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQY FLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKA IRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSKKKPLDGEYFTL QIRGRERFEMFRELNEALELKDAQAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQY FLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKA IRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS
EPKSCDKTHTAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEAL ELKDAQAGKEPG
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAG DKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKR LNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWA WESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVD
GYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCL PAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEA EKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMS LSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEK WRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVS NDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFN MLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVG WSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQY FLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKA IRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRE LNEALELKDAQAGKEPG
Table 25. Amino acid sequences of immunoglobulin (Ig) binding domains. Sequences are shown without affinity tag but any of the affinity tags outlined in Table 22 can be included at the N- or C-terminus of the Ig binding domain. Each of the Ig binding domains can be linked together without flexible linker as shown in this table or optionally be linked together via a flexible linker. Examples of flexible linker sequences are shown in Table 26. C1-C3 domains are Ig binding domains from Protein G. EDABC domains are Ig binding domains from Protein A. B1-B5 domains are Ig binding domains from Protein L. Each Ig binding domain combination shown in this table can be formatted into different Quad formats by linking them to sequences shown in Table 27 or as outlined schematically in
Each binding domain in this table may be used as a binding domain comprised by a polypeptide or multimer of the invention.
Table 32: Amino acid sequences of further engineered tandem VHH multivalent Quads and Quad formats of clinical stage mAbs targeting SARS-CoV-2.
The sequences of binding domains (eg, VH and VL) for use in the present invention are underlined and are intended to be read as written (ie, as part of the full sequence including non-underlined part) or alternatively without the futher underlined sequence.
In an embodiment, the polypeptide of the invention comprises a sequence that is shown in bold in this Table 32. In an embodiment, the polypeptide of the invention comprises, in N- to C-terminal direction, a binding domain or peptide; and a sequence that is shown in bold in this Table 32. The binding domain may be any binding domain disclosed herein, eg, a VH, VL, VHH, dAb or scFv.
The present disclosure further comprises each underlined sequence without the following bold sequence, eg, to provide a binding site or domain for use in any polypeptide or mutimer of the invention herein. The present disclosure further comprises each bold sequence without the immediately preceding underlined sequence, eg, to for use in any polypeptide or mutimer of the invention herein (such as as part of a polypeptide comprising a binding domain or peptide immediately preceding the bold sequence).
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD
DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSS
GGGGSGGG GSKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSKKK PLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEAL ELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDA
QAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSEPKSCDKTHTCPPCPAPELLGGKKKPLDGEYFTLQIRGRERF EMFRELNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSEPK SCDKTHTCPPCPAPELLGGKKKPLDGEYFTLQIRGRERFEMFRE LNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRERFEM FRELNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRERFEMFRELN EALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG GGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAM GWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNT VYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVT VSS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPG GGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAM GWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNT VYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVT VSSGGSGGSQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMG
WFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTV YLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTV SS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSEPKSCDKTHTCPPCPAPELLGGKKKPLDGEYFTLQIRGRERF EMFRELNEALELKDAQAGKEPGGGGGSGGGGSQVQLQESGGGLV QAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGG STYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAG LGTVVSEWDYDYDYWGQGTQVTVSS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSEPKSCDKTHTCPPCPAPELLGGKKKPLDGEYFTLQIRGRERF EMFRELNEALELKDAQAGKEPGGGGGSGGGGSQVQLQESGGGLV QAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGG STYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAG LGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQVQLQESGGGLVQ AGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGS TYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGL GTVVSEWDYDYDYWGQGTQVTVSS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG
GSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTKKKPLDGEYFTLQIRGRERFEM FRELNEALELKDAQAGKEPGGGGGSGGGGS
QVQLQESGGGLVQA GGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGST YYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLG TVVSEWDYDYDYWGQGTQVTVSSGGSGGSQVQLQESGGGLVQAG GSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTY YTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGT VVSEWDYDYDYWGQGTQVTVSS
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERF EMFRELNEALELKDAQAGKE PG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRE LNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKER EFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPD DTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGGGSGGG GSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGKKKPLDGEYF TLQIRGRERFEMFRELNEALELKDAQAGKEPG
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKE REFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKP DDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTVSSGGSGGSQ VQLQESGGGLVQAGGSLRLSCVASGSIFSINAMDWYRQAPGKQR ELVAGITSGGSTNYGDFVKGRFTISRDNAKNTVYLQMDSLKPED TAVYYCAAEVGGWGPPRPDYWGHGTQVTVSSGGGGSGGGGSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGKKKPLDGEYFTLQIR GRERFEMFRELNEALELKDAQAGKEPG
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMYWVRQAPGKG LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRT EDTAVYYCASGSDYGDYLLVYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKD AQAGKEPG
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMYWVRQAPGKG LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRT EDTAVYYCASGSDYGDYLLVYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGK EPG
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYAMYWVRQAPGKG LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRT EDTAVYYCASGSDYGDYLLVYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQ AGKEPG
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK APKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQSEDEADY YCNSLTSISTWVFGGGTKLTVL
GQPKAAPSVTLFPPSSEELQAN KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG LEWVSYITYSGSTIYYADSVKGRFTISRDNAKSSLYLQMNSLRA EDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKD AQAGKEPG
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG LEWVSYITYSGSTIYYADSVKGRFTISRDNAKSSLYLQMNSLRA EDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGK EPG
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG LEWVSYITYSGSTIYYADSVKGRFTISRDNAKSSLYLQMNSLRA EDTAVYYCARDRGTTMVPFDYWGQGTLVTVSS
ASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQ AGKEPG
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAP KLLIYAASNLETGVPSRFSGSGSGTDFTFTISGLQPEDIATYYC QQYDNLPLTFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKG LEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAE DTAVYYCARVLPMYGDYLDYWGQGTLVTVSS
ASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDA QAGKEPG
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKG LEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAE DTAVYYCARVLPMYGDYLDYWGQGTLVTVSS
ASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKE PG
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKG LEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAE DTAVYYCARVLPMYGDYLDYWGQGTLVTVSS
ASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQA GKEPG
DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPPEYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAP GKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQ MNSLKPDDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTQVTV SSGGSGGSQVQLQESGGGLVQAGGSLRLSCVASGSIFSINA MDWYRQAPGKQRELVAGITSGGSTNYGDFVKGRFTISRDNA KNTVYLQMDSLKPEDTAVYYCAAEVGGWGPPRPDYWGHGTQ VTVSSGGGGSGGGGSKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:268 [Homo sapiens] REGN10989_VH .
SEQ ID NO:269 [Homo sapiens] REGN10989_VL .
SEQ ID NO:270 [Homo sapiens] RENG10987_VH .
SEQ ID NO:271 [Homo sapiens] REGN10987_VL .
SEQ ID NO:272 [Homo sapiens] REGN10933_VH .
SEQ ID NO:273 [Homo sapiens] REGN10933_VK .
SEQ ID NO:274 [Homo sapiens] REGN10934_VH .
SEQ ID NO:275 [Homo sapiens] REGN10934_VK .
SEQ ID NO:276 [Homo sapiens] REGN10977_VH .
SEQ ID NO:277 [Homo sapiens] REGN10977_VK .
SEQ ID NO:278 [Homo sapiens] REGN10964_VH .
SEQ ID NO:279 [Homo sapiens] REGN10964_VK .
SEQ ID NO:280 [Homo sapiens] REGN10954_VH .
SEQ ID NO:281 [Homo sapiens] REGN10954_VK .
SEQ ID NO:282 [Homo sapiens] REGN10984_VH .
SEQ ID NO:283 [Homo sapiens] REGN10984_VL .
SEQ ID NO:284 [Homo sapiens] REGN10986_VH .
SEQ ID NO:285 [Homo sapiens] REGN10986_VL .
(a) The amino acid sequence of an anti-hRSV protein F nanobody for use as a binding domain in the present invention); there is provided a polypeptide or multimer as described herein comprising at least 4 copies of the nanobody (eg, for administration to a human or animal subject for treating or preventing RSV infection or a symptom thereof in the subject; also provided is a composition as described herein which comprises the multimer). SEQ ID NO: 293
(b) An antibody single variable domain (binding domain) herein may comprise a HCDR3 that comprises SEQ ID NO: 294 (which is the HCDR3 sequence of Nb1 1-59); there is provided a polypeptide or multimer as described herein comprising at least 4 copies of the domain (eg, for administration to a human or animal subject for treating or preventing SARS-CoV-2 infection or a symptom thereof in the subject; also provided is a composition as described herein which comprises the multimer). SEQ ID NO: 294
Number | Date | Country | Kind |
---|---|---|---|
2004153.9 | Mar 2020 | GB | national |
2005008.4 | Apr 2020 | GB | national |
2007280.7 | May 2020 | GB | national |
2007286.4 | May 2020 | GB | national |
2009847.1 | Jun 2020 | GB | national |
2017966.9 | Nov 2020 | GB | national |
2101331.3 | Jan 2021 | GB | national |
2102057.3 | Feb 2021 | GB | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2021/056576 | 3/15/2021 | WO |