The present invention relates to human anti-TNF-α antibodies with enhanced binding activity, and methods of producing and using such antibodies.
Tumor necrosis factor-α or TNF-α is cytokine recognized as the principle mediator of the body's response to gram-negative bacteria. The major source of TNF-α is LPS-activated mononuclear phagocytes, although the cytokine is also produced by antigen-activated T cells, activated NK cells, and activated mast cells (Abbas et al.). At low concentrations, TNF-α has a number of useful biological actions, including promotion of leukocyte accumulation at local sites of inflammation, activation of inflammatory leukocytes to kill microbes, and tissue remodeling, that are critical for local inflammatory responses to microbes. When TNF-α is present at higher concentrations, or under certain immune-response conditions, it can contribute to a variety of pathologies or disorders, including septic shock, autoimmune disorders, graft-versus-host diseases, transplantation rejection, and intravascular thrombosis.
Because TNF-α is associated with several pathological conditions in humans, it has been proposed to treat or ameliorate these conditions in human subjects by administration of a TNF-α antibody. To this end, several groups have reported the development of TNF-α antibodies. The earliest efforts along these lines were aimed at producing mouse monoclonal antibodies specific against human TNF-α (hTNF-α). Although these antibodies displayed high affinity for hTNF-α and neutralized hTNF-α activity, their use in humans was constrained by a number of known limitations associated with administering mouse antibodies to human subjects.
One solution to the limitation of mouse antibodies has been the development of partially humanized antibodies, typically by fusing variable regions of a mouse antibody with the constant regions of a human antibody. Another solution is to derive a fully human anti-TNF-α antibody using human hybridoma cell technology, although the latter approach has yet to produce anti-TNF-α antibodies with binding affinities suitable for therapeutic use. More recently, a fully human-derived TNF-α antibody made by recombinant technology and having binding and neutralization properties suitable for therapy has been reported (see U.S. Pat. Nos. 6,090,382, and 6,509,015).
Despite these advances, there remains a need for anti-TNF-α having enhanced binding affinity properties, e.g., a KD or Koff value that is at least 1.5 fold, preferably at least fold, lower than that of the highest affinity TNF-α antibodies available heretofore. Such enhanced-binding antibody would be effective at a substantially lower dose than currently available antibodies and/or would allow for more effective treatment at a comparable dose. These advantages have the potential to reduce the cost and/or improve the therapeutic result in treating a variety of TNF-α associated conditions.
The invention includes, in one aspect, an isolated human anti-TNF-α antibody, or antigen-binding portion thereof, containing at least one high-affinity VL or VH antibody chain that is effective, when substituted for the corresponding VL or VH chain of the anti-TNF-α scFv antibody having sequence SEQ ID NO: 1, to bind to human TNF-α with a KD dissociation constant or a Koff rate constant that is at least 1.5 fold lower, preferably at least two fold lower, than that of the antibody having SEQ ID NO: 1, when determined under identical conditions.
Exemplary sequences of the antibody VL and VH chains are identified by SEQ ID NOS 2 and 7. Exemplary sequences include those in which least one of the VL CDR1, CDR2, and CDR3 regions may have whose sequence is identified by SEQ ID NOS: 3, 4 and 5, respectively, and in which at least one of the VH CDR1, CDR2, and CDR3 regions whose a sequence is identified by SEQ ID NOS: 8, 9, and 10, respectively.
In a related aspect, the invention includes an isolated human anti-TNF-α antibody, or antigen-binding portion thereof, having VL and VH antibody chains whose sequences are identified by SEQ ID NOS 2 and 7, respectively. Exemplary sequences and embodiments are as noted above.
In another aspect of the invention, there is provided a method of treating a condition that is aggravated by TNF-α activity in a mammalian subject. In practicing the method, the above enhanced-affinity human anti-TNF-α antibody, or antigen-binding portion thereof is administered to the subject, in an amount sufficient to improve the condition in the subject. Exemplary sequences or embodiments of the antibody are as described above.
Also disclosed is a method of identifying human anti-TNF-α antibodies with enhanced binding affinity. In practicing the method, the amino-acid sequence variations contained in the SEQ ID NOS: 2 and 7 for the VL and VH CDRs, respectively, of the anti-TNF-α antibody defined by SEQ ID NO: 1, are used in constructing a library of antibody coding sequences encoding both VH and VL chains of the antibody. The library of coding sequences may include:
(a) a combinatorial library of coding sequences that encode combinations of the VL and VH CDR amino-acid sequence variations contained in at least one of the VL or VH sequences specified by SEQ ID NO: 2 or SEQ ID NO: 7,
(b) a walk-through mutagenesis library encoding, for at least one of the CDRs, the same amino acid substitution at multiple amino acid positions within that CDR, where the substituted amino acid corresponds to an amino acid variation found in at least one amino acid position of the VL or VH sequences specified by SEQ ID NO: 2 or SEQ ID NO: 7, for that CDR, or
(c) a library of localized saturation mutation sequences encoding, for at least one of said CDRs, all 20 natural L-amino acids at an amino acid position that admits to a sequence variation in at least one VL or VH sequences specified by SEQ ID NO: 2 or SEQ ID NO: 7.
The library of coding sequences is expressed in an expression system in which the encoded anti-TNF-α antibodies are expressed in a selectable expression system, and those antibodies having the lowest KD (or EC50) or Koff rate constants for human TNF-α are selected.
The library of coding sequences may constructed by identifying amino acid positions that are invariant within one or more selected CDRs, and retaining the codons for the invariant amino acid in the library antibody coding sequences.
The library of coding sequences may be a combinatorial library of coding sequences constructed by (i) producing a primary library of coding sequence encoding antibodies a single amino acid variation contained in at least one of the VL or VH sequences specified by SEQ ID NO: 2 or SEQ ID NO: 7, and (ii) shuffling the coding sequences in the primary library to produce a library of coding sequences having multiple amino acid variations contained in at least one of the VL or VH sequences specified by SEQ ID NO: 2 or SEQ ID NO: 7.
In a related embodiment, the library of coding sequences is a combinatorial library of coding sequences constructed by generating coding sequences having, at each amino acid variation position, codons for the wildtype amino acid and for each of the variant amino acids. In this embodiment, the CDR1-CDR3 coding regions of the library of coding sequences for the VL chain may have the sequences identified by SEQ ID NOS: 11-13, respectively. The CDR1-CDR3 coding regions of the library of coding sequences for the VH chain may have the sequences identified by SEQ ID NOS: 14-16, respectively.
The library of coding sequences may be constructed to encode multiple positively charged amino acids in the CDR-L1 domain or multiple polar amino acids in the CDR-H3 domain.
The expression system employed in the method may be a yeast expression system, and the library of coding sequences may encode scFv anti-TNF-α antibodies.
The library of coding sequences may include, for the CDR1, CDR2, and CDR3 regions of the VL chain, the sequences identified by SEQ ID NOS: 11-13, respectively, and those for the CDR1, CDR2, and CDR3 regions of VH chain may incorporate the sequences identified by SEQ ID NOS: 14-16, respectively. The antibody may be expressed in a scFv format, the expression system employed may be a yeast expression system, and the selection of high-affinity antibodies may be based on a kinetic selection to select antibodies on the basis of enhanced Koff binding constants.
In another aspect, the invention includes sequences selected from the group consisting of SEQ ID NOS: 11-16, for use in constructing coding sequences for generating human anti-TNF-α antibodies having one or more of the amino acid substitutions in the VL and VH CDR regions of mutations identified in SEQ ID NOS: 2 and 7, respectively.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
FIGS. 8 illustrates steps in the screening of anti-TNF-α antibodies formed in accordance with the presence invention for high binding affinity based on equilibrium binding to TNF-α;
g shows a Biacore determination of binding kinetics of anti-TNF-α D2E7 wild type (25A) and six affinity enhanced anti-TNF-α scFv clones (25B-25G);
I. Definitions
The terms below have the following definitions herein unless indicated otherwise.
The term “human TNF-α” or “TNF-α” refers to the human cytokine that exists as a 17 kD secreted form and a 26 kD membrane associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules., as described, for example, by Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989) Nature 338:225-228.
The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each chain consists of a variable portion, denoted VH and VL for variable heavy and variable light portions, respectively, and a constant region, denoted CH and CL for constant heavy and constant light portions, respectively. The CH portion contains three domains CH1, CH2, and CH3. Each variable portion is composed of three hypervariable complementarity determining regions (CDRs) and four framework regions (FRs).
The term “antibody” also encompasses antibody fragments, such as (i) an Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined by recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term antibody also encompasses antibodies having this scFv format.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
The term “humanized antibody” is intended to include antibodies in which one or more of the regions or domains of the antibody is derived from a non-human source, e.g., an antibody in which one of the heavy- or light-chain CDRs is derived from a mouse anti-TNF-α antibody, that is, has the same coding sequence or the same amino acid sequence or a sequence more closely related to a mouse anti-TNF-α than to a human anti-TNF-α antibody.
The term “recombinant antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell.
The term “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities.
A “neutralizing antibody”, as used herein refers to an antibody whose binding to TNF-α results in the inhibition of the biological activity of TNF-α, as assessed by measuring one or more indicators of TNF-α, such as TNF-α-induced cellular activation or TNF-α binding to TNF-α receptors. These indicators of biological activity can be assessed by standard in vitro or in vivo assays known in the art.
The term “Koff”, as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex, as determined from a kinetic selection set up.
The term “KD”, as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding sites present in a solution of antibody molecules at equilibrium, and is equal to Koff/Kon, the on and off rate constants for the antibody. The association constant KA of the antibody is 1/KD. The measurement of KD presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of KD.
The term “reference anti-TNF-α antibody” refers to the scFv antibody disclosed in U.S. Pat. Nos. 6,509,015 and 6,090,382. This antibody has a coding sequence derived exclusively from human germline. It is also identified herein as E2D7 scFv antibody, and by the amino acid sequence SEQ ID NO: 1.
The three-letter and one-letter amino acid abbreviations and the single-letter nucleotide base abbreviations used herein are according to established convention, as given in any standard biochemistry or molecular biology textbook.
II. Generating Enhanced-Affinity Anti-TNF-α Antibodies
This section describes methods for generating high-affinity anti-TNF-anti-TNF-α antibodies, in accordance with the invention. The general approach is to employ look-through mutagenesis (LTM) to produce a set of coding sequences that contain a selected amino acid substitution at each of the amino-acid residue positions in each of the light-chain and heavy-chain variable regions (CDRs).
Typically, the coding sequences encode an scFv anti-TNF-α antibody, and are contained in a vector used for transforming a suitable expression system such as a yeast expression system. For each of the VL and VH chains, the selected mutations may be placed at a selected position in one, two or all three CDRs of the variable chain. Anti-TNF-α antibodies produced by the expression system are then screened for high binding affinity, typically having a KD (EC50) or Koff that is substantially lower, typically at least 1.5 fold and preferably at least 2 fold lower than the D2E7 scFv antibody identified by SEQ ID NO:1, when measured under identical conditions. When measured according to the equilibrium (EC50) or kinetic binding (Koff) methods described below, the high-affinity antibodies have EC50 values less that about 10−8 M and/or Koff rate constants of less than 10−4 sec−1, the highest affinities yet reported for anti-TNF-α antibodies. The LTM method preferably employs a representative subset of nine amino acids, as described below.
Once CDR mutations associated with enhanced affinity are identified, by LTM, these mutations are used to guide the construction of a library of coding sequences from which even higher-affinity antibodies can be expressed and selected. Among the libraries that may be encoded are:
(a) a combinatorial library of coding sequences that encode combinations of the VL and VH CDR amino-acid sequence variations identified by the LTM method;
(b) a walk-through mutagenesis library encoding, for at least one of the CDRs, the same amino acid substitution at multiple amino acid positions within that CDR; and
(c) a library of localized saturation mutation sequences encoding, for at least one of said CDRs, all 20 natural L-amino acids at an amino acid position that admits to a sequence variation identified by the LTM method.
These libraries are used to encode antibodies in a suitable expression system, such as a yeast expression system allowing identification of the desired high-affinity antibodies.
A. Look through Mutagenesis (THM)
The purpose of look-through mutagenesis (LTM) is to introduce a selected substitution at each of target mutation positions in a region of a polypeptide, e.g., the CDR regions of the variable antibody chain. Unlike combinatorial methods or walk-through mutagenesis (WTM), which allow for residue substitutions at each and every position in a single polypeptide, LTM confines substitutions to a single selected position. This feature is illustrated in
B. Walk-through Mutagenesis (WTM)
The object of walk-through mutagenesis (WTM) is to investigate the effect on a polypeptide of substituting a selected amino acid, e.g., His, at each or substantially each of the amino acid positions in a selected portion of the polypeptide. In the usual case, the selected-amino acid substitutions are placed at each of a plurality of contiguous amino acid positions, where the target region for mutations is typically between 3-30 amino acid. The method is carried out so that the desired substitutions are produced with the minimum number of base substitutions in the coding sequences for target potion of the polypeptide, and the native (non-mutated) amino acid is preserved in at least coding sequence. That is, in the set of coding sequences needed to effect a single amino acid substitution at each target position, there is at least one coding sequence for the native polypeptide and at least one for each of the desired substitutions.
The walk-through method is illustrated in
The total number of different coding sequences is 213 or 8,192, and the total number of different peptide sequences is 46×2 or 8,192. These numbers are to be compared with the total possible number of coding sequences produced with randomly generated coding sequences (421) and the total number of different amino acid sequences that could be produced (207). Accordingly, the walk-through method also produces a much higher percentage of the desired mutants (25%-50% in the examples shown in
The walk-through method is illustrated in
The objective of WTM, as noted above, is to generate the smallest set of coding sequences that encode both the wildtype amino acid sequence, and sequences in which each residue in a selected region or regions of a polypeptide is substituted with a single selected amino acid. The amino acid selected for substitution within each CDR is preferably chosen from among those that are identified in the LTM approach above, that is, amino acids associated, in a particular CDR, with enhanced binding activity. In an exemplary embodiment, the one or more amino acids selected for substitution are those that represent beneficial mutations in more than one position of a CDR. For example, the CDR1 region of the VL chain contains lysine substitutions at each of three of the 11 CDR1 positions, suggesting that this region may benefit from multiple substitutions of a positive amino acid. A suitable WTM library would then contain codons for multiple Lys, His, or Arg substitutions within this CDR. The section below discusses doping techniques for controlling the total number of the selected amino acid that are substituted into an one CDR.
C. Combinatorial Methods
In the combinatorial approach, coding sequences are generated which represent combinations of the beneficial mutations identified by LTM. These combinations may be combinations of different beneficial mutations within a single CDR, mutations within two or more CDRs within a single antibody chain, or mutations within the CDRs of different antibody chains.
One combinatorial approach resembles the WTM method except that the selected codon substitutions within the CDRs are the different beneficial amino-acid substitutions identified by LTM. Thus, not every residue position in an antibody CDR will contain a mutation, and some positions will have multiple different amino acids substituted at that position. Overall, many if not all, combinations of beneficial mutations within a CDR or an antibody chain will be represented by at least one of the coding sequences in the library. As will be seen below, this coding-sequence library can be prepared by a modification of the WTM method, except that instead placing codons for a single amino acid at each different position in the variable coding region, the codons that are introduced are those corresponding to all beneficial mutations detected in the LTM method. In order to keep the size of this library manageable, the mutations may be confined to one of the two heavy or light chains only. This combinatorial approach is detailed below.
In a second approach, individual gene fragments containing a single CDR region, and having a codon variation encoding all combinations of beneficial mutations within CDR reconstructed, e.g., by gene shuffling methods, to produce VL and VH chain coding sequences having combinations of beneficial mutations in all CDRs of a given chain or all CDRs in both chains.
D. Localized Saturation Mutagenesis
In this approach, the beneficial mutations identified by LTM are used to identify “active” regions of the CDRs at which different types of amino acid substitutions are shown to produce beneficial mutations. The library of coding sequences in this approach are designed to encode up to and including each of the 20 amino acids at each of the identified “hot spots” in one or more of the six CDRs of the antibody. Conversely, the approach may be carried out by identifying the “cold spots” and designing coding sequences that saturate all CDR positions except the cold-spot sites.
E. scFv Coding Libraries
The synthesis of the coding sequence of the D2E7 scFv reference antibody having the amino-acid sequence identified by SEQ ID NO:1 is described in Example 1. Briefly, the D2E7 wild type scFv gene (approximately 1 kb) was assembled in vitro by PCR of 30 oligonucleotides shown in
As will be seen below, the LTM and WTM methods is applied to the coding and amino acid sequences of one or more of the D2E7 VH or VL chain CDR regions, for purposes of generating antibodies whose binding constant is substantially enhanced with respect to the reference scFv E2D7 antibody. More specifically, the LTM and WTM techniques described above are used to create pools of oligonucleotides with mutations in one or more CDRs of the light or heavy chain of the reference antibody. These oligonucleotides are synthesized to include some of the surrounding framework. These pools of oligonucleotides are utilized to generate all possible VL and VH chains in which there are mutations in single, double, and triple CDRs (CDR1, 2, and 3) using single overlap extension PCR (SOE-PCR). Methods for generating pools of LTM CDR oligonucleotides, and WTM oligonucleotides are detailed in Example 2. Methods for generating LTM and WTM libraries from these pools are detailed in Example 3.
For example, to create the pool of VH chains in which both VH CDR1 and VH CDR2 are mutated and VH CDR3 is wild-type, the CDR1 oligonucleotides are first used as templates and SOE-PCR is conducted to link the CDR2 oligonucleotides to generate the doubly mutated pool. Considering that each CDR may be either wild-type or mutant, there are eight possible combinations for each of the pools of VL and VH chains.
Combining the eight VL and eight VH pools creates 64 VL-VH combinations (scFvs), one of which is wild-type, and 63 of which are non wild-type. Each of the 64 VL-VH combinations (including the wild-type sequence) is termed a subset of the whole LTM™ or WTM™ scFv library. An LTM™ or WTM™ scFv library is generated for each amino acid selected for substitution. The number of amino acid sequences represented within each subset library depends on the length of the CDR, the amino acid sequence within the CDR, and the LTM™ or WTM™ oligonucleotide design strategy.
The individual scFv libraries are constructed using the splice overlap extension polymerase chain reaction (SOE-PCR) method (Horton, et al., 1989), providing a fast and simple method for combining DNA fragments that do not require restriction sites, restriction endonucleases, or DNA ligase. In SOE-PCR two oligonucleotides are first amplified by PCR using primers designed so that the PCR products share a complementary sequence at one end. Under PCR conditions the complementary sequences hybridize, forming an overlap. The complementary sequences then act as primers, allowing extension by DNA polymerase to produce a recombinant molecule. These methods are detailed in Example 3.
There are two additional constraints imposed on the WTM and LTM procedures discussed above. The first concerns the total number of amino acids whose substitution into the CDR regions of the antibody is examined. Rather than examine the effect of all 20 natural L-amino acids, it is more efficient to employ a subset of these that represent the chemical diversity of the entire group. One representative subset of L-amino acids that meets this criterion includes the alanine, aspartate, lysine, leucine, proline, glutamine, serine, tyrosine, and histidine. These amino acids display adequate chemical diversity in size, charge, hydrophobicity, and hydrogen bonding ability to provide meaningful initial information on the chemical functionality needed to improve antibody properties. The choice of a subset of amino acids may also be based on the frequency of certain amino acids in CDRs. For example, given a choice between tyrosine and phenylalanine to represent an amino acid with an aromatic side chain, tyrosine might be a better choice of its significantly higher preponderance in antibody binding sites.
Implicit in the selection of a representative subset of amino acids is that a beneficial mutation, that is, one that enhances binding activity or neutralizing activity of the antibody, produced by substitution of an amino acid in the representative subset will reasonably predict that the one or more amino acids that are related to the specific mutation in size, charge, hydrophobicity and/or hydrogen binding ability will also produce the same positive effect on antibody activity. In the present case, each of the nine representative subset amino acids will be taken to include the related amino acids given in parenthesis: Ala (Gly); Asp (Glu); Lys (Arg); Leu (Ile and Val); Pro; Gln (Asn); Ser (Thr); Tyr (Phe Trp); and His. Thus, a positive mutation from say, Asp to Tyr, will predict a similar effect by a Gly to Phe or Gly to Trp, and a positive mutation from, say Met to Ser, will predict a positive mutation from Met to Thr.
A second constraint imposed on coding sequences for WTM (but not LTM) involves the use of doping to control the percentage of sequences that code for either the wild-type or the mutation, with 12% to 50% of the sequences having the mutation. Doping the bases allows one to fine-tune the number of amino acid substitutions in the CDR of a WTM™ library member. In the above example for lysine substitutions, it is unlikely that it would be advantageous for a CDR to have lysine in all seven positions, or even in the majority of positions simultaneously. Utilizing doping, oligonucleotides are synthesized that maintain an average of 2-4 lysine substitutions per molecule or per CDR.
In the case of mixed-mutation WTM, doping can additionally be used to equalize the expected distribution of mutations at any given position. For example, if one base produces an expected level of a given substitution of 25%, and another, an expected level of a different amino acid of only 12.5%, the relative amounts of the two bases may be in a 1:2 ratio, to equalize the probabilities of seeing both mutations in equal amounts.
The relative molar amounts of each nucleotide in a two-nucleotide mix is indicated in the figures, and is typically either 4:1 (80:20) or 1:1 (50:50). The 4:1 ratios are “doping” ratios used to achieve an average of 3-4 mutations of the selected amino acid (for
The design of oligonucleotide WTM and LTM libraries is preferably carried out using software coupled with automated custom-built DNA synthesizers. Implementation of the LTM™ and WTM™ strategies involves the following steps. After selection of target amino acids to be incorporated into the CDRs, the software determines the codon sequence needed to introduce the targeted amino acids at the selected positions within the CDRs. Optimal codon usage is selected for expression in the selected display and screening host, e.g., the yeast expression system (see below). The software also eliminates any duplication of the wild-type sequence that may be generated by this design process. It then analyzes for potential stop codons, hairpins, loops and other problematic sequences that are then fixed. The software determines the ratios of bases added to each step in the synthesis (for WTM™) to fine tune the amino acid incorporation ratio. The completed LTM™ or WTM™ design plan is then sent to the DNA synthesizer, which performs automated synthesis.
F. Yeast Cell Expression and Surface Display
A variety of methods for selectable antibody expression and display are available. These include bacteriophage, Escherichia coli, and yeast. Other methods of antibody expression may include cell free systems such as ribosome display and array technologies which allow for the linking of the polynucleotide (i.e., a genotype) to a polypeptide (i.e., a phenotype) e.g., Profusion™ (see, e.g., U.S. Pat. Nos. 6,348,315; 6,261,804; 6,258,558; and 6,214,553). Convenient E. coli expression system, have been described by Pluckthun and Skerra. (Pluckthun, A. and Skerra, A., Meth. Enzymol. 178: 476-515 (1989); Skerra, A. et al., Biotechnology 9: 273-278 (1991)). By attaching a signal sequence, such as the ompA, phoA or pelB signal sequence to either the 5′ or 3′ end of the antibody coding sequence, the antibodies can be expressed for secretion into the periplasmic space of E. coli (Lei, S. P. et al., J. Bacteriol. 169: 4379 (1987)).
While each of these has been utilized for antibody improvement, the yeast display system affords several advantages (Boder and Wittrup 1997). Yeast can readily accommodate library sizes up to 107, with 103-105 copies of each antibody being displayed on each cell surface. Yeast cells are easily screened and separated using flow cytometry and fluorescence-activated cell sorting (FACS) or magnetic beads. Yeast also affords rapid selection and regrowth. The eukaryotic secretion system and glycosylation pathways of yeast allow for a much larger subset of scFv molecules to be correctly folded and displayed on the cell surface than prokaryotic display systems.
The yeast display system utilizes the a-agglutinin yeast adhesion receptor to display proteins on the cell surface. The proteins of interest, in this case, scFv WTM™ and LTM™ libraries, are expressed as fusion partners with the Aga2 protein. These fusion proteins are secreted from the cell and become disulfide linked to the Aga1 protein, which is attached to the yeast cell wall (see Invitrogen, pYD1 Yeast Display product literature). In addition, there are carboxyl terminal tags included which can be utilized to monitor expression levels and/or normalize binding affinity measurements. Methods for selecting expressed antibodies having substantially higher affinities for human TNF-α, relative to the reference D2E7 antibody, will now be described. Details of the yeast expression system and its use in antibody display are given in Example 4.
III. Selecting and Expressing Enhanced-Affinity Antibodies
This section describes methods for selecting enhanced affinity antibodies using either an equilibrium binding analysis method to measure KD (or EC50) or a kinetic binding analysis to determine a Koff constant. Several high-affinity antibodies produced by both binding criteria are disclosed. The two groups of enhanced-binding antibodies have many mutations in common and some that are unique to each method of affinity determination. The groups, when combined, provide a map of beneficial mutations in the VH and VL CDRs of the antibody that are associated with enhanced binding activity.
A Anti-TNF-α Antibodies with Enhanced EC50.
The antibodies disclosed in this section have EC50 values which are at least 1.5 and up to 2-5 fold lower than the measured EC50 for the reference D2E7 antibody, when both antibodies are expressed in scFv form, and measured under identical equilibrium binding conditions.
Initially, the yeast cells are equilibrated with biotinylated TNF-α, producing a mixture of cells having bound biotinylated TNF-α, indicated at 49, and low-affinity and non expressing cells. Following equilibration binding to TNF-α, streptavidin coated beads, such as beads 52, are added to the mixture, forming a binding complex 54 consisting of high-affinity expressing cells, biotinylated TNF-α, and magnetic beads. The complexes are isolated from the mixture using a magnet 56, and the bound complex is washed several times under stringent conditions to remove complexes of low-affinity cells and non-specifically bound cells. The resulting purified complexes are released from the complexes, by treatment with a suitable dissociation medium, to yield cells enriched for expression of high-affinity antibodies. In one exemplary screening method, the isolated cells are plated at low density, and clonal colonies are then suspended in medium at a known cell density. The cells are then titrated with biotinylated TNF-α by addition of known amounts of TNF-α, as indicated, e.g, from 10 pM to 1000 nM. After equilibration, the cells are pelleted by centrifugation and washed one or more times to remove unbound TNF-α, then finally resuspended in a medium containing fluoresceinated streptavidin. The fluoresceinated cells are scanned FACS to determine an average extent of bound fluorescein per cell. This method is described in Examples 5 and 6.
In the initial LTM study, LTM coding libraries for both the VH and VL chains were constructed, with the other chain containing a wildtype (D2E7) amino acid sequence. Each coding sequence in a VH or VL library contained a single mutation for a selected representative amino acid in one, two, or all three CDRs in that chain. The library sequences were used, as above, in constructing scFv coding sequences, and the scFv sequence used to transform the above yeast expression system, and antibodies having binding affinities, measured as EC50, of less than 0.05 nM (less than half the EC50 of D2E7) were selected and sequenced in the CDR regions. The individual amino acid mutations associated with the enhanced-affinity scFv antibodies are shown in
Collectively, the mutations shown in
It will be understood that a substitution mutation in the identified antibody sequences may represent the amino acid shown or its equivalent-class amino acid, as discussed above. Thus, in the above example, Xaa34=M or L will also cover, in one embodiment, the sequence Xaa34=M or L or I or V. Once high-affinity cells have been selected, the binding affinities of individual molecules displayed on the surface of clonal yeast cells is determined, as above. This allows for rapid identification of molecules with improved affinity.
B Anti-TNF-α Antibodies with Enhanced Koff.
The antibodies disclosed in this section have Koff values which are at least 1.5 and up to 2-5 fold lower than the measured measured Koff for the reference D2E7 antibody, when both antibodies are expressed in scFv form, and measured under identical kinetic binding conditions. The antibodies were generated using the LTM libraries above for each of the VL and VH chains, where the antibodies were expressed, as above, in scFv format.
The cells are then incubated with either non-biotinylated TNF-α, or with a competitive soluble antibody, e.g., D2E7, both at saturating conditions, for a selected time sufficient to reduce the percentage of biotinylated TNF-α bound to the cells, in both cases, as a function of the off rate of the antigen. Following incubation, the cells are centrifuged, and washed to remove unbound biotinylated TNF-α and/or soluble competitive antibody, yielding cells 62, each of which contains a ratio of biotinylated and native TNF-α in proportion of the antibody's Koff.
Details of the method are given in Example 7.
The koff values are then determined by incubating the cells with a fluoresceinated streptavidin (streptavidin-PE) and a fluoresceinted cell market (anti-his-fluorescein), washing the cells, and sorting with FACS. The koff value is determined from the ratio of the two fluorescent markers, according to known methods.
C. Production of Soluble Antibodies
Antibodies from high-affinity clones from above are sequenced to identify high-affinity mutations. Antibodies of interest are subcloned into a soluble expression system, such as Pichia pastoris or E. coli, and soluble antibody, e.g., scFv antibody, is produced. A number of commercially available vectors and cell lines for soluble antibody expression, including those from Invitrogen (i.e. pPIC9) are available. These systems are routinely used to generate soluble single chain or full-length antibody. Expression of high-affinity antibodies in accordance with the present invention has yielded greater than 1 mg per liter soluble scFv in the P. pastoris expression system (Invitrogen). Purification of proteins is facilitated by the presence of a His-tag at the C-terminus of the molecule, in the case of single chains or by protein A or protein G columns for full-length antibodies. Soluble single chain and full-length antibodies will be generated to obtain BIAcore affinity measurements and for use in the assays described below.
IV. Libraries of Antibody Coding Sequences
As noted above, beneficial mutations (yielding a substantially higher KD or koff) identified as above by LTM may be used to generate libraries of coding sequences useful for selecting combinations of mutations capable of producing additive beneficial binding effects. Ideally, the antibodies selected contain multiple mutations in at least one CDR, either the same or different amino acids, and/or amino acid substitutions in two or more CDRs or either the corresponding VH or VL antibody chain.
In one combinatorial approach, the beneficial mutations identified from both the equilibrium and kinetic binding selections were combined into one or both of the VH and VL chain sequences shown in
The above combinatorial libraries encoding each of the above VH chain CDR1, CDR2, and CDR3 regions are shown in
The combinatorial CDR coding regions above are incorporated into VH or VL coding regions, employing framework coding regions for the corresponding constant of framework coding regions on either side of each CDR coding region, according to methods described above for construction of the LTM libraries.
These VH and VL combinatorial WTM libraries are then combined with wildtype (D2E&) VL or VH coding regions, respectively to form a library of mutated VH or mutated VL antibody genes, e.g., genes expressing the scFv antibody format.
The libraries are used to transfer a suitable surface display system, e.g., yeast cells, and cells are then screened, by equilibrium or kinetic selection setups to identify cells expressing antibodies with enhanced binding KD or koff) antibodies. As indicated above, these antibodies will contain beneficial mutations in one or more of the CDR of either the VL or VH chain, may contain multiple mutation in any one CDR, and the mutations may include more than one type of amino acid. Once high-activity VL or VH chains are identified, the method may be further extended to select for mutations occurring simultaneously in both VL and VH chains, by generating more limited mixed-mutation WTM libraries covering both chain CDRs.
A combinatorial library of mutations may also be generated by known gene shuffling methods, such as detailed in U.S. patent application 2003/005439A1, and U.S. Pat. No.6,368,861, and (Stemmer WP (1994) Proc Natl Acad Sci 91(22):10747-51), all of which are incorporated herein by reference. The method involves limited DNase I digestion of the collected mixed mutation clones to produce a set of random gene fragments of various pre-determined sizes (e.g. 50-250 base pairs). The fragments are then first denatured and the various separate fragments are then allowed to re-associate based on homologous complementary regions. In this manner, the re-natured fragments may incorporate differing mixed mutation CDRs in the re-assembled segments which are then extended by SOE-PCR as above, and a re-assembled chimera may then incorporate, at a minimum, at least two sets of beneficial CDR mixed mutations from each parental DNA source donor. Other mix and match techniques for generating coding sequences from CDR oligonucleotide fragments may also be used.
Libraries of antibody coding sequences for a WTM may be constructed as above, employing a single selected amino acid substitution within each of the CDRs, and preferably also using doping to achieve an average amino substitution of 2-4 mutations in each CDR as described above. The amino acid that is selected for each CDR is preferably one corresponding to a beneficial amino acid substitution in at least two residues of that CDR, or having similar properties as beneficial mutations that occur in two or more residues. For example, looking at
Finally, the library of coding sequence constructed using the LTM beneficial mutations as a guide mutations can be a saturation sequence in which one or selected CDR positions, and preferably “hot spots”, are substituted for each of the up to and including 20 standard amino acids. These “hot spots” may be residue positions at which one or more substitutions appear in a large number of high-affinity mutants, such as the first and second CDR-H1, or the second, third, ninth, eleventh, and twelfth positions or at which several different beneficial mutations are found, such as or positions 4 and 5 of CDR-L1, positions 3, 5, and 6 or CDR-L2, position 5 of CDR-L3, position 1 of CDR-H1, and positions 2, 3, 11 and 12 of CDR-H3. The coding sequences are prepared, as above, by introducing codons for each amino acid at the one or selected beneficial mutation positions.
A. Construction of D2E7 Wild Type scFv Gene:
The D2E7 wild type scFv gene (approximately 1 kb) was assembled in vitro by PCR of 30 oligonucleotides (
The 30 primers were all incubated together as a mixture (5 μl of 10 uM oligonucleotide mix) and PCR assembled using 0.5 μl Pfx DNA polymerase (2.5 U/μl), 5 μl Pfx buffer (Invitrogen), 1 μl 10 mM dNTP, 1 μl 50 mM MgSO4 and 37.5 μl dH20 at 94 C for 2 min, followed by 24 cycles of 30 sec at 94 C, 30 sec at 50 C, and 1 min at 68 C and then incubated at 68 C for 5 min. The PCR assembly reaction permitted oligonucleotide overlap annealing, base-pair gap filling, and ligation of separate oligonucleotides on each strand of the DNA duplex to form a continuous full length D2E7 scFv gene. An aliquot (1 μl) of the above PCR assembly reaction was taken out for further D2E7 scFv full length amplification using an added pair of D2E7 5′ and 3′ end specific oligonucleotide primers (SEQ ID NO: 18 and 19) 2 μl each of 10 uM stock, 0.5 μl Pfx DNA polymerase (2.5 U/μl), 5 μl Pfx buffer, 1 μl 10 mM dNTP, 1 μl 50 mM MgSO4 and 37.5 μl dH20 at 94 C for 2 min, followed by 24 cycles of 30 sec at 94 C, 30 sec at 50 C, and 1 min at 68 C and then incubated for a 68 C for 5 min. The D2E7 scFv DNA from the PCR reaction was then extracted and purified (Qiagen PCR purification Kit) for subsequent Bam HI and Not I restriction endonuclease digestion as per manufacturer's directions (New England Biolabs). Full length D2E7 scFv was then subcloned into pYD1 vector and sequenced to verify that there were no mutations, deletions or insertions introduced (SEQ ID NO:1 and 6). Once verified, full length VH and VL D2E7 served as the wild type template for the subsequent strategies of building LTM and WTM libraries.
In the following examples, the predetermined amino acids of CDR-H2 segment (positions 56 to 69; TWNSGHIDYADSVE) from the D2E7 wild type VH section LDWVSAI-TWNSGHIDYADSVE-GRFTISR, was selected for both LTM and WTM analysis. The polypeptide sequences LDWVSAI and GRFTISR are portions of the VH frameworks 2 and 3 respectively flanking CDR-H2. In the design and synthesis of VH and VL CDR LTM and WTM oligonucleotides, flanking framework sequence lengths were approximately 21 base pairs for SOE-PCR complementary overlap. A reference oligonucleotide coding for the above CDR-H2 wild type sequence (in bold) (SEQ ID NO: 23) containing the flanking VH2 and VH3 portions (lowercase letters below) is below: 5′-gta gag tgg gtt tct gcg ata-ACT TGG AAT TCT GGT CAT ATT GAT TAT GCT GAT TCT GTT GAA-ggt aga ttt act att tcc cgt-3′.
A. Design of CDR LOOK THROUGH MUTAGENESIS (LTM) Oligonucleotides
Look Through Mutagenesis analysis introduces a predetermined amino acid into every position (unless the wildtype amino acid is the same as the LTM amino acid) within a defined region. In this VH CDR-H2 example, leucine LTM of VH CDR-H2 involves serially substituting only one leucine at a time, in every CDR-H2 position.
CDR-H2 LTM oligonucleotides for the other eight “subset” amino acids; alanine, aspartate, lysine, leucine, proline, glycine, serine, tyrosine, and histidine were designed and synthesized in analogous manner. For example, the first aspartate (codon in bold) LTM oligonucleotide (out of the fourteen for CDR H2) replacement was (SEQ ID 38): 5′-gtagagtgggtttctgcgata-GAC TGG AAT TCT GGT CAT ATT GAT TAT GCT GAT TCT GTT GAA-ggtagatttactatttcccgt-3′.
An example of oligonucleotides for CDR H1 leucine LTM is listed in SEQ ID NOS. 41-45. As in the CDR H2 design above, 17 base pairs of wild type D2E7 framework 1 and 2 sequences (lowercase lettering) flank the CDR H1 to allow SOE-PCR assembly into the remainder of the scFv construct.
B. Design of CDR WALK THROUGH MUTAGENESIS (WTM) Oligonucleotides
To perform a Walk Through Mutagenesis (WTM), a selected amino acid is multiply substituted in different positions and in various combinations with the wild type sequence of a predetermined region.
The LTM and WTM oligonucleotides described above were then used to create pools of mutations in a single CDR of the light or heavy chain. As shown, these LTM and WTM oligonucleotides are synthesized to include approximately 20 bases of flanking framework sequences to facilitate in overlap and hybridization during PCR.
A. Introduction of Oligonucleotides and Construction of LTM Libraries.
The approach in making the LTM CDR-H2 library is summarized in
T1 and T2 PCR reactions were then gel purified (as per instructions in Qiagen Gel purification kit) and equimolar aliquots from both were then combined for single overlap extension PCR (SOE-PCR). SOE-PCR is a fast and simple method for combining DNA fragments that does not require restriction sites, restriction endonucleases, or DNA ligase. The T1 and T2 PCR products were designed share end overlapping complementary sequences (
A set of D2E7 end specific 5′ Bam HI sense (SEQ ID NO: 18) and D2E7 3′ Not I antisense primers (SEQ ID NO: 19) was added to facilitate LTM D2E7 amplification and incorporate the restriction enzyme sites in the PCR amplicons (
B. PCR Product Cloning into Yeast Cell Expression Vector DYD1:
The plasmid pYD1, prepared from an E. coli host by plasmid purification (Qiagen), was digested with the restriction enzymes, Bam HI and Not I, terminally dephosphorylated with calf intestinal alkaline phosphatase. Ligation of the pYD1 vector and the above SOE-PCR products (also digested by BamHI and NotI), E. coli (DH50) transformation and selection on LB-ampicillin (50 mg/ml) plates were performed using standard molecular biology protocols.
C. Multiple LTM CDR Libraries.
Double and Triple CDR mutations (in different combinations of CDR1, 2, and 3) are created as above but instead of using the wild type D2E7 gene as PCR template, a previously generated LTM D2E7 library is chosen instead. For example, to create VH chains in which both CDR-H1 and CDR-H2 are mutated and CDR-H3 and VL are wild-type, the LTM CDR-H2 mutant genes were used as templates and then SOE-PCR was conducted to incorporate the CDR-H1 oligonucleotides to generate the Double LTM mutations and summarized in
In this case, the two separate PCR reactions, T3 used primer pairs FR1 sense (SEQ ID NO: 21) and FR5 antisense (SEQ ID NO: 20) to amplify the framework region 1 (FR 1). The T4 PCR reaction utilized the pooled CDR-H1 LTM oligonucleotides (SEQ ID NO: 27) with FR4 anti-sense primer ((SEQ ID NO 24) to amplify the remaining FR 2, CDR2 LTM, FR3, CDR3, FR4 and VL portions of D2E7 (
The double LTM CDR-H1, CDR-H2 library were then used as templates to incorporate LTM CDR-H3 oligonucleotides to make the Triple CDR H3 LTM libraries. By progressively utilizing the starting single and double LTM libraries, an more complex array of LTM library combinations in both the VH and VL CDR was developed (
pYD1 (
A. Transformation of Yeast Host Cells with PYD1 AGA2-scFv Constructs:
Competent yeast host cells (500 μl) was prepared as per instructions by Zymo Research Frozen-EZ yeast Kit (Catalogue #). Briefly, 500 μl of competent cells was mixed with 10-15 μg pYPD1 scFv library DNA after which 5 ml of EZ3 solution was added. The cell mixture was incubated for 45 minutes at 30° C. with occasional mixing (three times). The transformed cells were centrifuged and resuspended in Glucose select liquid media,
B. Induction of AGA2-scFv:
After grown in Glucose select media (see Invitrogen manual for composition) at 30° C. under shaking aeration conditions for 48 hours until the OD600=7 (OD600=1 represents 107 cells/ml). The cells were then collected, re-pelleted and re-suspended in the induction medium, Galactose select media (see Invitrogen manual for composition), to an OD600=0.9 at 20° C. for 48 hours. Expression of the Aga2-scFv fusion protein from pYD1 is tightly regulated by the GAL1 promoter and depends on galactose in the medium for promoter induction.
C. Biotinylated TNF-α Preparation:
Biotinylation of the TNF antigen can be accomplished by a variety of methods however; over-biotinylation is not desirable as it may block the epitope—antibody interaction site. The protocol used was adapted from Molecular Probes FluoReporter Biotin-XX Labeling Kit (cat# F-2610). Briefly, TNFα 300 μl of 1 mg/ml stock (Peprotech), was added to 30 μl 1M Sodium Bicarbonate Buffer at pH 8.3 and 5.8 μl of Biotin-XX solution (20 mg/ml Biotin-XX solution in DMSO). The mixture was incubated for 1 hour at 25° C. The solution was transferred to a micron centrifuge filter tube, centrifuged and washed repeatedly (four times) with PBS solution. The biotinylated-TNFα solution was collected and the protein concentration determined by OD 280.
D. FACS Monitoring of AGA2-scFv Expression and TNF□ Binding:
An aliquot of yeast cells (8×105 cells in 40 μl) from the culture medium was centrifuged for 5 minutes at 2300 rpm. The supernatant was aspirated and the cell pellet was washed with 200 μl of ice cold PBS/BSA buffer (PBS/BSA 0.5% w/v). The cells were re-pelleted and supernatant removed before re-suspending in 100 μl of buffer containing the biotinylated TNFα (200 nM). The cells were left to bind the TNF-α at 20° C. for 45 minutes after which they were washed twice with PBS/BSA buffer before the addition and incubation with streptavidin-FITC (2 mg/L) for 30 minutes on ice. Another round of washing in buffer was performed before final re-suspension volume of 400 μl in PBS/BSA. The cells were then analysed on FACSscan (Becton Dickinson) using CellQuest software as per manufacturers directions. The FACS plot (
A. Magnetic Sorting of TNF Binding (EC50
The tube was then removed from the magnetic holder whereupon 1 ml of Glucose select media was added and the recovered yeast cells to be incubated for 4 hours at 30° C. The magnet holder was re-applied to the culture tube to remove any remaining magnetic beads. The yeast culture was then grown in Glucose select media at 30° C. for 48 hours before scFv induction in Galactose select media. In the second selection round, TNF-α concentration was lowered from 50 nM to 0.5 nM. TNF-α binding, complex formation, yeast cell enrichment and re-growth were performed as described above. For the third selection round, the TNF-α concentration was further lowered to 0.1 nM.
TNF-α EC50 binding, or “fitness” from each round of enrichment was evaluated by FACS (Example 3 protocol).
B. FACS Sorting of TNF scFv Library (
In an alternative methodology, the LTM yeast cell libraries were also enriched for high affinity anti-TNF-α scFv clones by FACS. Library construction, transformation, liquid media propagation and induction were carried out as above for EC50 determination. After scFv induction, the cells were incubated with biotinylated TNF-α at saturating concentrations (400 nM) for 3 hours at 25 C under shaking. After washing the cells, a 40 hour cold chase using unlabelled TNF-α (1 uM) at 25° C. was performed. The cells were then washed twice with PBS/BSA buffer, labeled with Streptavidin PE (2 mg/ml) anti-HIS-FITC (25 nM) for 30 minutes on ice, washed and re-suspended as described in Example 3. The D2E7 wild type was initially FACS analyzed to provide a reference signal pattern for FACS sorting of the yeast LTM library (
FACS Measurement of TNF-α EC50 Binding:
A pre-determined amount of yeast cells (8×105 cells in 40 μl) D2E7 scFvs (wild type, LTM, WTM clones) were incubated with 1:4 serial dilutions of biotinylated TNF-α (200 nM, 50 nM, 12.5 nM, 3.1 nM, 0.78 nM, and 0.19 nM final concentrations in a total volume of 80 μl) and incubated at 20° C. for 45 minutes followed by 5-10 minutes on ice. The yeast cells were washed 3 times and resuspended in 5 ml of PBS/BSA buffer. Streptavidin-PE (2 mg/ml) and αHIS-FITC (25 nM) was added to label the cells during an 30 minute incubation on ice. The αHIS-FITC antibody allowed monitoring of yeast cell surface scFv expression. Another round of washing was performed before re-suspending in 400 μl of PBS/BSA buffer. The labeled cells were then analyzed on FACSscan using CellQuest software.
A. Individual scFv Clones:
From the FACS sorter, the pre-sorted clones were then grown overnight in Glucose select media and then plated on solid media to isolate single colonies.
From a single colony liquid cultures of clones were grown in Glucose select media at 30° C. with shaking for 48 hours. The cells were then pelleted and resuspended in Galactose select media for OD time period. Because the FACS pre-sort enriches (by approximately 80%) but does not eliminate all undesirable clones, it is necessary to characterize the EC50 of the isolated clones to eliminate those that display binding values inferior to D2E7 (as detailed in the procedure of Example 3). Those isolates with comparable or superior EC50 values were then selected for further analysis.
Pulse: Yeast cells (approximately 5×106) after induction in Galactose select media, were pelleted and re-suspended in PBS/BSA buffer (1 ml). Biotinylated TNF-α (400 nM final concentration) was then added to the re-suspended cells and allowed to incubate or 2 hours at 25° C. on a nutator for continuous gentle mixing.
Chase: The biotinylated-TNF-□ and yeast cell mixture was washed and re-suspended in PBS/BSA buffer. Unlabelled TNFα was then added (to a final concentration of 1 μM) and yeast cell mixture was further incubated for 24 hours at 25° C. with sample aliquots being taken every two hours for the next 24 hours. The cell mixtures were washed and re-suspended in chilled PBS/BSA buffer and staining antibody α-SA PE (2 μg/ml) added. After incubation for 30 minutes on ice with periodic mixing, the cell mixture was then twice washed and analyzed by FACS as above.
From these Koff assays,
B. Beneficial Library (Mixed Mutation) Construction
The sequence of the 6 degenerate CDR beneficial mutation oligonucleotides are listed in SEQ ID NOS: 46-51. For example, the CDR L1 beneficial mutation oligonucleotide coded for H164 A165 S166S/Y/K/Q167 G/K168L/K/I169 R170 N171 Y172 L173 A174. Two separate libraries were constructed, one composed of H1, H2, and H3 beneficial mutations (a triple VH CDRlibrary) and the other library composed of the triple L1, L2, and L3 beneficial mutations (triple VL CDR library). The incorporation of multiple degenerate CDRs into one was detailed above in Example 2 (
C. Beneficial Library (Mixed Mutation) Clones
pBAD Fab Construction
The scFv genes for D2E7 and those clones identified from the above Koff screens characterized as affinity-enhanced, were excised from pYD1 and sub-cloned into pBAD E. coli expression vector (Invitrogen pBAD expression system).
A. E. coli pBAD expression for production of soluble antibodies
Competent E. coli host cells were prepared as per manufacturer's instructions (Invitrogen pBAD expression system). Briefly, 40 μl LMG 194 competent cells and 0.5 μl pBAD scFv construct (approximately 1 μg DNA) was incubated together on ice for 15 minutes after which, a one minute 42° C. heat shock was applied. The cells were then allowed to recover for 10 minutes at 37° C. in SOC media before plating onto LB-Amp plates and 37° C. growth overnight. Single colonies were picked the next day for small scale liquid cultures to initially determine optimal L-arabinose induction concentrations for scFv production. Replicates of each clone after reaching an OD600=0.5 were test induced with serial (1:10) titrations of L-arabinose (0.2% to 0.00002% final concentration) after overnight growth at room temperature. Test cultures (1 ml) were collected, pelleted and100 μl 1× BBS buffer (10 mM, 160 mM NaCl, 200 mM Boric acid, pH=8.0) added to resuspend the cells before the addition of 50 μl of Lysozyme solution for 1 hour (37° C.). Cell supernatants from the lysozyme digestions were collected after centrifugation, and MgSO4 was added to final concentration 40 mM. This solution was applied to PBS pre-equilibrated Ni-NTA columns. His-tagged bound scFv samples were twice washed with PBS buffer upon which elution was accomplished with the addition of 250 mM Imidazole. Soluble scFvs expression was then examined by SDS-PAGE.
Purification of scFv from Large Scale E. coli Culture:
After determination of optimal growth conditions, large scale (volume) whole E. coli cell culture pellets were collected by centrifugation after overnight growth at 25° C. The pellets were then re-suspended in PBS buffer (0.1% tween) and subjected to 5 rounds of repeated sonication (Virtis Ultrasonic cell Disrupter) to lyse the bacterial cell membrane and release the cytoplasmic contents. The suspension was first clarified by high speed centrifugation to collect the supernatant for further processing. This supernatant was applied to PBS pre-equilibrated Ni-NTA columns. His-tagged bound scFv samples were twice washed with PBS buffer upon which elution was accomplished with the addition of 250 mM Imidazole. The pH of the supernatant was then adjusted to 5.5 with 6M HCl and before loading onto a SP Sepharose HP cation exchange column (Pharmacia). The scFv was eluted a salt (NaCl) gradient and fraction concentrations containing the scFv were determined by optical density at 280 nm and verified by PAGE. Fractions containing scFvs were then pooled and dialyzed with PBS.
Biacore Binding Analysis:
The TNF-α binding affinities (KD=kd/ka=koff/kon) of the scFv fragments were calculated from the resultant association (ka=kon) and dissociation (kd=koff) rate constants as measured using a BIAcore-2000 surface plasmon resonance system (BIAcore, Inc). To avoid valency problems due to the trimeric state of TNFα, the ligand was immobilized on the BIAcore chip sensor surface in effect, allows monitoring of the monomeric scFv binding from the flowed solution. BIAcore biosensor chip were activated for covalent coupling of TNF-α using N-ehtyl-N′-(3-dimethylaminopropyl)-carbo-diimide hydrochloride (EDC) and N-hydrosuccinimide (NHS) according to manufacturer's instructions. A solution of ethanolamine was injected as a blocking agent.
For the flow analysis, anti-TNF-α scFv were diluted into 20 mM Hepes buffered Saline pH 7.0 and diluted to approximately 50 nM. Aliquots of anti-TNF-α scFvs were injected at a flow rate of 2 ul/minute. For kinetic measurements, scFvs were injected at a flow rate of 10 ul/min. Dissociation was observed in running buffer without dissociating agents. The kinetic parameters of the binding reactions were determined using BIAevaluation 2.1 software.
Overall, as shown in Table 2, the association rate constants, ka, for all examined clones varied by 2.1 fold (2.8×105 to 5.8×105), whereas the dissociation rate, kd improved by 7.7 fold (1.15×10−4 to 1.49×10−5). Thus, the enhanced affinity shown by these anti-TNF-α clones is contributed mainly by their improved dissociation rate (kd) kinetics. ehtyl-N′9
The biological activity of the affinity enhanced CBM clones was measured using a TNF-α induced L929 cell cytotoxicity assay. Murine L929 cells after brief co-treatment with Actinomysin D are susceptible to TNF-α mediated cytotoxicity. If however, the soluble TNF-α is co-incubated with anti-TNF-α antibodies, the antibody bound cytokine unable to bind the TNF receptor and the cytotoxicity is neutralized. For a given concentration of anti-TNF-α antibody, the degree of cytotoxicity protection afforded by the anti-TNF-α antibody is therefore dependent upon its binding affinity for TNF-α. To determine the IC50, various TNF-α and antibody concentrations were co-incubated for 24 hours after which, a calorimetric metabolic dye was added to determine the extent of cell death and antibody mediated protection by measuring the resultant optical density generated by the substrate conversion in living cells.
Cell Culture:
L929 cells were propagated in the following growth medium: Minimal Essential Medium (Eagles), supplemented with 2 mM L-glutamine, and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, 10% FBS, 50 μg/mL gentamycin and cultivated in incubators at 37° C. in an atmosphere of 5% CO2. Before attaining confluence, L929 cell populations were sub-cultured at a ratio of 1:4 three times a week to maintain cells in the logarithmic phase of growth.
Neutralization Assay:
The neutralization assay that was performed was a modification of a procedure developed by Doring et al, (Molecular Immunology, 31:1059-1067 (1994)). In brief, L929 cells were plated 35,000 cells per well in a 96-well micro titer plate for overnight growth. The next day, the following six antibody drugs were serially diluted so that the final concentrations in the well would be as follows: Positive control Humira (IgG1) and D2E7 (scFv): 8100 pM, 2700 pM, 900 pM, 300 pM, 100 pM, 33.3 pM, 11.1 pM, 3.7 pM, 1.23 pM, 0.411 pM; CBM affinity enhanced clone Al (in scFv format): 1620 pM, 540 pM, 180 pM, 60 pM, 20 pM, 6.67 pM, 2.22 pM, 0.741 pM, 0.247 pM, 0.082 pM; CBM affinity enhanced clones 2-44-2, 1-3-3, 2-6-1 (all in scFv format): 810 pM, 270 pM, 90 pM, 30 pM, 10 pM, 3.33 pM, 1.11 pM, 0.370 pM, 0.123 pM, 0.0411 pM. The A1 sequence has the D2E7 mutations CDRH1:D31Q, CDRH3:S99P, and CDRL1:G28E. The 2-44-2, 1-3-3 and 2-6-1 antibodies have the mutations shown in
Given the higher affinity of the anti-TNF-α antibodies, CBM clones were started with dilutions tenfold lower, since preliminary experiments showed that if the CBM clone concentrations were of similar concentrations with the positive control Humira and D2E7, adding TNF-α at the IC50 value would not induce cytotoxicity. The diluent used for the antibody serial dilutions was the above MEM growth media. For the neutralization assay in the replicate wells of the above antibody control and clone dilutions, TNF-α was then added to yield two different final concentrations (175 pg/mL and 350 pg/mL). Therefore, one set of the antibody dilutions (e.g. 810 to 0.0411 pM) was incubated at a final TNF-α concentration of 175 pg/mL while another antibody dilution (e.g. 810 to 0.0411 pM) was incubated at 350 pg/mL TNF-α. To allow complex formation, these TNF-α and antibody co-incubations were performed at room temperature for 30 minutes prior to their addition to the cell culture plates.
As a negative binding control, an aliquot from each of the six test antibodies was boiled for 10 minutes, placed on ice for a few minutes then centrifuged (13,000 g) at 4° C. for 5 minutes to remove any precipitated material. One dilution concentration of the boiled, denatured antibodies was then co-incubated with TNF-α (175 pg/mL and 350 pg/mL) for 30 minutes at room temperature.
Prior to co-incubation of TNF-α and one of the test antibodies, the overnight media was aspirated from the L929 cell cultures and replaced with media containing 10% heat-inactivated serum and 1 μg/mL Actinomycin D. Exposure to Actinomycin D was no longer than 5-15 minutes prior to the addition of the TNF-α and antibody co-incubations. On the day that the neutralization experiments were run, a control TNF-α dose response curve was performed on a separate plate of L929 cells to ensure that the drug experiments are within the IC50 of cytotoxicity. The following TNF-α concentrations were used for the dose response curve: 0.08 pg/mL, 0.4 pg/mL, 2 pg/mL, 10 pg/mL, 25 pg/mL, 50 pg/mL, 100 pg/mL, 250 pg/mL, 500 pg/mL, and 1000 pg/mL. The TNF-α and antibody treated L929 cells were subsequently incubated for 20-24 hours at 37° C. The following day, a 1/10 volume ratio of WST-1 cell proliferation reagent was added to each well and the cells were allowed another 4 hours of incubation at 37° C. The introduced WST-1 reagent is taken in by the cell whereupon its' metabolized product causes an increase in OD 450 nm absorbance. Following WST-1 incubation, the culture plate was removed and placed upon a microplate reader where the absorbance at OD 450 nm was read and with a reference of 630 nm on a Wallac Victor2 plate reader. From the resulting plots, the IC50s were then determined by using Prism version 3.02 software. From the TNF control dose response experiments, it can be seen that greater levels of cytotoxicity through increasing TNF concentration exposure will result in decreased OD 450 nm readings (
Determination of the IC50 of TNF-α Treated L929 Cells
Table 3 and the associated
Neutralization of the Cytotoxic Effect of TNF-α on L929 Cells
Comparative neutralization experiments with four of the CBM affinity enhanced anti-TNF-α clones and the positive control anti-TNF-α Humira (IgG1) and D2E7 (scFv) were performed on the same day to eliminate the typical day to day variability. The TNF-α neutralization results for CBM clone 2-44-2, and representative of the other CBM experimental clones, are shown in Tables 4 and 5 and associated graphical plots
For CBM clone 2-44-2 (labeled as test drug 2 in the
Overall, the average IC50 for the TNF-α dose response curve was 248 pg/mL, well within the parameters of the values chosen by Bioren for the assay (175 pg/mL and 350 pg/mL). From their respective TNF-α neutralization assays, the average IC50 of affinity enhanced anti-TNF-α CBM clones (A1, 2-44-2, 1-3, 2-6-1) was determined to be approximately 5.11 pM (
Although the invention has been described with reference to particular embodiments and examples, it will be appreciated that various modifications and other applications may be made without departing from the spirit of the invention. For example, the selection of representative amino acids employed in LTM and WTM may be modified in a variety of ways that preserve the representation of basic physiocochemical properties of the 20 basic amino acids. Similarly, different antibody formats, and different reference sequences may be used. Instead of starting with all “human-derived” CDRs, for example, one or more of HV or HL chain CDRs could be based on mouse CDR sequence for the corresponding mouse anti-anti-TNF-α antibody sequence. Such a construction would be expected to provide additional structure-activity relationship information on the affect of amino acid sequence and binding activity.
Sequence Listing
SEQ ID NO: 1: (the amino acid sequence for D2E7 scFv antibody):
SEQ ID NO: 2: (the VL amino acid sequence of D2E7 with all VL CDR1-CDR3 mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1═R or H
X2=L, Q, R, K or Y
X3=G, E, R, S, Y or K
X4═I, L or K
X5=A or K
X6=L, S or Y
X7═S, A, K or T
X8═F, P or L
X9=Q, Y, K or L
X10═S, Q, H, R, K, N or P
X11═K or R
X12═N or D
X13═R, S, K, L, Q or D
X14=A, P or K
X15═P or Q
X16═Y or Q
X17=T or A
SEQ ID NO: 3: (the VL CDR1 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1AS X2X3X4RNYLA
X1═R or H
X2=L, Q, R, K or Y
X3=G, E, R, S, Y or K
X4═I, L or K
SEQ ID NO: 4: (the VL CDR2 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1X2X3X4X5X6
X1=A or K
X2=L, S or Y
X3═S, A, K or T
X4═F, P or L
X5=Q, Y, K or L
X6═S, Q, H, R, K, N or P
SEQ ID NO: 5: (the VL CDR3 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
QX1YX2X3X4X5X6X7
X1═K or R
X2═N or D
X3═R, S, K, L, Q or D
X4=A, P or K
X5═P or Q
X6═Y or Q
X7=T or A
SEQ ID NO: 6: (the amino acid sequence for D2E7 VH).
SEQ ID NO: 7: (the VH amino acid sequence of D2E7 with all VH CDR1-CDR3 mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X19WGQGTLVTV S
X1=D, Q, Y or H
X2═Y, S, or H
X3=M or L
X4=T, S, I, or A
X5═W or Y
X6═I, A, or H
X7=D, K or S
X8=A, S or K
X9═S or P
X10=A, K, S or V
X11═S, K, Q, H, R, or T
X12═Y, K, Q or H
X13═S or P
X14=A or S
X15═S, D or P
X16═S, Q or N
X17=L or H
X18=D, H, S or Q
X19═Y, N, S, L, Q, or H
SEQ ID NO: 8: (the VH CDR1 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1X2AX3H
X1=D, Q, Y or H
X2═Y, S, or H
X3=M or L
SEQ ID NO: 9: (the VH CDR2 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1X2NSGHX3X4YX5DX6VE
X1=T, S, I, or A
X2═W or Y
X3═I, A, or H
X4=D, K or S
X5=A, S or K
X6═S or P
SEQ ID NO: 10: (the VH CDR3 amino acid sequence with all mutations selected for enhanced affinity indicated as single or alternative-residue amino acids.)
X1X2X3LX4TX5X6X7X8X9X10
X1=A, K, S or V
X2═S, K, Q, H, R, or T
X3═Y, K, Q or H
X4═S or P
X5=A or S
X6═S, D or P
X7═S, Q or N
X8=L or H
X9=D, H, S or Q
X10═Y, N, S, L, Q, or H
SEQ ID NO: 11: the combinatorial coding sequences for the VL CDR1
X1=A or G
X2=A, C, or T
X3=A or G
X4=G or T
X5=A or G
X6=A or G
X7=A or C
X8=A or T
SEQ ID NO: 12: the combinatorial coding sequences for the VL CDR2
X1=A or C
X2=A or T
X3=A or T
X4=A or T
X5=A or C
X6=A, C or G
X7=T or G
SEQ ID NO: 13: the combinatorial coding sequences for the VL CDR3
X1=A or G
X2=A or G
X3=G or C
SEQ ID NO: 14: the combinatorial coding sequences for the VH CDR1
X1═C, G or T
X2=G or T
X3═C or T
X4=A or C
X5=A or C
SEQ ID NO: 15: the combinatorial coding sequences for the VH CDR2
SEQ ID NO: 16: the combinatorial coding sequences for the VH CDR3
X1=A or C
X2=A or G
X3=G or T
X4=A or C
X5=A or G
X6=G or T
X7═C or G
X8=G or T
X9═C or T
X10=A or C
X11=G or T
SEQ ID NO: 17: the complete nucleotide sequence of D2E7 scFV antibody;
SEQ ID NO: 18: 5′ Bam HI Forward sense oligonucleotide for D2E7 scFv
SEQ ID NO: 19: 3′ Not I Reverse flanking oligonucleotide for D2E7 scFv
SEQ ID NO: 20: FR5 anti-sense oligonucleotide
SEQ ID NO 21: FR1 sense oligonucleotide
SEQ ID NO 22: FR2 anti-sense oligonucleotide
SEQ ID NO 23: CDR H2 LTM oligonucleotides wildtype oligonucleotide
SEQ ID NO 24: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 25: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 26: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 27: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 28: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 29: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 30: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 31: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 32: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 33: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 34: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 35: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 36: CDR H2 LTM leucine oligonucleotides:
SEQ ID NO 37: FR4 anti-sense oligonucleotide
SEQ ID NO 38: CDR H2 LTM aspartate oligonucleotide (Asp codon are bold)
SEQ ID NO 39: CDR H2 WTM aspartate oligonucleotide:
SEQ ID NO 41: CDR-H1 LTM leucine oligonucleotides:
SEQ ID NO 42: CDR-H1 LTM leucine oligonucleotides:
SEQ ID NO 43: CDR-H1 LTM leucine oligonucleotides:
SEQ ID NO 44: CDR-H1 LTM leucine oligonucleotides:
SEQ ID NO 45: CDR-H1 LTM leucine oligonucleotides:
SEQ ID NO 46: CDR H1 beneficial mixed mutation oligonucleotide:
SEQ ID NO 47: CDR H2 beneficial mixed mutation oligonucleotide:
SEQ ID NO 48: CDR H3 beneficial mixed mutation oligonucleotide:
SEQ ID NO 49: CDR L1 beneficial mixed mutation oligonucleotide:
SEQ ID NO 50: CDR L2 beneficial mixed mutation oligonucleotide:
SEQ ID NO 51: CDR L3 beneficial mixed mutation oligonucleotide:
SEQ ID NO: 52: Sense strand oligonucleotide S1
SEQ ID NO: 53: Sense strand oligonucleotide S2
SEQ ID NO: 54: Sense strand oligonucleotide S3
SEQ ID NO: 55: Sense strand oligonucleotide S4
SEQ ID NO: 56: Sense strand oligonucleotide S5
SEQ ID NO: 57: Sense strand oligonucleotide S6
SEQ ID NO: 58: Sense strand oligonucleotide S7
SEQ ID NO: 59: Sense strand oligonucleotide S8
SEQ ID NO: 60: Sense strand oligonucleotide S9
SEQ ID NO: 61: Sense strand oligonucleotide S10
SEQ ID NO: 62: Sense strand oligonucleotide S11
SEQ ID NO: 63: Sense strand oligonucleotide S12
SEQ ID NO: 64: Sense strand oligonucleotide S13
SEQ ID NO: 65: Sense strand oligonucleotide S14
SEQ ID NO: 66: Sense strand oligonucleotide S15
SEQ ID NO: 67: Antisense strand oligonucleotide S1
AS1
SEQ ID NO: 68: Antisense strand oligonucleotide S2
SEQ ID NO: 69: Antisense strand oligonucleotide S3
SEQ ID NO: 70: Antisense strand oligonucleotide S4
SEQ ID NO: 71: Antisense strand oligonucleotide S5
SEQ ID NO: 72: Antisense strand oligonucleotide S6
SEQ ID NO: 73: Antisense strand oligonucleotide S7
SEQ ID NO: 74: Antisense strand oligonucleotide S8
SEQ ID NO: 75: Antisense strand oligonucleotide S9
SEQ ID NO: 76: Antisense strand oligonucleotide S10
SEQ ID NO: 77: Antisense strand oligonucleotide S11
SEQ ID NO: 78: Antisense strand oligonucleotide S12
SEQ ID NO: 79: Antisense strand oligonucleotide S13
SEQ ID NO: 80: Antisense strand oligonucleotide S14
SEQ ID NO: 81: Antisense strand oligonucleotide S15
This application claims priority to U.S. Provisional Patent Application No. 60/586,487 filed on Jul. 6, 2004, which is incorporated herein in its entirety by reference.
Number | Date | Country | |
---|---|---|---|
60586487 | Jul 2004 | US |