Rabbit-derived antigen binding protein nucleic acid libraries and methods of making the same
12435444 · 2025-10-07
Assignee
Inventors
- Anna Maria Sobieraj (Zürich, CH)
- Severin Oliver Wendelspiess (Zürich, CH)
- Fabian Bert Scheifele (Mägenwil, CH)
Cpc classification
C07K2317/32
CHEMISTRY; METALLURGY
C07K16/2809
CHEMISTRY; METALLURGY
C40B30/04
CHEMISTRY; METALLURGY
C12N15/1086
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C40B30/04
CHEMISTRY; METALLURGY
Abstract
Rabbit antigen binding protein nucleic acid libraries are provided (e.g., nucleic acid libraries encoding antigen binding proteins that specifically recognize a target peptide-MHC (pMHC)). Methods of producing the rabbit antigen binding protein nucleic acid libraries are also provided.
Claims
1. A method of producing an antigen binding protein that specifically recognizes a target antigen, the method comprising the steps of: (i) immunizing a rabbit with the target antigen; (ii) isolating a plurality of antigen binding protein encoding polynucleotide sequences from the rabbit, wherein the antigen binding protein encoding polynucleotide sequences encode for at least kappa VL; (iii) cloning the polynucleotide sequences into a nucleic acid library; (iv) mutagenizing the nucleic acid library to introduce an amino acid substitution at position C80 of the kappa VL, according to Kabat numbering; and (v) selecting the antigen binding protein that specifically recognizes a target antigen, wherein the library comprises polynucleotide sequences derived from one or more parental rabbit kappa VL genes IGKV1S1 to IGKVIS68.
2. The method of claim 1, comprising a C80A, a C80S, a C80P or a C80 germline amino acid substitution in the kappa VL, according to Kabat numbering.
3. The method of claim 1, wherein the target antigen comprises a pMHC.
4. The method of claim 1, wherein the kappa VL or lambda VL are operatively linked to the VH with an amino acid linker.
5. The method of claim 1, wherein the selecting step (v) is performed through biopanning against the target antigen.
6. The method of claim 2, wherein the amino acid substitution is introduced with a polymerase chain reaction (PCR).
7. The method of claim 6, wherein multiple PCRs with different pairs of primers specific for rabbit VL sequences are performed.
8. The method of claim 7, wherein 2, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 PCRs are performed, each with a different pair of primers.
9. The method of claim 7, wherein the said pair of primers comprises a first primer introducing a C80 substitution and a second primer.
10. The method of claim 9, wherein the second primer anneals with a conserved sequence stretch of a rabbit VL germline sequence.
11. The method of claim 6, wherein the PCR is performed with one or more primer pairs selected from F1/R1 (SEQ ID NO: 4/SEQ ID NO: 6), F1/R2 (SEQ ID NO: 4/SEQ ID NO: 7), F1/R3 (SEQ ID NO: 4/SEQ ID NO: 8), F1/R4 (SEQ ID NO: 4/SEQ ID NO: 9), F1/R5 (SEQ ID NO: 4/SEQ ID NO: 10), F1/R6 (SEQ ID NO: 4/SEQ ID NO: 11), F1/R7 (SEQ ID NO: 4/SEQ ID NO: 12), F1/R8 (SEQ ID NO: 4/SEQ ID NO: 13), F1/R9 (SEQ ID NO: 4/SEQ ID NO: 14), F1/R10 (SEQ ID NO: 4/SEQ ID NO: 15), F2/R1 (SEQ ID NO: 5/SEQ ID NO: 6), F2/R2 (SEQ ID NO: 5/SEQ ID NO: 7), F2/R3 (SEQ ID NO: 5/SEQ ID NO: 8), F2/R4 (SEQ ID NO: 5/SEQ ID NO: 9), F2/R5 (SEQ ID NO: 5/SEQ ID NO: 10), F2/R6 (SEQ ID NO: 5/SEQ ID NO: 11), F2/R7 (SEQ ID NO: 5/SEQ ID NO: 12), F2/R8 (SEQ ID NO: 5/SEQ ID NO: 13), F2/R9 (SEQ ID NO: 5/SEQ ID NO: 14), and F2/R10 (SEQ ID NO: 5/SEQ ID NO: 15).
12. The method of claim 6, wherein the PCR is performed with one or more primer pairs derived from the sequences of the group consisting of F1/R1 (SEQ ID NO: 4/SEQ ID NO: 6), F1/R2 (SEQ ID NO: 4/SEQ ID NO: 7), F1/R3 (SEQ ID NO: 4/SEQ ID NO: 8), F1/R4 (SEQ ID NO: 4/SEQ ID NO: 9), F1/R5 (SEQ ID NO: 4/SEQ ID NO: 10), F1/R6 (SEQ ID NO: 4/SEQ ID NO: 11), F1/R7 (SEQ ID NO: 4/SEQ ID NO: 12), F1/R8 (SEQ ID NO: 4/SEQ ID NO: 13), F1/R9 (SEQ ID NO: 4/SEQ ID NO: 14), F1/R10 (SEQ ID NO: 4/SEQ ID NO: 15), F2/R1 (SEQ ID NO: 5/SEQ ID NO: 6), F2/R2 (SEQ ID NO: 5/SEQ ID NO: 7), F2/R3 (SEQ ID NO: 5/SEQ ID NO: 8), F2/R4 (SEQ ID NO: 5/SEQ ID NO: 9), F2/R5 (SEQ ID NO: 5/SEQ ID NO: 10), F2/R6 (SEQ ID NO: 5/SEQ ID NO: 11), F2/R7 (SEQ ID NO: 5/SEQ ID NO: 12), F2/R8 (SEQ ID NO: 5/SEQ ID NO: 13), F2/R9 (SEQ ID NO: 5/SEQ ID NO: 14), and F2/R10 (SEQ ID NO: 5/SEQ ID NO: 15).
13. The method of claim 1, wherein each of said polynucleotide sequences is present in a circular DNA construct.
14. The method of claim 13, wherein multiple PCRs with different pairs of primers specific for rabbit VL sequences are performed, each pair of primers annealing specifically to rabbit kappa VL sequences, comprising a first and a second primer, wherein the first primer introduces an amino acid substitution at Kabat position 80 and the 5 ends of both primers anneal back-to-back on the template, and the primers hybridize with one or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or more allelic variant of rabbit kappa VL germline sequences.
15. The method of claim 1, wherein the antigen binding protein encoding polynucleotide sequences are isolated from a B cell population.
16. The method of claim 15, wherein the B cell population is a peripheral blood mononuclear cell (PBMC) population, a B cell population from spleen, a B cell population from lymph nodes, or a combination thereof.
17. The method of claim 1, wherein the antigen binding protein encoding polynucleotide sequences further encode for one or both of lambda VL and VH.
18. The method of claim 1, wherein the kappa VL and lambda VL is operatively linked to a CL domain and the VH is operatively linked to a CH1 domain.
19. The method of claim 1, wherein the antigen binding protein specifically binds to a tumor antigen.
20. The method of claim 19, wherein the tumor antigen is selected from the group consisting of: a melanoma-associated antigen A (MAGE-A), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), synovial sarcoma X (SSX), carcinoembryonic antigen (CEA), preferentially expressed antigen in melanoma (PRAME), melanoma antigen recognized by T cells 1 (MART-1), Kirsten rat sarcoma viral oncogene (K-ras), kinetochore NDC80 protein homolog (NDC80), Kita-Kyushu lung cancer antigen (KK-LC-1), and Wilms tumor 1 (WT1).
21. The method of claim 1, wherein the antigen binding protein specifically binds to a viral antigen.
22. The method of claim 21, wherein the viral antigen is selected from the group consisting of: Epstein-Barr virus derived EBNA1, EBNA2, EBNA3, LMP1, or LMP2; hepatitis B virus derived HBX; hepatitis C virus derived NS3 or NS5A; human papillomavirus derived type E5, E6, and E7 proteins; cytomegalovirus derived PP65; human immunodeficiency virus derived gag; and Kaposi sarcoma-associated herpesvirus derived vGPCR or vIRF-1.
23. The method of claim 1, wherein the nucleic acid library is selected from the group consisting of a ribosome display library, a phage display library, a yeast cell display library, a mammalian cell display library, and a DNA display library.
24. The method of claim 1, wherein the nucleic acid library is a phage display library.
25. The method of claim 1, wherein the nucleic acid library comprises a diversity of at least 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences.
26. The method of claim 1, wherein the one or more parental rabbit kappa VL genes IGKVIS1 to IGKV1S68 comprise a sequence of SEQ ID NOs 322-334, 333, 335, 330, 336, 330, 337, 333, 338-339, 332, 329, 333, 332, 340, 328, 332, 329, 332, 339, 328, 341, 338, 342, 331, 328, 330, 343-346, 330, 332, 347-348, 333, 332, 332, 332, 333, 328, 332, 349-350, 330, 330, 330, 351, 332, 332, 352, 332, 332, and 332, respectively.
27. The method of claim 1, wherein the nucleic acid library comprises polynucleotide sequences encoding about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of VL genes derived from parental rabbit kappa VL genes IGKVIS1 to IGKVIS68.
28. The method of claim 20, wherein the MAGE-A is selected from the group consisting of MAGE-A1, MAGE-A3 and MAGE-A4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.
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DETAILED DESCRIPTION
(14) Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein is well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
(15) Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of or means and/or unless stated otherwise. The use of the term including, as well as other forms, such as includes and included, is not limiting.
(16) So that the invention may be more readily understood, certain terms are first defined.
(17) Antigen Binding Proteins
(18) As used herein, the term antibody or antigen binding protein refers to an immunoglobulin molecule or immunoglobulin derived molecule that specifically binds to, or is immunologically reactive with an antigen or epitope, and includes both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to fragment antigen-binding (Fab) fragments, F(ab).sub.2 fragments, Fab fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH) fragments. The antibody may thus be a single domain antibody or comprise at least one variable light and at least one variable heavy chain. In one embodiment, the at least one variable light and at least one variable heavy chain are displayed as a single polypeptide chain. The term antibody or antigen binding protein includes germline derived antibodies. The term antibody or antigen binding protein includes genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv) and the like. Unless otherwise stated, the term antibody or antigen binding protein should be understood to encompass functional antibody fragments thereof. In certain embodiments, the antigen binding protein is multispecific (i.e., binds to two or more different target molecules or to two or more epitopes on the same target molecule). In certain embodiments, the antigen binding protein is bispecific and e.g., binds to two different target molecules or to two epitopes on the same target molecule. In certain embodiments, the antibody is trispecific and e.g., binds to at least three different target molecules.
(19) The antigen binding protein may be monovalent or multivalent, i.e., having one or more antigen binding sites. Non-limiting examples of monovalent antigen binding proteins include scFv, Fab, scFab, dAb, VHH, V (NAR), DARPins, affilins and nanobodies. A multivalent antigen binding protein can have two, three, four or more antigen binding sites. Non-limiting examples of multivalent antigen binding proteins include full-length immunoglobulins, F(ab).sub.2fragments, bis-scFv (or tandem scFv or BiTE), DART, diabodies, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-scFv, Fv2-Fc, FynomABs, quadroma, CrossMab, DuoBody, triabodies and tetrabodies. In some embodiments, the multivalent antigen binding protein is bivalent, i.e., two binding sites are present. In some embodiments, the multivalent antigen binding protein is bispecific, i.e., the antigen binding protein is directed against two different targets or two different target sites on one target molecule. In some embodiments, the multivalent antigen binding protein includes more than two, e.g., three or four different binding sites for three or four, respectively, different antigens. Such antigen binding protein is multivalent and multispecific, in particular tri- or tetra-specific, respectively.
(20) In some embodiments, the antigen binding proteins are multispecific (e.g., bispecific), such as, without being limited to, diabodies, single-chain diabodies, DARTs, BiTEs, tandem scFvs or IgG-like asymmetric heterobispecific antibodies. In certain embodiments, one of the binding specificities of the multispecific antigen binding protein is an immune cell engager (i.e., comprising binding affinity to a cell surface protein of an immune cell). Examples of immune cells that may be recruited include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophil cells, monocytes, and macrophages. Examples of surface proteins that may be used to recruit immune cells includes, but are limited to, CD3, TCR, TCR, CD16, NKG2D, CD89, CD64, and CD32. Such immune cell redirecting multispecific antigen binding proteins may in some embodiments comprise a Fc domain.
(21) In certain embodiments, the immune cell target antigen is CD3. An exemplary CD3 antigen binding domain is recited below in Table 7 and in WO2016086196 and WO2017201493, incorporated herein by reference.
(22) As used herein, a single-chain variable fragment (scFv) is an antigen binding protein comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL). The VH and VL domains of the scFv are linked via any appropriate art recognized linker. Such linkers include, but are not limited to, repeated GGGGS (SEQ ID NO: 188) amino acid sequences or variants thereof. The scFv is generally free of antibody constant domain regions, although an scFv of the disclosure may be linked or attached to antibody constant domain regions (e.g., antibody Fc domain) to alter various properties of the scFv, including, but not limited to, increased serum or tissue half-life. An scFv generally has a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm.
(23) As used herein, a Fab fragment or Fab is an antibody fragment comprising a light chain fragment comprising a variable light (VL) domain and a constant domain of the light chain (CL), and variable heavy (VH) domain and a first constant domain (CH1) of the heavy chain.
(24) As used herein, a VHH, nanobody, or heavy-chain only antibody is an antigen binding protein comprising a single heavy chain variable domain derived from the species of the Camelidae family, which includes camels, llama, alpaca. A VHH generally has a molecular weight of about 15 kDa.
(25) In one embodiment, the antigen binding protein comprises an Fc domain. The presence of an Fc domain may be advantageous to induce cytotoxic immune responses and/or activate complement (e.g., ADCC/ADCP or CDC effector function). Exemplary antibody formats including an Fc domain, without being limited to, are full-length immunoglobulins, DVD-Ig, scFv-Fc and scFv-Fc. scFv fusions, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions (such as e.g., bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab, Knob-into-Holes (KiHs)), DuoBody and/or CrossMabs. An active Fc domain may increase the likelihood of pro-inflammatory cytokine release by T cells and other effector cells in the tumor microenvironment which is believed to be part of the therapeutic mechanism of action. The Fc domain may be fully active or partly silenced to avoid over-stimulation of the immune system. In some embodiments, the Fc domain is inactive and does not stimulate pro-inflammatory cytokine release but does still improve half-life and/or stability of the antigen binding protein. In some embodiments, the antigen binding protein comprises a constant region selected from the group consisting of human IgG1, IgG2, IgG3 or IgG4 isotype. In other embodiments, the antigen binding protein comprises a constant region selected from the group consisting of murine IgG1, IgG2A, IgG2B or IgG3 isotype.
(26) The antigen binding proteins of the disclosure may comprise one or more linkers for linking the domains of the antigen binding protein (e.g., linking a VH and VL to form a scFv, or linking multiple binding domains to form a multispecific antigen binding protein).
(27) Illustrative examples of linkers include glycine polymers (Gly), (SEQ ID NO: 373); glycine-serine polymers (Gly.sub.nSer).sub.n (SEQ ID NO: 374), where n is an integer of at least one, two, three, four, five, six, seven, or eight; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.
(28) Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the antigen binding proteins described herein. Glycine accesses significantly more phi-psi space than other small side chain amino acids, and is much less restricted than residues with longer side chains (Scheraga, Rev. Computational Chem. 1:1173-142 (1992)). A person skilled in the art will recognize that design of a antigen binding protein in particular embodiments can include linkers that are all or partially flexible, such that the linker can include flexible linker stretches as well as one or more stretches that confer less flexibility to provide a desired structure.
(29) Linker sequences can however be chosen to resemble natural linker sequences, for example, using the amino acid stretches corresponding to the beginning of human CH1 and C sequences or amino acid stretches corresponding to the lower portion of the hinge region of human IgG.
(30) The design of the peptide linkers connecting VL and VH domains in the scFv moieties are flexible linkers generally composed of small, non-polar or polar residues such as, e.g., Gly, Ser and Thr. A particularly exemplary linker connecting the variable domains of the scFv moieties is the (Gly.sub.4Ser).sub.4 linker (SEQ ID NO: 375), where 4 is the exemplary number of repeats of the motif.
(31) Other exemplary linkers include, but are not limited to the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 376); TGEKP (SEQ ID NO: 377) (Liu et al, Proc. Natl. Acad. Sci. 94:5525-5530 (1997)); GGRR (SEQ ID NO: 378); (GGGGS).sub.n (SEQ ID NO: 379) wherein n=1, 2, 3, 4 or 5 (Kim et al, Proc. Natl. Acad. Sci.93:1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO: 380) (Chaudhary et al., Proc. Natl. Acad. Sci. 87:1066-1070 (1990)); KESGSVSSEQLAQFRSLD (SEQ ID NO: 381) (Bird et al., Science 242:423-426 (1988)), GGRRGGGS (SEQ ID NO: 382); LRQRDGERP (SEQ ID NO: 383); LRQKDGGGSERP (SEQ ID NO: 384); and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 385) (Cooper et al, Blood, 101(4): 1637-1644 (2003)). Alternatively, flexible linkers can be rationally designed using a computer program capable of modeling the 3D structure of proteins and peptides or by phage display methods.
(32) The antibodies may comprise a variable light (VL) domain and a variable heavy (VH) domain. Each VL and VH domain typically comprises a set of three CDRs.
(33) As used herein, the term complementarity determining region or CDR refers to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable domain (CDRH1, CDRH2, CDRH3) and three CDRs in each light chain variable domain (CDRL1, CDRL2, CDRL3). Framework regions or FRs are known in the art to refer to the non-CDR portions of the variable domains of the heavy and light chains. In general, there are four FRs in each heavy chain variable domain (HFR1, HFR2, HFR3, and HFR4), and four FRs in each light chain variable domain (LFR1, LFR2, LFR3, and LFR4). Accordingly, an antibody variable region amino acid sequence can be represented by the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Each segment of the formula, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, represents a discrete amino acid sequence (or a polynucleotide sequence encoding the same) that can be mutated, including one or more amino acid substitutions, deletions, and insertions. In certain embodiments, an antibody variable light chain amino acid sequence can be represented by the formula LFR1-CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR4. In certain embodiments, an antibody variable heavy chain amino acid sequence can be represented by the formula HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR4.
(34) The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (Chothia numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol. 262, 732-745. (Contact numbering scheme), Lefranc M P et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol, 2003 January; 27(1): 55-77 (IMGT numbering scheme), and Honegger A and Pluckthun A, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J Mol Biol, 2001 Jun. 8; 309(3): 657-70, (AHo numbering scheme).
(35) The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, 30a, and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (indels) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
(36) Table 1, below, lists exemplary position boundaries of CDRL1, CDRL2, CDRL3 and CDRH1, CDRH2, CDRH3 of an antibody, as identified by Kabat, Chothia, and Contact schemes, respectively. For CDRH1, residue numbering is listed using both the Kabat and Chothia numbering schemes. CDRs are located between FRs, for example, with CDRL1 located between LFR1 and LFR2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDRH1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
(37) TABLE-US-00001 TABLE 1 Exemplary Position Boundaries of CDRs CDR Kabat Chothia Contact LCDR1 L24--L34 L24--L34 L30--L36 LCDR2 L50--L56 L50--L56 L46--L55 LCDR3 L89--L97 L89--L97 L89--L96 HCDR1 H31--H35B H26--H32 . . . 34 H30--H35B (Kabat Numbering.sup.1) HCDR1 H31--H35 H26--H32 H30--H35 (Chothia Numbering.sup.2) HCDR2 H50--H65 H52--H56 H47--H58 HCDR3 H95--H102 H95--H102 H93--H101 .sup.1Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD .sup.2Al-Lazikani et al. (1997), J. Mol. Biol. 273:927-948
(38) Thus, unless otherwise specified, a CDR or complementary determining region, or individual specified CDRs (e.g., CDRH1, CDRH2), of a given antibody or fragment thereof, such as a variable domain thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the known schemes. Likewise, unless otherwise specified, an FR or framework region, or individual specified FRs (e.g., HFR1, HFR2) of a given antibody or fragment thereof, such as a variable domain thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR or FR is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.
(39) In certain embodiments, the rabbit antigen binding proteins disclosed here are humanized. As used herein, the term humanized or humanization refers to an antigen binding protein that has been altered to make it more like a human antibody. Non-human antigen binding proteins, such as the rabbit antigen binding proteins encoded in the nucleic acid libraries disclosed herein, would elicit a negative immune reaction if administered to a human for therapy. It is therefore advantageous to humanize the rabbit antigen binding proteins for later therapeutic use.
(40) In certain embodiments, the antigen binding proteins are humanized through resurfacing (i.e., remodel the solvent-accessible residues of the non-human framework such that they become more human-like). Resurfacing strategies are described in more detail in WO2004/016740, WO2008/144757, and WO2005/016950, each of which is incorporated herein by reference.
(41) In certain embodiments, the antigen binding proteins are humanized through CDR grafting (i.e., inserting the rabbit antigen binding protein CDRs into a human antibody acceptor framework). Grafting strategies and human acceptor frameworks are described in more detail in WO2009/155726, incorporated herein by reference.
(42) As used herein, the term affinity refers to the strength of the interaction between an antibody's antigen binding site and the epitope to which it binds. As readily understood by those skilled in the art, an antibody or antigen binding protein affinity may be reported as a dissociation constant (KD) in molarity (M). The antibodies of the disclosure may have KD values in the range of 10.sup.8 to 10.sup.14 M. High affinity antibodies have KD values of 10.sup.9 M (1 nanomolar, nM) and lower. For example, a high affinity antibody may have a KD value in the range of about 1 nM to about 0.01 nM. A high affinity antibody may have KD value of about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 nM. Very high affinity antibodies have KD values of 10.sup.12 M (1 picomolar, pM) and lower. Weak, or low, affinity antibodies may have KD values in the range of 10.sup.1 to 10.sup.4 M. Low affinity antibodies may have KD values of 10.sup.4 M and higher, such as 10.sup.4 M, 10.sup.3 M, 10.sup.2 M, or 10.sup.1 M.
(43) The ability of an antibody to bind to a specific antigenic determinant (e.g., a target peptide-MHC) can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
(44) As used herein, the term T cell receptor or TCR refers to a heterodimeric protein comprised of two different chains (TCR and TCR), which structurally belong to the immunoglobulin (Ig) superfamily. The extracellular portion of each chain is composed of variable (V and V) and constant (C and C) domains, and a hinge region, where the formation of a stabilizing disulfide bond occurs. The intracellular region forms a non-covalent interaction with another trans-membrane protein, CD3, which in the case of the correct target recognition leads to a series of conformational changes and a first T cell activation signal. Recognition and binding of peptide-MHC (pMHC) by a TCR is governed by the six hypervariable loops, termed complementarity determining regions (CDRs), located on the variable domains of the TCR (CDR1, CDR2, CDR3) and TCR (CDRB1, CDR2, CDR3). CDR3 loops (CDR3 and CDR3) lead the recognition of the processed antigen with the support of CDR1 and CDR1, that have been implicated in the recognition of the N- and C-terminal amino acids of the presented peptide, respectively (Rudolph et al. Annu Rev Immunol. 24:419-66. 2006). Recognition of the MHC is typically achieved through the interaction with CDR2 and CDR2. The high sequence diversity of the TCR is achieved through V(D)J recombination process, in which the variable domain is generated from a combination of genes: V (variable) and J (joining) for both TCR and TCR, and an additional D (diversity) gene for TCR. The high antigen specificity of the TCR is controlled by the thymic maturation process, in which the self-reacting T cells are negatively selected. TCR affinity towards the specific pMHC and the functional avidity are the key factors controlling T-cell activation. A critical role in antigen recognition, however, is played by the affinity, i.e., the strength of binding between the TCR and the cell-displayed pMHC (Tian et al. J Immunol. 179:2952-2960. 2007). The physiological affinities of TCRs range from 1 M to 100 M (Davis et al. Annu Rev Immunol. 16:523-544. 1998), which, in comparison to antibodies, is relatively low.
(45) As used herein, the term peptide-MHC refers to a major histocompatibility complex (MHC) molecule (MHC-I or -II) with an antigenic peptide bound in a peptide binding pocket of the MHC. In certain embodiments, the MHC is a human MHC.
(46) Nucleic Acid Libraries
(47) Provided herein are nucleic acid libraries encoding rabbit derived antigen binding proteins. The rabbit antibody nucleic acid libraries described herein possess several advantages over other antibody discovery platforms.
(48) Rabbits represent an ideal source for the generation of high affinity antibodies. The rabbit immune response is characterized by generation of a highly diverse B-lymphocyte repertoire, high variability in length and sequence of the CDRs provides rabbit antibodies with high specificity and the ability to recognize small epitopes, such as peptides. Therefore, rabbit antibodies may recognize the peptides in context of the HLA complex in a highly specific manner. Furthermore, rabbit antibodies possess very high affinity, typically 10- to 100-fold higher affinity values than mice and other monoclonal antibodies. This high affinity makes rabbit antibodies useful for targets that have low expression levels or amounts in a cell. The high affinity is particularly useful for targeting pMHC antigens, with natural presentation levels as low as 10 copies per cell.
(49) Isolation of rabbit antibodies is practically constrained to hybridoma generation and antigen-specific B cell sorting strategies. These technologies are characterized by low survival of B cells and a short time window to screen or immortalize the B cells resulting in loss of antibody diversity and therefore a reduced probability to find an antibody with the desired specificity and binding affinity (e.g., high specificity and binding affinity for a target pMHC). Furthermore, the antibodies produced through hybridoma technology or B cell culture are bivalent IgG isotype antibodies which during the screening process may exhibit an apparent increased affinity due to avidity (functional affinity) which is undesirable when targeting epitopes that may be expressed at low densities, such as epitopes on tumor-associated peptide-MHCs.
(50) Targeting pMHCs or other low density targets requires a more efficient process to explore the whole antibody repertoire in order to increase the probability for isolating antibodies with the right specificity. Immune libraries for phage display selection poses several advantages over conventional hybridoma or B cell sorting technologies. In such libraries the B-cells are isolated from the spleen of immunized animals and the genes encoding for the variable light and heavy chain domains of the antibody repertoire are cloned into a nucleic acid library (e.g., phage library vectors). This is advantageous for different reasons. First, the antibody genetic information of the B cell repertoire is instantly preserved in the library, therefore less losses in antibody diversity due to poor survival of B cells occur; second, it provides an unlimited time window for isolation of antibodies with the right properties; and third, the antibodies are screened and selected in monovalent format eliminating artifacts due to avidity effects.
(51) Most rabbit light chains of the K1 isotype have an additional disulfide bridge between variable and constant domains through cysteine residues at positions 80 and 171 which is unusual in other species. For the generation of nucleic acid libraries for display (e.g., phage display libraries) derived from immunized rabbits, the variable kappa light chain domains containing cysteine 80 results in a free thiol group, which is disadvantageous for the display and selection of antibody scFv fragments. Therefore, the free thiol group from cysteine 80 may significantly restrict the selectable diversity of the rabbit libraries coming from rabbits immunized with a target antigen (e.g., pMHC), thus reducing the chances for isolating antibodies with the desired specificity and affinity, more so, as the majority of total rabbit antibodies comprise a K1 light chain.
(52) To increase the probability of identifying high affinity and high specificity antibodies with the desired function, it is necessary to enable full access to the complete antibody repertoire of rabbits immunized with a target antigen (e.g., pMHCs). The present disclosure provides rabbit-derived nucleic acid libraries (and methods of producing the same) with the desired diversity, in part through eliminating the cysteine via genetic manipulation of nucleic acid libraries derived from the genes encoding for the variable light and heavy chain domains of the antibody repertoire of immunized rabbits. Therefore, the antibody libraries of the disclosure enable the display and thus the access to the complete rabbit antibody repertoire. This allows for isolation of larger amount and diversity of antibodies to the target antigen (e.g., the peptide target on the epitope surface on the pMHC). Such an approach increases the probability of isolating antibodies with more diverse properties, such as distinct affinities and specificities for the peptide target.
(53) The present disclosure solves the major challenges in the discovery of antibodies with high specificity and affinity to target pMHC by combining the strong immune response of rabbits, an antigen-based library construction and an optimized nucleic acid display system (e.g., a phage display system) that allows a complete access to the rabbit antibody gene repertoire.
(54) In one aspect, the disclosure provides a nucleic acid library comprising a plurality of polynucleotide sequences, each polynucleotide sequence in the plurality encoding for a rabbit antigen binding protein comprising a kappa variable light chain (VL), wherein the kappa VL comprises an amino acid substitution at position C80, according to Kabat numbering, and wherein at least a portion of the plurality of polynucleotide sequences encode for a rabbit antigen binding protein that specifically recognizes a target antigen (e.g., a peptide-MHC).
(55) In certain embodiments, the substitution at position C80, according to Kabat numbering, removes a free thiol from the kappa VL without substantially reducing binding affinity or stability of the kappa VL.
(56) Said cysteine amino acid residue at Kabat position 80 of the VL may e.g., be substituted by serine (Ser or S), alanine (Ala or A), proline (Pro or P) or a germline residue, such as the residue at the corresponding rabbit or human germline position, or any other amino residue other than cysteine.
(57) In certain embodiments, the kappa VL comprises a C80A, a C80P or a C80S amino acid substitution, according to Kabat numbering.
(58) In certain embodiments, the plurality of polynucleotide sequences are obtained from a rabbit immunized with the target antigen (e.g., a peptide-MHC).
(59) In certain embodiments, the target antigen comprises a peptide MHC (pMHC).
(60) In certain embodiments, the pMHC comprises a MAGE-A4 230-239 amino acid sequence of GVYDGREHTV (SEQ ID NO: 3).
(61) In certain embodiments, the pMHC comprises one or both of an HLA-A*02:01 extracellular domain amino acid sequence set forth in SEQ ID NO: 1, and a human 2m amino acid sequence set forth in SEQ ID NO: 2.
(62) In certain embodiments, the C80 amino acid substitution is introduced with a polymerase chain reaction (PCR).
(63) In certain embodiments, multiple PCR reactions with different sets of primers are performed as to cover as many rabbit VL sequences as possible. In certain embodiments, each pair of primers comprises one primer introducing a C80 substitution. The second primer may e.g., align with a conserved sequence stretch of a rabbit VL germline sequence, e.g., as selected from IGKV1S1 to IGKVIS68 (see
(64) In certain embodiments, the PCR is performed with one or more primer pairs selected from F1/R1, F1/R2, F1/R3, F1/R4, F1/R5, F1/R6, F1/R7, F1/R8, F1/R9, F1/R10, F2/R1, F2/R2, F2/R3, F2/R4, F2/R5, F2/R6, F2/R7, F2/R8, F2/R9, and F2/R10, as recited in Table 3.
(65) In certain embodiments, each polynucleotide sequence in the plurality encodes for a rabbit antigen binding protein (e.g., an antibody or fragment thereof comprising a variable light chain (VL) and a variable heavy chain (VH)). In certain embodiments, each polynucleotide sequence in the plurality encodes for one or both of a rabbit VL or a rabbit VH. For example, but in no way limiting, an individual polynucleotide sequence may only encode for a rabbit VL, a rabbit VH, or both a rabbit VL and rabbit VH, such as an scFv with the VH and VL linked.
(66) In certain embodiments, the rabbit antigen binding protein comprises a variable light chain (VL) and variable heavy chain (VH).
(67) In certain embodiments, the VL and VH are operatively linked with an amino acid linker (e.g., the VL and VH are operatively linked to form an scFv).
(68) In certain embodiments, the VL is operatively linked to a CL domain and the VH is operatively linked to a CH1 domain.
(69) In certain embodiments, the nucleic acid library further comprises a plurality of polynucleotide sequences, wherein each polynucleotide sequence in the plurality encodes for a rabbit lambda VL.
(70) In certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences.
(71) In certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences encoding rabbit kappa VL.
(72) In certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences encoding rabbit lambda VL.
(73) In certain embodiments, the library comprises polynucleotide sequences encoding matured rabbit kappa VL sequences derived from one or more (e.g., 1, 2, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or all 68) rabbit kappa VL germline genes selected from IGKV1S1 to IGKVIS68, as recited in
(74) In certain embodiments, the library comprises at least one polynucleotide sequence encoding a matured rabbit kappa VL sequence derived from the parental sequence IGKV1S1, IGKV1S2, IGKV1S3, IGKV1S4, IGKV1S5, IGKV1S6, IGKV1S7, IGKVIS8, IGKVIS9, IGKV1S10, IGKVIS11, IGKVIS12, IGKVIS13, IGKVIS14, IGKVIS15, IGKVIS16, IGKVIS17, IGKVIS18, IGKVIS19, IGKV1S20, IGKVIS21, IGKV1S22, IGKVIS23, IGKV1S24, IGKV1S25, IGKV1S26, IGKV1S27, IGKV1S28, IGKV1S29, IGKVIS30, IGKV1S31, IGKV1S32, IGKVIS33, IGKVIS34, IGKVIS35, IGKV1S36, IGKVIS37, IGKV1S38, IGKV1S39, IGKVIS40, IGKV1S41, IGKVIS42, IGKV1S43, IGKVIS44, IGKVIS45, IGKV1S46, IGKV1S47, IGKV1S48, IGKVIS49, IGKVIS50, IGKVIS51, IGKVIS52, IGKVIS53, IGKVIS54, IGKVIS55, IGKVIS56, IGKVIS57, IGKVIS58, IGKV1S59, IGKV1S60, IGKVIS61, IGKVIS62, IGKVIS63, IGKVIS64, IGKVIS65, IGKV1S66, IGKVIS67, or IGKVIS68.
(75) In certain embodiments, the library comprises a plurality of polynucleotide sequences encoding matured rabbit kappa VL sequences derived from the parental sequence IGKV1S1, IGKV1S2, IGKVIS3, IGKV1S4, IGKVIS5, IGKV1S6, IGKVIS7, IGKVIS8, IGKVIS9, IGKVIS10, IGKVIS11, IGKVIS12, IGKVIS13, IGKVIS14, IGKV1S15, IGKV1S16, IGKVIS17, IGKV1S18, IGKV1S19, IGKV1S20, IGKV1S21, IGKV1S22, IGKVIS23, IGKV1S24, IGKV1S25, IGKV1S26, IGKV1S27, IGKV1S28, IGKV1S29, IGKVIS30, IGKV1S31, IGKV1S32, IGKVIS33, IGKVIS34, IGKV1S35, IGKVIS36, IGKV1S37, IGKV1S38, IGKV1S39, IGKVIS40, IGKV1S41, IGKVIS42, IGKVIS43, IGKVIS44, IGKV1S45, IGKVIS46, IGKVIS47, IGKVIS48, IGKVIS49, IGKVIS50, IGKVIS51, IGKV1S52, IGKVIS53, IGKVIS54, IGKVIS55, IGKVIS56, IGKV1S57, IGKVIS58, IGKV1S59, IGKV1S60, IGKVIS61, IGKVIS62, IGKVIS63, IGKVIS64, IGKV1S65, IGKVIS66, IGKV1S67, IGKVIS68, or a combination thereof.
(76) In certain embodiments, the library comprises a plurality of polynucleotide sequences encoding matured rabbit kappa VL sequences derived from the parental sequence IGKV1S1, IGKVIS2, IGKV1S3, IGKV1S4, IGKVIS5, IGKVIS6, IGKVIS7, IGKV1S8, IGKVIS9, IGKV1S10, IGKV1S11, IGKVIS12, IGKV1S13, IGKVIS14, IGKV1S15, IGKVIS16, IGKV1S17, IGKVIS18, IGKVIS19, IGKV1S20, IGKV1S21, IGKVIS22, IGKVIS23, IGKV1S24, IGKV1S25, IGKV1S26, IGKV1S27, IGKV1S28, IGKV1S29, IGKVIS30, IGKV1S31, IGKV1S32, IGKVIS33, IGKV1S34, IGKVIS35, IGKVIS36, IGKVIS37, IGKVIS38, IGKVIS39, IGKVIS40, IGKVIS41, IGKVIS42, IGKVIS43, IGKVIS44, IGKV1S45, IGKV1S46, IGKVIS47, IGKV1S48, IGKVIS49, IGKV1S50, IGKVIS51, IGKVIS52, IGKVIS53, IGKVIS54, IGKV1S55, IGKVIS56, IGKV1S57, IGKVIS58, IGKVIS59, IGKVIS60, IGKVIS61, IGKVIS62, IGKVIS63, IGKV1S64, IGKV1S65, IGKV1S66, IGKV1S67, and IGKVIS68.
(77) In certain embodiments, the library comprises polynucleotide sequences encoding about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of rabbit kappa VL genes derived from the parental IGKV1S1 to IGKV1S68, as recited in
(78) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit VH CDRH3 amino acids with a length distribution of 5 amino acids to 25 amino acids.
(79) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit VH CDRH3 amino acids with an average length of about 8 amino acids to about 19 amino acids (e.g., an average length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids).
(80) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit kappa VL CDRL3 amino acids with a length distribution of 3 amino acids to 20 amino acids. In certain embodiments, the library comprises polynucleotide sequences encoding rabbit kappa VL CDRL3 amino acids with a length distribution of 5 amino acids to 16 amino acids. In certain embodiments, the library comprises polynucleotide sequences encoding rabbit kappa VL CDRL3 amino acids with a length distribution of 5 amino acids to 17 amino acids.
(81) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit kappa VL CDRL3 amino acids with an average length of about 8 amino acids to about 14 amino acids (e.g., an average length of 8, 9, 10, 11, 12, 13, or 14 amino acids).
(82) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit lambda VL CDRL3 amino acids with a length distribution of 5 amino acids to 16 amino acids.
(83) In certain embodiments, the library comprises polynucleotide sequences encoding rabbit lambda VL CDRL3 amino acids with an average length of about 8 amino acids to about 14 amino acids (e.g., an average length of 8, 9, 10, 11, 12, 13, or 14 amino acids).
(84) In certain embodiments, the nucleic acid library is selected from the group consisting of a ribosome display library, a phage display library, a yeast cell display library, a mammalian cell display library, and a DNA display library.
(85) In certain embodiments, the nucleic acid library is a phage display library.
(86) In one aspect, the disclosure provides a host cell population comprising the nucleic acid library described above. In certain embodiments, the host cell population is an E. coli cell population. In certain embodiments, the nucleic acid library is a phage display library introduced into an E. coli cell population.
(87) The library provided herein may be a nucleic acid library comprising a plurality of polynucleotide sequences, each polynucleotide sequence in the plurality encoding for a rabbit antigen binding protein, wherein at least a portion of the plurality of polynucleotide sequences encode for a rabbit antigen binding protein that specifically recognizes a target peptide-MHC (pMHC).
(88) The library provided herein may be a phage-displayed rabbit antigen binding protein library comprising a plurality of phage-displayed rabbit antigen binding proteins, each antigen binding protein comprising a variable heavy chain (VH) and variable light chain (VL), wherein at least a portion of the plurality of phage-displayed rabbit antigen binding proteins specifically recognize a target peptide-MHC (pMHC).
(89) As outlined above, in certain embodiments, the VL is a kappa VL. In certain embodiments, the kappa VL further comprises an amino acid substitution at position C80, according to Kabat numbering.
(90) Also provided is an antibody capable of binding to an antigen of interest, wherein at least one heavy chain or light chain CDR is derived from an antibody identified from a nucleic acid library comprising a plurality of polynucleotide sequences, such as the libraries described above, wherein each polynucleotide sequence in the plurality encodes for a rabbit antigen binding protein comprising a kappa variable light chain (VL), wherein the kappa VL comprises an amino acid substitution at position C80 (e.g., a C80A amino acid substitution), according to Kabat numbering, and wherein at least a portion of the plurality of polynucleotide sequences encode for a rabbit antigen binding protein that specifically recognizes a target antigen (e.g., a target pMHC).
(91) Methods of Producing Nucleic Acid Libraries
(92) In another aspect, the disclosure provides a method of producing a nucleic acid library encoding for a plurality of antigen binding proteins that specifically recognize a target antigen, the method comprising the steps of: (i) immunizing a rabbit with the target antigen; (ii) isolating antigen binding protein encoding polynucleotide sequences from a B cell population from the rabbit, wherein the antigen binding protein encoding polynucleotide sequences encode for at least kappa VL; (iii) cloning the polynucleotide sequences into a nucleic acid library; and (iv) mutagenizing the nucleic acid library to introduce an amino acid substitution at position C80 of the kappa VL, according to Kabat numbering, thereby producing a nucleic acid library encoding for a plurality of antigen binding proteins that specifically recognize a target antigen.
(93) In certain embodiments, the B cell population comprises a peripheral blood mononuclear cell (PBMC) population, a B cell population from spleen, a B cell population from lymph nodes, or a combination thereof.
(94) In some embodiments, the method involves isolating all lambda and kappa VL and VH from the B cell population.
(95) In certain embodiments, scFv libraries comprising lambda VL-VH and kappa VL-VH are constructed.
(96) The amino acid residue replacing C80 can be any amino acid residue. In certain embodiments, the method comprises introducing an amino acid substitution in the kappa VL (according to Kabat numbering) selected from the group consisting of C80A, C80S, C80P or a corresponding germline amino acid. A corresponding germline amino acid is an amino acid found at Kabat position 80 of a germline kappa VL sequence, e.g., as encoded by the germline genes selected from IGKV1S1 to IGKVIS68, as recited in
(97) Amino acid substitutions can be introduced with a polymerase chain reaction (PCR). Advantageously, multiple PCRs with different pairs of primers are performed on the sample comprising rabbit derived antigen binding protein encoding polynucleotide sequences. Each pair of primers is preferably specific for an allelic family. Performing multiple PCRs with different pairs of primers will thus allow to capture a bigger sequence diversity of antigen binding protein encoding polynucleotide sequences generated in the immunization procedure. The primers may e.g., be based on the rabbit germline sequences of the IMGT database. The more different PCRs will be performed, the higher the potential to grasp polynucleotide sequences encoding for highly specific antigen binding proteins with binding affinity. For example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 68 PCRs can be performed, each with a different pair of primers.
(98) In certain embodiments, the rabbit derived antigen binding protein encoding polynucleotide sequences are present in a circular DNA construct such as a phagemid or a plasmid. Such circular DNA constructs then serve as template for the PCR amplification.
(99) In certain embodiments, each pair of primers comprises a first primer introducing the C80 amino acid substitution and a second primer annealing closely to the first primer, optionally back-to-back. Accordingly, when the polynucleotide sequences are present in a closed circular DNA construct, a linear strand of DNA is produced by the PCR reaction. In some embodiments, the first primer is a reverse primer and the second primer is a forward primer. In other embodiments, the first primer is a forward primer and the second primer is a reverse primer.
(100) The primers may be designed such that they anneal back-to-back without overlap or gap. Upon re-circularization, the generated DNA construct will thus correspond to the template but having the C80 substitution. In some embodiments, the first primer (e.g., the forward primer) incorporates the C80 substitution in the center of the first primer, including at least 10, 11, 12, 13, 14 or 15 complementary nucleotides on the 3 side of the mutation, whereas the second primer (e.g., the reversed primer) is designed such that the 5 ends of the two primers anneal back-to-back.
(101) The linear strand of DNA may be circularized after the amplification step, either in a single-step DNA assembly and circulation reaction or in separate steps which may involve one or more of dephosphorylation, end repair and polishing, and ligation. Conveniently, the KLD Enzyme Mix (New England Biolabs) which comprises a kinase, a ligase and DpnI, may be used to perform these steps in a single reaction.
(102) In certain embodiments, the PCR is performed with one or more primer pairs selected from F1/R1, F1/R2, F1/R3, F1/R4, F1/R5, F1/R6, F1/R7, F1/R8, F1/R9, F1/R10, F2/R1, F2/R2, F2/R3, F2/R4, F2/R5, F2/R6, F2/R7, F2/R8, F2/R9, and F2/R10, as recited in Table 3.
(103) The antigen binding protein encoding polynucleotide sequences may further encode for one or both of lambda VL and VH. The kappa VL or lambda VL may be operatively linked to a VH with an amino acid linker. The kappa VL and lambda VL may be operatively linked to a CL domain and the VH operatively linked to a CH1 domain.
(104) Libraries of the invention have been described in detail above. Thus, in certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences.
(105) In certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences encoding rabbit kappa VL.
(106) In certain embodiments, the library comprises a diversity of at least about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 10.sup.14, or 10.sup.15 unique polynucleotide sequences encoding rabbit lambda VL.
(107) In certain embodiments, the library comprises polynucleotide sequences derived from one or more of parental rabbit kappa VL genes IGKV1S1 to IGKV1S68, as recited in
(108) In certain embodiments, the library comprises polynucleotide sequences encoding about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or 100% of parental rabbit kappa VL protein sequences IGKVIS1 to IGKVIS68, as recited in
(109) The library may comprise polynucleotide sequences encoding rabbit VH CDRH3 amino acids with a length distribution of 5 amino acids to 25 amino acids, such as having an average length of about 8 amino acids to about 19 amino acids.
(110) Alternatively or additionally, the library may comprise polynucleotide sequences encoding rabbit kappa VL CDRL3 amino acids with a length distribution of 3 amino acids to 20 amino acids, e.g., 5 amino acids to 16 amino acids or 5 amino acids to 16 amino acids, such as having an average length of about 8 amino acids to about 14 amino acids. In other embodiments, the library comprises polynucleotide sequences encoding rabbit lambda VL CDRL3 amino acids with a length distribution of 5 amino acids to 16 amino acids, such as having an average length of about 8 amino acids to about 14 amino acids.
(111) As described above, the nucleic acid library may be selected from the group consisting of a ribosome display library, a phage display library, a yeast cell display library, a mammalian cell display library, and a DNA display library, preferably a phage display library. In certain embodiments, the nucleic acid library is a phage display library introduced into an E. coli cell population.
(112) As outlined above, the target antigen may comprise a pMHC. Thus, in specific embodiments, the disclosure provides a method of producing a nucleic acid library encoding for a plurality of antigen binding proteins that specifically recognize a target peptide-MHC (pMHC), the method comprising the steps of: (i) immunizing a rabbit with the target pMHC; (ii) isolating antigen binding protein encoding polynucleotide sequences from a B cell population from the rabbit; and (iii) cloning the polynucleotide sequences into a nucleic acid library, thereby producing a nucleic acid library encoding for a plurality of antigen binding proteins that specifically recognize a target pMHC.
Methods of Producing an Antigen Binding Protein
(113) In another aspect, the disclosure provides a method of producing an antigen binding protein that specifically recognizes a target antigen which involves the creation of a library of the disclosure, the method comprising the steps of: (i) immunizing a rabbit with the target antigen; (ii) isolating a plurality of antigen binding protein encoding polynucleotide sequences from the rabbit, wherein the antigen binding protein encoding polynucleotide sequences encode for at least kappa VL; (iii) cloning the polynucleotide sequences into a nucleic acid library; (iv) mutagenizing the nucleic acid library to introduce an amino acid substitution at position C80 of the kappa VL, according to Kabat numbering; and (v) selecting the antigen binding protein that specifically recognizes a target antigen.
(114) The method may further comprise step (vi) comprising sequencing the polynucleotide sequence that encodes the antigen binding protein that specifically recognizes the target antigen.
(115) In certain embodiments, the method further comprises step (vii) comprising humanizing the antigen binding protein that specifically recognizes the target antigen. Humanizing may comprise grafting the CDR sequences of the antigen binding protein into an acceptable acceptor framework (e.g., a human antibody framework suitable for grafting rabbit antibody-derived CDR sequences).
(116) The method may comprise introducing an amino acid substitution selected from the group consisting of C80A, C80S, C80P and a corresponding germline amino acid in the kappa VL, according to Kabat numbering.
(117) The amino acid substitution may be introduced with a polymerase chain reaction (PCR). As outlined for the methods above, multiple PCRs are advantageously performed with different pairs of primers. Primer design and PCR amplifications are outlined in detail above for the methods of producing nucleic acid libraries and do also apply here. Thus, in some embodiments, the PCR is performed with one or more primer pairs selected from F1/R1, F1/R2, F1/R3, F1/R4, F1/R5, F1/R6, F1/R7, F1/R8, F1/R9, F1/R10, F2/R1, F2/R2, F2/R3, F2/R4, F2/R5, F2/R6, F2/R7, F2/R8, F2/R9, and F2/R10, as recited in Table 3.
(118) The antigen binding protein encoding polynucleotide sequences may be isolated from a B cell population, such as a peripheral blood mononuclear cell (PBMC) population, a B cell population from spleen, a B cell population from lymph nodes, or a combination thereof.
(119) The antigen binding protein encoding polynucleotide sequences may further encode for one or both of lambda VL and VH. The kappa VL or lambda VL may be operatively linked to the VH with an amino acid linker. The kappa VL and lambda VL may be operatively linked to a CL domain and the VH operatively linked to a CH1 domain.
(120) In certain embodiments, the selecting step (v) is performed through biopanning against the target antigen.
(121) Biopanning is a process which typically involves the step of preparing a display library, such as a phage display library. With respect to phage display, this involves providing a bacteriophage genome and inserting segments of a gene of interest therein. Upon protein expression, the resulting peptide product will be displayed on the surface of the bacteriophage virion. In the following panning step, the phage library is exposed to the desired target which may e.g., be bound to a solid surface and allowing the expressed peptide and the target to interact. Unbound phages are washed away; only the phages displaying the properly folded binding entity with good affinity are kept. Finally, in the elution step, the bound phages are eluted through changing of pH or other environment conditions.
(122) In one aspect, the disclosure provides an antigen binding protein that specifically recognizes a target antigen (e.g., a target pMHC), prepared by the methods described above. Such antigen binding protein may specifically recognize the target pMHC at a ratio of at least about 10:1 target pMHC:control (e.g., at least about 20:1, at least about 50:1, at least about 100:1, at least about 500:1, at least about 1000:1, or at least 10000:1). In some embodiments, the target peptide complex comprises the peptide of SEQ ID NO: 3. Said peptide may be presented by an HLA-A2 complex, as e.g., comprising SEQ ID NO: 1 and SEQ ID NO: 3. The control can be a non-target peptide-MHC complex, such as HLA-A2/Topoisomerase II a-b. Preferably, the antigen binding protein does not bind to the control. In some embodiments, the control is a peptide-MHC complex having the same HLA subtype as the peptide-MHC complex of interest. In some embodiments, the control peptide-MHC complex comprises a multitude of unrelated peptides.
(123) Thus, in specific embodiments, the disclosure provides a method of producing an antigen binding protein that specifically recognizes a target peptide-MHC (pMHC), the method comprising the steps of: (i) immunizing a rabbit with the target pMHC; (ii) isolating a plurality of antigen binding protein encoding polynucleotide sequences from the rabbit; (iii) cloning the plurality of polynucleotide sequences into a nucleic acid library; and (iv) selecting the antigen binding protein that specifically recognizes a target pMHC.
PCR Primers
(124) Provided herein are primers specific for rabbit kappa VL sequences, such as of kappa 1 (k1) isotype. As detailed in various passages above, the primers introduce an amino acid substitution in the rabbit kappa VL sequences, more specifically, substituting the cysteine at Kabat position 80. Said substitution may be selected from the group consisting of alanine (C80A), serine (C80S), proline (C80P), and a corresponding germline amino acid, such as the residue at the corresponding rabbit or human germline position. The primers can be used in any of the methods described herein.
(125) The primers provided herein may have a length of at least 11 nucleotides, such as about 11 to about 30 nucleotides (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
(126) In certain embodiments, the primer is selected from the group consisting of F1, F2, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10, as recited in Table 3. A primer may comprise a sequence as recited in any one of F1, F2, R1, R2, R3, R4, R5, R6, R7, R8, R9 or R10, as recited in Table 3.
(127) Primers F1 and F2 introduce a C80A substitution; in some embodiments, a primer comprises a sequence derived from F1 or F2, as recited in Table 3 with a nucleotide modification introducing a C80S, C80P or a germline residue at C80 (according to Kabat) in the kappa VL. Such primer may incorporate the C80 substitution in the center of its sequence and may include at least 10, 11, 12, 13, 14 or 15 complementary nucleotides on the 3 side of the mutation.
(128) A pair of primers typically includes a forward primer and a reverse primer. The pair may be designed such that both are specific for one allelic variant of rabbit kappa VL germline sequences of the IMGT databases. A first primer of said pair is designed such that it introduces the C80 substitution in the VL kappa sequence. A second primer of said pair may or may not span over a conserved area of such rabbit kappa VL germline sequence. The first or the second primer may be a universal primer which anneals with several or all rabbit kappa VL germline sequences. In some embodiments, the primer pair hybridizes with one or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or more allelic variant of rabbit kappa VL germline sequences of the IMGT databases. Such allelic variants include IGKVIS1 to IGKV1S68, as recited in
(129) In certain embodiments, the first and second primer are designed such that they anneal closely to each other on the template, preferably back-to-back, i.e., the 5 ends of the two primers anneal back-to-back on the template, without overlap or gap. This is advantageous when a circular DNA construct is used as template, so that the linear strand of DNA produced by the PCR reaction can be recirculated without gap or insertion.
(130) In some embodiments, the first primer is a reverse primer and the second primer is a forward primer. In other embodiments, the first primer is a forward primer and the second primer is a reverse primer.
(131) Expression of Antigen Binding Proteins
(132) In one aspect, polynucleotides or nucleic acids encoding the antigen binding proteins disclosed herein are provided. Methods of making an antigen binding protein comprising expressing these polynucleotides are also provided.
(133) Polynucleotides encoding the antigen binding proteins disclosed herein are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of the antigen binding proteins. Accordingly, in certain aspects, the invention provides expression vectors comprising polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.
(134) The term vector or expression vector is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may readily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
(135) Numerous expression vector systems may be employed for the purposes of this invention. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (e.g., RSV, MMTV, MOMLV or the like), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.
(136) In other embodiments, the antigen binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980, which is incorporated by reference herein in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
(137) More generally, once a vector or DNA sequence encoding an antibody, or fragment thereof, has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. Mammalian Expression Vectors Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host can be by electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
(138) As used herein, the term transformation shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
(139) Along those same lines, host cells refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of polypeptides from recombinant hosts, the terms cell and cell culture are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the cells may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
(140) In one embodiment, a host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte), 293 (human kidney) and the like. In one embodiment, the cell line provides for altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6 (Crucell) or FUT8-knock-out CHO cell lines (Potelligent cells) (Biowa, Princeton, N.J.)). Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from published literature.
(141) In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g., in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography.
(142) Genes encoding the antigen binding proteins featured in the invention can also be expressed non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed, i.e., those capable of being grown in cultures or fermentation. Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the proteins can become part of inclusion bodies. The proteins must be isolated, purified and then assembled into functional molecules.
(143) In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)), is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
(144) Target Peptide-MHC
(145) The antigen binding proteins described herein possess binding specificity to a variety of disease-relevant antigens presented on the surface of cells as peptide-MHC. Disease-relevant antigens include, but are not limited to, tumor antigens and viral antigens.
(146) Examples of tumor antigens include melanoma-associated antigen A (MAGE-A), such as MAGE-A1, MAGE-A3 and MAGE-A4, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), synovial sarcoma X (SSX), carcinoembryonic antigen (CEA), preferentially expressed antigen in melanoma (PRAME), melanoma antigen recognized by T cells 1 (MART-1), Kirsten rat sarcoma viral oncogene (K-ras), kinetochore NDC80 protein homolog (NDC80), Kita-Kyushu lung cancer antigen (KK-LC-1), and Wilms tumor 1 (WT1).
(147) Examples of viral antigens include Epstein-Barr virus derived EBNA1, EBNA2, EBNA3, LMP1, or LMP2; hepatitis B virus derived HBX; hepatitis C virus derived NS3 or NS5A; human papillomavirus derived type E5, E6, and E7 proteins; cytomegalovirus derived PP65; human immunodeficiency virus derived gag; and Kaposi sarcoma-associated herpesvirus derived vGPCR or vIRF-1.
(148) The target peptide may be presented on a MHC class I complex (such as of serotype HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L, or their respective subtypes) or an MHC class II complex (such as the serotypes HLA-DP, HLA-DQ, HLA-DR, DM or DO, or their respective subtypes). Each of the serotypes comprise different subtypes. In one embodiment, the antigen binding protein targets a peptide bound to an HLA-A2-MHC complex, also termed HLA-A*02, in particular HLA-A*02:01 comprising the extracellular domain of SEQ ID NO: 1.
(149) Engineering and Optimization of Antigen Binding Proteins
(150) The antigen binding proteins of the disclosure may be engineered or optimized. As used herein, optimized or optimization refers to the alteration of an antigen binding protein to improve one or more functional properties. Alteration includes, but is not limited to, deletions, substitutions, additions, and/or modifications of one or more amino acids within an antigen binding protein.
(151) As used herein, the term functional property is a property of an antigen binding protein for which an improvement (e.g., relative to a conventional antigen binding protein, such as an antibody) is desirable and/or advantageous to one of skill in the art, e.g., in order to improve the manufacturing properties or therapeutic efficacy of an antigen binding protein. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (e.g., under cellular conditions). In yet another embodiment, the functional property is aggregation behavior. In still another embodiment, the functional property is protein expression (e.g., in a prokaryotic cell). In yet another embodiment the functional property is refolding behavior following inclusion body solubilization in a manufacturing process. In certain embodiments, the functional property is not an improvement in antigen binding affinity. In another embodiment, the improvement of one or more functional properties has no substantial effect on the binding affinity of the antigen binding protein.
(152) In certain embodiments, the antigen binding protein of the disclosure is an scFv and is optimized by identifying preferred amino acid residues to be substituted, deleted, and/or added at amino acid positions of interest (e.g., amino acid positions identified by comparing a database of scFv sequences having at least one desirable property, e.g., as selected with Quality Control (QC) assay, versus a database of mature antibody sequences, e.g., the Kabat database) in an antigen binding protein. Thus, the disclosure further provides enrichment/exclusion methods for selecting a particular amino acid residue. Still further, the disclosure provides methods of engineering antigen binding proteins (e.g., scFvs) by mutating particular framework amino acid positions identified using the functional consensus approach described herein. In certain embodiments, the framework amino acid positions are mutated by substituting the existing amino acid residue by a residue which is found to be an enriched residue using the enrichment/exclusion analysis methods described herein. In one aspect, the disclosure provides a method of identifying an amino acid position for mutation in a single chain antibody (scFv), the scFv having VH and VL amino acid sequences, the method comprising: a) entering the scFv VH, VL or VH and VL amino acid sequences into a database that comprises a multiplicity of antibody VH, VL or VH and VL amino acid sequences such that the scFv VH, VL or VH and VL amino acid sequences are aligned with the antibody VH, VL or VH and VL amino acid sequences of the database; b) comparing an amino acid position within the scFv VH or VL amino acid sequence with a corresponding position within the antibody VH or VL amino acid sequences of the database; c) determining whether the amino acid position within the scFv VH or VL amino acid sequence is occupied by an amino acid residue that is conserved at the corresponding position within the antibody VH or VL amino acid sequences of the database; and d) identifying the amino acid position within the scFv VH or VL amino acid sequence as an amino acid position for mutation when the amino acid position is occupied by an amino acid residue that is not conserved at the corresponding position within the antibody VH or VL amino acid sequences of the database. ScFv optimization is described in further detail in WO2008110348, WO2009000099, WO2009000098, and WO2009155725, all of which are incorporated herein by reference.
(153) In certain embodiments, the antigen binding protein comprises an Fc domain which is modified such that it does not induce cytotoxic immune responses and/or does not activate complement. For example, one or more substitutions may be introduced into the Fc domain so that its ADCC/ADCP or CDC effector function is inactivated. Such antigen binding protein has the advantage of increased half-life when compared to antibody fragments with a molecular weight below 60 kDa, without mediating mediate cytotoxic immune responses.
(154) Chemical and/or Biological Modifications
(155) In one aspect, the antigen binding protein is chemically and/or biologically modified. For example, the antigen binding protein may be glycosylated, phosphorylated, hydroxylated, PEGylated, HESylated, PASylated, sulfated, labeled with dyes and/or radioisotopes, conjugated with enzymes and/or toxins, and/or Albumin fusion technology. Likewise, any nucleic acid sequence, plasmid or vector and/or host cell described herein may be modified accordingly.
(156) Such modification may for example be done to optimize pharmacodynamics, its water solubility or to lower its side effects. For example, PEGylation, PASylation, HESylation and/or the fusion to serum albumin may be applied to slow down renal clearance, thereby increasing plasma half-life time of the antigen binding protein. In one embodiment, a modification adds a different functionality to the antigen binding protein, for example, a detection label for diagnostics or a toxin to combat cancer cells even more efficiently.
(157) In one embodiment, the antigen binding protein is glycosylated. Glycosylation refers to a process that attaches carbohydrates to proteins. In biological systems, this process is performed enzymatically within the cell as a form of co-translational and/or post-translational modification. A protein can also be chemically glycosylated. The carbohydrates may be N-linked to a nitrogen of asparagine or arginine side-chains; O-linked to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains; employ xylose, fucose, mannose, and N-acetylglucosamine attached to a phospho-serine; and/or adding mannose sugar to a tryptophan residue found in a specific recognition sequence. Glycosylation patterns may, e.g., be controlled by choosing appropriate cell lines, culturing media, protein engineering manufacturing modes and process strategies (see., HOSSLER, P. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009, vol. 19, no. 9, p. 936-949.). In some embodiments, the glycosylation patterns of the antigen binding proteins described herein are modified to enhance ADCC and CDC effector function.
(158) The antigen binding protein may be engineered to control or alter the glycosylation pattern, e.g., by deleting and/or adding one or more glycosylation sites. The creation of glycosylation sites can e.g., be accomplished by introducing the corresponding enzymatic recognition sequence into the amino acid sequence of the antigen binding protein.
(159) In some embodiments, the antigen binding protein is PEGylated. PEGylation may alter the pharmacodynamic and pharmacokinetic properties of a protein. Additionally, PEGylation may reduce the immunogenicity by shielding the PEGylated antigen binding protein from the immune system and/or alter its pharmacokinetics by, e.g., increasing the in vivo stability of the antigen binding protein, protecting it from proteolytic degradation, extending its half-life time and by altering its biodistribution. Typically, polyethylene-glycol (PEG) of an appropriate molecular weight is covalently attached to the protein. Similar effects may be achieved using PEG mimetics, e.g., HESylating or PASylating the antigen binding protein. HESylation utilizes hydroxyethyl starch (HES) derivatives. During PASylation, the antigen binding protein is linked to conformationally disordered polypeptide sequences composed of the amino acids proline (P), alanine (A) and serine(S).
(160) In certain embodiments, the antigen binding protein is labelled with or conjugated to a second moiety which attributes one or more ancillary functions to the antigen binding protein. For example, the second moiety may have an additional immunological effector function, be effective in drug targeting or useful for detection. The second moiety can, e.g., be chemically linked or fused genetically to the antigen binding protein using known methods in the art. As used herein, the term label refers to any substance or ion which is indicative of the presence of the antigen binding protein when detected or measured by physical or chemical means, either directly or indirectly. For example, the label may be directly detectable by, without being limited to, light absorbance, fluorescence, reflectivity, light scatter, phosphorescence, or luminescence properties, molecules or ions detectable by their radioactive properties or molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Examples of indirect detection include light absorbance or fluorescence; for example, various enzymes which cause appropriate substrates to convert, e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules. A labelled antigen binding protein is particularly useful for in vitro and in vivo detection or diagnostic purposes. For example, an antigen binding protein labelled with a suitable radioisotope, enzyme, fluorophore or chromophore can be detected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), or flow cytometry-based single cell analysis (e.g., FACS analysis), respectively. Similarly, the nucleic acids and/or vectors disclosed herein can be labeled for detection or diagnostic purposes, e.g., using labelled fragments thereof as probes in hybridization assays.
(161) Non-limiting examples of second moieties include radioisotopes (35S, 32P, 14C, 18F, and/or 125I), apoenzymes, enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase and/or angiogenin), co-factors, peptide moieties (e.g., a HIS-tag), proteins (e.g. lectin, serum albumin), carbohydrates (e.g., mannose-6-phosphate tags), fluorophores (e.g., fluorescein isothiocyanate (FITC)), phycoerythrin, green/blue/red or other fluorescent proteins, allophycocyanin (APC), chromophores, vitamins (e.g., biotin), chelators, antimetabolites (e.g., methotrexate), toxins (e.g. a cytotoxic drug, or a radiotoxin).
(162) In one aspect, the invention relates to drug conjugates (in particular antibody-drug conjugates ADCs) comprising the antigen binding proteins described herein conjugated to a toxin which further enhances efficient killing of specific cells, such as e.g., MAGE-A4 positive cells. The toxin moiety is typically a small molecular weight moiety, such as anthracycline toxins, taxol, gramicidin D and/or colchicine which may be linked via a peptide linker to the antigen binding protein.
(163) The toxin may be conjugated non-site-specifically or site-specifically to the antigen binding protein. Non-site-specific conjugation typically involves the use of chemical linkers, e.g., with maleimide functionality, that mediate conjugation to lysine or cysteine amino acid side chains of the antibody. Site-specific conjugation may be achieved using chemical, chemo-enzymatic, or enzymatic conjugations known in the art, e.g., employing bifunctional linkers, bacterial transglutaminase or sortase enzymes, linkers allowing Pictet-Spengler chemistry on formyl-glycine forming enzyme modified antigen binding proteins, or glycan-remodeled antigen binding proteins.
(164) Chimeric Antigen Receptors
(165) In one aspect, the disclosure provides chimeric antigen receptors (CARs) and immune cells engineered to express such CARs, comprising the antigen binding proteins described herein. As used herein, the term chimeric antigen receptor or CAR refers to a receptor that is capable of activating an immune cell in response to antigen binding. CARs are recombinant membrane spanning molecules and are advantageously expressed on immune cells. Their structure typically comprises (i) an extracellular domain (ectodomain or antibody domain), (ii) a transmembrane domain and (iii) a cytoplasmic domain (endodomain or intracellular signaling domain).
(166) The ectodomain (i.e., antibody domain) typically comprises a scFv but other antigen binding proteins may also be used. A spacer connects the ectodomain and the transmembrane domain, which in turn is connected to an endodomain. Upon binding of the ectodomain to the antigen, the receptors cluster and an activation signal is transmitted to the cell which results in initiation of an immune response. First generation CARs have a simply structured endodomain comprising CD3-zeta. To increase the activation signal, a co-stimulatory domain was added in the second-generation CARs; and third generation CARs include two or more co-stimulatory domains (Maus M V et al (2014) Blood, 123:2625-2635). Said co-stimulatory domains may be selected from the group consisting of CD28, OX40 and/or 4-1BB. Apart from CD3-zeta, other ITAM-containing domains have been explored including the Fc receptor for IgE- domain.
(167) Suitable immune cells include, without being limited to, T cells, Natural Killer T (NKT) cells, natural killer (NK) cells, human embryonic stem cells, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS). Such T cell may be a cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an inflammatory T-lymphocytes, or a helper T-lymphocyte or a gamma-delta T cell. The T cell may be a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.
(168) In one aspect, the disclosure provides a chimeric antigen receptor (CAR) that specifically recognizes a target antigen (e.g., a target peptide-MHC), comprising: i) an antigen binding protein identified from the nucleic acid libraries or methods described herein; ii) a transmembrane domain; and iii) an intracellular signaling domain.
(169) In certain embodiments, the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domains of a type I transmembrane protein, an alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
(170) In certain embodiments, the intracellular signaling domain is selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcRIII, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
(171) The antibody domain may be any of the antigen binding proteins outlined above. Thus, in certain embodiments, the antibody domain comprises an antibody variable light domain (VL) comprising an amino acid sequence represented by the formula LFR1-CDRL1-LFR2-CDRL2-LFR3-CDRL3-LFR4. In certain embodiments, the antibody domain comprises an antibody variable heavy domain (VH) comprising an amino acid sequence represented by the formula HFR1-CDRH1-HFR2-CDRH2-HFR3-CDRH3-HFR4. In certain embodiments, the antibody domain comprises an scFv as described herein.
(172) Methods of Administering Antigen Binding Proteins
(173) Methods of preparing and administering antigen binding proteins of the disclosure as well as the nucleic acids described herein, the vectors described herein, the host cell cells described herein (in particular the immune cells bearing a CAR) or the compositions described herein to a subject are well known to or are readily determined by those skilled in the art. The route of administration of the antigen binding proteins of the current disclosure may e.g., be oral, parenteral, by inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The term intraocular as used herein includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or intracameral. The term topical as used herein includes, but is not limited to, administration with liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.
(174) While all these forms of administration are clearly contemplated as being within the scope of the current disclosure, a form for administration would be a solution for injection. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc. However, in other methods compatible with the teachings herein, the modified antibodies can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
(175) Effective doses of the compositions of the present disclosure, for the treatment of the related conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
(176) As previously discussed, the antigen binding proteins of the present disclosure, conjugates or recombinants thereof may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed antigen binding proteins will be formulated to facilitate administration and promote stability of the active agent.
(177) Pharmaceutical compositions in accordance with the present disclosure typically include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, nontoxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of the antigen binding proteins shall be held to mean an amount sufficient to achieve effective binding to an antigen and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the antigen binding proteins will typically be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the modified binding polypeptide.
(178) In keeping with the scope of the present disclosure, the antigen binding proteins of the disclosure may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The antigen binding proteins of the disclosure can be administered to such human or other animal in a conventional dosage form prepared by combining the antigen binding proteins of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of antigen binding proteins described in the current disclosure may prove to be particularly effective. Similarly, the nucleic acids described herein, the vectors described herein, the host cell cells described herein (in particular the immune cells bearing a CAR) or the compositions described herein may be administered to a human or other animal in accordance with the methods of treatment described above in an amount sufficient to produce a therapeutic or prophylactic effect.
(179) Efficacy or in vivo efficacy as used herein refers to the response to a therapy by the pharmaceutical composition of the disclosure, using e.g., standardized response criteria, such as standard ophthalmological response criteria. The success or in vivo efficacy of the therapy using a pharmaceutical composition of the disclosure refers to the effectiveness of the composition for its intended purpose, i.e., the ability of the composition to cause its desired effect. The in vivo efficacy may be monitored by established standard methods for the specific diseases. In addition, various disease specific clinical chemistry parameters and other established standard methods may be used.
(180) In some embodiments, the compounds and cells described herein are administered in combination with one or more different pharmaceutical compounds.
(181) Generally, therapeutic use of the compounds and cells described herein may be in combination with one or more therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, radiation therapy or vaccine therapy.
(182) Methods of Treating Peptide-MHC Mediated Diseases and Disorders
(183) In one aspect, the aforementioned antigen binding proteins, nucleic acids, vectors or host cells (in particular immune cells expressing CARs) or the vector, are useful as a medicament. Typically, such a medicament includes a therapeutically effective amount of a molecule or cell as provided herein. Accordingly, a respective molecule or host cell can be used for the production of a medicament useful in the treatment of one or more disorders, in particular target antigen expressing cancer (e.g., target pMHC expressing cancer).
(184) In one aspect, a method of treating a pMHC related or mediated disorder is provided. The method includes the steps of administering a pharmaceutically effective amount of a molecule or host cell as described herein, in particular the antigen binding proteins or host cell, to a subject in need thereof. In one embodiment, the pharmaceutical composition described above, which includes such pharmaceutically effective amount of the antigen binding protein, nucleic acid, vector or host cell is administered to the subject. The medicament referred to above may be administered to a subject.
(185) The subject in need of a treatment can be a human or a non-human animal. Typically, the subject is a mammal, e.g., a mouse, a rat, rabbit, a hamster, a dog, a cat, a monkey, an ape, a goat, a sheep, a horse, a chicken, a guinea pig or a pig. In typical embodiments, the subject is diagnosed with a pMHC related disorder or may acquire such a disorder. In case of an animal model, the animal might be genetically engineered to develop a pMHC related disorder. In an animal model, an animal may also be genetically engineered in such a way that it shows the characteristics of pMHC related disease.
(186) Use in Diagnostics and Detection Assays
(187) An antigen binding protein as disclosed herein may be used for detection or diagnostic purposes in vivo and/or in vitro. For example, a wide range of immunoassays using antibodies for detecting the expression in specific cells or tissues are known to the skilled person. For such purposes, it may be advantageous to use a antigen binding protein connected to a detectable label, such a biotin.
(188) In one embodiment, the described antigen binding proteins are useful for detecting the presence of a target peptide-MHC complex, in particular MAGE-A4, in a sample. The detection may be for quantitative or qualitative purposes. The sample is preferably of biological origin, such as blood, urine, cerebrospinal fluid, biopsy, lymph and/or non-blood tissues. In certain embodiments, a biological sample comprises a cell or tissue from a human patient. In certain embodiments, the method includes contacting a biological sample with an antigen binding protein under conditions permissive for binding of the inhibitor to the target peptide-MHC and then detecting the inhibitor-target complex. Such method may be an in vitro or in vivo method. In some embodiments, such method is performed to select subjects eligible for therapy with the antigen binding protein described herein.
(189) Kits
(190) Also contemplated are kits comprising at least one nucleic acid library or antigen binding protein or one or more pairs of primers as described herein, typically together with a packaged combination of reagents with instructions. In one embodiment, the kit includes a composition containing an effective amount of said antigen binding protein in unit dosage form. In one embodiment, the kit includes a composition comprising a one or more said pairs of primers, such as F1/R1, F1/R2, F1/R3, F1/R4, F1/R5, F1/R6, F1/R7, F1/R8, F1/R9, F1/R10, F2/R1, F2/R2, F2/R3, F2/R4, F2/R5, F2/R6, F2/R7, F2/R8, F2/R9, and F2/R10 (as recited in Table 3), or an oligonucleotide sequence derived thereof. Such derived sequence may comprise (i) additional nucleotides, such as 1, 2, 3, 4, 5 or more, such as 7, 8, 9, 10 or more nucleotides on the 5 and/or the 3 end of the sequence or (ii) a deletion of one or more nucleotides at the 5 and/or the 3 end of the sequence, such as 1, 2, 3, 4, 5 or more deletions. Preferably, the derived sequence does not change the amino acid sequence, but may or may not anneal at a different position than the parental sequence.
(191) Typically, a pair of primers comprises a forward primer and a reverse primer. In some embodiments, the kit additionally comprises one or more primers specific for a rabbit kappa and/or lambda VL sequence.
(192) Such kit may comprise a sterile container comprising the composition; non-limiting examples of such containers include, without being limited to, vials, ampoules, bottles, tubes, syringes, blister-packs. In some embodiments, the composition is a pharmaceutical composition and the containers is made of a material suitable for holding medicaments. In one embodiment, the kit may comprise in a first container the antigen binding protein in lyophilized form and a second container with a diluent (e.g., sterile water) for reconstitution or dilution of the antigen binding protein. In some embodiments, said diluent is a pharmaceutically acceptable diluent. In one embodiment, the kit is for diagnostic purposes and the antigen binding protein is formulated for diagnostic applications. In one embodiment, the kit is for therapeutic purposes and the antigen binding protein is formulated for therapeutic applications.
(193) In one embodiment, the kit comprises one or more containers, each container comprising a primer as disclosed herein. The primer may be provided freeze-dried state. In such case, the kit may comprise at least one container comprising a buffer suitable for resuspension. The kit may further comprise a container for a master mix for performing a PCR amplification, optionally freeze-dried.
(194) Typically, the kit will further comprise a separate sheet, pamphlet or card supplied in or with the container with instructions for use. If the kit is intended for pharmaceutical use, it may further comprise one or more of the following: information for administering the composition to a subject having a related disease or disorder (e.g., a MAGE-A4-mediated disease or disorder) and a dosage schedule, description of the therapeutic agent, precautions, warnings, indications, counter-indications, overdosage information and/or adverse reactions.
(195) It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.
EXAMPLES
Example 1Production of MHC Complexes as an Antigen for Immunization
(196) MHC class I heavy chain and 2m were cloned into a pET-24D(+) vector using standard molecular biology techniques (J Biol Chem. 1995 Jan. 13; 270(2): 971-7). E. coli BL-21 (DE3) were transformed with the expression vectors according to the supplier's protocols. Protein expression was performed for 16-18 hours at 37 C. with 220 rpm shaking in MagicMedium (Invitrogen), as described by the supplier. Cells were harvested and lysed with BugBuster (Invitrogen) and the inclusion bodies were washed twice with TBS supplemented with 0.5% LDAO and twice with TBS. Such prepared inclusion bodies were solubilized in a denaturing buffer (8 M urea, 100 mM Tris-HCl pH 8) using 5 mL buffer per 1 g inclusion body pellet. Refolding and purification of the MHC with the target peptides (HLA-A*02:01 extracellular domain, human 2M, and MAGE-A4 peptide 230-239) was performed essentially as described by Rodenko et al. (2006). The amino acid sequences for each component of the pMHC antigen are recited below in Table 2.
(197) TABLE-US-00002 TABLE2 AminoAcidSequencesOfpMHCAntigenComponents SequenceID Sequence HLA-A*02:01 GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFV extracellular RFDSDAASQRMEPRAPWIEQEGPEYVVDGETRKV domain KAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMY (SEQIDNO:1) GCDVGSDWRFLRGYHQYAYDGKDYIALKEDLR SWTAADMAAQTTKHKWEAAHVAEQLRAYLEG TCVEWLRRYLENGKETLQRTDAPKTHMTHHAV SDHEATLRCWALSFYPAEITLTWQRDGEDQTQD TELVETRPAGDGTFQKWAAVVVPSGQEQRYTCH VQHEGLPKPLTLRWE human2m IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDI (SEQIDNO:2) EVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTE FTPTEKDEYACRVNHVTLSQPKIVKWDRDM MAGE-A4.sub.230-239 GVYDGREHTV (SEQIDNO:3)
Example 2Rabbit Immunization
(198) To generate numerous antibodies able to specifically recognize the target peptides in the context of the HLA complex, 3 New Zealand white rabbits were immunized with the recombinantly produced MHC complex. Each animal received at different timepoints 4 injections of the pMHC complex with complete or incomplete Freund's adjuvant. The immune response of the animals was tested in ELISA to quantify anti-pMHC antibodies present in serum samples of the immunized animals. Antibody titers in sera indicated excellent immune responses.
Example 3Construction of Immune Libraries Derived from Rabbits
(199) scFv antibody cDNA libraries were constructed from the RNA extracted from isolated PBMCs and spleen lymphocytes from rabbits via PCR amplification. Coding sequences for the variable light- and heavy-domain were amplified separately and linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products. The amplified DNA sequences coding for the scFvs from rabbits were digested using appropriate restriction enzymes and were subsequently ligated into the phagemid vectors. The phagemid vectors were transformed into E. coli TG1 electrocompetent cells which are well suited for antibody phage display library creation. These processes resulted in two antibody libraries comprising a diversity of 5.210.sup.8 with a sequence accuracy of 87.5% for the kappa-based library and 2.010.sup.9 with an accuracy of 91.7% for the lambda-based library.
Example 4Alignment of the Kappa Light Chain Alleles
(200) 68 rabbit kappa light chain alleles are listed in the IMGT database. The DNA sequences of all 68 alleles were exported and aligned. Only 4 out of the 68 alleles do not have a cysteine at position 80 (according to Kabat numbering), which underlines the importance of optimizing scFv immune libraries comprising the rabbit kappa light chain repertoire. The nucleotide sequence in this cysteine flanking region shows a high sequence conservation. This allows the design of a primer set which covers the complete nave rabbit kappa light chain repertoire. The alignment of the sequences is shown in
Example 5Design of Primers
(201) Primers were designed to mutate the cysteine at position 80 in rabbit kappa light chains into an alanine. Two forward primers were designed comprising the nucleotide substitution C80A. In addition, 10 reverse primers are required to cover the full kappa light chain repertoire. See Table 3 below. Primer design was done according to Q5 site directed protocol of New England Biolabs.
(202) TABLE-US-00003 TABLE3 Primersetsusedtoremovethecysteine80, comprising2forwardprimersand10reverse primers.Thissetofprimersismeantto coverthefullnaverabbitVrepertoire. Primer Sequence(5to3) Tm forward_1(F1) GCTGACGATGCTGCCAC 62C. SEQIDNO:4 forward_2(F2) GCTGCCGATGCTGCC 63C. SEQIDNO:5 reverse_1(R1) CTCCACGCCACTGATG 63C. SEQIDNO:6 reverse_2(R2) CTGTACGCCACTGATGG 63C. SEQIDNO:7 reverse_3(R3) CTGCACACCGCTGATG 64C. SEQIDNO:8 reverse_4(R4) CTGCACGCCGCTG 65C. SEQIDNO:9 reverse_S(R5) CTGCACGCCACTGATG 64C. SEQIDNO:10 reverse_6(R6) CTGCACGCCGTTGATG 65C. SEQIDNO:11 reverse_7(R7) CTCCAGGTCGCTGATGG 65C. SEQIDNO:12 reverse_8(R8) CTGTGCACCGCTGATG 64C. SEQIDNO:13 reverse_9(R9) CTGCACGTCGCTGATG 64C. SEQIDNO:14 reverse_10(R10) CTGCACACCACTGATGG 63C. SEQIDNO:15
(203) For a proof of concept, 20 clones of an in-house rabbit immune library were randomly picked. These variants have been sequenced and aligned against the nave rabbit kappa light chains repertoire (IMGT database). Sequence alignment of the matured antibodies are listed in
(204) Within the 20 sequences which have been selected, 1/20 showed poor sequence quality. Of the 19 remaining sequences, 11/19 (58%) were fully covered by the primer set without any mismatches. From the remaining variants, 5/19 (26%) revealed 1 nucleotide mismatch in either the forward or the reverse primer. The other 3/19 (16%) showed two or three mismatches. With the assumption that a PCR would potentially still work for those with only 1 mismatch in the primer annealing region, a library recovery of 16/19 (84%) was found.
Example 6Optimization of an in-House Rabbit scFv Immune Library
(205) The DNA (Phagemid) of an in-house rabbit scFv immune library was used as template DNA to run all possible primer combinations of the explained primer set (20 PCR reactions). The Q5 Site-Directed Mutagenesis kit of New England Biolabs was used according to the provided protocol. The annealing temperature was set to 63 C. and 35 cycles were used with 1 ng of the original phagemid DNA as template. After PCR, the KLD reaction (a part of the Q5 Site-Directed Mutagenesis protocol) was done for each sample with incubating for 30 min at room temperature, followed by 30 min at 16 C. The KLD reactions were then purified using PCR purification followed by electroporation into TG1 cells. The transformed bacteria were plated on 2YT plates containing 100 g/ml ampicillin+1% glucose and incubated overnight at 37 C. After harvesting the bacteria, the phage amplification was initiated according to standard protocols. In addition, a serial dilution of bacteria was performed to determine the transformation titers which was indicating a library coverage of 8.5-fold above the original library. A few clones of each reaction were sequenced for quality control.
Example 7Quality of Optimized Library
(206) 4-5 variants for each of the 20 PCR reactions (96 in total) were sequenced to check the quality of the optimized library. For all PCR reactions, there were successfully optimized variants available. Overall, 64/96 (67%) correct insert with the foreseen substitution C80A were identified. The remaining 32 sequences exhibit different problems such as frameshifts, sequencing problems, and primer mismatches. Combined with the diversity of the original library of 8.5-fold within the bacteria transformation readout from which a correct insert percentage of 67% was identified, an overall library coverage of around 6-fold was determined.
(207) In addition, the sequenced variants (64/96) were further analyzed by designing a phylogenetic circle which indicated a good distribution of different rabbit kappa light chain subtypes, as shown in
Example 8Biopanning with Optimized Library
(208) The optimized in-house rabbit scFv immune library was used for biopanning against the specific pMHC target. In parallel, the original rabbit scFv immune library has been used as direct control for the quality and efficacy of the optimized library. Three rounds of phage display were performed, before the libraries were screened for specific hits. Screening was done with a monoclonal phage ELISA against specific and unspecific target. The ratio of the signal from the specific target binding to the unspecific binding was then calculated to find hits binding specifically to the target. The data can be found in Table 4 (original rabbit library) and Table 5 (optimized library).
(209) Specifically, Table 4 and Table 5 show the output of the monoclonal phage ELISA after three rounds of biopanning applied to the rabbit derived antibody library in which the C80 was removed. The values indicate the binding signal ratios to target peptide MAGE-A4 in context of the HLA complex/mix of 49 different unrelated peptides (SEQ ID NO: 268-316, as recited in Table 9) in context of the HLA complex. Ratios higher than 2.5 are highlighted in grey, each data point represents one phage displayed clone.
(210) Whereas for the original library after three rounds of biopanning only one binder could be identified, there are 13 binders found in the optimized library. This clearly shows the evidence of removing the free cysteine to use the full diversity from the rabbit immunization libraries.
(211) Additional rounds of panning have been executed by using the lambda library and the optimized kappa library. 19 unique and target specific antibodies were identified. The 19 antibody scFv sequences identified in the biopanning screen are recited below in Table 6.
(212) TABLE-US-00004 TABLE 4 Output of the panning of the phage display rabbit antibodies with Cys80. 1 2 3 4 5 6 7 8 9 10 11 12 A 0.89 0.97 1.00 0.89 0.96 0.89 0.86 0.75 0.90 0.75 0.86 0.56 B 0.71 0.97 0.95 0.65 0.75 0.94 0.63 0.96 0.72 1.00 0.93 0.63 C 0.74 0.66 0.67 0.90 0.90 0.79 0.61 1.11 0.82 0.81 0.86 0.77 D 0.66 0.76 0.76 0.64 0.69 0.71 1.06 0.82 0.80 0.76 0.65 0.65 E 0.66 0.59 1.11 0.64 0.88 1.02 1.06 0.59 0.96 0.84 1.07 1.08 F 0.79 0.68 0.72 1.04 0.49 0.64 1.06 0.68 1.13 0.62 0.70 0.68 G 0.84 0.68 3.04 0.51 0.94 0.92 0.57 0.57 0.69 0.65 0.60 0.70 H 0.57 0.71 0.54 0.60 0.47 0.59 0.53 0.95 0.90 0.54 0.88 1.15 Original rabbit library. Each data point A1-H12 represents on clone after three rounds of biopanning in a monoclonal phage ELISA for binding against HLA-A2/MAGE-A4 complex in relation to unspecific binding against HLA complex/mix of 49 different unrelated peptides (SEQ ID NO: 268-316). Ratios higher than 2.5 are highlighted in bold text.
(213) TABLE-US-00005 TABLE 5 Output of the panning of the phage display rabbit antibodies with Cys80. 1 2 3 4 5 6 7 8 9 10 11 12 A 0.73 0.94 0.67 0.80 0.94 11.64 0.83 0.95 15.58 0.99 1.73 12.60 B 0.88 10.18 11.26 0.75 0.87 22.48 1.13 10.85 0.94 1.02 0.88 0.94 C 0.97 0.27 0.65 0.89 0.89 0.89 3.74 0.87 0.84 0.76 0.97 1.11 D 0.95 0.83 0.90 0.94 17.06 0.70 0.97 0.87 0.70 19.62 0.96 1.02 E 0.86 0.94 0.75 0.60 0.84 0.88 0.67 0.92 0.89 0.57 0.76 11.32 F 0.53 0.92 0.96 2.66 0.95 1.31 0.64 0.92 12.50 0.98 0.94 0.76 G 0.97 0.96 0.93 0.92 0.68 0.56 1.67 0.71 0.73 0.81 0.68 0.62 H 1.12 0.75 0.82 0.60 0.95 0.93 0.59 0.92 0.80 0.74 0.91 4.51 Optimized library. Each data point A1-H11 represents on clone after three rounds of biopanning in a monoclonal phage ELISA for binding against HLA-A2/MAGE-A4 complex in relation to unspecific binding against HLA complex/mix of 49 different unrelated peptides. Ratios higher than 2.5 are highlighted in bold text. H12 represents a positive control.
(214) TABLE-US-00006 TABLE6 Rabbit-DerivedAntibodyscFvAminoAcidSequences.CDRsequencesare highlightedinboldunderlinedtext. AntibodyID Sequence M0848scFV QEQLVESGGGLVTPGTPLTLTCTVSGFSLSSYAMGWVRQ SEQIDNO:16 APGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLR VTSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTLV TVSSGGGGSGGGGSGGGGSGGGGASELDLTQTPASVEVA VGGTVTIKCQASQSIGSYLSWYQQKPGQRPKLLIFRASTL ASGVSSRFKGSGSGTQFTLTISGVECADAATYYCQQGYSS TNLDNVFGGGTEVVVK M0848VH QEQLVESGGGLVTPGTPLTLTCTVSGFSLSSYAMGWVRQ SEQIDNO:17 APGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLR VTSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTLV TVSS M0848VL ELDLTQTPASVEVAVGGTVTIKCQASQSIGSYLSWYQQKP SEQIDNO:18 GQRPKLLIFRASTLASGVSSRFKGSGSGTQFTLTISGVECA DAATYYCQQGYSSTNLDNVFGGGTEVVVK M0848CDRH1 SSYAMG SEQIDNO:19 M0848CDRH2 TINDGGTAFYASWVKG SEQIDNO:20 M0848CDRH3 AYGSNGDVYWGYENL SEQIDNO:21 M0848CDRL1 QASQSIGSYLS SEQIDNO:22 M0848CDRL2 RASTLAS SEQIDNO:23 M0848CDRL3 QQGYSSTNLDNV SEQIDNO:24 M0849scFv QEQLEESGGGLVTPGGTLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:25 APGKGLEWIGTINDGGTAFYAKWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTISSGGGGSGGGGSGGGGSGGGGASELVMTQTPSSVSEP VGGTVTIKCQASQSIGSNLAWYQQRPGQPPKLLIYSASTL ASGVSSRFKGSGSGTESTLTISGVQAADAATYYCQQGYSS SNVDNVFGGGTELEIL M0849VH QEQLEESGGGLVTPGGTLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:26 APGKGLEWIGTINDGGTAFYAKWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTISS M0849VL ELVMTQTPSSVSEPVGGTVTIKCOASQSIGSNLAWYQQRP SEQIDNO:27 GQPPKLLIYSASTLASGVSSRFKGSGSGTESTLTISGVQAA DAATYYCOQGYSSSNVDNVFGGGTELEIL M0849CDRH1 SNYAMG SEQIDNO:28 M0849CDRH2 TINDGGTAFYAKWLKG SEQIDNO:29 M0849CDRH3 AYGSNGDVYWGYFNL SEQIDNO:30 M0849CDRL1 QASQSIGSNLA SEQIDNO:31 M0849CDRL2 SASTLAS SEQIDNO:32 M0849CDRL3 QQGYSSSNVDNV SEQIDNO:33 M0850scFv QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:34 PGKGLEWIGTINDGGTAFYANWVKGRFTISRTSTTVDLK MTSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGASELVMTQTPASVSE PVGGTVTIKCQASQSIGSNLAWYQQKPGQPPKLLIYAAAN LASGVSSRFKGSRSGTEYTLTISGVQAADAATYYCQQGYS SSNVANVFGGGTELEIL M0850VH QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:35 PGKGLEWIGTINDGGTAFYANWVKGRFTISRTSTTVDLK MTSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTL VTVSS M0850VL ELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKP SEQIDNO:36 GQPPKLLIYAAANLASGVSSRFKGSRSGTEYTLTISGVQAA DAATYYCQQGYSSSNVANVFGGGTELEIL M0850CDRH1 SSYAMI SEQIDNO:37 M0850CDRH2 TINDGGTAFYANWVKG SEQIDNO:38 M0850CDRH3 AYGSNGDVYWGYVNL SEQIDNO:39 M0850CDRL1 QASQSIGSNLA SEQIDNO:40 M0850CDRL2 AAANLAS SEQIDNO:41 M0850CDRL3 QQGYSSSNVANV SEQIDNO:42 M0851scFv QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:43 PGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLKI TSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTLVT ISSGGGGSGGGGSGGGGSGGGGASELVMTQTPSSVSAAV GGTVTINCQASQNIGSVFAWYQQKPGQPPKLLIYKASSLA SGVPSRFKGSGSGTQFTLTISGVEAADAATYYCQQGASSS NVDNIFGGGTEVVVK M0851VH QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:44 PGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLKI TSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTLVT ISS M0851VL ELVMTQTPSSVSAAVGGTVTINCQASQNIGSVFAWYQQK SEQIDNO:45 PGQPPKLLIYKASSLASGVPSRFKGSGSGTQFTLTISGVEA ADAATYYCQQGASSSNVDNIFGGGTEVVVK M0851CDRH1 SSYAMI SEQIDNO:46 M0851CDRH2 TINDGGTAFYASWVKG SEQIDNO:47 M0851CDRH3 AYGSNGDVYWGYVNL SEQIDNO:48 M0851CDRL1 QASQNIGSVFA SEQIDNO:49 M0851CDRL2 KASSLAS SEQIDNO:50 M0851CDRL3 QQGASSSNVDNI SEQIDNO:51 M0852scFv QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:52 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGASELVMTQTASPVSA AVGGTVTINCQASQSISSRSLSWYQQKPGQPPKLLIYEAS KLASGVPSRFSGSGSGTQFTLTISGVQADDAATYYCQQGY SSSNVDNVFGGGTEVVVK M0852VH QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:53 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSS M0852VL ELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQQK SEQIDNO:54 PGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQA DDAATYYCQQGYSSSNVDNVFGGGTEVVVK M0852CDRH1 SNYAMG SEQIDNO:55 M0852CDRH2 TINDGGTAFYANWLKG SEQIDNO:56 M0852CDRH3 AYGSNGDVYWGYFNL SEQIDNO:57 M0852CDRL1 QASQSISSRSLS SEQIDNO:58 M0852CDRL2 EASKLAS SEQIDNO:59 M0852CDRL3 QQGYSSSNVDNV SEQIDNO:60 M0853scFv QQQLVESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:61 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGASELVMTQTASPVSA AVGGTVTINCQASQSISSRSLSWYQQKPGQPPKLLIYEAS KLASGVPSRFSGSGSGTQFTLTISGVQADDAATYYCQQGY SSSNVDNFGGGTEVVVK M0853VH QQQLVESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:62 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSS M0853VL ELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQQK SEQIDNO:63 PGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGVQA DDAATYYCQQGYSSSNVDNFGGGTEVVVK M0853CDRH1 SNYAMG SEQIDNO:64 M0853CDRH2 TINDGGTAFYANWLKG SEQIDNO:65 M0853CDRH3 AYGSNGDVYWGYFNL SEQIDNO:66 M0853CDRL1 QASQSISSRSLS SEQIDNO:67 M0853CDRL2 EASKLAS SEQIDNO:68 M0853CDRL3 QQGYSSSNVDN SEQIDNO:69 M0854scFv QSVKESWGRLVTPGGSLTLTCTVSGIDLNNYAMGWVRQA SEQIDNO:70 PGKGLEWIGTINNDGATYYPSWARGRFTISKTSTTVDLKI TSPTTEDTATYFCARTYGSNGDVYWGYFNLWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGASALELTQTPASVEVAV GGTVTINCQASQSIGGALNWYQQKSGQPPKLLIYLASTLA SGVSSRFKGSGSGTQFTLTISGVEAADAATYYCQQGYSAS NIDNAFGGGTEVVVK M0854VH QSVKESWGRLVTPGGSLTLTCTVSGIDLNNYAMGWVRQA SEQIDNO:71 PGKGLEWIGTINNDGATYYPSWARGRFTISKTSTTVDLKI TSPTTEDTATYFCARTYGSNGDVYWGYFNLWGQGTLVT VSS M0854VL ALELTQTPASVEVAVGGTVTINCQASQSIGGALNWYQQK SEQIDNO:72 SGQPPKLLIYLASTLASGVSSRFKGSGSGTQFTLTISGVEA ADAATYYCQQGYSASNIDNAFGGGTEVVVK M0854CDRH1 NNYAMG SEQIDNO:73 M0854CDRH2 TINNDGATYYPSWARG SEQIDNO:74 M0854CDRH3 TYGSNGDVYWGYFNL SEQIDNO:75 M0854CDRL1 QASQSIGGALN SEQIDNO:76 M0854CDRL2 LASTLAS SEQIDNO:77 M0854CDRL3 QQGYSASNIDNA SEQIDNO:78 M0855scFv QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:79 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TISSGGGGSGGGGSGGGGSGGGGASELVMTQTPASVSEPV GGTVTIKCQASQSIGSNLAWYQQKPGQPPKLLIYYESILA SGVPSRFSGSGSGTEYTLTISGAQADDAATYYCQQGYSSS NIDNAFGGGTEVVVK M0855VH QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:80 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TISS M0855VL ELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKP SEQIDNO:81 GQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQADD AATYYCQQGYSSSNIDNAFGGGTEVVVK M0855CDRH1 SSYAMG SEQIDNO:82 M0855CDRH2 TINDGGSAFYASWVKG SEQIDNO:83 M0855CDRH3 TYGTNGDVYWGYFNL SEQIDNO:84 M0855CDRL1 QASQSIGSNLA SEQIDNO:85 M0855CDRL2 YESILAS SEQIDNO:86 M0855CDRL3 QQGYSSSNIDNA SEQIDNO:87 M0856scFv QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:88 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TISSGGGGSGGGGSGGGGSGGGGASELVMTQTPASVSEPV GGTVTIKCQASQSIGSNLAWYQQKPGQPPKLLIYYESILA SGVPSRFSGSGSGTEYTLTISGAQADDAATYYCQQGYSSS NILNAFGGGTEVVVK M0856VH QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:89 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TISS M0856VL ELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKP SEQIDNO:90 GQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQADD AATYYCQQGYSSSNILNAFGGGTEVVVK M0856CDRH1 SSYAMG SEQIDNO:91 M0856CDRH2 TINDGGSAFYASWVKG SEQIDNO:92 M0856CDRH3 TYGTNGDVYWGYFNL SEQIDNO:93 M0856CDRL1 QASQSIGSNLA SEQIDNO:94 M0856CDRL2 YESILAS SEQIDNO:95 M0856CDRL3 QQGYSSSNILNA SEQIDNO:96 M0857scFv QQQLVESGGRLVTPGTPLTLTCTASGIDLNSNAMSWVRQ SEQIDNO:97 GPGKGLEWIGDIWSGGYTDYASWAKGRFTISKTSTTVDL KMTSLTAADTATYFCARDRLAGDGVVDYDLWGQGTLVT ISSGGGGSGGGGSGGGGSGGGGASELDMTQTPASVEVAV GGTVTIKCQASQNIYSNLAWYQQKPGQRPKLLIYGASTL ASGVPSRFKGSGSGTEYTLTINGVQAADAATYYCQQGFSS SNVDNVFGGGTEVVVK M0857VH QQQLVESGGRLVTPGTPLTLTCTASGIDLNSNAMSWVRQ SEQIDNO:98 GPGKGLEWIGDIWSGGYTDYASWAKGRFTISKTSTTVDL KMTSLTAADTATYFCARDRLAGDGVVDYDLWGQGTLVT ISS M0857VL ELDMTQTPASVEVAVGGTVTIKCQASQNIYSNLAWYQQK SEQIDNO:99 PGQRPKLLIYGASTLASGVPSRFKGSGSGTEYTLTINGVQA ADAATYYCQQGFSSSNVDNVFGGGTEVVVK M0857CDRH1 NSNAMS SEQIDNO:100 M0857CDRH2 DIWSGGYTDYASWAKG SEQIDNO:101 M0857CDRH3 DRLAGDGVVDYDL SEQIDNO:102 M0857CDRL1 QASQNIYSNLA SEQIDNO:320 M0857CDRL2 GASTLAS SEQIDNO:103 M0857CDRL3 QQGFSSSNVDNV SEQIDNO:104 M0858scFv QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:105 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGASELVLTQPQSVSGSLGQTVSI SCKRARNNIEDYYVHWYQQHPGRSPTIVIHKDDQRPSGV PDRFSGSIDSTSNSASLTITGLLAEDEADYFCQSFDNNANP VFGGGTQLTVTG M0858VH QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:106 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSS M0858VL ELVLTQPQSVSGSLGQTVSISCKRARNNIEDYYVHWYQQ SEQIDNO:107 HPGRSPTIVIHKDDQRPSGVPDRFSGSIDSTSNSASLTITGL LAEDEADYFCQSFDNNANPVFGGGTQLTVTG M0858CDRH1 SNYAMS SEQIDNO:108 M0858CDRH2 IVSSGGTTYYASWAKG SEQIDNO:109 M0858CDRH3 DLYYGPTTYSAFNL SEQIDNO:110 M0858CDRL1 KRARNNIEDYYVH SEQIDNO:111 M0858CDRL2 KDDORPS SEQIDNO:112 M0858CDRL3 QSFDNNANPV SEQIDNO:113 M0859scFv QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:114 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISSG GGGSGGGGSGGGGSGGGGASELVLTQPQSVSGSLGQTVSI SCKRARDNIEDYYVHWYQQHPGKTPTIVIHKDDORPSGV PDRFSGSIDSTSNSASLTITGLLAEDEADYFCQSFDNDASP VFGGGTQLTVTG M0859VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:115 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISS M0859VL ELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWYQQ SEQIDNO:116 HPGKTPTIVIHKDDQRPSGVPDRFSGSIDSTSNSASLTITGL LAEDEADYFCQSFDNDASPVFGGGTQLTVTG M0859CDRH1 SNYAMS SEQIDNO:117 M0859CDRH2 IVSSGGTTYYASWAKG SEQIDNO:118 M0859CDRH3 DLYYGPTTYSAFNL SEQIDNO:119 M0859CDRL1 KRARDNIEDYYVH SEQIDNO:120 M0859CDRL2 KDDQRPS SEQIDNO:121 M0859CDRL3 QSFDNDASPV SEQIDNO:122 M0860scFv QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:123 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISSG GGGSGGGGSGGGGSGGGGASELVLTQPQSVSGSLGQTVSI SCKRARDNIEDYYVHWYQQYPGKTPTIVIYKDDQRPSGV PDRFSGSIDSTSNSASLTITGLLAEDEADYFCQSFDNNANV VFGGGTQLTVTG M0860VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:124 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISS M0860VL ELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWYQQ SEQIDNO:125 YPGKTPTIVIYKDDQRPSGVPDRFSGSIDSTSNSASLTITGL LAEDEADYFCQSFDNNANVVFGGGTQLTVTG M0860CDRH1 SNYAMS SEQIDNO:126 M0860CDRH2 IVSSGGTTYYASWAKG SEQIDNO:127 M0860CDRH3 DLYYGPTTYSAFNL SEQIDNO:128 M0860CDRL1 KRARDNIEDYYVH SEQIDNO:129 M0860CDRL2 KDDORPS SEQIDNO:130 M0860CDRL3 QSFDNNANVV SEQIDNO:131 M0861scFv QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:132 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISSG GGGSGGGGSGGGGSGGGGASELVLTQPASVQVNLGQTVS LTCTADTLSRSYASWYQLKPGQAPVLLIYRDTSRPSGVPD RFSGSSSGNTATLTISGAQAGDEGDYVCATSDGSGSNFOL FGGGTQLTVTG M0861VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:133 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTISS M0861VL ELVLTQPASVQVNLGQTVSLTCTADTLSRSYASWYQLKP SEQIDNO:134 GQAPVLLIYRDTSRPSGVPDRFSGSSSGNTATLTISGAQAG DEGDYVCATSDGSGSNFQLFGGGTQLTVTG M0861CDRH1 SNYAMS SEQIDNO:135 M0861CDRH2 IVSSGGTTYYASWAKG SEQIDNO:136 M0861CDRH3 DLYYGPTTYSAFNL SEQIDNO:137 M0861CDRL1 TADTLSRSYAS SEQIDNO:138 M0861CDRL2 RDTSRPS SEQIDNO:139 M0861CDRL3 ATSDGSGSNFQL SEQIDNO:140 M0862scFv PEQLMESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQ SEQIDNO:141 APGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDL KMTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGASAQVLTQTPASVSAAVGG TVSISCOSSOSVVNNNWLAWYQQKPGQPPKLLIYKASTL ESGVPSRFKGSGSGTQFTLTISGVQADDAATYYCLGEFSC SSADCHAFGGGTELEIL M0862VH PEQLMESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQ SEQIDNO:142 APGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDL KMTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVS S M0862VL AQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAWYQ SEQIDNO:143 QKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTISGV QADDAATYYCLGEFSCSSADCHAFGGGTELEIL M0862CDRH1 SYGVN SEQIDNO:144 M0862CDRH2 FIFGDGTTYYANWAKG SEQIDNO:145 M0862CDRH3 DGYGGYDYIINL SEQIDNO:146 M0862CDRL1 OSSQSVVNNN SEQIDNO:147 M0862CDRL2 KASTLES SEQIDNO:148 M0862CDRL3 LGEFSCSSADCHA SEQIDNO:149 M0863scFv PEQLMESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQ SEQIDNO:150 APGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDL KMTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGASAQVLTQTPASVSAAVGG TVSISCOSSOSVVNNNWLAWYQQKPGQPPKLLIYKASTL ESGVPSRFKGSGSGTQFTLTISGVQADDAATYYCOGAYSG NIYYNAFGGGTEVVVK M0863VH PEQLMESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQ SEQIDNO:151 APGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDL KMTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVS S M0863VL AQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAWYQ SEQIDNO:152 QKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTISGV QADDAATYYCOGAYSGNIYYNAFGGGTEVVVK M0863CDRH1 SSYGVN SEQIDNO:153 M0863CDRH2 FIFGDGTTYYANWAKG SEQIDNO:154 M0863CDRH3 DGYGGYDYIINL SEQIDNO:155 M0863CDRL1 OSSOSVVNNN SEQIDNO:156 M0863CDRL2 KASTLES SEQIDNO:157 M0863CDRL3 QGAYSGNIYYNA SEQIDNO:158 M0864scFv QSVKESGGGLVTPGTPLTLTCTVSGFSLSTYAISWVRQAP SEQIDNO:159 GKGLEWIGFIDTVDSAYYASWAKGRFTISKTSSTTVDLK MTSPTTEDTATYFCAKLRYGDYGDYTLWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGASELVMTQTPSPVSGAVGGT VTIKCOASONIYSYLAWYQQKPGQPPKLLIYKASTLASGV PSRVKGSGSGTEYTLTISGVQAADAATYYCOCTYYDSNTF GGGTEVVVK M0864VH QSVKESGGGLVTPGTPLTLTCTVSGFSLSTYAISWVRQAP SEQIDNO:160 GKGLEWIGFIDTVDSAYYASWAKGRFTISKTSSTTVDLK MTSPTTEDTATYFCAKLRYGDYGDYTLWGQGTLVTVSS M0864VL ELVMTQTPSPVSGAVGGTVTIKCOASONIYSYLAWYQQK SEQIDNO:161 PGQPPKLLIYKASTLASGVPSRVKGSGSGTEYTLTISGVQA ADAATYYCOCTYYDSNTFGGGTEVVVK M0864CDRH1 STYAIS SEQIDNO:162 M0864CDRH2 FIDTVDSAYYASWAKG SEQIDNO:163 M0864CDRH3 LRYGDYGDYTL SEQIDNO:164 M0864CDRL1 QASQNIYSYLA SEQIDNO:165 M0864CDRL2 KASTLAS SEQIDNO:166 M0864CDRL3 QCTYYDSNT SEQIDNO:167 M0865scFv PAALMESGGRLVTPGTPLTLTCTVSGIDLSTFAMTWVRQA SEQIDNO:168 PGKGLEWLGIINTGGSAYYTSWAKGRFTISRTSTTVDLKI TSPTTEDTATYFCARGDWSSATDLWGQGTLVTISSGGGGS GGGGSGGGGSGGGGASDPDMTQTPSSVSAAVGGTVTINC QASQSVYDNKVLAWYRQKPGQPPKLLIYKASTLASGVPS RFKGRGSGTQFTLTISGVQADDAATYYCLGEFSCSSADCH AFGGGTELEIL M0865VH PAALMESGGRLVTPGTPLTLTCTVSGIDLSTFAMTWVRQA SEQIDNO:169 PGKGLEWLGIINTGGSAYYTSWAKGRFTISRTSTTVDLKI TSPTTEDTATYFCARGDWSSATDLWGQGTLVTISS M0865VL DPDMTQTPSSVSAAVGGTVTINCOASOSVYDNKVLAWY SEQIDNO:170 RQKPGQPPKLLIYKASTLASGVPSRFKGRGSGTQFTLTISG VQADDAATYYCLGEFSCSSADCHAFGGGTELEIL M0865CDRH1 STFAMT SEQIDNO:171 M0865CDRH2 IINTGGSAYYTSWAKG SEQIDNO:172 M0865CDRH3 GDWSSATDL SEQIDNO:173 M0865CDRL1 QASQSVYDNKVLA SEQIDNO:174 M0865CDRL2 KASTLAS SEQIDNO:175 M0865CDRL3 LGEFSCSSADCHA SEQIDNO:176 M0866scFv QSVKESGGRLVTPGTPLTLTCTASGFTISSSAISWVRQAPG SEQIDNO:177 KGLEYIGIIRSGGTTDYASWAKGRFAISKTSTTVDLKITSP TTEDTATYFCARDPPYITSTYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGASELVLTQPQSVSGSLGQTVSISCKR ARDSVESYDVHWYQQHPGKTPTIVIYKDDORPSGVPDRF SGSIDSTSNSASLTITGLLAEDEADYFCOSFDGDAVVFGGG TQLTVTG M0866VH QSVKESGGRLVTPGTPLTLTCTASGFTISSSAISWVRQAPG SEQIDNO:178 KGLEYIGIIRSGGTTDYASWAKGRFAISKTSTTVDLKITSP TTEDTATYFCARDPPYITSTYFDLWGQGTLVTVSS M0866VL ELVLTQPQSVSGSLGQTVSISCKRARDSVESYDVHWYQQ SEQIDNO:179 HPGKTPTIVIYKDDORPSGVPDRFSGSIDSTSNSASLTITGL LAEDEADYFCOSFDGDAVVFGGGTQLTVTG M0866CDRH1 SSSAIS SEQIDNO:180 M0866CDRH2 HIRSGGTTDYASWAKG SEQIDNO:181 M0866CDRH3 DPPYITSTYFDL SEQIDNO:182 M0866CDRL1 KRARDSVESYDVH SEQIDNO:183 M0866CDRL2 KDDQRPSG SEQIDNO:184 M0866CDRL3 QSFDGDAVV SEQIDNO:185 M0700HC QEQLVESGGGLVTPGTPLTLTCTVSGFSLSSYAMGWVRQ SEQIDNO:190 APGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLR VTSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC M0700LC ASELDLTQTPASVEVAVGGTVTIKCQASQSIGSYLSWYQQ SEQIDNO:191 KPGQRPKLLIFRASTLASGVSSRFKGSGSGTQFTLTISGVEC ADAATYYCOOGYSSTNLDNVFGGGTEVVVKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC M0700VH QEQLVESGGGLVTPGTPLTLTCTVSGFSLSSYAMGWVRQ SEQIDNO:192 APGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLR VTSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTLV TVSS M0700VL ASELDLTQTPASVEVAVGGTVTIKCOASQSIGSYLSWYQQ SEQIDNO:193 KPGQRPKLLIFRASTLASGVSSRFKGSGSGTQFTLTISGVEC ADAATYYCOOGYSSTNLDNVFGGGTEVVVK M0701HC QEQLEESGGGLVTPGGTLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:194 APGKGLEWIGTINDGGTAFYAKWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC M0701LC ASELVMTQTPSSVSEPVGGTVTIKCOASOSIGSNLAWYQQ SEQIDNO:195 RPGQPPKLLIYSASTLASGVSSRFKGSGSGTESTLTISGVQA ADAATYYCOQGYSSSNVDNVFGGGTELEILRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC M0701VH QEQLEESGGGLVTPGGTLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:196 APGKGLEWIGTINDGGTAFYAKWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSS M0701VL ASELVMTQTPSSVSEPVGGTVTIKCOASOSIGSNLAWYQQ SEQIDNO:197 RPGQPPKLLIYSASTLASGVSSRFKGSGSGTESTLTISGVQA ADAATYYCOQGYSSSNVDNVFGGGTELEIL M0702HC QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:198 PGKGLEWIGTINDGGTAFYANWVKGRFTISRTSTTVDLK MTSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC M0702LC ASELVMTQTPASVSEPVGGTVTIKCOASOSIGSNLAWYQQ SEQIDNO:199 KPGQPPKLLIYAAANLASGVSSRFKGSRSGTEYTLTISGVQ AADAATYYCOQGYSSSNVANVFGGGTELEILRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC M0702VH QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:200 PGKGLEWIGTINDGGTAFYANWVKGRFTISRTSTTVDLK MTSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTL VTVSS M0702VL ASELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQ SEQIDNO:201 KPGQPPKLLIYAAANLASGVSSRFKGSRSGTEYTLTISGVQ AADAATYYCOQGYSSSNVANVFGGGTELEIL M0703HC QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:202 PGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLKI TSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC M0703LC ASELVMTQTPSSVSAAVGGTVTINCOASONIGSVFAWYQ SEQIDNO:203 QKPGQPPKLLIYKASSLASGVPSRFKGSGSGTQFTLTISGV EAADAATYYCOQGASSSNVDNIFGGGTEVVVKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC M0703VH QEQLEESGGGLVTPGTPLTLTCTASGFSLSSYAMIWVRQA SEQIDNO:204 PGKGLEWIGTINDGGTAFYASWVKGRFTISRTSTTVDLKI TSPTTEDTATYFCARAYGSNGDVYWGYVNLWGQGTLVT VSS M0703VL ASELVMTQTPSSVSAAVGGTVTINCOASONIGSVFAWYQ SEQIDNO:205 QKPGQPPKLLIYKASSLASGVPSRFKGSGSGTQFTLTISGV EAADAATYYCQQGASSSNVDNIFGGGTEVVVK M0704HC QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:206 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC M0704LC ASELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQ SEQIDNO:207 KPGQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQA DDAATYYCOOGYSSSNIDNAFGGGTEVVVKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC M0704VH QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:208 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TVSS M0704VL ASELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQ SEQIDNO:209 KPGQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQA DDAATYYCOQGYSSSNIDNAFGGGTEVVVK M0705HC QQQLVESGGRLVTPGTPLTLTCTASGIDLNSNAMSWVRQ SEQIDNO:210 GPGKGLEWIGDIWSGGYTDYASWAKGRFTISKTSTTVDL KMTSLTAADTATYFCARDRLAGDGVVDYDLWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC M0705LC ASELDMTQTPASVEVAVGGTVTIKCOASONIYSNLAWYQ SEQIDNO:211 QKPGQRPKLLIYGASTLASGVPSRFKGSGSGTEYTLTINGV QAADAATYYCQQGFSSSNVDNVFGGGTEVVVKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC M0705VH QQQLVESGGRLVTPGTPLTLTCTASGIDLNSNAMSWVRQ SEQIDNO:212 GPGKGLEWIGDIWSGGYTDYASWAKGRFTISKTSTTVDL KMTSLTAADTATYFCARDRLAGDGVVDYDLWGQGTLVT VSS M0705VL ASELDMTQTPASVEVAVGGTVTIKCOASONIYSNLAWYQ SEQIDNO:213 QKPGQRPKLLIYGASTLASGVPSRFKGSGSGTEYTLTINGV QAADAATYYCOQGFSSSNVDNVFGGGTEVVVK M0706HC QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:214 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC M0706LC ASELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQ SEQIDNO:215 QKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGV QADDAATYYCQQGYSSSNVDNVFGGGTEVVVKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC M0706VH QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:216 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSS M0706VL ASELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQ SEQIDNO:217 QKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGV QADDAATYYCOQGYSSSNVDNVFGGGTEVVVK M0707HC QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:218 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC M0707LC ASELVMTQTPASVSEPVGGTVTIKCOASOSIGSNLAWYQQ SEQIDNO:219 KPGQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQA DDAATYYCOQGYSSSNILNAFGGGTEVVVKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC M0707VH QQQLEESGGGLVTPGTPLTLTCTVSGIDLSSYAMGWVRQ SEQIDNO:220 APGKGLEWIGTINDGGSAFYASWVKGRFTISRTSTTVDLK ITSPTAEDTATYFCAKTYGTNGDVYWGYFNLWGQGTLV TVSS M0707VL ASELVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQ SEQIDNO:221 KPGQPPKLLIYYESILASGVPSRFSGSGSGTEYTLTISGAQA DDAATYYCOOGYSSSNILNAFGGGTEVVVK M0708HC QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:222 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC M0708LC ASELVLTQPQSVSGSLGQTVSISCKRARNNIEDYYVHWY SEQIDNO:223 QQHPGRSPTIVIHKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNNANPVFGGGTQLTVTGRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC M0708VH QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:224 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSS M0708VL ASELVLTQPQSVSGSLGQTVSISCKRARNNIEDYYVHWY SEQIDNO:225 QQHPGRSPTIVIHKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNNANPVFGGGTQLTVTG M0709HC QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:226 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC M0709LC ASELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWY SEQIDNO:227 QQHPGKTPTIVIHKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNDASPVFGGGTQLTVTGRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC M0709VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:228 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSS M0709VL ASELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWY SEQIDNO:229 QQHPGKTPTIVIHKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNDASPVFGGGTQLTVTG M0710HC QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:230 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC M0710LC ASELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWY SEQIDNO:231 QQYPGKTPTIVIYKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNNANVVFGGGTQLTVTGRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC M0710VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:232 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSS M0710VL ASELVLTQPQSVSGSLGQTVSISCKRARDNIEDYYVHWY SEQIDNO:233 QQYPGKTPTIVIYKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDNNANVVFGGGTQLTVTG M0762HC QSVKESGGRLVTPGTPLTLTCTASGFTISSSAISWVRQAPG SEQIDNO:234 KGLEYIGIIRSGGTTDYASWAKGRFAISKTSTTVDLKITSP TTEDTATYFCARDPPYITSTYFDLWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC M0762LC ASELVLTQPQSVSGSLGQTVSISCKRARDSVESYDVHWY SEQIDNO:235 QQHPGKTPTIVIYKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCQSFDGDAVVFGGGTQLTVTGRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC M0762VH QSVKESGGRLVTPGTPLTLTCTASGFTISSSAISWVRQAPG SEQIDNO:236 KGLEYIGIIRSGGTTDYASWAKGRFAISKTSTTVDLKITSP TTEDTATYFCARDPPYITSTYFDLWGQGTLVTVSS M0762VL ASELVLTQPQSVSGSLGQTVSISCKRARDSVESYDVHWY SEQIDNO:237 QQHPGKTPTIVIYKDDORPSGVPDRFSGSIDSTSNSASLTIT GLLAEDEADYFCOSFDGDAVVFGGGTQLTVTG M0763HC QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:238 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC M0763LC ASELVLTQPASVQVNLGQTVSLTCTADTLSRSYASWYQL SEQIDNO:239 KPGQAPVLLIYRDTSRPSGVPDRFSGSSSGNTATLTISGAQ AGDEGDYVCATSDGSGSNFOLFGGGTQLTVTGRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC M0763VH QSVKESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP SEQIDNO:240 GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSS M0763VL ASELVLTQPASVQVNLGQTVSLTCTADTLSRSYASWYQL SEQIDNO:241 KPGQAPVLLIYRDTSRPSGVPDRFSGSSSGNTATLTISGAQ AGDEGDYVCATSDGSGSNFOLFGGGTQLTVTG M0764HC QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:242 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC M0764LC ASELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQ SEQIDNO:243 QKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGV QADDAATYYCQQGYSSSNVDNFGGGTEVVVKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC M0764VH QQQLEESGGGLVTPGTPLTLTCTVSGFSLSNYAMGWVRQ SEQIDNO:244 APGKGLEWIGTINDGGTAFYANWLKGRFTISRTSTTVDL KITSPTTEDTATYFCARAYGSNGDVYWGYFNLWGQGTL VTVSS M0764VL ASELVMTQTASPVSAAVGGTVTINCQASQSISSRSLSWYQ SEQIDNO:245 QKPGQPPKLLIYEASKLASGVPSRFSGSGSGTQFTLTISGV QADDAATYYCOQGYSSSNVDNFGGGTEVVVK M0765HC QSVKESWGRLVTPGGSLTLTCTVSGIDLNNYAMGWVRQA SEQIDNO:246 PGKGLEWIGTINNDGATYYPSWARGRFTISKTSTTVDLKI TSPTTEDTATYFCARTYGSNGDVYWGYFNLWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC M0765LC ASALELTQTPASVEVAVGGTVTINCOASOSIGGALNWYQ SEQIDNO:247 QKSGQPPKLLIYLASTLASGVSSRFKGSGSGTQFTLTISGV EAADAATYYCOOGYSASNIDNAFGGGTEVVVKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC M0765VH QSVKESWGRLVTPGGSLTLTCTVSGIDLNNYAMGWVRQA SEQIDNO:248 PGKGLEWIGTINNDGATYYPSWARGRFTISKTSTTVDLKI TSPTTEDTATYFCARTYGSNGDVYWGYFNLWGQGTLVT VSS M0765VL ASALELTQTPASVEVAVGGTVTINCQASQSIGGALNWYQ SEQIDNO:249 QKSGQPPKLLIYLASTLASGVSSRFKGSGSGTQFTLTISGV EAADAATYYCOOGYSASNIDNAFGGGTEVVVK M0766HC PEQLEESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQA SEQIDNO:250 PGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDLK MTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSC M0766LC ASAQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAW SEQIDNO:251 YQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTIS GVQADDAATYYCLGEFSCSSADCHAFGGGTELEILRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC M0766VH PEQLEESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQA SEQIDNO:252 PGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDLK MTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVSS M0766VL ASAQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAW SEQIDNO:253 YQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTIS GVQADDAATYYCLGEFSCSSADCHAFGGGTELEIL M0767HC PEQLEESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQA SEQIDNO:254 PGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDLK MTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSC M0767LC ASAQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAW SEQIDNO:255 YQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTIS GVQADDAATYYCOGAYSGNIYYNAFGGGTEVVVKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC M0767VH PEQLEESGGGLVTPGGVLTLTCTASGFSFSSYGVNWVRQA SEQIDNO:256 PGKGLEWIGFIFGDGTTYYANWAKGRFTISKTSTTVDLK MTSPTTEDTATYFCARDGYGGYDYIINLWGQGTLVTVSS M0767VL ASAQVLTQTPASVSAAVGGTVSISCOSSOSVVNNNWLAW SEQIDNO:257 YQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTIS GVQADDAATYYCOGAYSGNIYYNAFGGGTEVVVK M0768HC QSVKESGGGLVTPGTPLTLTCTVSGFSLSTYAISWVRQAP SEQIDNO:258 GKGLEWIGFIDTVDSAYYASWAKGRFTISKTSSTTVDLK MTSPTTEDTATYFCAKLRYGDYGDYTLWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSC M0768LC ASELVMTQTPSPVSGAVGGTVTIKCOASONIYSYLAWYQ SEQIDNO:259 QKPGQPPKLLIYKASTLASGVPSRVKGSGSGTEYTLTISGV QAADAATYYCOCTYYDSNTFGGGTEVVVKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC M0768VH QSVKESGGGLVTPGTPLTLTCTVSGFSLSTYAISWVRQAP SEQIDNO:260 GKGLEWIGFIDTVDSAYYASWAKGRFTISKTSSTTVDLK MTSPTTEDTATYFCAKLRYGDYGDYTLWGQGTLVTVSS M0768VL ASELVMTQTPSPVSGAVGGTVTIKCOASQNIYSYLAWYQ SEQIDNO:261 QKPGQPPKLLIYKASTLASGVPSRVKGSGSGTEYTLTISGV QAADAATYYCOCTYYDSNTFGGGTEVVVK M0769HC PAALEESGGRLVTPGTPLTLTCTVSGIDLSTFAMTWVRQA SEQIDNO:262 PGKGLEWLGIINTGGSAYYTSWAKGRFTISRTSTTVDLKI TSPTTEDTATYFCARGDWSSATDLWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC M0769LC ASDPDMTQTPSSVSAAVGGTVTINCOASOSVYDNKVLAW SEQIDNO:263 YRQKPGQPPKLLIYKASTLASGVPSRFKGRGSGTQFTLTIS GVQADDAATYYCLGEFSCSSADCHAFGGGTELEILRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC M0769VH PAALEESGGRLVTPGTPLTLTCTVSGIDLSTFAMTWVRQA SEQIDNO:264 PGKGLEWLGIINTGGSAYYTSWAKGRFTISRTSTTVDLKI TSPTTEDTATYFCARGDWSSATDLWGQGTLVTVSS M0769VL ASDPDMTQTPSSVSAAVGGTVTINCOASOSVYDNKVLAW SEQIDNO:265 YRQKPGQPPKLLIYKASTLASGVPSRFKGRGSGTQFTLTIS GVQADDAATYYCLGEFSCSSADCHAFGGGTELEIL CDR4-bispecific QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYAMSWVRQAP 01(M0719HC) GKGLEYIGIVSSGGTTYYASWAKGRFTISKTSTTVDLKITS SEQIDNO:266 PTTEDTATYFCAKDLYYGPTTYSAFNLWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC CDR4-bispecific ASELVLTQPQSVSGSLGQTVSISCKRARNNIEDYYVHWY 01(M0719LC) QQHPGRSPTIVIHKDDORPSGVPDRFSGSIDSTSNSASLTIT SEQIDNO:267 GLLAEDEADYFCOSFDNNANPVFGGGTQLTVTGRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECGGGGSAVVTQEPSLTVSPG GTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTN KRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALW YSNHWVFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSE VQLVESGGGSVQPGGSLRLSCAASGFTFSTYAMNWVRQA PGKGLEWVGRIRSKANNYATYYADSVKGRFTISRDDSKN TLYLQMNSLRAEDTATYYCVRHGNFGDSYVSWFAYWG QGTTVTVSS
Example 9Expression of Antibodies as Monovalent Monospecific Fabs or Bispecific Antibodies
(215) The monovalent monospecific antibodies were expressed in a Fab format. Additionally, bispecific antibodies including a CD3 binding moiety were expressed based on a Fab format, which is highly stable and an efficient heterodimerization scaffold. scFvs or sdAbs were fused to the C-terminal regions of the Fab. The rabbit variable domains were paired with human constant domains (heavy chain and kappa light chain) to generate the chimeric Fab, which binds to the target pMHC. An scFv with binding specificity to CD3 was linked to the C terminus of the Fab light chain constant region. The amino acid sequences of the constant domains, amino acid linker, and CD3 scFv are recited below in Table 7.
(216) TABLE-US-00007 TABLE7 AminoAcidSequencesForGeneratingChimericFab SequenceID Sequence Humanconstantkappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR SEQIDNO:186 EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC Humanconstantheavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE SEQIDNO:187 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Linker(linkingtheCLto GGGGS thescFv) SEQIDNO:188 CD3scFv(CDRsequences AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYA arehighlightedinbold, NWVQQKPGKSPRGLIGGTNKRAPGVPARFSGSLL underlinedtext) GGKAALTISGAQPEDEADYYCALWYSNHWVFG SEQIDNO:189 GGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQL VESGGGSVQPGGSLRLSCAASGFTFSTYAMNWV RQAPGKGLEWVGRIRSKANNYATYYADSVKGR FTISRDDSKNTLYLQMNSLRAEDTATYYCVRHGN FGDSYVSWFAYWGQGTTVTVSS
(217) Synthetic genes encoding for the different antibody chains (i.e., heavy chain and light chain) were constructed at Twist Bioscience Corporation and were separately cloned into the expression vectors for transient expression in HEK 293 6E cells. Expression vector DNA was prepared using conventional plasmid DNA purification methods (for example Qiagen HiSpeed plasmid maxi kit, cat. #12662).
(218) The monospecific antigen binding proteins and bispecific antigen binding proteins including a CD3 binding moiety were expressed by transient co-transfection of the respective mammalian expression vectors in HEK293-6E cells, which were cultured in suspension using polyethylenimine (PEI 40 kD linear). The HEK293-6E cells were seeded at 1.710.sup.6 cells/mL in Freestyle F17 medium supplemented with 2 mM L-Glutamine. The DNA for every mL of the final production volume was prepared by adding DNA and PEI separately to 50 L medium without supplement. Both fractions were mixed, vortexed and rested for 15 minutes, resulting in a DNA:PEI ratio of 1:2.5 (1 g DNA/mL cells). The cells and DNA/PEI mixture were put together and then transferred into an appropriate container which was placed in a shaking device (37 C., 5% CO.sub.2, 80% RH). After 24 hours, 25 L of Tryptone N1 was added for every mL of final production volume.
(219) After 7 days, cells were harvested by centrifugation and sterile filtered. The antigen binding proteins were purified by an affinity step. For the affinity purification of Fab-based constructs, the supernatant was loaded on a protein CHI column (Thermo Fisher Scientific, #494320005) equilibrated with 6 CV PBS (pH 7.4). After a washing step with the same buffer, the antigen binding protein was eluted from the column by step elution with 100 mM Citric acid (pH 3.0). The fractions with the desired antigen binding protein were immediately neutralized by 1 M Tris Buffer (pH 9.0) at 1:10 ratio, then pooled, dialyzed and concentrated by centrifugation.
(220) After concentration and dialysis against PBS buffer, content and purity of the purified proteins were assessed by SDS-PAGE and size-exclusion HPLC. After expression in HEK293-6E cells, the proteins were purified by a single capture step and analyzed by analytical size exclusion chromatography.
Example 10Characterization of Hits
(221) Phylogenetic analysis of the selected HLA-A2/MAGE-A4 binding hits originating from the rabbit immunization libraries was performed using the Maximum Likelihood method based on a Jones-Taylor-Thornton (JTT) model (MEGAX software). Sequence diversity of the selected binders is depicted in
(222) All available hits were evaluated for their ability to bind MAGE-A4/HLA-A2 complex and a control peptide/HLA-A2 complex in a direct binding ELISA assay. The control peptide/HLA-A2 complex in this assay comprised an HLA-A2 complex loaded with a mixture of 49 unrelated peptides, as recited in Table 9 (SEQ ID NOs: 268-316). Briefly, 96 well ELISA plates were coated with purified human MAGE-A4/HLA-A2 complex or control HLA-A2 complex. Serial dilutions of antibody molecules were added to the plate and detected by an anti-kappa light chain-HRP (Invitrogen) followed by goat anti-rabbit IgG (H+L) HRP (Southern Biotech). Binders were considered for further characterization when showing high binding to MAGE-A4/HLA-A2 complex and no binding to control peptide/HLA-A2 complex. Binding of M0700-M0710 and M0762-M0766 to HLA-A2/MAGE-A4 complex, as determined by ELISA, is shown in
(223) Binding of the specific antibodies M0709 and M0763 to the MAGE-A4 peptide-HLA-A2 complex presented on cells was determined. Briefly, T-B hybrid T2 cells were incubated with serum-free RPMI1640 medium containing MAGE-A4 or control peptides. Control peptides constituted sequences with high identity to MAGE-A4 and had previously been identified in healthy human tissues, i.e., Ctrl.1 (GLADGRTHTV; SEQ ID NO: 317), Ctrl.2 (GLYDGPVHEV; SEQ ID NO: 318) and Ctrl.3 (GVFDGLHTV; SEQ ID NO: 319) (US20180171024, incorporated herein by reference). Peptide loading efficiency was verified by using the ratio between median fluorescent intensity (MFI) of HLA-A2-binding antibody BB7.2 on peptide loaded T2 cells and MFI of unloaded T2 cells (>1). T2 cells were incubated with each of the specific antibodies followed by fluorophore-labeled detection antibodies (anti-kappa light chain or anti-Flag). The cells were fixed and fluorescence was measured by flow cytometry. Binding and specificity of the selected antibodies M0709 and M0763 to the T2 cells displaying MAGE-A4 or control peptides 1, 2 and 3 is presented in
Example 11Optimization of Rabbit Immunization Libraries Leads to Higher Library Diversity
(224) Immunization of rabbits with a different specific pMHC complex was performed as described previously (Example 2). Constructed libraries were optimized using the methods described herein. Biopanning of the native and optimized kappa libraries was conducted according to the method described in Example 8. Monoclonal phage ELISA was performed with phages displaying scFvs from the native and optimized kappa libraries to identify pMHC-specific hits following the second and third biopanning round. The ratio of the signal from the specific target binding to the unspecific binding was then calculated to find hits binding specifically to the target. 73 and 35 hits from the native and optimized kappa libraries, respectively, were identified and sequenced.
(225) Phylogenetic analysis of the selected alternative pMHC binding hits was performed using the Maximum Likelihood method based on a Jones-Taylor-Thornton (JTT) model (MEGAX software). Sequence diversity of the selected binders originating from the native and optimized kappa libraries is depicted in
Example 12Redirected T Cell Killing of Antigen-Positive and -Negative Cell Lines Using pHLA-Targeting Bispecific Antibodies
(226) Redirected T cell killing of tumor cell lines by peptide-HLA (pHLA) targeting bispecific antibodies was determined by endpoint cytotoxicity measurements (LDH release) and real-time imaging (IncuCyte).
(227) The Lactate Dehydrogenase release assay was performed. Briefly, target cells were co-cultured with effector cells (e.g., PBMCs) at an ET ratio of about 10:1. Solutions of the CDR4-bispecific 01 antibody, M0719 covering a concentration range from 0.4 nM to 40 nM were added to the relevant wells. Cytotoxicity was quantified by colorimetric absorbance measurements of the amount of LDH released from damaged cells into the medium after 48 h. The analysis was performed on HLA-A2 expressing antigen-positive cell lines (e.g., A375 (melanoma), U20S (osteosarcoma), SCaBER (bladder carcinoma) and NCI-H1703 (non-small cell lung adenocarcinoma). The obtained data is presented in
(228) Moreover, CDR4-bispecific 01 was also tested in an LDH assay in combination with an immune checkpoint inhibitor pembrolizumab (anti-PD-1 antibody). Briefly, LDH assay was performed as described above. EC50 for cell killing was determined by LDH release after 48 h co-incubation of PBMCs and MAGE-A4 positive cell lines A375, U20S, SCaBER and NCI-H1703 at E:T ratio 10:1 in the presence of MAGE-A4 bispecific 01 (concentrations ranging from 0.078 to 40 nM) with or without 300 nM anti-PD-1 antibody (pembrolizumab). The EC50 values for cell killing by CDR4-bispecific 01 and pembrolizumab with CDR4-bispecific 01 combination were plotted and are shown in
(229) In addition, MAGE-A4 positive/HLA-A2 positive NCI-H1703 cells and MAGE-A4 negative/HLA-A2 positive cells (NCI-H441 (lung adenocarcinoma) and MRC5 (normal human fibroblasts)) were co-incubated with PBMCs (E:T 10:1) and CDR4-bispecific 01 at a concentration of 0.8 nM. Images were recorded with the IncuCyte S3 system for up to 72 h and the respective cytotoxicity is depicted in
(230) TABLE-US-00008 TABLE9 HLAcomplexcontrolpeptides SEQIDNO: PeptideSequence SEQIDNO:268 GVRGRVEEI SEQIDNO:269 AVLDGLLSL SEQIDNO:270 FLYDDNQRV SEQIDNO:271 YMLDLQPETT SEQIDNO:272 ELAGIGILTV SEQIDNO:273 EAAGIGILTV SEQIDNO:274 LLGDLFGV SEQIDNO:275 FLWGPRALV SEQIDNO:276 SLYNTVATL SEQIDNO:277 SLYSYFQKV SEQIDNO:278 GLCTLVAML SEQIDNO:279 GILGFVFTL SEQIDNO:280 VLAGGFFLL SEQIDNO:281 FVGEFFTDV SEQIDNO:282 FLYALALLL SEQIDNO:283 YMDDVVLGV SEQIDNO:284 ALLTSRLRFI SEQIDNO:285 FLPSDFFPSV SEQIDNO:286 KIFGSLAFL SEQIDNO:287 SLLMWITQV SEQIDNO:288 RMFPNAPYL SEQIDNO:289 YMDGTMSQV SEQIDNO:290 VLFGLGFAI SEQIDNO:291 SLPPPGTRV SEQIDNO:292 VLEETSVML SEQIDNO:293 RMPEAAPPV SEQIDNO:294 ILKEPVHGV SEQIDNO:295 KTWGQYWQV SEQIDNO:296 SLLPIMWQL SEQIDNO:297 NLVPMVATV SEQIDNO:298 VLQELNVTV SEQIDNO:299 CINGVCWTV SEQIDNO:300 LMLGEFLKL SEQIDNO:301 VLDFAPPGA SEQIDNO:302 LTLGEFLKL SEQIDNO:303 IMDQVPFSV SEQIDNO:304 CLGGLLTMV SEQIDNO:305 VTEHDTLLY SEQIDNO:306 FLLTKILTI SEQIDNO:307 WLSLLVQFV SEQIDNO:308 LLLLTVLTV SEQIDNO:309 FLLTRILTI SEQIDNO:310 ITDQVPFSV SEQIDNO:311 YMCSFLFNL SEQIDNO:312 ILSLELMKL SEQIDNO:313 YLEYRQVPV SEQIDNO:314 RLPLVLPAV SEQIDNO:315 KLQVFLIVL SEQIDNO:316 YLGSYGFRL