Enhanced anti-HVEM antibodies and uses thereof

Abstract

Antibodies or antigen-binding fragments thereof that bind HVEM, inhibit HVEM-BTLA interaction, and enable binding to LIGHT are provided. Methods of treating diseases using these antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding these antibodies or antigen-binding fragments thereof, and kits comprising these antibodies or antigen-binding fragments thereof are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 169,335, filed April 1, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THEINVENTION The present invention is in the field of immunotherapy. [Background technology]

[0003] 2. Background of the Invention Immunotherapies designed to enhance immune responses are considered as activating immunotherapies and are at the forefront of cancer treatment. Currently, immune checkpoint blockade therapy has been successfully used to treat advanced non-small cell lung cancer (NSCLC), metastatic melanoma, advanced renal cell carcinoma (RCC), metastatic urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), MSI-high tumors, Merkel cell carcinoma and many others. Several such drugs developed and manufactured by different companies, including antibodies against immune checkpoints programmed cell death protein 1 receptor (PD1), programmed cell death protein 1 ligand (PDL1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), have shown great promise in different cancer types and have been approved by the FDA. Patient response rates, however, are still suboptimal, as only 10%-40% of treated patients usually benefit, and at the same time, patients may suffer from side effects of post-immunotherapy treatment. In addition, treatment with these antibodies or their antigen-binding fragments may induce resistance via upregulation of further immune checkpoints. Although anti-PD1 and anti-CTLA-4 combination therapy in melanoma patients demonstrated a high response rate (60%) compared with single antibodies or their antigen-binding fragments, this combination therapy is also associated with severe treatment-related side effects. Therefore, there is a clear need for new anti-tumor immune-activating antibodies or their antigen-binding fragments. Summary of the Invention

[0004] Herpes virus entry mediator (HVEM) is a protein found on the surface of various cell types, including hematopoietic and non-hematopoietic cells. HVEM acts as a receptor for canonical TNF-related ligands such as LIGHT and LTα, and therefore functions as a signaling receptor. However, it also functions as a ligand for immunoglobulin (Ig) superfamily molecules such as the inhibitory receptors BTLA and CD160. Thus, bidirectional signaling is possible in the HVEM-mediated signaling network, which can participate in positive or negative immune responses under different circumstances. Dysregulation of this network is involved in the pathogenesis of autoimmune diseases, inflammatory diseases and cancer, making HVEM a target for immunotherapy. There is a great need for antibodies or antigen-binding fragments thereof that can block HVEM inhibitory signaling, particularly via BTLA interactions, while maintaining activation of signaling.

[0005] The present invention provides antibodies or antigen-binding fragments thereof that bind HVEM, inhibit HVEM-BTLA interaction, and inhibit downstream BTLA signaling. Methods of treating diseases using these antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding these antibodies or antigen-binding fragments thereof, and kits containing these antibodies or antigen-binding fragments thereof are also provided.

[0006] According to a first aspect, there is provided an antibody or antigen-binding fragment thereof, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS) and CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 49 (X1IX2X3X4X5X 18 X7X 19 YYADSVX 20 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, and X 18 is any amino acid other than C, X7 is S, Y, G or R, and X 19 is any amino acid other than S or C, and X 20is any amino acid, and CDR-H3 is the amino acid sequence set forth in SEQ ID NO: 18 (AX9X 10 X 11 X 12 X 13 X 14 YX 15 DY), in which X9 is P or S, and X 10 is G or Y, and X 11 is D or R, and X 12 is Y, N, P or S, and X 13 is T or Y, and X 14 is A or N, and X 15 is F, G or Y, CDR-L1 comprises the amino acid sequence (RASQSVSSYLA) set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence (GASSRAT) set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence (QQYGSX 16 PPX 17 T), in which X 16 is S or Y, and X 17 is Y or L; and does not include any of CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 (APGDYTAYFDY), and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 (QQYGSSPPYT).

[0007] According to another aspect, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention and a pharma- ceutically acceptable carrier, excipient, or adjuvant.

[0008] According to another aspect, there is provided a method for treating a disease or condition characterized by HVEM-positive cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention or an antibody or antigen-binding fragment of the invention, thereby treating the disease or condition.

[0009] According to another aspect, there is provided a method for determining the suitability of a subject to be treated by the method of the invention, comprising obtaining a disease sample from a subject and determining the level of HVEM in the sample, wherein positive expression of HVEM indicates that the subject is suitable for the treatment method of the invention.

[0010] According to another aspect, a method for detecting HVEM in a sample is provided, the method comprising contacting the sample with an antibody or antigen-binding fragment of the invention, thereby detecting HVEM.

[0011] In another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the invention is provided.

[0012] According to another aspect, the pharmaceutical composition of the present invention and a.Anti-PD-1 / PD-L1 based immunotherapy; b. A label indicating that the pharmaceutical composition of the invention is for use with anti-PD-1 / PD-L1-based immunotherapy; and c. A secondary detection molecule for detecting at least one antibody or antigen-binding fragment thereof of the present invention. A kit is provided that includes at least one of the following:

[0013] According to some embodiments, the CDR-H2 has the amino acid sequence set forth in SEQ ID NO: 17 (X1IX2X3X4X5X6X7X 21 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, X6 is G, D, S, E, Q or N, X7 is S, Y, G or R, and X 21 is T, A, E, G or N and X8 is E or K.

[0014] According to some embodiments, X 20 is any amino acid other than C or S.

[0015] According to some embodiments, X 20is any non-positively charged amino acid.

[0016] According to some embodiments, X 20 is K or E.

[0017] According to some embodiments, CDR-H2 is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:20 (GINGNGDYTYYADSVKG), SEQ ID NO:21 (AIGGSGSGTYYADSVKG), SEQ ID NO:22 (NIYSNPNRTYYADSVEG), SEQ ID NO:23 (NINGPGNGTYYADSVEG), SEQ ID NO:47 (NIYSNPNRTYYADSVKG), SEQ ID NO:48 (AISGSGGSTYYADSVEG), SEQ ID NO:57 (NIYSNPDRTYYADSVEG), SEQ ID NO:58 ( NIYSNPERTYYADSVEG), SEQ ID NO:59 (NIYSNPGRTYYADSVEG), SEQ ID NO:60 (NIYSNPQRTYYADSVEG), SEQ ID NO:61 (NIYSNPSRTYYADSVEG), SEQ ID NO:62 (NIYSNPNRAYYADSVEG), SEQ ID NO:63 (NIYSNPNREYYADSVEG), SEQ ID NO:64 (NIYSNPNRGYYADSVEG) and SEQ ID NO:65 (NIYSNPNRNYYADSVEG).

[0018] According to some embodiments, the CDR-H3 comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:24 (ASYRNYNYGDY), SEQ ID NO:25 (ASYDPTNYYDY) and SEQ ID NO:26 (ASYRSTNYFDY).

[0019] According to some embodiments, the CDR-L3 comprises an amino acid sequence selected from SEQ ID NO:6 and SEQ ID NO:27 (QQYGSYPPLT).

[0020] According to some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0021] According to some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2, CDR-H3 comprises the amino acid sequences set forth in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5 and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0022] According to some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:27.

[0023] According to some embodiments, the antibody or antigen-binding fragment of the invention comprises: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINGNGDYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 28), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIGGSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 29), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 30), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNINGPGNGTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 31), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 50), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO:51), VQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPDRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 66), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPERTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 67), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPGRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 68), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPQRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 69), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPSRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 70), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRAYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 71), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNREYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 72), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRGYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 73), VQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRNYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 74), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRNYNYGDYWGQGTLVTVSS (SEQ ID NO: 32), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYDPTNYYDYWGQGTLVTVSS (SEQ ID NO: 33), and QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRSTNYFDYWGQGTLVTVSS (SEQ ID NO: 34) The heavy chain comprises a sequence selected from:

[0024] According to some embodiments, the antibody or antigen-binding fragment of the invention comprises: ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8) ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSYPPLTFGQGTKVEIK (SEQ ID NO: 35) The light chain comprises a sequence selected from the group consisting of:

[0025] According to some embodiments, the antibody or antigen-binding fragment of the invention comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 85-103.

[0026] According to some embodiments, the antibody or antigen-binding fragment of the invention comprises a light chain comprising an amino acid sequence selected from SEQ ID NO:104 and SEQ ID NO:105.

[0027] According to some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain comprising SEQ ID NO:97 and a light chain comprising SEQ ID NO:104.

[0028] According to some embodiments, the methods of the invention further comprise inhibiting or blocking a non-HVEM immune checkpoint protein.

[0029] According to some embodiments, the methods of the invention further comprise administering an anti-PD-1 / PD-L1 based immunotherapy.

[0030] According to some embodiments, the methods of the invention further comprise administering adoptive cell therapy.

[0031] According to some embodiments, the adoptive cell therapy comprises adoptive TIL therapy.

[0032] According to some embodiments, adoptive cell therapy involves administering immune cells that express a chimeric antigen receptor (CAR), where the CAR targets a non-HVEM protein on the surface of an HVEM-expressing cell.

[0033] According to some embodiments, the disease or condition is HVEM-positive cancer or precancerous lesion.

[0034] In some embodiments, the disease or condition is an infectious disease and the infected cells comprise HVEM expression.

[0035] According to some embodiments, positive expression of HVEM comprises elevated levels of HVEM compared to a healthy sample or a predefined threshold.

[0036] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0037] [Figure 1] Figure 1: Photographs of SDS-PAGE of various purified antibodies under non-reducing and reducing conditions. The heavy chain band of the H4 antibody migrates higher due to glycosylation, which is lost in three mutant antibodies in which the NXS / T glycosylation sites have been removed. [Diagram 2] FIG. 2: Bar graph of binding to hHVEM-coated plates by two antibodies of the invention (H4 and Par-K64E), as well as an isotype control as a negative control and a parental antibody as a positive control. [Diagram 3] Figures 3A-3D: (3A-3B) Biacore (3A) multi-cycle kinetics and (3B) single-cycle kinetics raw sensorgrams and 1:1 model fitted curves for binding to hHVEM of (3A) SEC purified antibodies H4, H4 T57A, H4 T57G, H4 T57N and parental antibodies, and (3B) H4 N55G, H4 N55S, H4 N55D, H4 N55Q, H4N55E, H4T57E supernatants and parental antibodies. (3C) Histogram overlay of binding of the four antibodies to hHVEM on the CHO cell surface. (3D) Bar graph of hHVEM expression on CHO cells detected by flow cytometry using parental or H4 T57A antibodies at various concentrations. [Figure 4]4A-4H: (4A-4E) Plates coated with recombinant hHVEM in the presence of (4A) parental, H4 T57A, H4 T57G and H4 T57N anti-HVEM antibodies, (4B) parental, H4 T57A, H4 T57G and H4 T57N anti-HVEM antibodies and recombinant hBTLA, (4C) parental, H4 T57A, H4 T57G and H4 T57N anti-HVEM antibodies and recombinant hLIGHT, (4D) parental, H4 and Par-K64E anti-HVEM antibodies and recombinant hBTLA, (4E) parental, H4 and Par-K64E anti-HVEM antibodies and recombinant hLIGHT, or (4F-4G) (4F) parental, H4 T57A, H4 T57G and H4 T57N anti-HVEM antibodies and (4G) parental, H4 Bar graphs of absorbance readings at 450 nm and 570 nm of ELISA on recombinant cHVEM-coated plates in the presence of T57A, H4 T57G and H4 T57N anti-HVEM antibodies and recombinant cBTLA. In 4A and 4F, detection of antibody. In 4B, 4D and 4G, detection of BTLA. In 4C and 4E, detection of LIGHT. (4H) Line graph of absorbance at increasing concentrations of antibody in the presence of recombinant hBTLA. [Diagram 5] Figures 5A-5B: Line graphs of specific cell death of melanoma cells co-cultured with TILs after preincubation with (5A) anti-HVEM antibody or (5B) anti-HVEM antibody in combination with anti-PD-1 antibody. **=p<0.01, ***=p<0.001. [Figure 6] Figures 6A-6B: Line graphs of CD107a positive TILs co-cultured with (6A) the first HVEM positive melanoma cell line, M-001, and (6B) the second HVEM positive melanoma cell line, M-004. *=p<0.05, **=p<0.01, ***=p<0.001. [Figure 7] 7A-7B: Line graphs of specific cell death of primary ovarian cancer cells co-cultured with autologous PBMCs in the presence of (7A) anti-HVEM or anti-PD1 antibodies, or (7B) anti-HVEM antibody in combination with anti-PD-1 antibody. [Figure 8]Figure 8: Photomicrographs (magnification 63x, zoom 1.5) of FFPE CHO cells overexpressing hHVEM fluorescently stained with parental (left) or H4 T57A (right) antibodies and secondary Cy3 antibody (red fluorescence). Nuclei are counterstained blue with DAPI. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention In some embodiments, the present invention provides antibodies or antigen-binding fragments thereof that bind HVEM, inhibit HVEM-BTLA interaction, and do not significantly inhibit HVEM-LIGHT interaction. The present invention further relates to methods of treating HVEM-positive disease in a subject in need thereof by administering these antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding these antibodies or antigen-binding fragments thereof, and kits comprising these antibodies or antigen-binding fragments thereof.

[0039] It is well known that HVEM-positive cancers can evade immune surveillance by binding BTLA on the surface of immune cells. BTLA binding generates an inhibitory signal in immune cells, which reduces T cell activation and increases cancer survival. It is known that this signaling can be inhibited by antibodies that bind HVEM or that bind BTLA. International Patent Publication WO2020222235, the entirety of which is incorporated herein by reference, provides antibodies that specifically bind HVEM and do not inhibit the binding of other HVEM ligands, such as LIGHT. HVEM is also expressed on the surface of immune cells, where it plays both inhibitory and activating roles depending on the context. These antibodies block HVEM-BTLA interaction by binding to HVEM on pathogenic cells, thus not only relieving immune cells from inhibition, but also allowing HVEM to bind activating ligands (e.g., LIGHT). The present invention is based on the discovery of antibodies that are superior to those presented in WO2020222235. The antibodies of the present invention have higher potency (lower K D ) to bind to HVEM.

[0040] According to a first aspect, there is provided an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS) and CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 17 (X1IX2X3X4X5X6X7X 21 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, X6 is G, D, S, E, Q or N, X7 is S, Y, G or R, and X 21 is T, A, E, G or N, and X8 is E or K, and CDR-H3 is the amino acid sequence set forth in SEQ ID NO: 18 (AX9X 10 X 11 X 12 X 13 X 14 YX 15 DY), in which X9 is P or S, and X 10 is G or Y, and X 11 is D or R, and X 12 is Y, N, P or S, and X 13 is T or Y, and X 14 is A or N, and X 15 is F, G or Y, CDR-L1 comprises the amino acid sequence (RASQSVSSYLA) set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence (GASSRAT) set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence (QQYGSX 16 PPX 17 T), in which X 16 is S or Y, and X 17 is Y or L.

[0041] According to another aspect, an antibody or antigen-binding fragment thereof is provided, comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS) and CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 49 (X1IX2X3X4X5X18 X7X 19 YYADSVX 20 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, and X 18 is any amino acid other than C, X7 is S, Y, G or R, and X 19 is any amino acid other than S or C, and X 20 is any amino acid, and CDR-H3 is the amino acid sequence set forth in SEQ ID NO: 18 (AX9X 10 X 11 X 12 X 13 X 14 YX 15 DY), in which X9 is P or S, and X 10 is G or Y, and X 11 is D or R, and X 12 is Y, N, P or S, and X 13 is T or Y, and X 14 is A or N, and X 15 is F, G or Y, CDR-L1 comprises the amino acid sequence (RASQSVSSYLA) set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence (GASSRAT) set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence (QQYGSX 16 PPX 17 T), in which X 16 is S or Y, and X 17 is Y or L.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof binds to HVEM. In some embodiments, the antibody or antigen-binding fragment thereof has superior HVEM binding compared to antibodies known in the art. In some embodiments, the antibody known in the art is a parent antibody. In some embodiments, the antibody known in the art is an antibody comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), where CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6 (QQYGSSPPYT). Those skilled in the art will appreciate that there are multiple ways to calculate CDRs. The heavy chain CDRs of SEQ ID NOs: 1-3 and the light chain CDRs of SEQ ID NOs: 4-6 are numbered according to the KABAT numbering system. In some embodiments, an antibody known in the art comprises the following CDRs: CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 11 (GFTFSSYA), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 12 (ISGSGGST), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 13 (AKAPGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 14 (QSVSSY), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 15 (GAS), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 16 (QQYGSSPPYT). The heavy chain CDRs of SEQ ID NOs: 11-13 and the light chain CDRs of SEQ ID NOs: 14-16 are numbered according to the IMGT numbering system. In some embodiments, an antibody known in the art is an antibody that comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 7 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, an antibody known in the art is an antibody having a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.

[0043] In some embodiments, better binding is stronger binding. In some embodiments, better binding is binding with a higher affinity. In some embodiments, better binding is binding with a lower K D In some embodiments, the antibody or antigen-binding fragment thereof is an anti-HVEM antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody that blocks HVEM. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between HVEM and BTLA. In some embodiments, the antibody or antigen-binding fragment thereof has a better blocking ability than antibodies known in the art. In some embodiments, better is stronger. In some embodiments, better is longer lasting. In some embodiments, better is more specific. In some embodiments, the antibody or antigen-binding fragment thereof activates downstream signaling through HVEM. In some embodiments, the antibody or antigen-binding fragment thereof has a better activation than antibodies known in the art. In some embodiments, the antibody or antigen-binding fragment thereof inhibits downstream signaling through BTLA. In some embodiments, the antibody or antigen-binding fragment thereof has a better inhibitory effect than antibodies known in the art. In some embodiments, the antibody or antigen-binding fragment thereof activates downstream signaling through HVEM and inhibits downstream signaling through BTLA. In some embodiments, inhibition of the interaction between HVEM and BTLA inhibits downstream signaling through BTLA.

[0044] In some embodiments, the HVEM is mammalian HVEM. In some embodiments, the HVEM is rodent HVEM. In some embodiments, the HVEM is monkey HVEM. In some embodiments, the HVEM is human HVEM. In some embodiments, the HVEM is any one of mouse, monkey and human HVEM. In some embodiments, the HVEM is membrane-bound HVEM. In some embodiments, the HVEM is HVEM on a cell. In some embodiments, the HVEM is HVEM on the cell surface. In some embodiments, the HVEM is soluble HVEM.

[0045] In some embodiments, the cell is a pathogenic cell. In some embodiments, the cell is a cancerous cell. In some embodiments, the cell is a pathogen cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a pathogen-infected eukaryotic cell. In some embodiments, the cell is a bacterially-infected cell. In some embodiments, the cell is a virally-infected cell. In some embodiments, the pathogen is selected from a bacterium, a virus, and a fungus.

[0046] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8 positive T cell. In some embodiments, the T cell is a cytotoxic CD8 positive T cell. In some embodiments, the T cell is a CD4 positive T cell. In some embodiments, the T cell is a CD4 positive helper T cell. In some embodiments, the T cell is selected from a CD8 positive T cell and a CD4 positive T cell. In some embodiments, the T cell is a CD8 positive T cell, a CD4 positive T cell, or both. In some embodiments, the immune cell is a gamma / delta T cell. In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL). In some embodiments, the immune cell is not a peripheral blood immune cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a myeloid-derived suppressor cell (MDSC). In some embodiments, the cell is selected from a T cell, a B cell, a NK cell, a neutrophil, a dendritic cell, an MDSC, and a macrophage. In some embodiments, the cell is selected from a T cell, a B cell, a NK cell, a neutrophil, a dendritic cell, and a macrophage.

[0047] In some embodiments, the immune cells are immune cells expressing a chimeric antigen receptor (CAR). In some embodiments, the CAR is a CAR-T cell. In some embodiments, the CAR is a CAR-NK cell. As used herein, the term "CAR" refers to an engineered receptor that has specificity for at least one protein of interest (e.g., a protein expressed by an HVEM-expressing cell) and is engrafted into an immune effector cell (such as a T cell or an NK cell). In some embodiments, the CAR-T cell has the specificity of a monoclonal antibody engrafted into a T cell. In some embodiments, the CAR-NK cell has the specificity of a monoclonal antibody engrafted into an NK cell. In some embodiments, the T cell is selected from a cytotoxic T lymphocyte and a regulatory T cell. MART1 is an example of a target protein that is co-expressed with HVEM on a target cell. In some embodiments, the CAR targets a protein expressed by an HVEM-expressing cell. In some embodiments, the protein is not HVEM. In some embodiments, the CAR targets a protein on the surface of a cell expressing HVEM. In some embodiments, the protein is an antibody. In some embodiments, the CAR targets an antibody. In some embodiments, the CAR targets an antibody of the invention. In some embodiments, the CAR targets a cytotoxic antibody. In some embodiments, the CAR targets an antibody constant domain. In some embodiments, the CAR targets an Fc domain. In some embodiments, the CAR therapy further comprises administering an antibody that targets cells expressing HVEM. In some embodiments, the antibody targeted by the CAR is not an antibody of the invention.

[0048] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target and are cytotoxic to proteins that are bound by receptors. In some embodiments, CAR-T or CAR-NK cells target at least one cancer protein. In some embodiments, CAR-T or CAR-NK cells target multiple cancer proteins.

[0049] The construction of CAR-T cells is well known in the art. In one non-limiting example, a monoclonal antibody against a cancer protein can be made, and then a vector encoding the antibody is constructed. The vector also includes a costimulatory signal region. In some embodiments, the costimulatory signal region includes an intracellular domain of a known T cell or NK cell stimulatory molecule. In some embodiments, the intracellular domain is selected from at least one of the following: CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD38. In some embodiments, the vector also includes a CD3Z signaling domain. This vector is then transfected into T cells, for example, by lentiviral infection.

[0050] In some embodiments, HVEM is tumor necrosis factor receptor superfamily member 14 (TNFRSF14). In some embodiments, HVEM is CD270. In some embodiments, HVEM is a receptor for BTLA. In some embodiments, HVEM is a ligand for BTLA. In some embodiments, BTLA is CD272. In some embodiments, HVEM is a receptor for tumor necrosis factor superfamily member 14 (TNFSF14). In some embodiments, HVEM is a ligand for TNFSF14. In some embodiments, TNFSF14 is LIGHT. In some embodiments, TNFSF14 is CD258. In some embodiments, HVEM is a receptor for CD160. In some embodiments, HVEM is a ligand for CD160. In some embodiments, HVEM is a receptor for lymphotoxin alpha (LTα). In some embodiments, HVEM is a ligand for LTα. In some embodiments, LTα is TNF-β. In some embodiments, HVEM is a receptor for SALM5. In some embodiments, HVEM is a ligand for SALM5.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to HVEM. In some embodiments, the antibody or antigen-binding fragment thereof does not bind any other protein other than HVEM. In some embodiments, the antibody or antigen-binding fragment thereof binds the extracellular domain of HVEM. In some embodiments, the antibody or antigen-binding fragment thereof binds at the ligand-binding domain of HVEM. In some embodiments, the antibody or antigen-binding fragment thereof binds at the BTLA-binding domain of HVEM. In some embodiments, the antibody or antigen-binding fragment thereof occludes the BTLA-binding domain of HVEM. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and BTLA. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between HVEM and BTLA. In some embodiments, the antibody or antigen-binding fragment thereof inhibits HVEM-mediated and BTLA-induced immunosuppression. In some embodiments, the antibody or antigen-binding fragment thereof binds HVEM and prevents the bound HVEM from further binding BTLA. In some embodiments, the antibody or antigen-binding fragment thereof inhibits downstream signaling via BTLA, hi some embodiments, the antibody or antigen-binding fragment thereof reduces downstream signaling via BTLA.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit the interaction between HVEM and TNFSF14. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and TNFSF14. In some embodiments, the TNFSF14 is membrane TNFSF14 (mTNFS14). In some embodiments, the TNFSF14 is soluble TNFSF14 (sTNFS14). In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit the interaction between one of mTNFS14 and sTNFS14 and HVEM, but inhibits the interaction with the other. In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit the interaction between both mTNFS14 and sTNFS14 and HVEM. In some embodiments, the inhibition is a substantial inhibition. In some embodiments, the inhibition is at least a 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99, or 100% reduction. Each possibility represents a separate embodiment of the invention. In some embodiments, the antibody or antigen-binding fragment thereof does not block the interaction between HVEM and TNFSF14. In some embodiments, the antibody or antigen-binding fragment thereof does not substantially block the interaction between HVEM and TNFSF14. In some embodiments, the antibody or antigen-binding fragment thereof does not block / inhibit TNFSF14-mediated signaling. In some embodiments, the antibody or antigen-binding fragment thereof does not block / inhibit TNFSF14-mediated cell survival.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof activates signaling through HVEM. In some embodiments, the antibody or antigen-binding fragment thereof induces superior activation compared to antibodies known in the art. In some embodiments, the antibody or antigen-binding fragment thereof is an HVEM agonist. In some embodiments, the antibody or antigen-binding fragment thereof is a superior agonist compared to antibodies known in the art. In some embodiments, the antibody or antigen-binding fragment thereof induces signaling through HVEM. In some embodiments, the signaling through HVEM is HVEM downstream signaling. In some embodiments, the antibody or antigen-binding fragment thereof binds HVEM on a cell and activates / induces HVEM signaling in the cell. In some embodiments, the HVEM signaling comprises activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) signaling. In some embodiments, the NF-κB signaling comprises regulation of genes with NF-κB response elements in their promoters. In some embodiments, the signaling comprises increased cytotoxicity of the cell. In some embodiments, the signaling comprises increased cytotoxicity of cells expressing HVEM contacted by the antibody or antigen-binding fragment thereof. In some embodiments, the increased cytotoxicity comprises increased secretion of inflammatory cytokines. In some embodiments, the inflammatory cytokines are selected from IL-1, IL-1B, IL-4, IL-6, TNFα, IFNγ, MCP1, IL-12, IL-18, IL-23, and CM-CSF. In some embodiments, the inflammatory cytokine is IFNγ. In some embodiments, the increase is compared to cells not contacted by an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the increase is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500% increase. Each possibility represents a separate embodiment of the present invention.

[0054] In some embodiments, the cell is an immune cell and the signaling comprises immune activation. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a CD8+ T cell. In some embodiments, the immune cell is a CD4+ T cell. In some embodiments, the immune cell is not a gamma / delta T cell. In some embodiments, the immune cell is a gamma / delta T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the activation of HVEM signaling comprises activation of the immune cell. In some embodiments, the immune activation comprises increased proliferation. In some embodiments, the immune activation comprises increased cytotoxicity. In some embodiments, the immune activation comprises increased migration. In some embodiments, the immune activation comprises increased homing. In some embodiments, the immune activation comprises increased cell clustering. In some embodiments, the immune activation is T cell activation. In some embodiments, the immune activation comprises an increase in the number of Th1 T cells. In some embodiments, the increase is a relative increase compared to Th2 T cells. In some embodiments, immune activation comprises an increase in CD8+ T cells. In some embodiments, immune activation comprises a decrease in regulatory T cells. In some embodiments, immune activation comprises increased expression of a marker selected from 41BB, CD69, CD25, CD107a, HLA-DR, and secretion of a cytokine. In some embodiments, the cytokine is a proinflammatory cytokine. In some embodiments, T cell activation comprises increased T cell clustering.

[0055] In some embodiments, the cells are cancerous cells and the signaling comprises an anti-tumor effect. In some embodiments, the anti-tumor effect comprises increased apoptosis. In some embodiments, the anti-tumor effect comprises decreased proliferation. In some embodiments, the anti-tumor effect comprises increased chemotherapy sensitivity. In some embodiments, the anti-tumor effect comprises decreased motility. In some embodiments, the anti-tumor effect comprises decreased invasion. In some embodiments, the anti-tumor effect comprises decreased metastasis. In some embodiments, the anti-tumor effect comprises decreased self-renewal. In some embodiments, the effect is compared to cancer cells not contacted by an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the reduction is at least a 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 95, 97, 99, or 100% reduction. Each possibility represents a separate embodiment of the present invention.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof does not induce apoptosis. In some embodiments, the induction of apoptosis is direct apoptosis induction. In some embodiments, the antibody or antigen-binding fragment thereof does not directly induce apoptosis. As used herein, "direct induction" refers to a result that occurs immediately as a result of binding of the antibody or antigen-binding fragment thereof and is not characterized by downstream signaling in the bound cell. In some embodiments, the antibody or antigen-binding fragment thereof is not cytotoxic. In some embodiments, the antibody or antigen-binding fragment thereof is not cytotoxic per se. In some embodiments, the antibody or antigen-binding fragment thereof does not induce antibody-directed cell cytotoxicity (ADCC). In some embodiments, the antibody or antigen-binding fragment thereof does not induce complement-dependent cytotoxicity (CDC). In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a cytotoxic moiety. In some embodiments, the antibody or antigen-binding fragment thereof does not induce apoptosis of a cell expressing HVEM upon binding of HVEM expressed by the cell. In some embodiments, the antibody or antigen-binding fragment thereof does not induce apoptosis through interaction with another cell. In some embodiments, the antibody or antigen-binding fragment thereof does not directly induce death of a cell expressing HVEM. In some embodiments, the antibody or antigen-binding fragment thereof does not target the cell for killing. In some embodiments, the antibody or antigen-binding fragment thereof induces apoptosis indirectly. In some embodiments, indirect apoptosis is apoptosis induced by downstream signaling through the HVEM receptor that results in apoptosis. In some embodiments, indirect apoptosis is apoptosis that requires signaling. In some embodiments, indirect apoptosis is apoptosis that does not require the involvement of a second cell. In some embodiments, the antibody or antigen-binding fragment thereof does not induce apoptosis in immune cells. In some embodiments, the antibody or antigen-binding fragment thereof induces apoptosis in cancer cells.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and CD160. In some embodiments, the antibody or antigen-binding fragment thereof induces superior inhibition compared to antibodies known in the art. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and LTα. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and herpes simplex virus type 1 glycoprotein D (HSV1-gD). In some embodiments, the antibody or antigen-binding fragment thereof inhibits the interaction between HVEM and at least two of CD160, HSV1-gD, and LTα. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between HVEM and CD160. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between HVEM and LTα. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between HVEM and HSV1-gD. In some embodiments, the antibody or antigen-binding fragment thereof blocks the interaction between at least two of CD160, HSV1-gD, and LTα and HVEM. In some embodiments, the antibody or antigen-binding fragment thereof inhibits / blocks the interaction between all of CD160, HSV1-gD, and LTα and HVEM. In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit or block the interaction between at least one of CD160, HSV1-gD, and LTα and HVEM.

[0058] In some embodiments, the antibody or fragment thereof is a fab fragment. In some embodiments, the antibody or fragment thereof is a single chain antibody (scFv). In some embodiments, the antibody or fragment thereof is a single domain antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a blocking antibody. In some embodiments, the antibody is an affinity matured antibody.

[0059] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that includes at least one binding domain formed from the folding of polypeptide chains with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant characteristics of the antigen. Antibodies typically have a tetrameric form, including two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelid, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic and antibodies and fragments that may be labeled in soluble or conjugated form, including epitope-binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies may be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single chain antibodies (svFC), dimeric variable regions (diabody y) and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further recognize that other fusion products may be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) to Fc fusions, and scFv-scFv-Fc fusions.

[0060] The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the antibody comprises an IgG2 or IgG4. In some embodiments, the antibody comprises an IgG2. In some embodiments, the antibody comprises an IgG4. In some embodiments, the antibody comprises an IgG1. In some embodiments, the antibody comprises an IgG3. In some embodiments, the antibody comprises a modified IgG1 or IgG3 with reduced toxicity.

[0061] The basic unit of naturally occurring antibody structure is a heterotetrameric glycoprotein complex of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains linked together by both non-covalent and disulfide bonds. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. There are five human antibody classes (IgG, IgA, IgM, IgD, and IgE), and within these classes, various subclasses are recognized based on structural differences such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in the length and sequence of the chains. The class and subclass of an antibody is its isotype.

[0062] The amino-terminal regions of the heavy and light chains are more diverse in sequence than the carboxy-terminal regions and are therefore called the variable domains. This part of the antibody structure confers the antigen-binding specificity of the antibody. The heavy variable (VH) domain and the light variable (VL) domain together form a single antigen-binding site, so that a basic immunoglobulin unit has two antigen-binding sites. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber, (1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

[0063] The carboxy-terminal portions of the heavy and light chains form the constant domains, namely CH1, CH2, CH3, and CL. There is much less diversity in these domains, but there are differences between animal species, and moreover, within the same individual there are several different antibody isotypes, each with a different function.

[0064] The term "framework region" or "FR" refers to amino acid residues in the variable domain of an antibody, other than those of the hypervariable region as defined herein. The term "hypervariable region" as used herein refers to amino acid residues in the variable domain of an antibody that are responsible for antigen binding. A hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". The CDRs are primarily responsible for binding to an epitope of an antigen. The extent of FRs and CDRs has been precisely defined (see Kabat et al.). In some embodiments, the positions of the CDRs are determined by a numbering system. In some embodiments, the positions of the CDRs are determined by the EU numbering system.

[0065] Immunoglobulin variable domains can also be analyzed using the IMGT information system (www: / / imgt.cines.fr / ) (IMGT® / V-Quest) to identify variable region segments that contain the CDRs. See, e.g., Brochet, X. et al., Nucl. Acids Res. J6:W503-508 (2008).

[0066] As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to human antibodies. A humanized antibody may be produced by the production of recombinant DNA encoding the CDRs of a non-human antibody surrounded by sequences similar to human antibodies. In some embodiments, a humanized antibody is a chimeric antibody. In some embodiments, humanization involves the insertion of the CDRs of the invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art, and any method of producing an antibody that retains the CDRs of the invention may be employed.

[0067] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during the generation of the monoclonal antibody, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as being produced by any particular method of preparation. The monoclonal antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0068] The mAbs of the invention can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, or IgA. Hybridomas producing mAbs can be cultured in vitro or in vivo. High titer mAbs can be obtained by in vivo production, in which cells from individual hybridomas are injected intraperitoneally into pristine-primed Balb / c mice to produce ascites fluid containing high concentrations of the desired mAb. mAbs of isotype IgM or IgG can be purified from such ascites fluid or from culture supernatants using column chromatography methods well known to those skilled in the art.

[0069] "Antibody fragment" includes a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDbs), linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); one-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFv)4-Fc, di-scFv, bi-scFv, or tandem (di, tri)-scFv); and bispecific T cell engagers (BiTEs).

[0070] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0071] An "Fv" is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. The three faces of the VH-VL dimer are in this configuration. Together, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only the three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0072] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0073] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0074] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, delta, e, gamma, and micro, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0075] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general description of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994).

[0076] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, generating two antigen binding sites. Diabody production is known in the art and described in Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0077] The monoclonal antibodies of the present invention may be prepared using methods well known in the art, including a variety of techniques such as those of Kohler, G. and Milstein, C, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0078] In addition to the traditional methods of making antibodies in vivo, antibodies can be made in vitro using phage display technology. Such recombinant antibody production is much faster compared to traditional antibody production, and they can be made against a huge number of antigens. Moreover, using traditional methods, many antigens have been found to be non-immunogenic or highly toxic, and therefore cannot be used to make antibodies in animals. Moreover, affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, a large number of different antibodies against a specific antigen can be made in one selection procedure. To make recombinant monoclonal antibodies, a large pool of antibodies with different antigen recognition sites can be made using all the different methods based on display libraries. Such libraries can be made in several ways: synthetic repertoires can be made by cloning synthetic CDR3 regions into a pool of heavy chain germline genes, thus making a large antibody repertoire, from which recombinant antibody fragments with different specificities can be selected. A pool of human lymphocytes can be used as starting material for the construction of antibody libraries. It is possible to construct a naive repertoire of human IgM antibodies and thus generate a highly diverse human library. This method has been successfully and widely used to select large numbers of antibodies against different antigens. Protocols for the construction of bacteriophage libraries and the selection of recombinant antibodies are described in the well-known reference Current Protocols in Immunology, Colligan et al. (eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

[0079] In some embodiments, antibodies and portions thereof include, but are not limited to, antibodies, antibody fragments, Fab and F(ab')2, single domain antigen-binding recombinant fragments, and natural nanobodies. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, scFv, scFv2, or scFv4 fragments.

[0080] In some embodiments, the invention provides nucleic acid sequences encoding an antibody or antigen-binding portion of the invention.

[0081] For example, a polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (VH), but also a heavy chain constant region (CH), which typically includes three constant domains, CH1, CH2, and CH3; and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0082] Other polypeptides that may be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and CHI, where the CK or CL domains have been truncated. Because minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic applications, but because they are bivalent, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context dictates otherwise, the term "antibody" as used herein encompasses whole antibody molecules as well as antigen-binding antibody fragments of the types discussed above. Each framework region present in the encoded polypeptide may contain at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the acceptor framework region. Some reasonable substitutions will be recognized by those skilled in the art, taking into account the properties of the individual amino acids that comprise the disclosed protein products. Amino acid substitutions, or "conservative substitutions," may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0083] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0084] As used herein, the term "non-naturally occurring" material, composition, entity, and / or any combination of materials, compositions, or entities, or grammatical variations thereof, is a qualified term that expressly excludes, but only excludes, those forms of material, composition, or entity, and / or any combination of materials, compositions, or entities that are well understood by those of skill in the art to be "naturally occurring" or that have been determined or interpreted, or may be determined or interpreted at any time, as "naturally occurring" by a judge or administrative or judicial body.

[0085] In some embodiments, the antibody comprises IgG4. In some embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG2 or IgG4. In some embodiments, the antibody comprises IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody does not comprise IgG1. In some embodiments, the antibody does not comprise IgG3. In some embodiments, the antibody does not comprise IgG1 or IgG3. In some embodiments, the antibody comprises a mutated IgG1 and / or IgG3, wherein the mutation inhibits induction of ADCC, CDC or both. In some embodiments, the mutation is in an FcR gamma binding motif.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof is not an antibody present in WO2020222235. In some embodiments, the antibody or antigen-binding fragment thereof does not include all of CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:1 (SYAMS), CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:5 (GASSRAT), and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:6 (QQYGSSPPYT). In some embodiments, the antibody or antigen-binding fragment thereof does not include all of CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:2, CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:3, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise both a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2 and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof does not include any of CDR-H1 consisting of the amino acid sequence (SYAMS) set forth in SEQ ID NO: 1, CDR-H2 consisting of the amino acid sequence (AISGSGGSTYYADSVKG) set forth in SEQ ID NO: 2, CDR-H3 consisting of the amino acid sequence (APGDYTAYFDY) set forth in SEQ ID NO: 3, CDR-L1 consisting of the amino acid sequence (RASQSVSSYLA) set forth in SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence (GASSRAT) set forth in SEQ ID NO: 5, and CDR-L3 consisting of the amino acid sequence (QQYGSSPPYT) set forth in SEQ ID NO: 6.In some embodiments, the antibody or antigen-binding fragment thereof does not include all of CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:2, CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:3, and CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding fragment thereof does not include both CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:2 and CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the antibody or antigen-binding fragment thereof does not include CDR-H2 consisting of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the antibody or antigen-binding fragment thereof does not include CDR-H3 consisting of the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the antibody or antigen-binding fragment thereof does not include CDR-L3 consisting of the amino acid sequence set forth in SEQ ID NO:6.

[0087] In some embodiments, the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 17 (X1IX2X3X4X5X6X7X 21 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, X6 is G, D, S, E, Q or N, X7 is S, Y, G or R, and X 21 is T, A, E, G or N, and X8 is E or K. In some embodiments, CDR-H2 has the amino acid sequence set forth in SEQ ID NO: 49 (X1IX2X3X4X5X 18 X7X 19 YYADSVX 20 X1 is A, G or N, X2 is S, N, G or Y, X3 is G or S, X4 is S, N or P, X5 is G or P, and X 18 is any amino acid other than C, X7 is S, Y, G or R, and X 19 is any amino acid other than S or C, and X 20 is any amino acid.

[0088] In some embodiments, X 18is any amino acid other than C. In some embodiments, X 18 is selected from any amino acid other than C. In some embodiments, X 18 is X6. In some embodiments, X 18 is G, D, S, or N. In some embodiments, X 18 is any amino acid other than G. In some embodiments, X 18 is selected from any amino acid other than G.

[0089] In some embodiments, X 19 is any amino acid other than C. In some embodiments, X 19 is selected from any amino acid other than C. In some embodiments, X 19 is any amino acid other than S. In some embodiments, X 19 is selected from any amino acid other than S. In some embodiments, X 19 is any amino acid other than C and S. In some embodiments, X 19 is selected from any amino acid other than C and S. In some embodiments, X 19 is any amino acid other than T. In some embodiments, X 19 is selected from any amino acid other than T. In some embodiments, X 19 is T.

[0090] In some embodiments, X 20 is any amino acid. In some embodiments, X 20 is selected from any amino acid. 20 is an amino acid other than K. In some embodiments, X 20 is any amino acid other than C or S. In some embodiments, X 20 is an amino acid other than K, C, or S. In some embodiments, X 20is a non-positively charged amino acid. In some embodiments, the positively charged amino acid is K, R, or H. In some embodiments, the positively charged amino acid is any one of K, R, and H. In some embodiments, the charge is at pH 7.0. In some embodiments, the charge is at neutral pH. In some embodiments, the charge comprises a charged side chain. In some embodiments, X 20 is a negatively charged amino acid. In some embodiments, the negatively charged amino acid is E or D. In some embodiments, the negatively charged amino acid is selected from E and D. In some embodiments, X 20 is K or E. In some embodiments, X 20 is selected from K and E. In some embodiments, X 20 is a negatively charged amino acid or a polar amino acid. 20 is selected from negatively charged amino acids and polar amino acids. In some embodiments, the polar amino acid is Y, S, T, N, or Q. In some embodiments, the polar amino acid is selected from Y, S, T, N, and Q. In some embodiments, X 20 is K. In some embodiments, X 20 is E.

[0091] In some embodiments, X1 is A, G, or N. In some embodiments, X1 is selected from A, G, and N. In some embodiments, X2 is S, N, G, or Y. In some embodiments, X2 is selected from S, N, G, and Y. In some embodiments, X3 is G or S. In some embodiments, X3 is selected from G and S. In some embodiments, X4 is S, N, or P. In some embodiments, X4 is selected from S, N, and P. In some embodiments, X5 is G or P. In some embodiments, X5 is selected from G and P. In some embodiments, X6 is G, D, S, or N. In some embodiments, X6 is selected from G, D, S, and N. In some embodiments, X7 is S, Y, G, or R. In some embodiments, X7 is selected from S, Y, G, and R. In some embodiments, X8 is E or K. In some embodiments, X8 is selected from E and K.

[0092] In some embodiments, CDR-H2 is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:20 (GINGNGDYTYYADSVKG), SEQ ID NO:21 (AIGGSGSGTYYADSVKG), SEQ ID NO:22 (NIYSNPNRTYYADSVEG), SEQ ID NO:23 (NINGPGNGTYYADSVEG), SEQ ID NO:47 (NIYSNPNRTYYADSVKG), SEQ ID NO:48 (AISGSGGSTYYADSVEG), SEQ ID NO:57 (NIYSNPDRTYYADSVEG), SEQ ID NO:58 (N In some embodiments, the CDR-H2 comprises an amino acid sequence selected from SEQ ID NO: 59 (NIYSNPGRTYYADSVEG), SEQ ID NO: 60 (NIYSNPQRTYYADSVEG), SEQ ID NO: 61 (NIYSNPSRTYYADSVEG), SEQ ID NO: 62 (NIYSNPNRAYYADSVEG), SEQ ID NO: 63 (NIYSNPNREYYADSVEG), SEQ ID NO: 64 (NIYSNPNRGYYADSVEG) and SEQ ID NO: 65 (NIYSNPNRNYYADSVEG). In some embodiments, the CDR-H2 comprises SEQ ID NO: 2. In some embodiments, the CDR-H2 consists of SEQ ID NO: 2. In some embodiments, the CDR-H2 comprises SEQ ID NO: 20. In some embodiments, the CDR-H2 consists of SEQ ID NO: 20. In some embodiments, the CDR-H2 comprises SEQ ID NO: 21. In some embodiments, the CDR-H2 consists of SEQ ID NO: 21. In some embodiments, the CDR-H2 comprises SEQ ID NO: 22. In some embodiments, the CDR-H2 consists of SEQ ID NO: 22. In some embodiments, the CDR-H2 comprises SEQ ID NO: 23. In some embodiments, the CDR-H2 consists of SEQ ID NO: 23. In some embodiments, the CDR-H2 is SEQ ID NO: 17. In some embodiments, the CDR-H2 comprises SEQ ID NO: 17. In some embodiments, the CDR-H2 consists of SEQ ID NO: 17. In some embodiments, the CDR-H2 comprises SEQ ID NO: 47. In some embodiments, the CDR-H2 comprises SEQ ID NO: 47. In some embodiments, the CDR-H2 comprises SEQ ID NO: 48. In some embodiments, the CDR-H2 consists of SEQ ID NO: 48. In some embodiments, the CDR-H2 comprises SEQ ID NO: 57.In some embodiments, the CDR-H2 consists of SEQ ID NO:57. In some embodiments, the CDR-H2 comprises SEQ ID NO:58. In some embodiments, the CDR-H2 consists of SEQ ID NO:58. In some embodiments, the CDR-H2 comprises SEQ ID NO:59. In some embodiments, the CDR-H2 consists of SEQ ID NO:59. In some embodiments, the CDR-H2 comprises SEQ ID NO:60. In some embodiments, the CDR-H2 consists of SEQ ID NO:60. In some embodiments, the CDR-H2 comprises SEQ ID NO:61. In some embodiments, the CDR-H2 consists of SEQ ID NO:61. In some embodiments, the CDR-H2 comprises SEQ ID NO:62. In some embodiments, the CDR-H2 consists of SEQ ID NO:62. In some embodiments, the CDR-H2 comprises SEQ ID NO:63. In some embodiments, the CDR-H2 consists of SEQ ID NO:63. In some embodiments, the CDR-H2 comprises SEQ ID NO:64. In some embodiments, the CDR-H2 consists of SEQ ID NO:64. In some embodiments, the CDR-H2 comprises SEQ ID NO:65. In some embodiments, the CDR-H2 consists of SEQ ID NO:65.

[0093] In some embodiments, CDR-H3 comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:24 (ASYRNYNYGDY), SEQ ID NO:25 (ASYDPTNYYDY), and SEQ ID NO:26 (ASYRSTNYFDY). In some embodiments, CDR-H3 comprises SEQ ID NO:3. In some embodiments, CDR-H3 consists of SEQ ID NO:3. In some embodiments, CDR-H3 comprises SEQ ID NO:24. In some embodiments, CDR-H3 consists of SEQ ID NO:24. In some embodiments, CDR-H3 comprises SEQ ID NO:25. In some embodiments, CDR-H3 consists of SEQ ID NO:25. In some embodiments, CDR-H3 comprises SEQ ID NO:26. In some embodiments, CDR-H3 consists of SEQ ID NO:26. In some embodiments, CDR-H3 is SEQ ID NO:18. In some embodiments, CDR-H2 comprises SEQ ID NO:18. In some embodiments, CDR-H2 consists of SEQ ID NO:18.

[0094] In some embodiments, CDR-L3 comprises an amino acid sequence selected from SEQ ID NO:6 and SEQ ID NO:27 (QQYGSYPPLT). In some embodiments, CDR-L3 comprises SEQ ID NO:6. In some embodiments, CDR-L3 consists of SEQ ID NO:6. In some embodiments, CDR-L3 comprises SEQ ID NO:27. In some embodiments, CDR-L3 consists of SEQ ID NO:27. In some embodiments, CDR-L3 is SEQ ID NO:19. In some embodiments, CDR-H2 comprises SEQ ID NO:19. In some embodiments, CDR-H2 consists of SEQ ID NO:19.

[0095] In some embodiments, the CDR is not prone to instability. In some embodiments, the CDR(s) are not prone to instability. In some embodiments, the instability tendency is a tendency to acid instability. In some embodiments, the tendency is a tendency to degradation. In some embodiments, the tendency is a tendency to cleavage. In some embodiments, the tendency is a DP diamino acid. In some embodiments, X 11 X 12 is not DP. In some embodiments, X 11 If D, then X 12 is not P, G, D, S, T or H. In some embodiments, X 11 If D, then X 12is not P. In some embodiments, the propensity is a propensity towards aspartate isomerization. In some embodiments, the propensity is a DG diamino acid. In some embodiments, the propensity is a DT diamino acid. In some embodiments, the propensity is a DS diamino acid. In some embodiments, the propensity is a DD diamino acid. In some embodiments, the propensity is a DH diamino acid. In some embodiments, the propensity is a propensity towards deamination. In some embodiments, the propensity is a NG diamino acid. In some embodiments, the propensity is a NS diamino acid. In some embodiments, the propensity is a NT diamino acid. In some embodiments, the propensity is a NH diamino acid. In some embodiments, the propensity is a NN diamino acid. In some embodiments, the propensity is a propensity towards N-linked glycosylation. In some embodiments, the propensity is NXS / T (SEQ ID NO: 84). In some embodiments, the propensity is NXS. In some embodiments, the propensity is NXT. It will be understood that X in these propensity sequences can be any amino acid. In some embodiments, the propensity is a free cysteine ​​residue. In some embodiments, all CDRs are free of free cysteine ​​residues. In some embodiments, the propensity is oxidation propensity. In some embodiments, the propensity is surface exposed methionine or tryptophan residues. In some embodiments, the propensity is surface exposed methionine residues. In some embodiments, the propensity is surface exposed tryptophan residues. In some embodiments, the CDR comprises a single amino acid change that eliminates the propensity. In some embodiments, the CDR comprises a single amino acid change that eliminates a diamino acid sequence.

[0096] In some embodiments, the CDR-H2 lacks an N-glycosylation site. In some embodiments, the N-glycosylation site comprises NXS / T, where X is any amino acid and the third amino acid is S or T. In some embodiments, the CDR-H2 has SEQ ID NO: 106 (NIYSNPNRX 22 In some embodiments, X 22is any amino acid other than S and T. In some embodiments, CDR-H2 is SEQ ID NO: 107 (NIYSNPNRX 23 In some embodiments, X 23 is any amino acid other than S, T, and C. In some embodiments, CDR-H2 consists of SEQ ID NO: 106. In some embodiments, CDR-H2 consists of SEQ ID NO: 107. In some embodiments, CDR-H2 consists of SEQ ID NO: 112 (NIYSNPX 24 In some embodiments, X 24 is any amino acid other than N. In some embodiments, CDR-H2 is SEQ ID NO: 113 (NIYSNPX 25 In some embodiments, X 25 is any amino acid other than N and C. In some embodiments, CDR-H2 is SEQ ID NO: 114 (NIYSNPX 26 In some embodiments, X 26 is G, D, S, E, Q or N. In some embodiments, CDR-H2 consists of SEQ ID NO: 112. In some embodiments, CDR-H2 consists of SEQ ID NO: 113. In some embodiments, CDR-H2 consists of SEQ ID NO: 114.

[0097] In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:20, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 21, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 22, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:23, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 47, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 48, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 57, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 58, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:59, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 60, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 61, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 62, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 63, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:64, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:65, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0098] In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 24, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 25, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 26, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0099] In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:27.

[0100] In some embodiments, CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, CDR-H3 comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises an amino acid sequence selected from SEQ ID NO:6 and SEQ ID NO:27.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a heavy chain comprising QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7). In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a heavy chain variable region comprising or consisting of SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 7 and a light chain comprising a CDR-L3 comprising SEQ ID NO: 27.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINGNGDYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 28). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIGGSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 29). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 30). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNINGPGNGTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 31). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 50). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 51).In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPDRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 66). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPERTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 67). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPGRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 68). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPQRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 69). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPSRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 70). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRAYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 71).In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNREYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 72). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRGYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 73). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRNYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 74). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRNYNYGDYWGQGTLVTVSS (SEQ ID NO: 32). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYDPTNYYDYWGQGTLVTVSS (SEQ ID NO: 33). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that comprises QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRSTNYFDYWGQGTLVTVSS (SEQ ID NO: 34).In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 28-34, 50-51, and 66-74. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 7, 28-34, 50-51, and 66-74. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 28-34, 50-51, and 66-74. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 7, 28-34, 50-51, and 66-74. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region consisting of a sequence selected from SEQ ID NOs: 28-34, 50-51, and 66-74. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region consisting of a sequence selected from SEQ ID NOs: 7, 28-34, 50-51, and 66-74.

[0103] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a sequence selected from ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPYTFGQGTKVEIK (SEQ ID NO: 8) and ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSYPPLTFGQGTKVEIK (SEQ ID NO: 35). In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of a sequence selected from SEQ ID NO: 8 and SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a sequence selected from SEQ ID NO: 8 and 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region comprising a sequence selected from SEQ ID NO: 8 and 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region consisting of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment comprises a light chain variable region consisting of a sequence selected from SEQ ID NO: 8 and 35.

[0104] In some embodiments, the heavy chain comprises an N-terminal peptide comprising MGWSCIILFLVATATGVHS (SEQ ID NO:36). In some embodiments, the heavy chain comprises an N-terminal peptide consisting of SEQ ID NO:36. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:36. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:36 at the N-terminus of any one of SEQ ID NO:7, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. In some embodiments, the N-terminal peptide is a signal peptide. In some embodiments, the heavy chain comprises a signal peptide. In some embodiments, the heavy chain is free of a signal peptide. In some embodiments, the heavy chain lacks a signal peptide. In some embodiments, the heavy chain comprises a signal peptide when initially expressed in a cell, the signal peptide is cleaved, and the secreted heavy chain lacks the signal peptide. Signal peptides are well known in the art, and any signal peptide that functions to effect secretion of the antibody chain may be used.

[0105] In some embodiments, the heavy chain comprises a C-terminal peptide comprising ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 37). In some embodiments, the heavy chain comprises a C-terminal peptide consisting of SEQ ID NO: 37. In some embodiments, the C-terminal peptide is a heavy chain constant region. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:37. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:37 at the C-terminus of any one of SEQ ID NO:7, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. In some embodiments, the C-terminal peptide is an IgG backbone. In one embodiment, the antigen-binding fragment lacks the C-terminal peptide. In some embodiments, the antigen-binding fragment is devoid of the C-terminal peptide. In some embodiments, the C-terminal peptide is not cytotoxic. In some embodiments, the C-terminal peptide is mutated to reduce cytotoxicity. In some embodiments, the C-terminal peptide comprises an antibody hinge domain. One skilled in the art will appreciate that disulfide bridges present in the hinge domain of the heavy chain are sufficient for heavy chain dimerization. The hinge region itself does not confer cytotoxicity. In some embodiments, the C-terminal peptide comprises a CH1 domain.One skilled in the art will appreciate that a disulfide bridge formed between the CH1 domain of the heavy chain and the CL domain of the light chain is sufficient for heavy-light chain dimerization and formation of a functional antibody or antigen-binding fragment. In some embodiments, the C-terminal peptide comprises a dimerization domain. In some embodiments, the dimerization domain is sufficient to induce dimerization between two heavy chains. In some embodiments, the dimerization domain is sufficient to induce dimerization between a heavy chain and a light chain. In some embodiments, the C-terminal peptide comprises two dimerization domains, a first dimerization domain sufficient to induce dimerization between two heavy chains and a second dimerization domain sufficient to induce dimerization between a heavy chain and a light chain. In some embodiments, the dimerization is by forming a disulfide bond.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof is MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP In some embodiments, the heavy chain comprises the sequence of SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO: 9.SEQ ID NO:9 may also lack the signal peptide (SEQ ID NO:36), which is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP It will be appreciated that the present invention can be practiced to generate the signal peptide APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 85), or can contain a different signal peptide.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSX1IX2X3X4X5X6X7TYYADSVX8GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:54). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO:54. It will be appreciated that SEQ ID NO:54 can also include the signal peptide of the invention (SEQ ID NO:36) or contain a different signal peptide.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof is 18 X7X 19 YYADSVX 20GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:55). In some embodiments, the heavy chain consists of the sequence of SEQ ID NO: 55. It will be understood that SEQ ID NO: 55 can also include the signal peptide of the invention (SEQ ID NO: 36) or contain a different signal peptide.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof is 10 X 11 X 12 X 13 X 14 YX 15In some embodiments, the heavy chain comprises a heavy chain comprising the sequence of SEQ ID NO:56. It will be understood that SEQ ID NO:56 may also include the signal peptide of the present invention (SEQ ID NO:36) or may contain a different signal peptide.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINGNGDYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 86). It will be appreciated that SEQ ID NO: 86 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 39 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 86 or 39.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIGGSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 87). It will be appreciated that SEQ ID NO: 87 also includes the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 40 or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 87 or 40.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 88). It will be appreciated that SEQ ID NO: 88 also includes the signal peptide of the invention (SEQ ID NO: 36) which generates SEQ ID NO: 41, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 88 or 41.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNINGPGNGTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 89). It will be understood that SEQ ID NO: 89 also includes the signal peptide of the invention (SEQ ID NO: 36) which generates SEQ ID NO: 42, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 89 or 42.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 90). It will be appreciated that SEQ ID NO: 90 also includes the signal peptide of the invention (SEQ ID NO: 36) which generates SEQ ID NO: 52, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 90 or 52.In some embodiments, the antibody or antigen-binding fragment thereof is SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:91). It will be appreciated that SEQ ID NO:91 also includes the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:53, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:91 or 53.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPDRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:92). It will be understood that SEQ ID NO:92 can also include the signal peptide of the invention (SEQ ID NO:36) generating SEQ ID NO:75 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:92 or 75.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPERTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:93). It will be appreciated that SEQ ID NO:93 can also include the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:76 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:93 or 76. In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPGRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL. YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:94). It will be appreciated that SEQ ID NO:94 can also include the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:77 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:94 or 77. In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPQRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 95). It will be appreciated that SEQ ID NO: 95 also includes the signal peptide of the invention (SEQ ID NO: 36) which generates SEQ ID NO: 78, or can contain a different signal peptide.In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 95 or 78. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPSRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:96). It will be appreciated that SEQ ID NO:96 also includes the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:79 or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:96 or 79.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRAYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:97). It will be understood that SEQ ID NO:97 also includes the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:80, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:97 or 80.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNREYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:98). It will be appreciated that SEQ ID NO:98 also includes the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:81, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:98 or 81.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRGYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:99). It is understood that SEQ ID NO:99 also includes the signal peptide of the invention (SEQ ID NO:36) which generates SEQ ID NO:82, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO:99 or 82.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRNYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 100). It will be appreciated that SEQ ID NO: 100 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 83 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 100 or 83.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRNYNYGDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 101). It will be appreciated that SEQ ID NO: 101 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 43 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 101 or 43.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYDPTNYYDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 102). It will be appreciated that SEQ ID NO: 102 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 44 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 102 or 44.In some embodiments, the antibody or antigen-binding fragment thereof is QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRSTNYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 103). It will be appreciated that SEQ ID NO: 103 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 45 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of SEQ ID NO: 103 or 45.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof is 22107, wherein X is a cyclic nucleotide sequence selected from the group consisting of aryl, aryl stearate ... 22 is any amino acid other than S and T. It will be understood that SEQ ID NO:108 can also include the signal peptide of the invention (SEQ ID NO:36) generating SEQ ID NO:109 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof has the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRX 23YYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 110), 23 is any amino acid other than S, T, and C. It will be understood that SEQ ID NO:110 can also include the signal peptide of the invention (SEQ ID NO:36) generating SEQ ID NO:111, or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof has the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPX 24RTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 115), 24 is any amino acid other than N. It will be understood that SEQ ID NO:115 can also include the signal peptide of the invention (SEQ ID NO:36) generating SEQ ID NO:116 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof has the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPX 25RTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 117), 25 is any amino acid other than N and C. It will be understood that SEQ ID NO:117 can also include the signal peptide of the invention (SEQ ID NO:36) generating SEQ ID NO:118 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof has the sequence QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPX 26RTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 119), 26 is G, D, S, E, Q or N. It will be understood that SEQ ID NO: 119 can also include the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 120 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of any one of SEQ ID NOs: 108-111. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of any one of SEQ ID NOs: 108-111. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of any one of SEQ ID NOs: 115-120. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain consisting of any one of SEQ ID NOs: 108-111 and 115-120.

[0113] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 39-45, 52-53, and 75-83. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 86-103. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 9, 39-45, 52-53, and 75-83. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising a sequence selected from SEQ ID NOs: 85-103. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 39-45, 52-53, and 75-83. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 86-103. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 9, 39-45, 52-53, and 75-83. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain consisting of a sequence selected from SEQ ID NOs: 85-103.

[0114] In some embodiments, the light chain comprises an N-terminal peptide comprising SEQ ID NO: 36. In some embodiments, the light chain comprises an N-terminal peptide consisting of SEQ ID NO: 36. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 36 at the N-terminus of any one of SEQ ID NO: 8 and SEQ ID NO: 35. In some embodiments, the N-terminal peptide is a signal peptide. In some embodiments, the light chain comprises a signal peptide. In some embodiments, the light chain is free of a signal peptide. In some embodiments, the light chain lacks a signal peptide. In some embodiments, the light chain comprises a signal peptide when initially expressed in a cell, the signal peptide is cleaved, and the secreted light chain lacks the signal peptide.

[0115] In some embodiments, the light chain comprises a C-terminal peptide comprising RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 38). In some embodiments, the light chain comprises a C-terminal peptide consisting of SEQ ID NO: 38. In some embodiments, the C-terminal peptide is a light chain constant region. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO: 38 C-terminal to any one of SEQ ID NO: 8 and SEQ ID NO: 35. In some embodiments, the C-terminal peptide comprises a CL domain. In some embodiments, the CL is a kappa CL domain. In some embodiments, the CL is a lambda CL domain. One of skill in the art will appreciate that disulfide bridges formed between the CH1 domain of the heavy chain and the CL domain of the light chain are sufficient for heavy-light chain dimerization and formation of a functional antibody or antigen-binding fragment. In some embodiments, the C-terminal peptide comprises a dimerization domain. In some embodiments, the dimerization domain is sufficient to induce dimerization between a heavy chain and a light chain.

[0116] In some embodiments, the antibody comprises a light chain comprising the sequence ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 104). It will be appreciated that SEQ ID NO: 104 can also include the signal peptide of the invention (SEQ ID NO: 36) which generates SEQ ID NO: 10, or can contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEDFAVYYCQQYGSYPPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 105). It will be appreciated that SEQ ID NO: 105 can also comprise the signal peptide of the invention (SEQ ID NO: 36) generating SEQ ID NO: 46 or contain a different signal peptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain consisting of SEQ ID NO: 104 or 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain consisting of SEQ ID NO: 105 or 46. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a sequence selected from SEQ ID NO: 10 and SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising a sequence selected from SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the antibody or antigen-binding fragment comprises a light chain consisting of a sequence selected from SEQ ID NO: 10 and SEQ ID NO: 46. In some embodiments, the antibody or antigen-binding fragment comprises a light chain consisting of a sequence selected from SEQ ID NO: 104 and SEQ ID NO: 105.

[0117] In some embodiments, the heavy and light chains are joined by a linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24, or 25 amino acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 22, 24, or 25 amino acids in length. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker is 1-25, 5-25, 10-25, 15-25, 20-25, 1-20, 5-20, 10-20, 15-20, 1-15, 5-15, 10-15, 1-10, 5-10, or 1-5 amino acids in length, with each possibility representing a separate embodiment of the present invention.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof is selected from those provided in Table 1. Table 1: Antibodies of the invention [Table 1]

[0119] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 32 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 101 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 43 and a light chain that comprises or consists of SEQ ID NO: 10.

[0120] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 33 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 102 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 44 and a light chain that comprises or consists of SEQ ID NO: 10.

[0121] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 34 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 103 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 45 and a light chain that comprises or consists of SEQ ID NO: 10.

[0122] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 30 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 88 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 41 and a light chain that comprises or consists of SEQ ID NO: 10.

[0123] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 50 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 90 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 52 and a light chain that comprises or consists of SEQ ID NO: 10.

[0124] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 66 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 92 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 75 and a light chain that comprises or consists of SEQ ID NO: 10.

[0125] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 67 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 93 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 76 and a light chain that comprises or consists of SEQ ID NO: 10.

[0126] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 68 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 94 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 77 and a light chain that comprises or consists of SEQ ID NO: 10.

[0127] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 69 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 95 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 78 and a light chain that comprises or consists of SEQ ID NO: 10.

[0128] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 70 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 96 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 79 and a light chain that comprises or consists of SEQ ID NO: 10.

[0129] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 71 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 97 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 80 and a light chain that comprises or consists of SEQ ID NO: 10.

[0130] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 72 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 98 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 81 and a light chain that comprises or consists of SEQ ID NO: 10.

[0131] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 73 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 99 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 82 and a light chain that comprises or consists of SEQ ID NO: 10.

[0132] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 74 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 100 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 83 and a light chain that comprises or consists of SEQ ID NO: 10.

[0133] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 31 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 89 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 42 and a light chain that comprises or consists of SEQ ID NO: 10.

[0134] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 28 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 86 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 39 and a light chain that comprises or consists of SEQ ID NO: 10.

[0135] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 29 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 87 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 40 and a light chain that comprises or consists of SEQ ID NO: 10.

[0136] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 51 and a light chain variable region that comprises or consists of SEQ ID NO: 8. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 91 and a light chain that comprises or consists of SEQ ID NO: 104. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 53 and a light chain that comprises or consists of SEQ ID NO: 10.

[0137] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO:7 and a light chain variable region that comprises or consists of SEQ ID NO:35. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO:85 and a light chain that comprises or consists of SEQ ID NO:105. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO:9 and a light chain that comprises or consists of SEQ ID NO:46.

[0138] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 30 and a light chain variable region that comprises or consists of SEQ ID NO: 35. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 88 and a light chain that comprises or consists of SEQ ID NO: 105. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 41 and a light chain that comprises or consists of SEQ ID NO: 46.

[0139] In some embodiments, the antibody comprises a heavy chain variable region that comprises or consists of SEQ ID NO: 31 and a light chain variable region that comprises or consists of SEQ ID NO: 35. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 89 and a light chain that comprises or consists of SEQ ID NO: 105. In some embodiments, the antibody comprises a heavy chain that comprises or consists of SEQ ID NO: 42 and a light chain that comprises or consists of SEQ ID NO: 46.

[0140] According to another embodiment, an antibody or antigen-binding fragment is provided that contains at least 70% amino acid identity to an antibody or antigen-binding fragment provided in Table 1 and contains the same CDRs as that antibody.

[0141] Those skilled in the art will appreciate that the CDRs define the binding specificity of the antibody / binding fragment, and therefore the sequences surrounding the CDRs may be modified / altered as long as the CDRs are retained. In some embodiments, at least 70% is at least 75%, 80%, 85%, 90%, 92%, 95%, 97%, 99% or 100%. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 70% is at least 80%. In some embodiments, at least 70% is at least 90%.

[0142] Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and / or VL, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived.

[0143] For example, in the 4C7 VH region, framework amino acids at positions 25, 68 and 82a all differ from germline and can be backmutated to germline by substitutions of H25S, S68T and T82aT, respectively.

[0144] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0145] In addition to, or as an alternative to, modifications made within the framework or CDR regions, the antibodies of the invention can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Further, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of the residues in the Fc region is that of the EU index of Kabat.

[0146] In a preferred embodiment, the antibody is an IgG4 isotype antibody that contains a serine to proline mutation (S228P; EU index) in the hinge region of the heavy chain constant region at a position corresponding to position 228. This mutation has been reported to eliminate the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al. supra; position 241 is based on the Kabat numbering system).

[0147] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0148] In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcus protein A (SpA) binding relative to the native Fc hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745.

[0149] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be modified in the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in US Patent Nos. 5,869,046 and 6,121,022.

[0150] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260.

[0151] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551.

[0152] In another example, one or more amino acid residues within amino acid positions 231 to 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94 / 29351.

[0153] In yet another example, the Fc region may be modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to modify the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 365, 367, 368, 369, 370, 372, 376, 378, 380, 383, 7, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication WO 00 / 42072. Additionally, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and mutants with improved binding have been described (see Shields et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcγRIII. Additionally, the following combination mutants: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A have been shown to improve FcγRIII binding.

[0154] In yet another embodiment, the glycosylation of the antibody is altered. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). The glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such sugar modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can enhance the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0155] Additionally or alternatively, antibodies can be made with altered types of glycosylation, e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased GlcNac bisecting structures. Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such sugar modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), such that antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose on their sugars. Ms704, Ms705, and Ms709 FUT8 - / -The cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes cell lines with functional disruption of the FUT8 gene encoding fucosyltransferase, and antibodies expressed in such cell lines exhibit hypofucosylation due to reduced or eliminated α-1,6 bond-related enzymes. EP 1,176,195 also describes cell lines with reduced or no enzyme activity that adds fucose to N-acetylglucosamine attached to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC® CRL 1662). PCT Publication WO 03 / 035835 describes a mutant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked sugars, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies with altered glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies with altered glycosylation profiles can be produced in plant cells such as Lemna (U.S. Patent No. 7,632,983). Methods for the production of antibodies in plant systems are disclosed in U.S. Patent Nos. 6,998,267 and 7,388,081. PCT Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines have increased GlcNac bisecting structures, which results in enhanced ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, fucose residues of antibodies can be cleaved off using a fucosidase enzyme.For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

[0156] Another modification of the antibodies herein contemplated by the present disclosure is pegylation. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups become attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0154316 and EP 0401384.

[0157] According to another aspect, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention.

[0158] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable carrier, excipient, or adjuvant, hi some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention.

[0159] As used herein, the terms "carrier," "excipient," or "adjuvant" refer to any component of a pharmaceutical composition that is not an active antibody or antigen-binding fragment thereof. As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium such as physiological saline. Some examples of substances which can function as pharma- ceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers for antibodies or antigen-binding fragments thereof such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffers, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers and other non-toxic medicamentously compatible materials used in pharmaceutical formulations. Lubricants such as wetting antibodies or antigen-binding fragments thereof and sodium lauryl sulfate, as well as colorants for antibodies or antigen-binding fragments thereof, flavorants for antibodies or antigen-binding fragments thereof, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein.In this regard, suitable pharma- ceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, such as those described in The Merck Index, 13th Edition, Budavari et al. (eds.), Merck & Co., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and the "Inactive Ingredient Guide," U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Administration, all of which are incorporated herein by reference in their entireties. Examples of pharma-ceutically acceptable carriers, excipients, and diluents useful in the present compositions include distilled water, saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman: The Pharmacological Bases of Therapeutics, 8th Edition, Gilman et al. (eds.) Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated herein by reference in its entirety. The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, sustained release particles, and other vehicles that increase the half-life of the peptide or polypeptide in serum.Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the peptides described herein are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and blood stability. A variety of methods are available for preparing liposomes, as reviewed, for example, by Coligan, JE et al., Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, see also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0160] The carriers may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0161] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and dosing regimen will be determined by a physician depending on the patient's condition. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of an antibody or antigen-binding fragment of the invention. In some embodiments, a pharmaceutical composition is a therapeutic composition.

[0162] In some embodiments, the antibody or antigen-binding fragment thereof is for use in treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In some embodiments, the disease or condition is characterized by a cell expressing HVEM. In some embodiments, the cell is a diseased cell characterized as expressing HVEM. In some embodiments, the cell is characterized as overexpressing HVEM. In some embodiments, the overexpression is compared to a healthy cell. In some embodiments, the healthy cell is a non-diseased cell. In some embodiments, the overexpression is increased expression. In some embodiments, the disease or condition is an HVEM-positive disease or condition. In some embodiments, the disease or condition is a BTLA-positive disease or condition.

[0163] In some embodiments, the disease or condition is an HVEM positive cancer. In some embodiments, the disease or condition is a cancer that comprises BTLA positive resident cells. In some embodiments, the disease or condition is an HVEM positive precancerous lesion. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is selected from melanoma, renal cancer, cervical cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, breast cancer, hepatocellular carcinoma, cholangiocarcinoma, thymoma, and head and neck cancer. In some embodiments, the renal cancer is renal cell carcinoma. One of skill in the art will recognize that any cancer that expresses HVEM may be a therapeutic target. In some embodiments, the disease is an infectious disease. In some embodiments, the infected cells comprise HVEM expression. In some embodiments, the infected cells comprise HVEM expression. In some embodiments, the infectious disease is a bacterial infection and the bacterial cells comprise HVEM expression. In some embodiments, the infectious disease is a fungal infection and the fungal cells comprise HVEM expression. In some embodiments, the infectious disease is a viral infection and cells of the virally infected subject contain HVEM expression. In some embodiments, the virus is a herpes virus. In some embodiments, the HVEM expression is HVEM overexpression. In some embodiments, the HVEM expression is increased HVEM expression.

[0164] According to another aspect, there is provided a method of treating an HVEM-positive disease or condition in a subject in need thereof, the method comprising administering to the subject an antibody, or antigen-binding fragment thereof, of the invention, thereby treating the subject.

[0165] According to another aspect, there is provided a method of treating an HVEM-positive disease or condition in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the invention, thereby treating the subject.

[0166] In some embodiments, the method further comprises not inhibiting HVEM-TNFSF14 interaction. In some embodiments, the method further comprises not substantially inhibiting HVEM-TNFSF14 interaction. In some embodiments, the method further comprises not completely inhibiting HVEM-TNFSF14 interaction.

[0167] In some embodiments, the method further comprises immune checkpoint inhibition of a non-HVEM checkpoint protein. In some embodiments, the non-HVEM checkpoint protein is PD-1. In some embodiments, the method further comprises immune checkpoint inhibition of a non-HVEM checkpoint protein. In some embodiments, the method further comprises inhibiting an interaction between PD-1 and one of its ligands. In some embodiments, the PD-1 ligand is selected from PD-L1 and PD-L2. In some embodiments, the method further comprises inhibiting PD-1, PD-L1 or PD-L2. In some embodiments, the method further comprises inhibiting a PD-1 to PD-L1 interaction. In some embodiments, the method further comprises inhibiting a PD-1 to PD-L2 interaction. In some embodiments, the method further comprises inhibiting a PD-1 to PD-L1 or PD-L2 interaction. In some embodiments, inhibiting the interaction comprises administering an antibody or antigen-binding fragment thereof that inhibits a PD-1 to PD-L1 interaction or a PD-1 to PD-L2 interaction. In some embodiments, inhibiting the interaction comprises administering an antibody or antigen-binding fragment thereof that inhibits the interaction between PD-1 and at least one of its ligands. In some embodiments, the at least one ligand is two ligands. In some embodiments, inhibiting the interaction comprises PD-1 / PD-L1 blockade. In some embodiments, inhibiting the interaction comprises PD-1 / PD-L1 or PD-L2 blockade. In some embodiments, inhibiting the interaction comprises anti-PD-1 / PD-L1 therapy. In some embodiments, inhibiting the interaction comprises anti-PD-1 / PD-L1 or PD-L2 therapy. In some embodiments, the therapy is immunotherapy. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits the interaction is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits the interaction is an anti-PD-1, anti-PD-L1 or anti-PD-L2 antibody.In some embodiments, the antibody or antigen-binding fragment thereof that inhibits the interaction is a PD-1 / PD-L1 inhibitor. In some embodiments, the antibody or antigen-binding fragment thereof that inhibits the interaction is a PD-1 / PD-L1 / PD-L2 inhibitor. In some embodiments, the antibody is a blocking antibody. PD-L1 / L2 and PD-1 therapies are known in the art and include, but are not limited to, nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab, avelumab, durvalumab, cemiplimab (Libtayo), pidilizumab, AMP-224, AMP-514, and PDR001.

[0168] In some embodiments, the method of treatment further comprises administering another therapeutic agent to the HVEM-expressing cells. In some embodiments, the other therapeutic agent is an anti-cancer therapeutic agent. In some embodiments, the therapeutic agent is a non-autologous immune cell. In some embodiments, the other therapeutic agent is an autologous immune cell. In some embodiments, the immune cell is a CAR-expressing immune cell. In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the autologous immune cell is TIL therapy. In some embodiments, the non-autologous immune cell is adoptive cell therapy. In some embodiments, the autologous immune cell is adoptive cell therapy. In some embodiments, the adoptive cell is a CAR cell. In some embodiments, the adoptive cell is a TIL.

[0169] In some embodiments, a composition of the invention and another therapeutic agent are administered simultaneously, hi some embodiments, a composition of the invention is administered before, after, or simultaneously with another therapeutic agent.

[0170] In some embodiments, the subject is a human. In some embodiments, the subject is a subject suffering from an HVEM positive disease or condition. In some embodiments, the subject is suffering from a disease characterized by HVEM positive cells. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is an HVEM positive cancer. In some embodiments, the subject is therapy naive. In some embodiments, the subject is anti-HVEM therapy naive. In some embodiments, the subject is immunotherapy naive.

[0171] As used herein, the terms "administer", "administration" and similar terms refer to any method of delivering a composition containing an active agent to a subject to provide a therapeutic effect in sound medical practice. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of the subject composition to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, intrathecal, or intraperitoneal.

[0172] The dosage administered will depend upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0173] According to another aspect, there is provided a method of determining the suitability of a subject to be treated by the method of the present invention, the method comprising obtaining a sample from the subject and determining the level of HVEM in the sample, wherein positive expression of HVEM indicates that the subject is suitable for the treatment method of the present invention.

[0174] According to another aspect, a method for detecting HVEM in a sample is provided, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof of the invention, thereby detecting HVEM.

[0175] In some embodiments, determining the HVEM level comprises detecting HVEM. In some embodiments, detecting comprises quantifying the amount of HVEM. In some embodiments, the contacting is performed under conditions suitable for the antibody or antigen-binding fragment to bind to HVEM. In some embodiments, the conditions are suitable for specific binding to HVEM. In some embodiments, binding is hybridizing. The conditions under which the antibody / antibody or antigen-binding fragment thereof binds are sample dependent. One of skill in the art will recognize that the conditions required for binding to tissue samples (which depend on the method / type of fixation) are different from the conditions for binding in solution, such as whole cell lysates. Such binding conditions are well known in the art and one of skill in the art can select appropriate conditions for a given sample.

[0176] In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a paraffin-embedded sample. In some embodiments, the sample is a perfused sample. In some embodiments, the sample is from a subject. In some embodiments, the sample is a healthy sample. In some embodiments, the sample is a diseased sample. In some embodiments, the sample is a diagnostic sample.

[0177] In some embodiments, the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention. In some embodiments, the detection is immunohistochemical detection. In some embodiments, the detection is immunofluorescence detection. In some embodiments, the detection is FACS detection. In some embodiments, the detection further comprises contacting the sample with a secondary antibody that recognizes the antibody or antigen-binding fragment thereof of the invention. In some embodiments, the detection comprises detecting the secondary antibody. In some embodiments, the detection comprises microscopy.

[0178] In some embodiments, the subject is suffering from a disease or condition. In some embodiments, the subject is suspected of suffering from a disease or condition. In one embodiment, the subject is at risk of developing a disease or condition. In some embodiments, the subject is suffering from a disease or condition that may include increased HVEM expression. In some embodiments, the subject is suffering from a disease or condition that may be characterized by increased HVEM expression. In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer is a cancer that has failed to respond to first line therapy. In some embodiments, the cancer is a cancer recurrence. In some embodiments, the cancer is a cancer that has failed to respond to PD-1 / PD-L1 therapy.

[0179] In some embodiments, the sample is a disease sample. In some embodiments, the sample is a biological fluid. In some embodiments, the biological fluid is selected from blood, serum, plasma, urine, feces, bile, semen, tumor fluid, and cerebrospinal fluid. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a cell sample. In some embodiments, the sample comprises cells. In some embodiments, the sample comprises disease cells. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy sample.

[0180] In some embodiments, the positive expression of HVEM comprises HVEM expression. In some embodiments, the positive expression of HVEM comprises elevated HVEM levels. In some embodiments, the positive expression of HVEM comprises increased HVEM levels. In some embodiments, the positive expression of HVEM comprises overexpression of HVEM. In some embodiments, the positive expression of HVEM is compared to a healthy sample. In some embodiments, the healthy sample is from healthy tissue and / or healthy cells adjacent to diseased tissue and / or diseased cells. In some embodiments, the healthy sample comprises uninfected cells. In some embodiments, the healthy sample is of a healthy donor. In some embodiments, the positive expression of HVEM is compared to a pre-defined threshold.

[0181] According to another aspect, there is provided a kit comprising a pharmaceutical composition of the invention, or an antibody or antigen-binding fragment thereof of the invention.

[0182] In some embodiments, the kit further comprises a PD-1 based therapy. In some embodiments, the kit further comprises a PD-L1 based therapy. In some embodiments, the therapy is an immunotherapy. In some embodiments, the therapy is an anti-PD-1 therapy. In some embodiments, the therapy is an anti-PD-L1 therapy. In some embodiments, the therapy is a PD-1 / PD-L1 blockade therapy. In some embodiments, the therapy is a blocking antibody. In some embodiments, the therapy is an anti-PD-1 antibody. In some embodiments, the therapy is an anti-PD-L1 antibody. In some embodiments, the antibody is a blocking antibody.

[0183] In some embodiments, the kit further comprises a label indicating that the pharmaceutical composition of the invention is for use with a PD-1 and / or PD-L1 based therapy. In some embodiments, the kit further comprises a label indicating that the antibody, or antigen-binding fragment thereof, of the invention is for use with a PD-1 and / or PD-L1 based therapy.

[0184] In some embodiments, the kit further comprises a secondary detection molecule. In some embodiments, the detection molecule is for detecting the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the secondary detection molecule is an antibody that binds to the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the detection molecule comprises a detectable moiety. Examples of detectable moieties include, but are not limited to, fluorescent moieties, tags, radiolabels, dyes, and chemiluminescent moieties. In some embodiments, the detectable moiety is a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to, GFP, RFP, YFP, APC, CY5, CY7, and Pacific Blue. Secondary detection molecules are well known in the art, and any such molecule that will detect the antibody or antigen-binding fragment thereof of the present invention may be used.

[0185] According to another aspect, a nucleic acid molecule encoding an antibody or antigen-binding fragment of the invention is provided.

[0186] According to another aspect, there is provided a nucleic acid molecule encoding the heavy chain of an antibody or antigen-binding fragment of the invention, and a nucleic acid molecule encoding the light chain of an antibody or antigen-binding fragment of the invention.

[0187] In some embodiments, the nucleic acid molecule is configured for expression. In some embodiments, the expression is in a cell. In some embodiments, the expression is in an in vitro expression system. As used herein, the term "expression" refers to the biosynthesis of a coding region, including transcription and / or translation of the coding region. Thus, expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription resulting in an mRNA or other functional RNA) and / or the translation of the RNA into a precursor or mature protein (polypeptide). In some embodiments, the coding region encodes an antibody or antigen-binding fragment of the invention. In some embodiments, the coding region encodes a heavy chain. In some embodiments, the coding region encodes a light chain.

[0188] Expression of a coding region in a cell is well known to those of skill in the art. This can be done by transfection, viral infection, or direct modification of the genome of the cell, among many other methods. In some embodiments, the coding region is in an expression vector, such as a plasmid or viral vector. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector.

[0189] The nucleic acid sequence of a vector generally contains at least an origin of replication for propagation in a cell, and optionally additional elements, such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selection markers (e.g., antibiotic resistance), polyadenine sequences, etc.

[0190] The vector can be a DNA plasmid delivered via non-viral or viral methods. The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a pox virus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0191] In some embodiments, the coding region is operably linked to a regulatory element. In some embodiments, the coding region is operably linked to a promoter. In some embodiments, the regulatory element is a protein. In some embodiments, the regulatory element is an enhancer. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element or elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0192] In some embodiments, vectors are introduced into cells by standard methods including electroporation (as described, for example, in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with viral vectors, high velocity ballistic penetration by small particles carrying the nucleic acid either within the matrix of small beads or particles or on their surface (Klein et al., Nature 327.70-73 (1987)).

[0193] As used herein, the term "promoter" refers to a group of transcriptional control modules clustered around the initiation site of RNA polymerase, i.e., RNA polymerase II. Promoters are composed of individual functional modules, each consisting of about 7-20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0194] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0195] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myC / cyto, pCMV / myC / cyto, pCR3.1, pSinRep5, DH26s, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0196] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses, such as retroviruses, are used in the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of a protein under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0197] In some embodiments, recombinant viral vectors that offer advantages such as lateral infection and targeting specificity are used for in vivo expression. In one embodiment, lateral infection is, for example, inherent in the retroviral life cycle, a process whereby a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, this results in a large area becoming rapidly infected, most of which were not originally infected by the original viral particle. In one embodiment, a viral vector that cannot spread laterally is generated. In one embodiment, this feature can be useful when the desired goal is to introduce a specific gene into only a localized number of target cells.

[0198] A variety of methods can be used to introduce the expression vectors of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al., Biotechniques 4(6):504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see U.S. Patent Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0199] In one embodiment, a plant expression vector is used. In one embodiment, expression of the polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)] or the coat protein promoter for TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, a plant promoter is used, such as the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or a heat shock promoter, such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques known to those skilled in the art. See, for example, Weissbach & Weissbach, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988). Other expression systems, such as insect and mammalian host cell systems, known in the art, may also be used according to the present invention.

[0200] It will be appreciated that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also contain sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0201] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0202] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides, a reference to a "polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a predicate for using exclusive language, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0203] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and / or a combination of A, B, and C, etc.). One of ordinary skill in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0204] It is recognized that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination was individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and expressly disclosed herein.

[0205] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0206] Additionally, various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0207] Working Example Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques, such techniques being fully explained in the literature. See, for example, Molecular Cloning: A laboratory Manual, Sambrook et al. (1989); Current Protocols in Molecular Biology, vols. I-III, Ausubel, RM, (ed.) (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds.) Genome Analysis: A Laboratory Manual Series, vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); the methodologies described in U.S. Patents Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057; Cell Biology: A Laboratory Handbook, Vols. I-III, Cellis, JE, (ed.) (1994); Culture of Animal Cells-A Manual of Basic Technique, Freshney, Wiley-Liss, NY (1994), 3rd ed.; Current Protocols in Immunology, Vols. I-III, Coligan JE, (eds.) (1994); Stites et al. (eds.), Basic and Clinical Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), Strategies for Protein Purification and Characterization-A Laboratory Course Manual, CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0208] Materials and Methods Affinity maturation: To improve binding affinity, the parent antibodies were affinity matured using phage display. The heavy and / or light chain CDRs of the variable domain regions were semi-randomized using oligonucleotide mutagenesis. Selections were performed using separate heavy and light chain phage libraries and decreasing concentrations of biotinylated His-tagged HVEM target to select the highest affinity binders from each library. After four rounds of selection, individual clones were screened as scFv supernatants in a binding ELISA to identify lead candidates with higher signals than the parent scFv expressed in the same assay. Identified hits were converted to IgG and cloned into mammalian expression vectors. Plasmids encoding the affinity-improved mutants paired with the appropriate parent heavy or light chain and the parent antibodies were transiently transfected at small scale into HEK EBNA adherent cells (LGC Standards, Teddington, UK). Five days after transfection, supernatants were harvested, quantified on an Octet (serial number FB-30203) and analyzed by Biacore single cycle kinetics.

[0209] Single cycle kinetics: To evaluate the binding of affinity matured IgG, single cycle kinetic analysis was performed on the supernatant using a Biacore T200 (serial number 1909913). Kinetic experiments were performed on a Biacore T200 running at 25°C using Control Software V2.0.1 and Evaluation Software V3.0 (Cytiva, Marlborough, USA). Antibodies were diluted to a final concentration of 1.0 μg / ml in HBS-P+ (Cytiva, Marlborough, USA) containing 1 mg / ml BSA. Antibodies were loaded onto Fc2, Fc3 and Fc4 of a Protein A capture sensor chip (Cytiva, Marlborough, USA). IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 50 RU. The surface was then allowed to stabilize. Single cycle kinetic data were acquired using His-tagged HVEM (Acro Biosystems, Newark, USA) as the analyte at a flow rate of 40 μL / min. Two-fold dilutions of His-tagged antigen ranging from 6.125 nM to 100 nM were used with no regeneration between each concentration.

[0210] Multi-cycle kinetics: Multi-cycle kinetic analysis was performed on purified antibodies. Kinetic experiments were performed at 25°C on a Biacore 8K. HBS-P + 0.1% BSA (Cytiva, Marlborough, USA) was used as the running buffer. Purified antibodies were diluted to 1.0 μg / mL in running buffer and loaded onto Fc2, Fc3, and Fc4 of a Protein A sensor chip (Cytiva, Marlborough, USA) at the beginning of each cycle. Antibodies were captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 100 RU. The surface was then allowed to stabilize. Multi-cycle kinetic data were acquired using His-tagged human HVEM as the analyte injected at a flow rate of 50 μl / min. Eight two-fold dilutions of antigen ranging from 0.78 nM to 100 nM were prepared in running buffer. For each concentration, the association phase was monitored for 180 s and the dissociation phase was measured for 300 s. Regeneration of the sensor chip surface was performed between cycles using two injections of 10 mM Glycine-HCl, pH 1.5.

[0211] Cytotoxicity assay: Nuclear RFP stained melanoma cells were seeded for overnight incubation to allow them to attach. Melanoma cells were then preincubated with 20 μg / ml of parental mAb of the present invention or affinity matured mAb or hIgG4 isotype control antibody (Invivogen, France), and TILs were preincubated with or without 20 μg / ml of anti-PD1 mAb (Nivolumab, BMS) or hIgG4 isotype control antibody (Invivogen, France). Preincubated TILs or medium with only anti-PD1 or hIgG4 isotype control mAb was added to melanoma cells with CellEvent caspase-3 / 7 green detection reagent (Invivogen, USA). Plates were placed in an Incucyte ZOOM system (ESSEN Bioscience, USA) and scanned every hour. Caspase 3 / 7 positive events, reflecting specific killing of melanoma, were counted.

[0212] T cell activation assay with Incucyte ZOOM system: Nuclear RFP stained melanoma cells were seeded for overnight incubation to allow them to attach. After overnight incubation, melanoma cells were incubated with 20 μg / ml of the mAb of the present invention, parental mAb or hIgG4 isotype control at 37° C. for 1 hour. TILs or media were then added to the melanoma cells along with conjugated CD107a antibody. Plates were placed in the Incucyte ZOOM system and scanned every hour.

[0213] Example 1: Affinity maturation of anti-HVEM antibodies The lead antibody of WO2020222235 consisted of a heavy chain as provided in SEQ ID NO:9 and a light chain as provided in SEQ ID NO:10. The CDRs were in a human backbone, including an IgG4 backbone carrying the S228P mutation. The antibody could bind human HVEM, as well as mouse and cynomolgus monkey HVEM, and bind proteins in solution and on the cell surface. The antibody inhibited the binding of BTLA to HVEM, but did not inhibit the binding of LIGHT to HVEM. Furthermore, the antibody itself had a low level of activating effect, inducing signaling through HVEM upon its binding. Most importantly, the antibody could enhance the cytotoxicity of immune cells against cancer and increase the cytotoxicity of non-autologous immune cells, thus enhancing standard adoptive immune cell therapy.

[0214] To improve the function of this antibody, affinity maturation procedures were performed on the CDR regions of the antibody, which are known to improve affinity, reduce immunogenicity, overcome propensities in structure, and any combination of the above.

[0215] Affinity maturation was performed on the CDR regions of VH and VL. The resulting clones were sequenced and their KpAp to HVEM extracellular domain peptides were determined. D The K observed with the parent antibody was DThe fold improvement over the 100% CDRs is presented in Table 2. The affinity matured clones showed enhanced binding compared to the parent antibody, demonstrating their superiority. Various mutant CDRs are combined to create antibodies with improved CDR combinations. The binding affinity of these combined antibodies is evaluated.

[0216] The H4 antibody was found to have the highest improvement compared to the parent antibody (9.57-fold increase). However, its CDR2 contains an N-linked glycosylation site (NXS / T glycosylation site) that was found to be glycosylated in the final antibody (Figure 1). Glycosylation can lead to variability in batch production of antibodies and affect function. Therefore, several mutants were specifically created in which the asparagine at position 55 or the threonine at position 57 were mutated to different amino acids. As expected, these mutants of H4 were no longer glycosylated (Figure 1). Notably, two of these antibodies, T57A and T57N, had even better binding than the unmodified H4 (20.66- and 23.51-fold increase compared to the parent).

[0217] Table 2: K of antibody binding to HVEM D evaluation [Table 2]

[0218] Example 2: Further characterization of affinity matured antibodies The newly generated antibodies were compared to the parental antibodies to further evaluate their functionality. Plates were coated without protein or with 2.5 μg / ml human recombinant HVEM (hHVEM) diluted in PBS by overnight incubation at 4° C. Plates were washed, blocked with 2% BSA for 1 h at room temperature and washed again. Four antibodies were then tested separately for binding to the plates: isotype control, parental, H4 and Par-K64E. Antibodies were diluted to a concentration of 10 μg / ml in blocking buffer and incubated for 1 h at room temperature. Plates were then washed and incubated with peroxidase and anti-human Fc for 30 min at room temperature. After washing again, TMB was added along with stop solution. Absorbance was read at 450 nm and 570 nm. Both modified antibodies showed better binding than the parental antibodies (Figure 2). Indeed, H4 showed a significantly enhanced binding and is clearly the superior option.

[0219] Example 3: Characterization of H4 antibodies and H4 derivative antibodies It was decided to proceed with the H4 antibody that showed the best HVEM binding, specifically the mutant with the putative glycosylation site (NRT) removed. First, a multi-cycle kinetic Biacore affinity assay was performed to confirm the improved binding of the H4, T57A, T57G and T57N antibodies (Figure 3A). Each purified antibody was captured on a Protein A sensor chip prior to injection of increasing concentrations of antigen (hHVEM). The fold improvement in affinity was calculated as the average K of the parent antibodies. D The average K of each mutant antibody D It was calculated by dividing by K D The K values ​​and fold improvements are summarized in Table 3. As can be seen, the H4 antibody is indeed better than the parent antibody, and both the T57A and T57N mutants are even better binders than H4. Single cycle kinetic Biacore assays were also performed using the H4 N55G, H4 N55S, H4 N55D, H4 N55Q, H4 N55E, and H4 T57E antibodies (Figure 3B). The K values ​​for these antibodies were DThe values ​​and fold improvements are summarized in Table 4. These antibodies were also found to be superior to the parental antibodies.

[0220] Table 3: Multi-cycle kinetic Biacore binding assay [Table 3]

[0221] Table 4: Single cycle kinetic Biacore binding assay [Table 4]

[0222] Next, binding to HVEM on the surface of cells was examined. CHO-K1 cells were engineered to overexpress human HVEM (hHVEM). Cells were then incubated with 20 μg / ml of antibody for 1 hour on ice and then stained with anti-human IgG Fc-biotin and anti-biotin-FITC. Expression was analyzed by flow cytometry. All four antibodies were able to bind hHVEM and importantly detected surface expression (Figure 3C). We hypothesized that such high concentrations (20 μg / ml) would mask differences in binding between the parental and affinity matured antibodies, and therefore binding was compared between the parental and T57A antibodies at various antibody concentrations. The T57A antibody was indeed found to bind stronger than the parent and at lower concentrations, indicating that it is a superior binder of surface hHVEM (Figure 3D).

[0223] Next, the ability of the antibodies to block the interaction with BTLA was evaluated. For this purpose, a binding ELISA system was set up. Plates were coated with 2.5 μg / ml recombinant hHVEM diluted in PBS overnight at 4° C. Plates were washed and blocked with 2% BSA for 1 h at room temperature. The different antibodies and isotype controls were diluted in blocking buffer to a concentration of 10 μg / ml. First, binding in this system was evaluated. The antibodies were incubated for 1 h at room temperature, followed by washing and further incubation with peroxidase anti-human FC for 30 min at room temperature. Finally, after further washing, TMB was added, followed by the addition of stop solution. Finally, the absorbance was read at 450 nm and 570 nm.

[0224] As can be seen in Figure 4A, all four antibodies bind hHVEM in this setting. Moreover, all four antibodies were found to block the interaction of hBTLA with hHVEM (Figure 4B) and did not substantially block the interaction with hLIGHT (Figure 4C) (assays performed using hBTLA-Fc and hLIGHT-His and anti-BTLA-biotin or anti-LIGHT, respectively). Similar results were seen for the original H4 and Par-K64E antibodies (Figures 4D-4E). Interestingly, when monkey HVEM (cHVEM) was evaluated, all tested antibodies showed binding to the recombinant protein (Figure 4F), but the parental antibodies showed poor blocking of the cHVEM-cBTLA interaction and all affinity matured antibodies showed better blocking (Figure 4G).

[0225] It was hypothesized that the similarity in BTLA blockade between the parent and affinity matured antibodies was due to the high antibody concentrations. Therefore, IC50s for blocking hHVEM and hBTLA interaction were calculated by serial dilutions of the parent and H4 T57A antibodies. As can be seen in Figure 4H, the affinity matured variants were indeed superior blockers of hBTLA compared to the parent. IC50s were calculated by serial dilutions for the other variant antibodies, and similar results were observed for the other affinity matured antibodies.

[0226] Example 4: Functional evaluation of H4 antibody and H4 derivative antibody Alleviating the HVEM-BTLA T cell inhibitory axis promotes cancer cell killing by T cells. To test the cytotoxic effect of affinity matured antibodies, RFP-positive melanoma cells (HVEM positive) were cultured overnight and then incubated with anti-HVEM antibody (20 μg / ml) for 1 h at 37°C. At the end of that time, autologous tumor infiltrating lymphocytes (TILs) were added to the melanoma cells and cell death events were counted every hour using CellEvent caspase 3 / 7 detection reagent and Incucyte ZOOM system scanning. After 9 h of co-culture, the parental antibody increased specific killing events in cancer cells by 9% compared to the isotype control (Figure 5A). While the H4-T57N mutant showed similar killing to the parent (9% increase), both the T57A and T57G antibodies showed significantly more killing events (19% increase, p<0.01) and superior cytotoxic effects (Figure 5A).

[0227] The T57A mutant lacks the glycosylation site and was found to bind hHVEM more than twice as strongly as H4 (superior binding compared to T57G) and induce an increased rate of cell killing compared to the parent (superior killing compared to T57N), so this mutant antibody was selected for all further studies.

[0228] It is known that parental antibodies can enhance anti-PD-1 mediated T cell activation and thereby enhance cancer cell killing. Therefore, the above killing assay was also performed in the presence of anti-PD-1 antibody. Again, the H4 T57A antibody was found to be superior to the parental antibody, increasing specific cell killing by 64% compared to the isotype control, while the parental increase was only 44% (Figure 5B). Specific killing in the presence of anti-PD-1 antibody was also measured for other affinity matured antibodies, and similar superiority is observed.

[0229] To better quantify the activation in TILs caused by anti-HVEM antibodies, the activation marker CD107a was measured in melanoma cells co-cultured with these cells in the presence of isotype control, parent anti-HVEM antibody, or H4 T57A mutant antibody. Both anti-HVEM antibodies generated more CD107a-positive cells compared to the isotype control, but the affinity matured antibody generated significantly more activated cells (Figure 6A). When a second melanoma cell line was tested, the same trend was observed, but with an even larger difference between the mutant and parent antibodies (Figure 6B). Activation, as assessed by CD107a levels, was also measured in the presence of other affinity matured antibodies, and similar advantages were observed.

[0230] Finally, ex vivo killing assays were also performed with primary cancer cells in a similar manner as performed with cell lines. Freshly isolated cells from ovarian cancer were co-cultured with autologous PBMCs in the presence of isotype control, anti-PD1 antibody, parental mAb or H4 T57A mutant antibody. Specific cancer cell killing was monitored over a period of 55 hours. The anti-PD-1 antibody provided a very modest improvement (9% improvement at 55 hours) compared to the isotype control, whereas the parental antibody provided a 48% improvement and the H4 T57A antibody provided a 68% improvement (Figure 7A). This again demonstrates the superiority of affinity matured antibodies. Cell killing remained roughly stable in co-culture with the parental antibody, whereas a steady and continuing increase was observed with the mutant antibody. The same assay was performed but with anti-PD-1 in combination with the parental and mutant antibodies. As before, the inclusion of anti-PD-1 enhanced the difference between the parental and mutant antibodies. Combining anti-PD-1 with the parental antibody produced only a modest increase in specific killing compared to anti-PD-1 and the isotype control (a 13% increase at 55 hours), whereas combining anti-PD-1 with the H4 T57A antibody produced a robust increase of 79% (Figure 7B), demonstrating that the mutant antibodies have superior therapeutic efficacy compared to the parental antibody, both alone and in combination with anti-PD-1.

[0231] Other affinity matured antibodies of the invention are also tested in the same manner as above, and similar results are observed, showing improved binding and potency compared to the parent antibody.

[0232] In addition, mouse cancer cells expressing human HVEM are implanted into transgenic immune-competent mice expressing human BTLA and allowed to grow to form tumors. Affinity matured antibodies, anti-PD-1 antibodies, combinations thereof, or IgG isotype controls are administered. Tumor growth inhibition is measured. The new antibodies are found to be superior to the parental and at least comparable to PD-1 alone in inhibiting cancer growth. Furthermore, the new antibodies produced enhanced synergy with anti-PD-1.

[0233] Example 5: Diagnostic and Laboratory Uses of Mutant Antibodies Since the H4 antibody and its derivatives proved to be excellent binders for recombinant HVEM and HVEM bound on cell surface, we hypothesized that affinity matured antibodies could be used for clinical evaluation of HVEM as well as laboratory staining / detection. Formalin-fixed paraffin-embedded (FFPE) cell blocks of CHO cells overexpressing hHVEM were sliced ​​and mounted on slides for immunofluorescence detection. The parental and H4 T57A antibodies were used along with Cy3 (red signal) secondary antibodies. Significantly more positively stained cells and stronger staining were observed when the mutant antibody was used, indicating that it is also an excellent antibody for diagnostic / testing purposes (Figure 8).

[0234] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1 (SYAMS) and CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:49 (X １ IX ２ X ３ X ４ X ５ X １８ X ７ X １９ YYADSVX ２０ G) in which the sequence X １ is A, G or N, and X ２ is S, N, G or Y; X ３ is G or S, and X ４ is S, N or P, and X ５ is G or P, and X １８ is any amino acid other than C, and X ７ is S, Y, G or R; X １９ is any amino acid other than S or C, and X ２０ is any amino acid, and CDR-H3 is the amino acid sequence set forth in SEQ ID NO: 18 (AX ９ X １０ X １１ X １２ X １３ X １４ YX １５ DY) in the sequence ９ is P or S, and X １０ is G or Y, and X １１ is D or R, and X １２ is Y, N, P or S, and X １３ is T or Y, and X １４ is A or N, and X １５ is F, G or Y, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5 (GASSRAT), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 19 (QQYGSX １６ PPX １７ T) in the sequence １６ is S or Y and X １７ is Y or L; and does not include any of CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 2 (AISGSGGSTYYADSVKG), CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 3 (APGDYTAYFDY), and CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 6 (QQYGSSPPYT).

2. CDR-H2 is the amino acid sequence set forth in SEQ ID NO: 17 (X １ IX ２ X ３ X ４ X ５ X ６ X ７ X ２１ YYADSVX ８ G) in which the sequence X １ is A, G or N, and X ２ is S, N, G or Y; X ３ is G or S, and X ４ is S, N or P, and X ５ is G or P, and X ６ is G, D, S, E, Q or N, and X ７ is S, Y, G or R; X ２１ is T, A, E, G or N and X ８ is E or K. The antibody or antigen-binding fragment of claim 1 .

3. X ２０ is any amino acid other than C or S.

4. X ２０ The antibody or antigen-binding fragment thereof of claim 1 or 3, wherein is any non-positively charged amino acid.

5. X ２０ The antibody or antigen-binding fragment thereof according to claim 1 or 3, wherein is K or E.

6. CDR-H2 is NIYSNPNRX ２３ YYADSVEG (SEQ ID NO: 107) ２３ is any amino acid other than S, T and C.

7. CDR-H2 is SEQ ID NO: 2, SEQ ID NO: 20 (GINGNGDYTYYADSVKG), SEQ ID NO: 21 (AIGGSGSGTYYADSVKG), SEQ ID NO: 22 (NIYSNPNRTYYADSVEG), SEQ ID NO: 23 (NINGPGNGTYYADSVEG), SEQ ID NO: 47 (NIYSNPNRTYYADSVKG), SEQ ID NO: 48 (AISGSGGSTYYADSVEG), SEQ ID NO: 57 (NIYSNPDRTYYADSVEG), SEQ ID NO: 58 (NIYSNPERTYYADSVEG), SEQ ID NO:

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising an amino acid sequence selected from SEQ ID NO: 59 (NIYSNPGRTYYADSVEG), SEQ ID NO: 60 (NIYSNPQRTYYADSVEG), SEQ ID NO: 61 (NIYSNPSRTYYADSVEG), SEQ ID NO: 62 (NIYSNPNRAYYADSVEG), SEQ ID NO: 63 (NIYSNPNREYYADSVEG), SEQ ID NO: 64 (NIYSNPNRGYYADSVEG) and SEQ ID NO: 65 (NIYSNPNNRNYYADSVEG).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein CDR-H3 comprises an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 24 (ASYRNYNYGDY), SEQ ID NO: 25 (ASYDPTNYYDYDY) and SEQ ID NO: 26 (ASYRSTNYFDY).

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein CDR-L3 comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 27 (QQYGSYPPLT).

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:1, CDR-H2 comprises an amino acid sequence selected from SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:

6.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises an amino acid sequence selected from SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:

6.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 5, and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:

27.

13. QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINGNGDYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 28), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIGGSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 29), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 30), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNINGPGNGTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 31), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO:50), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO:51), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPDRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 66), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPERTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 67), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPGRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 68), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPQRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 69), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPSRTYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 70), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRAYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 71), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNREYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 72), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNRGYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 73), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNIYSNPNNRNYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 74), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRNYNYGDYWGQGTLVTVSS (SEQ ID NO: 32), QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYDPTNYYDYWGQGTLVTVSS (SEQ ID NO: 33), and QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKASYRSTNYFDYWGQGTLVTVSS (SEQ ID NO: 34) The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a heavy chain comprising a sequence selected from the group consisting of:

14. ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8) and ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSYPPLTFGQGTKVEIK (SEQ ID NO: 35) The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a light chain comprising a sequence selected from the group consisting of:

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 85 to 103.

16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a light chain comprising an amino acid sequence selected from SEQ ID NO: 104 and SEQ ID NO:

105.

17. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a heavy chain comprising SEQ ID NO:97 and a light chain comprising SEQ ID NO:

104.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharma- ceutical acceptable carrier, excipient or adjuvant.

19. A method for treating a disease or condition characterized by HVEM-positive cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition described in claim 18, thereby treating the disease or condition.

20. 20. The method of claim 19, further comprising inhibiting or blocking a non-HVEM immune checkpoint protein.

21. 21. The method of claim 20, comprising administering an anti-PD-1 / PD-L1 based immunotherapy.

22. The method of any one of claims 19 to 21, further comprising administering adoptive cell therapy.

23. 23. The method of claim 22, wherein the adoptive cell therapy comprises adoptive TIL therapy.

24. 23. The method of claim 22, wherein the adoptive cell therapy comprises administering immune cells expressing a chimeric antigen receptor (CAR), wherein the CAR targets a non-HVEM protein on the surface of the HVEM-expressing cell.

25. The method of any one of claims 19 to 21, wherein the disease or condition is an HVEM-positive cancer or precancerous lesion.

26. 22. The method of any one of claims 19 to 21, wherein the disease or condition is an infectious disease and the infected cells comprise HVEM expression.

27. 22. A method for determining the suitability of a subject to be treated by the method of any one of claims 19 to 21, comprising obtaining a disease sample from the subject and determining the level of HVEM in the sample, wherein positive expression of HVEM indicates that the subject is suitable for the treatment method of any one of claims 19 to 21.

28. 28. The method of claim 27, wherein positive expression of HVEM comprises elevated HVEM levels compared to a healthy sample or a predefined threshold.

29. 4. A method for detecting HVEM in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, thereby detecting HVEM.

30. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

31. The pharmaceutical composition according to claim 18, a. Anti-PD-1 / PD-L1 based immunotherapy; b. A label indicating that the pharmaceutical composition of the invention is for use with anti-PD-1 / PD-L1-based immunotherapy; and c. A secondary detection molecule for detecting at least one antibody or antigen-binding fragment thereof of the pharmaceutical composition of claim 18. A kit comprising at least one of the following:

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