TW202436354A - Anti-cldn6 antibodies and methods of use - Google Patents

Abstract

The present disclosure provides for antibodies and antigen-binding fragments thereof that bind to human CLDN6, a pharmaceutical composition comprising said antibody or antigen-binding fragments thereof, and use of the antibody or antigen-binding fragments thereof or the composition for treating a disease, such as cancer.

Description

Anti-CLDN6 antibodies and methods of use thereof

Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human claudin 6 (CLDN6), multispecific antibodies or antigen-binding fragments thereof that bind to CLDN6 and human cluster of differentiation 3 (human CD3), and methods for producing the same. Specifically, the present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof and methods for treating cancer.

The following description of the background of the present technology is provided only as an aid to understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

The claudin (CLDN) gene family encodes integral membrane proteins that are important structural and functional components of tight junctions (TJs). CLDNs display a four-span transmembrane topology with two extracellular loops and both the N- and C-termini located in the cytoplasm (Krause et al. , Biochimica et Biophysica Acta (BBA) - Biomembranes. 2008). There are 26 human CLDNs expressed in epithelial and endothelial cells in a tissue-specific manner (Günzel et al. , Physiol Rev. 2013). By interacting with each other via cis (intracellular) and trans (intercellular) interactions, CLDNs play an important role in regulating paracellular permeability and maintaining cell polarity (Tsukita et al. , Trends in Biochemical Sciences. 2019). In addition, CLDN can serve as a protein scaffold for assembling complexes at cell junctions and transmit signals to the interior of the cell to regulate gene expression and cell behavior (Matter et al. , Nat Rev Mol Cell Biol. 2003; Singh et al ., Pflugers Arch. 2017).

CLDN6 was first identified and characterized in 2001 (Turksen et al. , Developmental Dynamics. 2001). The expression of CLDN6 is dynamically regulated by various factors and mechanisms (Du et al. , Mol Med Rep. 2021). CLDN6 is one of the earliest proteins expressed in embryonic stem cells that determines epithelial fate and is a cell surface marker of human pluripotent stem cells (hPSCs) (Ben-David et al. , Nat Commun. 2013). Interestingly, CLDN6 expression can be detected in fetal tissues (including stomach, pancreas, lung, and kidney), but not in corresponding adult tissue samples (Reinhard et al ., Science. 2020; Abuazza et al. , Am J Physiol Renal Physiol. 2006; Hashizume et al. , Dev Dyn. 2004). Notably, although transcriptionally silent in normal adult tissues, CLDN6 has been reported to be upregulated in a variety of cancer types including ovarian cancer, endometrial cancer, testicular cancer, lung cancer, gastric cancer, etc. (Kohmoto et al. , Gastric Cancer. 2020; Kojima et al. , Cancers (Basel). 2020; Micke et al. , Int J Cancer. 2014; Sullivan et al. , Am J Surg Pathol. 2012; Ushiku et al. , Histopathology. 2012). The differential expression of CLDN6 between cancer and normal tissues and its membrane localization make it an attractive target for cancer immunotherapy.

An important consideration for targeting CLDN6 is that many members of the CLDN family share high sequence identity, with claudin 9 (CLDN9) having the highest similarity to CLDN6. Only 3 of the 76 residues in the extracellular loops of CLDN6 and CLDN9 differ. Considering that CLDN9 is highly expressed in some normal tissues, achieving high selectivity for CLDN6 over CLDN9 is critical for any therapeutic based on CLDN6-targeted antibodies.

The present disclosure provides anti-CLDN6 antibodies and antigen-binding fragments thereof. The present disclosure includes the following embodiments.

In some aspects, the present disclosure provides an antibody or an antigen-binding fragment thereof, comprising an antigen-binding domain that specifically binds to human claudin 6 (CLDN6).

In some embodiments, the antigen binding domain does not bind to other claudin (CLDN) protein family members.

In some embodiments, the antigen binding domain does not bind to human claudin 9 (CLDN9).

In some embodiments, the first antigen binding domain is highly selective for human CLDN6 over human CLDN9.

In some embodiments, the antigen binding domain that specifically binds to human CLDN6 comprises: (a) a heavy chain variable region comprising: (i) a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 2, (iii) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (iv) a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; (b) a heavy chain variable region comprising: (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 23, (iii) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; (c) a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 39, (iii) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 of SEQ ID NO: 40, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; or (d) a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 1, (ii) HCDR2 of SEQ ID NO: 45, (iii) HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 of SEQ ID NO: 40, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6.

In some embodiments, the antigen binding domain comprises: (a) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8; (b) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a light chain variable region; (c) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 42; (d) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 43, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 44 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a light chain variable region; (e) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 47; or (f) a heavy chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46, and a light chain variable region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 42 has a light chain variable region having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46 or 47 have been inserted, deleted or substituted.

In some embodiments, the antigen-binding domain comprises: (a) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 7, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 8; (b) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 24, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 12; (c) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 41, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 42; (d) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 43, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 44; (e) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 46, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: or (f) the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 46, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 42.

In some embodiments, the antibodies or antigen-binding fragments disclosed herein are monoclonal antibodies, chimeric antibodies, humanized antibodies, human engineered antibodies, single chain antibodies (scFv), Fab fragments, Fab' fragments or F(ab')2 fragments.

In some embodiments, the antibody is a multispecific antibody.

In some embodiments, the antibody is a bispecific antibody.

In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein have antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein have reduced glycosylation or no glycosylation or are hypofucosylated.

In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise an increased bisecting GlcNac structure.

In some embodiments, the Fc domain of the antibodies or antigen-binding fragments thereof disclosed herein is IgG1.

In some embodiments, the Fc domain is IgG1 with reduced effector function.

In some embodiments, the Fc domain is IgG4.

In some aspects, the disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments disclosed herein.

In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises histidine/histidine HCl, trehalose dihydrate and/or polysorbate 20.

In some aspects, the present disclosure provides methods for treating cancer, comprising administering to a patient in need thereof an effective amount of an antibody or antigen-binding fragment as disclosed herein.

In some embodiments, the cancer is a solid cancer.

In some embodiments, the cancer is selected from gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor and/or neuroglioma.

In some embodiments, the antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agents.

In some embodiments, the one or more therapeutic agents are selected from paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

In some embodiments, the one or more therapeutic agents is paclitaxel, lelizumab, or 5-azacytidine.

In some embodiments, the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

In some embodiments, the anti-PD1 antibody is Tislelizumab.

In some aspects, the disclosure provides isolated nucleic acids encoding the antibodies or antigen-binding fragments disclosed herein.

In some aspects, the disclosure provides vectors comprising a nucleic acid.

In some aspects, the disclosure provides host cells comprising a nucleic acid or a vector.

In some aspects, the disclosure provides methods for producing an antibody or antigen-binding fragment disclosed herein, comprising culturing a host cell disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

In some embodiments, the antibodies or antigen-binding fragments disclosed herein are used in a method of treating cancer.

In some aspects, the disclosure provides uses of the antibodies or antigen-binding fragments disclosed herein for the manufacture of a medicament for the treatment of cancer.

In some embodiments, the pharmaceutical compositions disclosed herein are used in a method of treating cancer.

An antibody or an antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to human CLDN6.

Antibodies or antigen-binding fragments, wherein the antigen-binding domain does not bind to other CLDN family members.

An antibody or antigen-binding fragment, wherein the antigen-binding domain does not bind to human CLDN9.

Antibodies or antigen-binding fragments, wherein the antigen-binding domain is highly selective for human CLDN9.

An antibody or antigen-binding fragment, wherein the antigen-binding domain that specifically binds to human CLDN6 comprises: (i).         A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii).        A heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 23, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6 SEQ ID NO: 5 LCDR2, and (f) SEQ ID NO: 6 LCDR3; (iii).       A heavy chain variable region comprising (a) SEQ ID NO: 1 HCDR1, (b) SEQ ID NO: 39 HCDR2, (c) SEQ ID NO: 3 HCDR3, and a light chain variable region comprising: (d) SEQ ID NO: 40 LCDR1, (e) SEQ ID NO: 5 LCDR2, and (f) SEQ ID NO: 6 LCDR3; or (iv).       A heavy chain variable region comprising (a) SEQ ID NO: 1 HCDR1, (b) SEQ ID NO: 45 HCDR2, (c) SEQ ID NO: 3 HCDR3, and a light chain variable region comprising: (d) SEQ ID NO: 40 LCDR1, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

The antibody or antigen-binding fragment of the present invention, wherein the antigen-binding domain comprises: (i).         a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8; (ii).        a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence; (iii).       A heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 42; (iv).       A heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44; (v).        a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 47; or (vi). A heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 42.

The antibody or antigen-binding fragment of the present invention, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46 or 47 have been inserted, deleted or substituted.

The antibody or antigen-binding fragment of the present invention, wherein the antigen-binding domain comprises: (i).         comprising a heavy chain variable region (VH) of SEQ ID NO: 7, and a light chain variable region (VL) of SEQ ID NO: 8; (ii).        comprising a heavy chain variable region (VH) of SEQ ID NO: 24, and a light chain variable region (VL) of SEQ ID NO: 12; (iii).       comprising a heavy chain variable region (VH) of SEQ ID NO: 41, and a light chain variable region (VL) of SEQ ID NO: 42; (iv).       comprising a heavy chain variable region (VH) of SEQ ID NO: 43, and a light chain variable region (VL) of SEQ ID NO: 44; (v).        comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or (vi).       A heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42.

The antibody or antigen-binding fragment of the present invention is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

The antibody of the present invention is a multispecific antibody.

The antibody of the present invention is a bispecific antibody.

The antibody or antigen-binding fragment of the present invention has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

The antibody or antigen-binding fragment of the present invention, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or is low in fucosylation.

The antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises an increased bisecting GlcNac structure.

The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG1.

The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG1 with reduced effector function.

The antibody or antigen-binding fragment of the present invention, wherein the Fc domain is IgG4.

A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention further comprises a pharmaceutically acceptable carrier.

A pharmaceutical composition further comprising histidine/histidine HCl, trehalose dihydrate and polysorbate 20.

A method for treating cancer comprises administering an effective amount of the antibody or antigen-binding fragment of the present invention to a patient in need thereof.

The method, wherein the cancer is gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma.

The method wherein the antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

The method, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, cranberry, lelidomide or 5-azacytidine.

The method, wherein the therapeutic agent is paclitaxel, lelizumab, or 5-azacytidine.

The method, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

The method, wherein the anti-PD1 antibody is tislelizumab.

An isolated nucleic acid encoding an antibody or antigen-binding fragment of the invention.

A vector containing the nucleic acid of the present invention.

A host cell containing the nucleic acid or vector of the present invention.

A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing host cells and recovering the antibody or antigen-binding fragment from the culture.

In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more complementary determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45.

In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising one or more complementation determining regions (HCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 23, SEQ ID NO: 39 and SEQ ID NO: 45, and/or (b) a light chain variable region comprising one or more complementation determining regions (LCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 40.

In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementary determining regions (HCDRs), wherein the one or more HCDRs are HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 45; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and/or (b) a light chain variable region comprising three complementary determining regions (LCDRs), wherein the one or more LCDRs are LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 40; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

In another embodiment, the antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising three complementary determining regions (HCDRs), which are HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; or HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 45 and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and/or (b) a light chain variable region comprising three complementary determining regions (LCDRs), which are LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; or LCDR1 comprising the amino acid sequence of SEQ ID NO: 40, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

In another embodiment, the antibody or antigen-binding fragment comprises: an antigen-binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 2, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

In another embodiment, the antibody or antigen-binding fragment comprises: an antigen-binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 23, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 4, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

In another embodiment, the antibody or antigen-binding fragment comprises: an antigen-binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 39, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

In another embodiment, the antibody or antigen-binding fragment comprises: an antigen-binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 1, (b) HCDR2 of SEQ ID NO: 45, (c) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 40, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6.

In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: (a) a heavy chain variable region having the amino acid sequence of HCDR or VH listed in Table 1; and/or (b) a light chain variable region comprising the amino acid sequence of LCDR or VL listed in Table 1.

In another embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) an amino acid sequence comprising one, two or three amino acid substitutions of the amino acid sequences of HCDR or VH listed in Table 1; and/or (b) a light chain variable region comprising an amino acid sequence comprising one, two, three, four or five amino acid substitutions of the amino acids of LCDR or V listed in Table 1. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

In one embodiment, the antibodies disclosed herein are of IgG1, IgG2, IgG3 or IgG4 isotype. In a more specific embodiment, the antibodies disclosed herein comprise the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2.

In one embodiment, the antibodies of the disclosure bind to CLDN6 with a binding affinity ( _KD ) of 1× ^10-6 M to 1× ^10-10 M. In another embodiment, the antibodies of the disclosure bind to CLDN6 with a binding affinity ( _KD ) of about 1× ^10-6 M, about 1× ^10-7 M, about 1× ^10-8 M, about 1× ^10-9 M, or about 1× ^10-10 M.

In another embodiment, the anti-human CLDN6 antibody disclosed herein exhibits cross-species binding activity to cynomolgus macaque CLDN6.

In one embodiment, the antibodies disclosed herein have strong Fc-mediated effector functions. Such antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing CLDN6.

Cross-references to related applications

This application claims priority to international application PCT/CN2023/079815 filed on March 6, 2023, the entire contents of which are incorporated herein by reference .

Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

As used herein, including in the appended claims, singular forms of words such as "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" is used to mean and is used interchangeably with the term "and/or", unless the context clearly dictates otherwise. As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in an alternative manner ("or").

As used herein, "about" when used with a numerical value means the stated numerical value and the value plus or minus 10%. For example, "about 10" should be understood as "10" and "9-11".

As used herein, a phrase in the form “A/B” or “A and/or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

As used herein, the term "anticancer agent" refers to any agent useful in treating a cell proliferative disorder, such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, and immunotherapeutic agents.

The term "claudin 6" or "CLDN6" refers to a member of the CLDN family. The molecular weight of CLDN6 is 23 kDa. CLDN6 has four transmembrane domains and a PDZ binding region located at the cytoplasmic carboxyl terminus. The amino acid sequence of human CLDN6 can be found in UniPort ID P56747. An exemplary human CLDN6 sequence is SEQ ID NO: 87.

The term "claudin 9" or "CLDN9" refers to another member of the CLDN family. The molecular weight of CLDN9 is 23 kDa, and its amino acid sequence can be found in UniPort ID 095484. An exemplary human CLDN9 sequence is SEQ ID NO: 88.

As used herein, the term "cluster of differentiation 3" or "CD3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3ε, CD3γ, CD3α, and CD3β chain. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring CD3 variants, including, for example, splice variants or allelic variants. CD3 includes, for example, a human CD3ε protein having a length of 207 amino acids (NCBI RefSeq No. NP_000724) and a human CD3γ protein having a length of 182 amino acids (NCBI RefSeq No. NP_000064).

As used herein, the terms "administration," "administring," "treating," and "treatment" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, mean contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of cells encompasses contact between a reagent and a cell, and contact between a reagent and a fluid, wherein a fluid is in contact with a cell. The terms "administering" and "treating" also mean in vitro and ex vivo treatment of cells, for example, by an agent, a diagnostic agent, a binding compound, or by another cell. The term "subject" herein includes any organism. Non-limiting examples include animals. In any embodiment, the animal is a mammal (e.g., a primate, a higher primate, a human, a rat, a mouse, a dog, a cat, a rabbit). In any embodiment, the mammal is a human. In any embodiment, the subject is a patient suffering from or at risk for a disorder described herein. In any embodiment, treating any disease or disorder refers to ameliorating the disease or disorder (i.e., slowing or preventing or reducing the development of the disease or at least one clinical symptom thereof). In another aspect, "treat," "treating," or "treatment" refers to the reduction or improvement of at least one physical parameter, including those parameters that may not be discernible by the patient. In another aspect, "treat," "treating," or "treatment" refers to regulating a disease or condition physically (e.g., stabilization of an identifiable symptom), physiologically (e.g., stabilization of a physical parameter), or both. In another aspect, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or condition. In one aspect, the terms "prevent," "preventing," and "prevention" as used herein with respect to cancer refer to eliminating or reducing the risk of developing cancer. Prevention may also refer to preventing recurrence or secondary cancers once the initial cancer has been treated or cured.

The terms "individual", "subject" and "patient" are used interchangeably herein and refer to any individual mammalian subject, such as a cow, dog, cat, horse or human. In certain embodiments, the individual, subject or patient is a human.

As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. The variable region of an antibody interacts with the antigen at multiple sites within the antigen via non-covalent forces. Generally speaking, the more interactions, the stronger the affinity.

The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that can bind to the corresponding antigen non-covalently, reversibly and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementation determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs) arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies. Antibodies may be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

The term "chimeric" antibody refers to an antibody in which one portion of the heavy chain and/or light chain is derived from a particular source or species, while the remainder of the heavy chain and/or light chain is derived from a different source or species.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region.

In some embodiments, the anti-CLDN6 antibody comprises at least one antigen binding site, at least one variable region. In some embodiments, the anti-CLDN6 antibody comprises an antigen binding fragment of a CLDN6 antibody described herein. In some embodiments, the anti-CLDN6 antibody is isolated or recombinant.

The term "monoclonal antibody" or "mAb" or "Mab" herein means a population of substantially homogeneous antibodies, that is, the antibody molecules contained in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include many different antibodies that have different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epitopes. The modifier "monoclonal" indicates the property of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, e.g., Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein may be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclass thereof such as IgG1, IgG2, IgG3, IgG4. The hybridoma producing the monoclonal antibody may be cultured in vitro or in vivo. High titer monoclonal antibodies can be obtained in vivo in which cells from individual fusion tumors are injected intraperitoneally into mice, such as priming presensitized Balb/c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from culture supernatants using column chromatography methods well known to those skilled in the art.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, human light chains are divided into kappa light chains and lambda light chains. In addition, human heavy chains are typically divided into α, δ, ε, γ or μ, and the isotype of the antibody is defined as IgA, IgD, IgE, IgG and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

The variable regions of each light chain/heavy chain (VL/VH) pair form the antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, the primary sequences of the two binding sites are usually identical.

Typically, both the heavy and light chain variable domains contain three hypervariable regions, also called "complementary determining regions" or "CDRs," which are located between relatively conserved framework regions (FRs). CDRs are usually aligned by the framework regions, thereby enabling binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The positions of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136). (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). The definition of antigen binding sites is also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M. P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., Sternberg M. J. E. (eds.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, according to Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (according to Kabat numbering). According to IMGT, the program IMGT/DomainGap Align can be used to determine the CDR regions of an antibody.

The term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from the "CDRs" (e.g., LCDR1, LCDR2, and LCDR3 in the light chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain). See Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies, and multispecific antibodies formed from antibody fragments.

As used herein, an antibody "specifically binds" to a target protein means that the antibody exhibits preferential binding to the target as compared to other proteins, but such specificity does not require absolute binding specificity. Antibody "specifically binds" or "selectively binds" is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment to refer to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological products, such as in a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least two-fold when compared to background levels and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, the antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least ten (10) fold when compared to background levels under specified immunoassay conditions and does not specifically bind to other antigens present in a sample in significant amounts.

As used herein, an "antigen binding domain" comprises at least three CDRs and specifically binds to an epitope. The "antigen binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen binding domain that specifically binds to a first epitope and a second antigen binding domain that specifically binds to a second epitope, also comprising at least three CDRs. Multispecific antibodies may be bispecific, trispecific, tetraspecific, etc., with antigen binding domains directed to each specific epitope. Multispecific antibodies may be multivalent (e.g., a bispecific tetravalent antibody), comprising multiple antigen binding domains, e.g., 2, 3, 4 or more antigen binding domains that specifically bind to a first epitope and 2, 3, 4 or more antigen binding domains that specifically bind to a second epitope.

The term "human antibody" herein means an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, mouse cell, or a hybridoma derived from a mouse cell, a human antibody may contain a murine carbohydrate chain. Similarly, a "mouse antibody" or a "rat antibody" means an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

The term "humanized" or "humanized antibody" means an antibody form that contains sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin sequence. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. Where necessary, the prefix "hum", "hu", "Hu" or "h" is added to the antibody homologous name to distinguish the humanized antibody from the parent rodent antibody. Humanized forms of haloperidol antibodies generally comprise the same CDR sequences as the parent haloperidol antibody, although certain amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

The term "epitope" refers to a specific site on an antigen to which an antibody binds. The specific site on an antigen to which an antibody binds can be determined, for example, by crystallography. Methods such as hydroxyl radical protein footprinting and alanine scanning mutagenesis can also be used, but may have lower resolution.

The term "monospecific antibody" refers to an antibody that specifically binds to only one antigen. A monospecific antibody may bind to only one epitope of an antigen or may bind to two or more epitopes of an antigen. A monospecific antibody that binds to two or more epitopes of an antigen is a monospecific multi-epitope antibody.

The term "multispecific antibody" or "multi-specific antibody" refers to an antibody that specifically binds to two or more antigens ( e.g., bispecific antibodies, trispecific antibodies, etc.). Non-limiting examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH/VL unit has multiple epitope specificities; antibodies having two or more VL and VH domains, wherein each VH/VL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; diabodies; terabodies, etc., as well as full-length antibodies and/or antibody fragments that have been covalently or non-covalently linked.

The terms "polyepitopic antibody" and "antibody with polyepitopic specificity" are used interchangeably herein to refer to an antibody that binds to two or more epitopes on the same or different antigens.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Eu numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Therefore, the composition of intact antibodies may include antibody populations with all Lys447 residues removed, antibody populations with no Lys447 residues removed, and antibody populations having a mixture of antibodies with and without Lys447 residues.

A "functional Fc region" has the effector function of a native sequence Fc region. Exemplary effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays disclosed herein or otherwise known in the art. A functional Fc region may have effector functions that are substantially similar to wild-type IgG, reduced effector functions compared to wild-type IgG, or enhanced effector functions compared to wild-type IgG. For antibodies comprising a human Fc region, comparisons are typically made to wild-type human IgG1.

A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region and naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification (e.g., about one to about ten amino acid modifications, and in some embodiments, about one to about five amino acid modifications), preferably one or more amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with a native sequence Fc region and/or an Fc region of a parent polypeptide, preferably at least about 90% homology therewith, or preferably at least about 95% homology therewith. In some embodiments, the variant Fc region may have reduced or enhanced effector function compared to wild-type IgG. For antibodies comprising a human Fc region, comparisons are typically made to wild-type human IgG1.

The term "Fc component" used herein refers to the hinge region, CH2 domain or CH3 domain of the Fc region.

The term "hinge region" is generally defined as stretching from about residues 216 to 230 of IgG (Eu numbering), from about residues 226 to 243 of IgG (Kabat numbering), or from about residues 1 to 15 of IgG (IMGT unique numbering).

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') ₂ , F(ab) _c , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), trias, tetrabodies, single-chain antibodies, scFv, scFv dimers, single domain antibodies, single-domain antibodies, and multivalent domain antibodies. Typically, the binding fragment competes for specific binding with the intact antibody from which it is derived. Binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

The term "Fab" refers to the portion of an antibody composed of a single light chain (both variable and first constant regions) joined by disulfide bonds to a single heavy chain.

The term "Fab'" refers to the Fab fragment including a portion of the hinge region.

The term "F(ab') ₂ " refers to a dimer of Fab'. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The term "Fv" refers to the smallest fragment of an antibody with a complete antigen-binding site. The Fv fragment consists of a single light chain variable region combined with a single heavy chain variable region.

The term "single-chain antibody" refers to an antibody consisting of a heavy chain variable region and a light chain variable region connected by a linker. In most cases, but not all cases, the linker can be a peptide. The length of the linker varies depending on the type of single-chain antibody. Covalently or non-covalently linking two or more single-chain antibodies together will produce higher-order forms. Single-chain antibodies and their higher-order forms may include but are not limited to single-domain antibodies, multivalent domain antibodies, single-chain variant fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), biantibodies, and triabodies and tetrabodies.

The terms "single-chain Fv antibody" and "scFv" are used interchangeably herein and refer to a single-chain antibody consisting of a heavy chain variable region and a light chain variable region connected by a linker. In most cases, but not all cases, the linker can be a peptide. The length of the linker peptide is preferably about 5 to 30 amino acids, or about 10 to 25 amino acids. Typically, the linker stabilizes the variable domain without interfering with the correct folding and the generation of active binding sites. In a preferred embodiment, the linker peptide is rich in glycine and serine or threonine. Two or more scFvs are covalently or non-covalently linked together to produce higher-order forms such as di-scFv, tri-scFv, tetra-scFv, etc. The antigen binding sites of each scFv in a higher order format can target the same or different antigens or epitopes.

The term "single-chain Fv-Fc antibody" or "scFv-Fc" refers to a full-length antibody consisting of a scFv linked to an Fc region.

"Diabodies" are higher-order variants of single-chain antibodies composed of two single-chain antibodies. For each single-chain antibody, a linker is used that is too short to allow pairing between two domains on the same chain, forcing the domains to pair with complementary domains of the other chain, thereby generating two antigen-binding sites. In most cases, but not all, the linker can be a peptide. The antigen-binding sites can target the same or different antigens or epitopes. Triabodies (three single-chain antibodies assembled to form three antigen-binding sites), tetrabodies (four single-chain antibodies assembled to form four antigen-binding sites) and higher-order variants can be similarly generated. See, e.g., Holliger P. et al., Proc Natl Acad Sci USA . Jul 15;90(14):6444-8 (1993); EP404097; WO93/11161.

"Single domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In certain instances, two or more _VH domains are covalently linked with a peptide linker to generate a multivalent domain antibody. The two or more _VH domains of a multivalent domain antibody may be directed against the same or different antigens or epitopes.

The term "heavy chain antibody" refers to an antibody composed of two heavy chains. Heavy chain antibodies may be IgG-like antibodies from camels, camels, alpacas, sharks, etc., or IgNARs from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94/04678; WO94/25591; or U.S. Patent No. 6,005,079. Heavy chain antibodies were originally derived from camelids (camels, dromedaries and camels). Despite the lack of light chains, camelized antibodies have a veritable antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics . Apr;54(1):39-47 (2002); Nguyen VK et al. Immunology . May;109(1):93-101 (2003)). The variable domain (VHH domain) of a heavy chain antibody represents the smallest known antigen-binding unit produced by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)).

The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A corresponding human germline sequence may be only a framework region, only a complementary determining region, a framework and complementary determining region, a variable segment (as defined above), or other combinations of sequences or subsequences comprising a variable region. Sequence identity may be determined using the methods described herein, such as by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences, or mutant forms of human germline sequences, or antibodies comprising a common framework sequence derived from human framework sequence analysis, such as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time-1) divided by the association rate constant (ka, time-1, M1). The equilibrium dissociation constant can be measured by any method known in the art. The antibodies disclosed herein will typically have an equilibrium dissociation constant of less than about 10-7 or 10-8 M, such as less than about 10-9 M or 10-10 M, in some aspects, less than about 10-11 M, 10-12 M or 10-13 M.

The term "cancer" or "tumor" herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of the present disclosure, cancer is not limited to a particular type or location.

In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the replacement of an original amino acid with a new amino acid that does not substantially change the chemical, physical and/or functional properties of the antibody or fragment, such as its binding affinity for CLDN6. In particular, common conservative amino acid substitutions are well known in the art.

As used herein, the term "knobs-into-holes" technology refers to the amino acid pairing of two polypeptides together by introducing a spatial protrusion (knob) into one polypeptide and a socket or cavity (hole) into the other polypeptide at the interface where the polypeptides interact, either in vitro or in vivo. For example, knobs-into-holes have been introduced into the Fc:Fc binding interface, CL:CHI interface, or VH/VL interface of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knobs-into-holes ensure that two different heavy chains are correctly paired together during the production of multispecific antibodies. For example, a multispecific antibody with knobs-in-hole amino acids in its Fc region may further comprise a single variable domain linked to each Fc region, or further comprise different heavy chain variable domains paired with similar or different light chain variable domains. Knobs-in-hole technology can also be used for VH or VL regions to ensure correct pairing.

The term "knob" as used herein in the context of the "knob-into-hole" technique refers to an amino acid change that introduces a protrusion (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

The term "hole" as used herein in the context of "knob into hole" refers to an amino acid change that introduces a socket or cavity (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, other polypeptides have a knob mutation.

An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying shorter words of length W in the query sequence that match or satisfy some positive value threshold score T when aligned with a word of the same length in a database sequence. T is called the neighborhood word score threshold. These initial neighborhood word hits serve as initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence to the extent that the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score drops to zero or lower due to the accumulation of one or more negative residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the defaults wordlength (W) of 11, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLAST program uses the defaults wordlength of 3 and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software suite, using either the BLOSUM62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

In the context of nucleic acids, the term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to a functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, it is operably linked to a coding sequence. In general, a promoter transcriptional regulatory sequence that is operably linked to a transcribed sequence is physically contiguous with the transcribed sequence, that is, it acts in sequence. However, some transcriptional regulatory sequences, such as enhancers, are not necessarily physically contiguous or located in close proximity to the coding sequence whose transcription they enhance.

In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-CLDN6 multispecific antibody as described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents and the like that are physiologically compatible. Excipients may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions disclosed herein may be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The suitable form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable solutions or infusible solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment of the disease, disorder or condition. The "therapeutically effective amount" may vary with the antibody, the disease, disorder, and/or symptoms of the disease or disorder, the severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. In any given case, the appropriate amount will be apparent to one skilled in the art or can be determined by routine experimentation. In the case of combination therapy, the "therapeutically effective amount" refers to the total amount of the composition used to effectively treat the disease, disorder or condition.

The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the treatment conditions or disorders described in this disclosure. Such administration encompasses the co-administration of such therapeutic agents in a substantially simultaneous manner. Such administration also encompasses the co-administration of each active ingredient in multiple containers or in separate containers (e.g., capsules, powders, and liquids). The powders and/or liquids may be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also encompasses the use of various types of therapeutic agents in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen provides the beneficial effects of the drug combination in the treatment of the conditions or disorders described herein.

As used herein, the phrase "in combination with" means that the anti-CLDN6xCD3 multispecific antibody is administered to a subject simultaneously with, just before, or just after the administration of an additional therapeutic agent. In certain embodiments , the anti-CLDN6xCD3 multispecific antibody is administered as a co-formulation with the additional therapeutic agent.

The present disclosure provides antibodies, antigen-binding fragments, and anti-CLDN6 multispecific antibodies. In addition, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable properties, and therefore can be used to reduce the likelihood of cancer or to treat cancer. The present disclosure also provides pharmaceutical compositions comprising the antibodies and methods of preparing and using such pharmaceutical compositions for the prevention and treatment of cancer and related disorders. Anti- CLDN6 Antibodies

The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CLDN6. The antibodies or antigen-binding fragments disclosed herein include but are not limited to antibodies or antigen-binding fragments thereof generated as described below.

The present disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence listed in Table 1. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises a HCDR having an amino acid sequence of any HCDR listed in Table 1. In one aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody comprises (or consists of) one, two, three or more HCDRs having an amino acid sequence of any HCDR listed in Table 1.

The present disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises a VL domain having an amino acid sequence listed in Table 1. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises a LCDR having an amino acid sequence of any LCDR listed in Table 1. In a specific aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to CLDN6, wherein the antibody or antigen-binding fragment comprises (or consists of) one, two, three or more LCDRs having an amino acid sequence of any LCDR listed in Table 1.

Other antibodies or antigen-binding fragments thereof disclosed herein include amino acids that have been altered but have at least 60%, 70%, 80%, 90%, 95% or 99% identity in the CDR regions with the CDR regions disclosed in Table 1. In some embodiments, the amino acid sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some aspects, they include amino acid alterations wherein no more than 1, 2, 3, 4 or 5 amino acids in the CDR regions are altered compared to the CDR regions depicted in the sequences described in Table 1.

Other antibodies disclosed herein include those in which the amino acids or nucleic acids encoding the amino acids have been altered, but have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences described in Table 1. In some embodiments, the amino acid sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some aspects, they include changes in the amino acid sequence, wherein no more than 1, 2, 3, 4 or 5 amino acids in the variable region are altered compared to the variable region depicted in the sequence described in Table 1, while retaining substantially the same therapeutic activity.

The present disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chain and full-length light chain of antibodies that specifically bind to CLDN6. Such nucleic acid sequences can be optimized for expression in mammalian cells.

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CLDN6. In certain aspects, the antibody and the antigen-binding fragment may bind to the same epitope of CLDN6.

The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CLDN6 antibodies described in Table 1. Thus, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in a binding assay (e.g., competitively inhibit the binding of other antibodies in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CLDN6 demonstrates that the test antibody can compete with the antibody or antigen-binding fragment thereof for binding to CLDN6. Without being bound by any theory, such antibodies may bind to the same or related (e.g., structurally similar or spatially adjacent) epitope on CLDN6 as the antibody or antigen-binding fragment thereof that they compete with. In one aspect, the antibody that binds to the same epitope on CLDN6 as the antibody or antigen-binding fragment thereof disclosed herein is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

In one embodiment, the anti-CLDN6 antibody disclosed herein may be an anti-CLDN6 multispecific antibody. The antibody molecule is a multispecific antibody molecule, for example, comprising a plurality of antigen binding domains, wherein at least one antigen binding domain sequence specifically binds to CLDN6 as a first epitope, and the second antigen binding domain sequence specifically binds to a second epitope. In one embodiment, the multispecific antibody comprises a third, fourth or fifth antigen binding domain. In one embodiment, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody. In each example, the multispecific antibody comprises at least one anti-CLDN6 antigen binding domain and at least one anti-CD3 antigen binding domain.

In one embodiment, the multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds only two antigens. A bispecific antibody comprises a first antigen binding domain that specifically binds to CLDN6 and a second antigen binding domain that specifically binds to another epitope. This includes a bispecific antibody comprising a heavy chain variable domain and a light chain variable domain that specifically bind to CLDN6 as a first epitope and a heavy chain variable domain that specifically binds to CD3 as a second epitope. The bispecific antibody comprises an antigen binding fragment, which may be a Fab, F(ab')2, Fv, or a single chain Fv (ScFv) or scFv.

Previous experiments (Coloma and Morrison, Nature Biotech. 15: 159-163 (1997)) describe engineering tetravalent bispecific antibodies by fusing DNA encoding a single-chain anti-tansyl antibody Fv (scFv) to the C-terminus (CH3-scFv) or hinge (hinge-scFv) of an IgG3 anti-tansyl antibody. The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen binding domains that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibodies. The multivalent antibodies herein comprise three to eight, but preferably four, antigen binding domains that specifically bind to at least two antigens. Linker

It is also understood that the domains and/or regions of the polypeptide chains of the bispecific tetravalent antibody can be separated by linker regions of various lengths. In some embodiments, the antigen binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3 or the entire Fc region by linker regions. For example, VL1-CL-(linker) VH2-CH1, VH-linker-VL. Such linker regions may comprise randomly assorted amino acids or a restricted set of amino acids. Such linker regions may be flexible or rigid (see US2009/0155275).

Multispecific antibodies are constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments with or without a flexible linker (Mallender et al., J. Biol. Chem. 1994 269:199-206; Mack et al., Proc. Natl. Acad. Sci. USA. 1995 92:7021-5; Zapata et al., Protein Eng. 1995 8.1057-62); via dimerization apparatus such as leucine zipper (Kostelny et al., J. Immunol. 1992 148:1547-53; de Kruifetal J. Biol. Chem. 1996 271:7630-4) and Ig C/CH1 domains (Muller et al., FEBS Lett. 422:259-64); by bifunctional antibodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., Bio/Technology (NY) 1996 14:192-6); Fab-scFv fusion (Schoonjans et al., J. Immunol. 2000 165:7050-7); and miniantibody formats (Pack et al., Biochemistry 1992. 31:1579-84; Pack et al., Bio/Technology 1993 11:1271-7).

The bispecific tetravalent antibodies disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more amino acid residues between one or more of the antigen binding domain, CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain or Fc region. In some embodiments, the amino acids glycine and serine comprise amino acids within the linker region. In another embodiment, the linker can be GS, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGSGS, GGGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA(G4S) )4, SAKTTP, SAKTTPKLGG, SAKTTPKLEEGEFSEARV, ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALMALS, GPAKELTPLKEAKVS and GHEAAAVMQVQYPAS or any combination thereof (see WO2007/024715). Dimerization-specific amino acids

In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change results in a "knobs into holes" interaction and increases the assembly of the correct multivalent antibody. The dimerization-specific amino acid may be located in the CH1 domain or the CL domain or a combination thereof. Dimerization-specific amino acids for pairing CH1 domains with other CH1 domains (CH1-CH1) and for pairing CL domains with other CL domains (CL-CL) may be found in at least the disclosures of WO2014082179, the WO2015181805 family, and WO2017059551. The dimerization-specific amino acid may also be located in the Fc domain and may be combined with dimerization-specific amino acids in the CH1 or CL domains. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair. Further changes in the Fc region framework

In other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced by different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of a complement. This method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260, both filed by Winter et al.

In another aspect, one or more amino acid residues can be replaced by one or more different amino acid residues, such that the antibody has altered C1q binding and/or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

In another aspect, one or more amino acid residues are changed to change the ability of the antibody to fix complement. This method is described in, for example, the publication WO 94/29351 of Bodmer et al. In a specific aspect, one or more amino acids of the antibody or antigen-binding fragment thereof disclosed herein are replaced by one or more heterotypic amino acid residues of the IgG1 subclass and the κ isotype. Heterotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgG1, IgG2 and IgG3 subclasses and the constant region of the light chain of the κ isotype, as described by Jefferis et al., MAbs. 1:332-338 (2009).

In another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This approach is described, for example, in publication WO00/42072 by Presta. In addition, the binding sites of FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

In another aspect, the glycosylation of the multispecific antibody is modified. For example, a non-glycosylated antibody can be prepared (i.e., the antibody lacks glycosylation or has reduced glycosylation). For example, the glycosylation can be changed to increase the affinity of the antibody for the "antigen". Such carbohydrate modifications can be accomplished by, for example, changing 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 the site. Such non-glycosylation can increase the affinity of the antibody for the antigen. Such methods are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered types of glycosylation can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in this technology and can be used as host cells in which recombinant antibodies are expressed, thereby producing antibodies with altered glycosylation. For example, Hang et al. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in this cell line are hypofucosylated. Presta's publication WO 03/035835 describes a mutant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al. WO99/54342 describes a cell line engineered to express a glycoprotein modifying glycosyltransferase, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

In another aspect, if it is desired to reduce ADCC, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and almost no CDC effector function (Moore GL et al., 2010 MAbs, 2:181-189). However, it was found that native IgG4 is less stable under stress conditions, such as in acidic buffer or at elevated temperatures (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al., 2002 Immunol, 105:9-19). Reduction of ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with a combination of changes that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which leads to the generation of bispecific antibodies in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten M et al., 2007 Science, 317:1554-157). The mutation of serine to proline at position 228 (EU numbering system) appears to have an inhibitory effect on IgG4 heavy chain dissociation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19). It has been reported that some amino acid residues in the hinge and γFc region affect the interaction between antibodies and Fcγ receptors (Chappel SM et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev Immunol, 25:21-50). In addition, some IgG4 isoforms that are rarely found in the human population may also give rise to different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To generate multispecific antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce many changes. Such modified IgG4 Fc molecules can be found in U.S. Patent No. 8,735,553 to Li et al., which is incorporated herein by reference. Antibody Generation

Antibodies and antigen-binding fragments thereof can be produced by any method known in the art, including but not limited to recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

The present disclosure further provides polynucleotides encoding antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising complementary determining regions as described herein. In some aspects, the polynucleotides encoding the heavy chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 56, SEQ ID NO: 60 and SEQ ID NO: 64. In some aspects, the polynucleotides encoding the light chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO: 10, 57, 61 or 65.

The polynucleotides disclosed herein can encode the variable region sequences of anti-CLDN6 antibodies. They can also encode the variable regions and constant regions of antibodies. Some polynucleotide sequences encode polypeptides comprising the heavy chain and light chain variable regions of exemplary anti-CLDN6 antibodies.

The present disclosure also provides expression vectors and host cells for producing anti-CLDN6 antibodies. The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-CLDN6 antibody chain or antigen-binding fragment. In some aspects, an inducible promoter is used to prevent expression of the inserted sequence unless under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population toward coding sequences whose expression products are more easily tolerated by host cells. In addition to the promoter, other regulatory elements may be required or desired for efficient expression of the anti-CLDN6 antibody or antigen-binding fragment. Such elements typically include the ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be enhanced by including an enhancer suitable for the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

Host cells used to carry and express anti-CLDN6 antibody chains can be prokaryotic or eukaryotic. E. coli is a suitable prokaryotic host for cloning and expressing the polynucleotides disclosed herein. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cells. In addition, there will be any number of various well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the promoter system from bacteriophage lambda. Promoters typically control expression together with an operator sequence as appropriate, and have ribosome binding site sequences and the like for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-CLDN6 antibodies. Combinations of insect cells and bacillivirus vectors can also be used. In other aspects, mammalian host cells are used to express and produce the anti-CLDN6 antibodies of the present disclosure. For example, it can be a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line carrying an exogenous expression vector. These include any normal mortal cell or normal or abnormal immortal animal or human cell. For example, a variety of suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences, such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Such expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type specific, stage specific, and/or regulatable or controllable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as the human immediate early CMV promoter), constitutive CMV promoters, and promoter-enhancer combinations known in the art. Generation of Bispecific Antibodies

The current standard for engineering heterodimeric antibody Fc domains is the knobs-into-hole (KiH) design, which introduces mutations at the core CH3 domain interface. The resulting heterodimers have a reduced CH3 unbinding temperature (69°C or lower). In contrast, the ZW heterodimeric Fc design has a thermal stability of 81.5°C, comparable to the wild-type CH3 domain. Detection and diagnostic methods

The antibodies or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for detecting CLDN6. In one aspect, the antibodies or antigen-binding fragments can be used to detect the presence of CLDN6 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain aspects, the biological sample comprises cells or tissues. In other aspects, such tissues include normal and/or cancerous tissues that express CLDN6 at higher levels relative to other tissues.

In one aspect, the present disclosure provides a method for detecting the presence of CLDN6 in a biological sample. In certain aspects, the method comprises contacting the biological sample with an anti-CLDN6 antibody under conditions that allow the antibody to bind to the antigen and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, urine, tissue, saliva, or blood samples.

Also included are methods for diagnosing a disorder associated with CLDN6 expression. In certain aspects, the method comprises contacting a test cell with an anti-CLDN6 antibody; determining the expression level of CLDN6 expressed by the test cell (quantitatively or qualitatively) by detecting binding of the anti-CLDN6 antibody to a CLDN6 polypeptide; and comparing the expression level of the test cell with the expression level of CLDN6 in a control cell (e.g., a normal cell or a non-CLDN6 expressing cell of the same tissue origin as the test cell), wherein a higher level of CLDN6 expression in the test cell compared to the control cell indicates the presence of a disorder associated with CLDN6 expression. Treatment Methods

The antibodies or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for treating CLDN6-related disorders or diseases. In one aspect, the CLDN6-related disorder or disease is cancer.

In one aspect, the present disclosure provides a method for treating cancer. In certain aspects, the method comprises administering an effective amount of an anti-CLDN6 antibody or antigen-binding fragment to a patient in need thereof. In some embodiments, the cancer is a solid tumor. The cancer may include, but is not limited to, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdomyosarcoma and/or neuroglioma.

The antibodies or antigen-binding fragments disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if local treatment is required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulse infusion.

The antibodies or antigen-binding fragments disclosed herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to the practicing physician. The antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used in the same dosages and by administration routes as described herein, or about 1 to 99% of the dosages described herein, or in any dosage and by any route determined to be appropriate based on experience/clinical practice.

For the prevention or treatment of disease, the appropriate dose of the antibodies or antigen-binding fragments disclosed herein will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 100 mg/kg of the antibody may be an initial candidate dose for administration to the patient, whether by, for example, one or more separate administrations or by continuous infusion. A typical daily dose range may be about 1 μg/kg to 100 mg/kg or more, depending on the above factors. For repeated administrations over several days or longer, depending on the condition, treatment is generally continued until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives about two to about twenty, or, for example, about six doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of such therapy is readily monitored by known techniques and assays. Combination Therapy

In one embodiment, the anti-CLDN6 antibodies disclosed herein can be used in combination with other therapeutic agents. Other therapeutic agents that can be used together with the anti-CLDN6 antibodies disclosed herein include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel; (e.g., Abraxane®), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, isocyclophosphamide, oxaliplatin, pemetrexed disodium; disodium), cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g. EGFR inhibitors (e.g. erlotinib), multikinase inhibitors (e.g. MGCD265, RGB-286638), CD-20 targeted agents (e.g. rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeted agents (e.g. alemtuzumab), prednisolone, darbepoetin alfa alfa), lelidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19 targeting agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR/ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g. CS-1008), EGEN-001, Polo-like kinase 1 inhibitors (e.g. BI 672).

The anti-CLDN6 antibodies disclosed herein can be used in combination with other therapeutic agents, such as immune checkpoint antibodies. Such immune checkpoint antibodies may include anti-PD1 antibodies. Anti-PD1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab or nivolumab. Tislelizumab is disclosed in US 8,735,553. Pembrolizumab (formerly known as MK-3475) is disclosed in US 8,354,509 and US 8,900,587 and is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is in clinical studies for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clonal 5C4) is disclosed in U.S. Patent No. US 8,008,449 and WO 2006/121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer and Hodgkin's lymphoma.

Other immune checkpoint antibodies combined with anti-CLDN6 antibodies may include anti-TIGIT antibodies. Such anti-TIGIT antibodies may include, but are not limited to, anti-TIGIT antibodies as disclosed in WO2019/129261.

Other immune checkpoint antibodies combined with anti-CLDN6 antibodies may include anti-OX40 antibodies. Such anti-OX40 antibodies may include, but are not limited to, anti-OX40 antibodies as disclosed in WO2019/223733.

Other immune checkpoint antibodies combined with anti-CLDN6 antibodies may include anti-TIM3 antibodies. Such anti-TIM3 antibodies may include, but are not limited to, anti-TIM3 antibodies as disclosed in WO2018/036561. Pharmaceutical Compositions and Formulations

Compositions, including pharmaceutical formulations, are also provided, which include anti-CLDN6 antibodies or antigen-binding fragments thereof, or polynucleotides comprising sequences encoding anti-CLDN6 antibodies or antigen-binding fragments thereof. In certain embodiments, the composition includes one or more anti-CLDN6 antibodies or antigen-binding fragments, or one or more polynucleotides comprising sequences encoding one or more anti-CLDN6 antibodies or antigen-binding fragments. Such compositions may also include a suitable carrier, such as a pharmaceutically acceptable excipient well known in the art, including a buffer.

Pharmaceutical formulations of anti-CLDN6 antibodies or antigen-binding fragments as described herein are prepared by mixing such antibodies or antigen-binding fragments having the desired purity with one or more pharmaceutically acceptable carriers, as appropriate (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersions, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Nos. US 7,871,607 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

In one embodiment, the formulation consists of L-histidine/L-histidine hydrochloride monohydrate, trehalose and polysorbate 20. In another embodiment, after reconstitution with sterile water for injection, the concentration of the anti-CLDN6 antibody drug product is an isotonic solution consisting of 10 mg/mL anti-CLDN6 antibody, 20 mM histidine/histidine HCl, 240 mM trehalose dihydrate and 0.02% polysorbate 20 (pH about 5.5).

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles such as films or microcapsules.

Formulations for intravenous administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

The sequence listings of the present disclosure are provided in Tables 1 to 3 below. Table 1. Sequence Listing (Kabat Numbering ) antibody SEQ ID NO Notes sequence BG87P SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 2 VH CDR2 WIYPGSGYTKYNEKFKV SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 7 V A QIQLQQSGPELVKPGASVKISCKASGYSFTDYHINWVKQRPGQGLEWIGWIYPGSGYTKYNEKFKVKATLTVDTSSSTAYMHLTSLTSEDSAVYFCARSGIYHYVSSYYFDHWGQGTTLTVSS SEQ ID NO: 8 V L DIVMTQSHTFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYTFGGGTKLEIK SEQ ID NO: 9 VH DNA CAGATTCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCTCCGGTGAAGATCAGCTGCAAGGCCTCCGGCTACAGCTTCACCGACTACCACATCAACTGGGTGAAGCAGAGACCCGGCCAAGGCCTGGAGTGGATCGGCTGGATCTACCCGGCAGCGGCTACACCAAGTACAACGAGAAGTTCAAGGTGAAGGCCACCCTGACCGTCGACACAAGCAGCAGCACCGCCTACATGCACCTGACAAGCCT GACAAGCGAGGACAGCGCCGTGTACTTCTGCGCTAGAAGCGGCATCTACCACTACGTGAGCAGCTACTACTTCGACCACTGGGGCCAAGGCACCACCCTCACCGTGAGCAGC SEQ ID NO: 10 VL DNA GACATCGTGATGACACAGAGCCACACCTTCATGAGCACAAGCGTGGGCGACAGAGTGAGCATCACCTGCAAGGCCTCCCAAGACGTGGGCACCGCCGTCGCCTGGTATCAGCAGAAGCCCGGGCAGAGCCCCAAGCTGCTGATCTACTGGGCCTCCACAAGACACACCGGCGTGCCCGACAGATTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAACGTGCAGAGCGAGGACCTGGCCGACTACT TCTGTCAGCAGTACAGCAGCTACACCTTCGGCGGGGGCACCAAGCTGGAGATCAAG ChBG87P SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 2 VH CDR2 WIYPGSGYTKYNEKFKV SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT BG87P-z0 SEQ ID NO: 11 V A QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-Bz0 SEQ ID NO: 13 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz1 SEQ ID NO: 15 V A QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz2 SEQ ID NO: 16 V A QIQLVQSGAEVKKPGASVKVSCKASGYTFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz3 SEQ ID NO: 17 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTITVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz4 SEQ ID NO: 18 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTRDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz5 SEQ ID NO: 19 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz6 SEQ ID NO: 13 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 20 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz7 SEQ ID NO: 13 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 21 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-Bz8 SEQ ID NO: 13 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 22 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-21 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 24 V A QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-22 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 25 V A QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-23 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 26 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-24 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 27 V A QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTLTRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-25 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 28 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTLTRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-26 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 29 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-27 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 23 VH CDR2 WIYPGSGYTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 4 VL CDR1 KASQDVGTAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 30 V A QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTLTRDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 12 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-m1 SEQ ID NO: 31 V A QIQLQQSGPELVKPGASVKISCKASGYSFTDYHINWVKQRPGQGLEWIGWIYPGSGYTKYNEKFKVKATLTVDTSSSTAYMHLTSLTSEDSAVYFCARSGIYHYVSSYYFEHWGQGTTLTVSS SEQ ID NO: 8 V L DIVMTQSHTFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYTFGGGTKLEIK BG87P-m2 SEQ ID NO: 32 V A QIQLQQSGPELVKPGASVKISCKASGYSFTDYHINWVKQRPGQGLEWIGWIYPGSGYTKYNEKFKVKATLTVDTSSSTAYMHLTSLTSEDSAVYFCARSGIYHYVSSYYFQHWGQGTTLTVSS SEQ ID NO: 8 V L DIVMTQSHTFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYTFGGGTKLEIK BG87P-m3 SEQ ID NO: 33 V A QIQLQQSGPELVKPGASVKISCKASGYSFTDYHINWVKQRPGQGLEWIGWIYPGSGYTKYNEKFKGKATLTVDTSSSTAYMHLTSLTSEDSAVYFCARSGIYHYVSSYYFDHWGQGTTLTVSS SEQ ID NO: 8 V L DIVMTQSHTFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYTFGGGTKLEIK BG87P-m4 SEQ ID NO: 34 V A QIQLQQSGPELVKPGASVKISCKASGYSFTDYAINWVKQRPGQGLEWIGWIYPGSGYTKYNEKFKVKATLTVDTSSSTAYMHLTSLTSEDSAVYFCARSGIYHYVSSYYFDHWGQGTTLTVSS SEQ ID NO: 8 V L DIVMTQSHTFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYTFGGGTKLEIK BG87P-m5 SEQ ID NO: 35 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFEHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-m6 SEQ ID NO: 36 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFQHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-m7 SEQ ID NO: 37 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYHINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-m8 SEQ ID NO: 38 V A QIQLVQSGAEVKKPGASVKVSCKASGYSFTDYAINWVRQAPGQRLEWMGWIYPGSGYTKYNEKFKVRVTLTVDTSASTAYMELSSLRSEDTAVYFCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 14 V L DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKSPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSVQPDDFATYFCQQYSSYTFGGGTKVEIK BG87P-33 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 39 VH CDR2 YIYPGSGHTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 40 VL CDR1 KASQDAGSAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 41 V A EVQLQESGGGLVQPGGSLRLSCAASGYSFTDYHINWVRQAPGKGLEWVSYIYPGSGHTKYNEKFKGRFTISRDTSKNTAYLQMNSLRAEDTAVYYCARSGIYHYVSSYYFDHWGQGTLVTVSS SEQ ID NO: 42 V L DIQMTQSPSTLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-34 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 39 VH CDR2 YIYPGSGHTKYNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 40 VL CDR1 KASQDAGSAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 43 V A EVQLQESGGGLVQPGGSLRLSCAASGYSFSDYHINWVRQAPGKGLEWVGYIYPGSGHTKYNEKFKGRFTISRDTSSNTAYLQMNSLRAEDTAVYYCARSGIYHYVSSYYFDHWGQGTLVTVSS SEQ ID NO: 44 V L DIQMTQSPSSSLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYTFGGGTKLEIK BG87P-31 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 45 VH CDR2 YIYPGSGHTKKNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 40 VL CDR1 KASQDAGSAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 46 V A QVQLQQSGAETKKPGASVKVSCKASGYSFTDYHINWVRQASGQRLEWMGYIYPGSGHTKKNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 47 V L DIQMTQSPSTLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIKWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK BG87P-32 SEQ ID NO: 1 VH CDR1 DYHIN SEQ ID NO: 45 VH CDR2 YIYPGSGHTKKNEKFKG SEQ ID NO: 3 VH CDR3 SGIYHYVSSYYFDH SEQ ID NO: 40 VL CDR1 KASQDAGSAVA SEQ ID NO: 5 VL CDR2 WASTRHT SEQ ID NO: 6 VL CDR3 QQYSSYT SEQ ID NO: 46 V A QVQLQQSGAETKKPGASVKVSCKASGYSFTDYHINWVRQASGQRLEWMGYIYPGSGHTKKNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGIYHYVSSYYFDHWGKGTTVTVSS SEQ ID NO: 42 V L DIQMTQSPSTLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSYTFGGGTKVEIK Table 2. Sequence Listing (Kabat Number ) Sp34 SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 49 HCDR2 (Kabat) RIRSKYNNYATYYADSVKD SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 54 V A EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 55 V L QAVVTQESALTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL SEQ ID NO: 56 VH DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCAAGGGCAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGAGACGACTCTCAGAGCATCCTGTACCTGCAGA TGAACAACCTGAAGACCGAGGACACCGCCATGTACTACTGCGTGAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACAGTAAGCAGC SEQ ID NO: 57 VL DNA CAAGCCGTGGTGACCCAAGAGAGCGCCCTGACCACAAGCCCCGGCGAGACCGTGACCCTGACCTGCAGAAGCAGCACCGGCCCGTGACCACAAGCAACTACGCCAACTGGGTGCAAGAGAAGCCCGACCACCTGTTCACCGGTCTGATCGGCGGTACCAACAAGAGAGCCCCCGGCGTGCCCGCTAGATTCAGCGGCTCACTGATCGGCGACAAGGCCGCCCTGACCATCACCGGCGCTCAGACCGAGGACGAGGCCATCTACT TCTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTG BG53P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 49 HCDR2 (Kabat) RIRSKYNNYATYYADSVKD SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 58 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKSSLYLQMNSLRAEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 59 V L QAVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKPGQLFTGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAIYFCALWYSNLWVFGGGTKVEIK SEQ ID NO: 60 VH DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGAGACGACGCCAAGAGCAGCCTGTACCTGCAGAT GAACAGCCTGAGAGCCGAGGACACCGCCATGTACTACTGCGTGAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC SEQ ID NO: 61 VL DNA CAAGCCGTGGTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCTCCGTGACCACAAGCAACTACGCCAACTGGGTGCAAGAGAAGCCCGGGCAGCTGTTCACCGGCCTGATCGGCGGTACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCGACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGC CATCTACTTCTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG BG56P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 49 HCDR2 (Kabat) RIRSKYNNYATYYADSVKD SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 62 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 63 V L EIVLTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 64 VH DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGTTAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC SEQ ID NO: 65 VL DNA GAAATCGTGCTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCTCCGTGACCACAAGCAACTACGCCAACTGGGTGCAACAGAAGCCCGGGCAGGCCCCGCGGCCTGATCGGCGGTACCAACAAGAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCGACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGC CGTCTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG BG561P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 49 HCDR2 (Kabat) RIRSKYNNYATYYADSVKD SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 62 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 63 V L EIVLTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 66 scFv AA EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLT ISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 67 scFv DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGTTAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC GGCGGCGGTGGCGGAGGCGGCTCTGGCGGCGGAGGATCCGAAATCGTGCTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCCGTGACCACAAGCAACTACGCCAACTGGGTGCACAGAAGCCCGGGCAGGCGCCCCGCGGCCTGATCGGCGGTACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCG ACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTCTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG BG562P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 49 HCDR2 (Kabat) RIRSKYNNYATYYADSVKD SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 62 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 68 V L EIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 69 ScFv AA EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLT ISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 70 ScFv DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGCTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGTTAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC GGCGGCGGTGGCGGAGGCGGCTCTGGCGGCGGAGGATCCGAAATCGTGGTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCGCCGTGACCACAAGCAACTACGCCAACTGGGTGCACAGAAGCCCGGGCAGGCGCCCCGCGGCCTGATCGGCGGTACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCG ACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTCTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG BG563P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 71 HCDR2 (Kabat) RIRSKYNNYATYYADSVKG SEQ ID NO: 50 HCDR3 (Kabat) HGNFGNSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 72 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 68 V L EIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 73 scFv AA EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEAT LTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 74 scFv DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGGTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGTTAGACACGGCAACTTCGGCAACAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC GGCGGCGGTGGCGGAGGCGGCTCTGGCGGCGGAGGATCCGAAATCGTGGTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCGCCGTGACCACAAGCAACTACGCCAACTGGGTGCACAGAAGCCCGGGCAGGCGCCCCGCGGCCTGATCGGCGGTACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCG ACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTCTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG BG564P SEQ ID NO: 48 HCDR1 (Kabat) TYAMN SEQ ID NO: 71 HCDR2 (Kabat) RIRSKYNNYATYYADSVKG SEQ ID NO: 75 HCDR3 (Kabat) HGNFGSSYVSWFAY SEQ ID NO: 51 LCDR1 (Kabat) RSSTGAVTTSNYAN SEQ ID NO: 52 LCDR2 (Kabat) GTNKRAP SEQ ID NO: 53 LCDR3 (Kabat) ALWYSNLWV SEQ ID NO: 76 V A EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGSSYVSWFAYWGQGTLVTVSS SEQ ID NO: 68 V L EIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 77 scFv AA EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGSSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLT ISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK SEQ ID NO: 78 scFv DNA GAGGTGCAGCTGGTGGAGTCCGGCGGTGGCCTTGTGCAGCCCGGCGGCAGTCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGGTAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGTTAGACACGGCAACTTCGGCAGCAGCTACGTGAGCTGGTTCGCCTACTGGGGCCAAGGCACCCTGGTAACGGTGAGCAGC GGCGGCGGTGGCGGAGGCGGCTCTGGCGGCGGAGGATCCGAAATCGTGGTGACCCAAAGTCCCGCGACCCTTAGCGTAAGCCCCGGCGAGAGAGCTACCCTGAGCTGCAGAAGCAGCACCGGCGCCGTGACCACAAGCAACTACGCCAACTGGGTGCACAGAAGCCCGGGCAGGCGCCCCGCGGCCTGATCGGCGGTACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCAGCGGCAGCCTGAGCGGCG ACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTCTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGCGGCACCAAGGTGGAGATCAAG Table 3. Sequence Listing (Kabat Number ) BG143P SEQ ID NO: 79 Heavy Pestle Chain AA EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRHGNFGSSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWQQKPGQAPRGLIGGTNKRAPGIPARFSGSLSGDEATLT ISSLQSEDFAVYYCALWYSNLWVFGGGTKVEIK EPKSSDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK BG143P SEQ ID NO: 80 Heavy chain DNA GAGGTGCAACTCGTCGAGAGCGGCGGGGGCCTCGTGCAGCCTGGGGGCAGCCTGAGACTCAGCTGCGCCGCTAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCAGAATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACGCCAAGAACAGCCTGTACCTGCAGATGA ACAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGTGAGACACGGCAACTTCGGCAGCAGCTACGTGAGCTGGTTCGCTTATTGGGGGCAAGGCACACTGG TGACCGTCAGCAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAGATTGTGGTGACACAGAGCCCCGCCACCCTGAGCGTGAGCCCCGGCGAGAGAGCTACACTGAGCTGCAGAAGCAGCACCGGCGCCGTGACCACAAGCAACTACGCCAACTGGGTGCAGCAGAAGCCCGGCCAAGCCCCTAGAGGCCTGATCGGGGGCACCAACAAGAGCCCCCGGCATCCCCGCTAGATTCTCC GGCAGCCTGAGCGGCGACGAGGCCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTC GGCGGGGGCACCAAGGTCGAGATCAAGGAGCCCAAAAGCAGCGATAAGACCCACACCTGTCCTCCCTGCCCCGCCCCTCCTGCCGCCGGCCCTAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAG CAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCC TGGCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCCCAAGTGTACACCCTGCCCCCTAGCAGAGACGAGCTCACCAAGAACCAAGTGAGCCTGTGGTGCCTGGTGAAGGGCTTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTCCTGTACAGCAAGCTGACCGTGGACAAGAGCA GATGGCAGCAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCCCGGCAAGTGA BG143P SEQ ID NO: 81 Heavy Mortar Chain AA EVQLQESGGGLVQPGGSLRLSCAASGYSFSDYHINWVRQAPGKGLEWVGYIYPGSGHTKYNEKFKGRFTISRDTSSNTAYLQMNSLRAEDTAVYYCARSGIYHYVSSYYFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK BG143P SEQ ID NO: 82 Recombinant DNA GAAGTGCAGCTGCAAGAGTCCGGGGGGGGGCTCCGTGCAACCCGGGGGCTCCCTGAGACTGAGCTGCGCCGCTAGCGGCTACAGCTTCAGCGACTACCACATCAACTGGGTGAGACAAGCCCCCGGCAAGGGCCTGGAGTGGGTGGGCTACATCTACCCCGGCAGCGGCCACACCAAGTACAACGAGAAGTTCAAGGGCAGATTCACCATCAGCAGAGACACAAGCAGCAACACCGCCTACCTGCAGATGAACAGCCTGAGA GCCGAGGACACCGCCGTGTACTACTGCGCTAGAAGCGGCATCTACCACTACGTGAGCAGCTACTACTTCGACCACTGG GGCCAAGGCACCCTGGTGACAGTGTCCTCCGCCTCCACAAAGGGGCCTAGCGTGTTCCCCCTGGCCCCTAGCAGCAAGAGCACAAGCGGCGGCACCGCCGCCCTCGGCTGCCTGGTGAAGGACTATTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTCACAAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCTAGCAGCAGCCTGG GCACACAGACCTACATCTGCAACGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCG ATAAGACCCACACCTGCCCCCCTTGTCCCGCTCCCCCTGCTGCCGGGCCTAGCGTGTTCCTGTTTCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTCGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTACAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTG CTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGGCCGCCCCCATCGAGAAGAC CATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCCCAAGTGTACACCCTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAAGTGAGCCTGTCCTGCGCCGTCAAGGGCTTCTACCCTAGCGACATCGCCGTGGAGTGGGAGCAACGGGCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTCCTGGTGAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAAGGCAACGTGTTTCAG CTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCCCGGCAAGTGA BG143P SEQ ID NO: 83 Kappa chain AA DIQMTQSPSSSLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC BG143P SEQ ID NO: 84 Kappa DNA GACATTCAGATGACACAGAGCCCTAGCAGCCTGTCCGCTAGCGTGGGCGACAGAGTGACCATCACCTGCAAGGCTAGCCAAGACGCCGGGAGCGCCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTGGGCTAGCACAAGACACACCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCT ACTACTGTCAGCAGTACAGCAGCTACACCTTCGGCGGGGGCACCAAGCTGGAGATCAAGCGTA CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA Connector SEQ ID NO: 85 AA GGGGSGGGGSGGGGSGGGGS Connector SEQ ID NO: 86 AA GGGGSGGGGSGGGGS CLDN6 SEQ ID NO: 87 AA MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDSKARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEAQKRELGASLYLGWAASGLLLLGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRG PSEYPTKNYV CLDN9 SEQ ID NO: 88 AA MASTGLELLGMTLAVLGWLGTLVSCALPLWKVTAFIGNSIVVAQVVWEGLWMSCVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLLALLGLVAITGAQCTTCVEDEGAKARIVLTAGVILLLAGILVLIPVCWTAHAIIQDFYNPLVAEALKRELGASLYLGWAAAALLMLGGGLLCCTCPPPQVERPRGPRLGYSIPSRSGASGLDKRDYV Examples Example 1. Production of mouse anti- CLDN6 antibodies

To generate antibodies against CLDN6, groups of 20-25 inbred mice of BALB/C, SJL strains were immunized with human CLDN6 overexpressing cells (L929/human CLDN6, prepared in-house), and each group received an immunization strategy that included a unique combination of CLDN6 antigen, dose, injection route, adjuvant, and timing of immunization. A total of 5 animals in 4-5 groups were immunized. Animals were immunized at different times between 0 and 56 days. To monitor the immune response, titrated sera were screened by FACS after 2-4 immunizations, typically on days 21-56. Sera were screened for antibodies that bind to CLDN6 overexpressing cells CHOK1/human CLDN6. The CLDN6-specific antibody response of each animal was measured, and animals with sufficient titers of anti-CLDN6 Ig were selected for a final boost over 4 days.

Lymphoid organs including spleen and lymph nodes were isolated from mice immunized as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2/0 by PEG-based fusion. The resulting cells were plated in 96-well cell culture plates using conventional 1640 medium supplemented with HAT for selection of hybridomas. Example 2. Screening and selection of anti -CLDN6 antibodies

Fusion tumors were generated as described in Example 1. After 10-13 days of culture and growth medium change, fusion tumor culture supernatants were collected from individual wells and screened to identify wells with secreted CLDN6-specific antibodies. All supernatants were initially screened against at least two overexpressing cell lines, including CHOK1/human CLDN6 and CHOK1/human CLDN9 (made in-house). Antibody binding on overexpressing cell lines was measured by FACS. Supernatants from more than approximately 20,000 culture wells from 4 fusion tumor fusions were screened for CLDN6 antibodies. Briefly, 100 μL of fusion tumor culture supernatant was incubated with cancer cell lines expressing CLDN6 (e.g. PA-1 or CHOK1/human CLDN6 stable cell lines) or control cells (e.g. parental CHOK1) for 30-60 min, washed, and incubated with anti-mouse IgG Fc secondary antibody conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

Fusion tumors from positive wells were transferred to 24-well plates containing fresh medium and grown for 2-3 days, then screened again by flow cytometry to confirm antibody binding to cynomolgus macaque CLDN6 overexpressing cell lines and human CLDN6 positive cancer cell line (PA-1). Antibody binding to cynomolgus macaque CLDN6 overexpressing cell lines and human CLDN6 positive cancer cell line (PA-1) was measured by flow cytometry. Briefly, 100 μL of fusion tumor culture supernatant was co-incubated with CLDN-expressing cancer cell lines (e.g., PA-1 or CHOK1/human CLDN6 and CHOK1/human CLDN9 stable cell lines) or control cells (e.g., parental CHOK1) for 30-60 min, washed, and incubated with anti-mouse IgG Fc secondary antibody conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry. Example 3. Secondary selection of CLDN6 Ab- secreting fusion tumors

Selected CLDN6 antibody secreting fusion tumors were subcloned once or twice to ensure monoclonal selection. Briefly, approximately 80-100 viable fusion tumor cells were plated in 3 mL of semisolid methylcellulose medium (Stem Cell Technologies) in a 6-well plate. After 7-10 days, fusion tumor colonies that were viable clones were picked to 96-well plates and further cultured in fresh medium for 2-4 days. Culture supernatants were screened by ELISA and flow cytometric analysis as described above to confirm human and cynomolgus macaque CLDN6 binding. Stable fusion tumor subclones were cultured in vitro for cell cryopreservation and antibody VH and VL gene cloning and sequencing. Example 4. Determination of CLDN6 Binding EC50 Value of Mouse Anti- CLDN6 Antibody

The anti-CLND6 antibody-secreting fusion tumors selected after secondary selection were inoculated into T75 flasks containing 40 ml of fresh 1640 medium supplemented with 2% FBS for antibody production. After 7-10 days of culture, the fusion tumor supernatant was harvested and used to purify the antibody using a protein A column. Flow cytometry was then used to characterize the binding activity of mouse anti-CLDN6 antibodies to CLDN6-positive cells. The EC50 values of pure BG87P are presented in Tables 4-6. The data show that pure BG87P binds to human CLDN6, but not to human CLDN9. In addition, BG87P binds to mouse CLDN6 and cynomolgus macaque CLDN6. Table 4. Binding activity of BG87P to CHOK1- stabilized cells expressing human CLDN6 and human CLDN9 Pure CHOK1/human CLDN6-GFP binding EC50 (nM) CHOK1/human CLDN6-GFP binding Emax (MFI) CHOK1/human CLDN9-GFP binding EC50 (nM) CHOK1/human CLDN9-GFP binding Emax (MFI) BG87P 3.41 26576 - 30.7 Human IgG1 isotype control - 31.1 - 26.9 Table 5. Cross-species binding activity of BG87P to mouse CLDN6 and cynomolgus macaque CLDN6 Pure CHOK1/mouse CLDN6-GFP binding EC50 (nM) CHOK1/mouse CLDN6-GFP binding Emax (MFI) CHOK1/Cynomolgus macaque CLDN6-GFP binding EC50 (nM) CHOK1/Cynomolgus macaque CLDN6-GFP binding Emax (MFI) BG87P 94.69 27700 6.08 43400 Human IgG1 isotype control - 41.8 - 225 Table 6. Binding activity of BG87P to the cancer cell line PA-1 expressing endogenous CLDN6 Pure PA-1 binding EC50 (nM) PA-1 binding E maximum (MFI) BG87P 3.38 74200 Human IgG1 isotype control - 34 Example 5. Cloning and sequencing of antibody VH and VL genes

After removing the supernatant, the fusion tumors secreting CLDN6 antibodies selected after secondary selection were lysed by adding 100 mL of RLT buffer in a 96-well round-bottom plate. The lysate containing mRNA was then transferred to a 96-well deep-well plate for mRNA isolation, cDNA synthesis, and DNA sequencing by standard sequencing techniques (Sanger sequencing and next-generation sequencing). In general, total RNA from cell lysates is prepared using a total RNA isolation kit according to the manufacturer's instructions. cDNA was generated by mRNA reverse transcription using Super Script III First Strand Synthesis SuperMix (Invitrogen®) according to the manufacturer's instructions. The nucleic acid and amino acid sequences of BG87P are shown in Figure 1 (SEQ ID NO: 1-10). Generation of chimeric BG87P antibody (chBG87P)

The ChBG87P antibody was produced by subcloning the variable regions of mouse BG87P (SEQ ID NO: 7 and 8) into an in-house developed expression vector containing the constant regions of human wild-type IgG1 and κ chains. The antibody was expressed by co-transfecting the two constructs into HEK293T cells and purified using a protein A column (Catalog No.: 17-5438-02, GE Life Sciences®). The purified chimeric antibody was concentrated to 0.5-10 mg/ml in PBS and stored in aliquots in a -80°C freezer. Example 6. Humanization of anti- CLDN6 chimeric BG87P antibody (chBG87P) [ Humanization method

For humanization of chBG87P, human germline IgG genes were searched for sequences that shared high homology with the protein sequence of the chBG87P variable region by sequence comparison against the human immunoglobulin gene database in IMGT. Human IGHV and IGKV genes, which are present at high frequency in the human antibody repertoire and are highly homologous to chBG87P, were selected as templates for humanization. Design of humanized variants

Humanization was performed by CDR grafting followed by incorporation of key back mutations. Humanized antibodies were engineered to the human IgG1 wild-type format using an in-house developed expression vector. In the initial first round of humanization, mutations from mouse variable region to human framework amino acid residues were guided by 3D structural analysis, and mouse framework residues that were structurally important for maintaining the CDR canonical structure were retained in the first round of humanization design. Five reversion mutations on the heavy chain and three mutations on the light chain were selected and single-site mutations were performed to explore key reversion mutations: BG87P-Bz1 (VH SEQ ID NO: 15 and VL: SEQ ID NO: 14), BG87P-Bz2 (VH SEQ ID NO: 16 and VL: SEQ ID NO: 14), BG87P-Bz3 (VH SEQ ID NO: 17 and VL: SEQ ID NO: 14), BG87P-Bz4 (VH SEQ ID NO: 18 and VL: SEQ ID NO: 14), BG87P-Bz5 (VH SEQ ID NO: 19 and VL: SEQ ID NO: 14), BG87P-Bz6 (VH SEQ ID NO: 13 and VL: SEQ ID NO: 20), BG87P-Bz7 (VH SEQ ID NO: 14 and VL: SEQ ID NO: 21), BG87P-Bz8 (VH SEQ ID NO: 15 and VL: SEQ ID NO: 14), BG87P-Bz9 (VH SEQ ID NO: 16 and VL: SEQ ID NO: 25), BG87P-Bz10 (VH SEQ ID NO: 17 and VL: SEQ ID NO: 14), BG87P-Bz (VH SEQ ID NO: 13 and VL: SEQ ID NO: 20) and BG87P-Bz8 (VH SEQ ID NO: 13 and VL SEQ ID NO: 22). BG87P-Bz0 (VH: SEQ ID NO: 13 and VL: SEQ ID NO: 14) is a variant that incorporates all theoretical reversion mutations, and the binding ability of BG87P-Bz0 should be comparable to that of the parent chBG87P. Comparison of binding data revealed which reversion mutations significantly affected binding. Specifically, the LCDR of chBG87P (SEQ ID NOs: 4 to 6) was transplanted into the framework of the human germline variable genes IGKV1-5 and 01-IGKJ4*01 with A43S, L78V and Y87F mouse framework residues (result is SEQ ID NO: 14). The HCDRs of chBG87P (SEQ ID NOs: 1 to 3) were transplanted into the frameworks of human germline variable genes IGHV1-3 and 01-JH6c, retaining the V2I, T28S, I69L, R71V and Y91F mouse framework residues (resulting in SEQ ID NO: 13); BG87P-z0 (VH: SEQ ID NO: 11 and VL: SEQ ID NO: 12) is the resulting humanized variant with the above HCDR and LCDR transplants, but without any reversion mutations from the mouse VH and VL frameworks. Expression and purification of chBG87P and humanized antibodies

All first-round BG87P humanized variants (BG87P-z0, BG87P-Bz0, BG87P-Bz1, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7, and BG87P-Bz8) were constructed as humanized full-length antibodies using in-house developed expression vectors containing the constant regions of human wild-type IgG1 and κ chains, respectively, with readily adaptable secondary cloning sites. All humanized variants were expressed by co-transfecting the two constructs into HEK293T cells and purified using a protein A column (Cat. No. 17-5438-02, GE Life Sciences). The purified antibodies were concentrated to 0.5-10 mg/ml in PBS and stored in aliquots in a -80°C freezer. Cell binding activity assay of the first round humanized BG87P variant (hBG87P) and PTM- removed variants

For affinity determination, CLDN6 overexpressing HEK293T cells and PA-1, a cancer cell line expressing high levels of human CLDN6, were used to assess the binding activity of BG87P-related engineered variants. Live cells were seeded in 96-well plates and incubated with a series of dilutions of chBG87P and its engineered variants. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC50 values for dose-dependent binding to cell lines expressing CLDN6 were determined by fitting the dose-response data to a four-parameter logical model using GraphPad Prism. The cell binding activity of round 1 BG87P humanized variants against HEK293T/human CLDN6 was compared with chBG87P and is shown in Figure 1 A. The cell binding affinity (EC50) and Emax (MFI) of round 1 humanized variants were normalized to chBG87P for direct comparison and ranking (Table 7).

Starting with the chBG87P antibody and BG87P-Bz0, some additional amino acid changes were made in the CDR regions of the VH and VL to further improve the biophysical properties of the therapeutic for human use. Considering factors including removal of post-translational modification (PTM), improved thermal stability (Tm), while maintaining binding activity, the resulting variants are BG87P-m1 (VH SEQ ID NO: 31 and VL SEQ ID NO: 8), BG87P-m2 (VH SEQ ID NO: 32 and VL SEQ ID NO: 8), BG87P-m3 (VH SEQ ID NO: 33 and VL SEQ ID NO: 8), and BG87P-m4 (VH SEQ ID NO: 34 and VL SEQ ID NO: 8) and BG87P-m5 (VH SEQ ID NO: 35 and VL SEQ ID NO: 14), BG87P-m6 (VH SEQ ID NO: 36 and VL SEQ ID NO 14), BG87P-m7 (VH SEQ ID NO: 37 and VL SEQ ID NO: 14), and BG87P-m8 (VH SEQ ID NO: 38 and VL SEQ ID NO: 14). The cell binding activity of chBG87P and BG87P-Bz0 related PTM removal variants against HEK293T/human CLDN6 was compared with chBG87P and BG87-Bz0, respectively (Figure 1F). Cell binding affinity (EC50) and Emax (MFI) were normalized to chBG87P for direct comparison and ranking (Table 7). The results showed that, except for the H33A mutation leading to BG87P-m4 and BG87P-m8, other substitutions of potentially deleterious residues maintained the binding ability of the respective parental residues. Table 7. Summary of the binding activity of humanized and PTM- removed variants against CLDN6 - overexpressing HEK293T cells HEK293T-human CLDN6 Ec50 Ranking E Maximum Ranking Antibody Name Highest MFI average Ec50 (nM) Antibody Name Highest MFI average Ec50 (nM) Ratio of Emax to BG87P BG87P-Bz8 130650.51 1.71 BG87P-m3 227257.19 2.32 1.18 BG87P-Bz4 109529.57 1.88 BG87P-m1 206987.02 3.56 1.08 BG87P-27 131381.01 2 BG87P-m2 202202.02 2.51 1.05 BG87P-Bz0 106974.58 2.1 chBG87P 192332.83 2.72 1.00 BG87P-22 136523.4 2.12 BG87P-Bz7 154833.51 2.54 0.81 BG87P-24 141013.73 2.16 BG87P-21 149494.66 3.07 0.78 BG87P-Bz1 99692.6 2.19 BG87P-26 147913.6 2.3 0.77 BG87P-26 147913.6 2.3 BG87P-23 147866.77 2.43 0.77 BG87P-m3 227257.19 2.32 BG87P-Bz6 143672.43 2.79 0.75 BG87P-25 142863.99 2.33 BG87P-m4 143182.68 11.76 0.74 BG87P-m6 118322.89 2.34 BG87P-25 142863.99 2.33 0.74 BG87P-m7 134492.69 2.43 BG87P-24 141013.73 2.16 0.73 BG87P-23 147866.77 2.43 BG87P-Bz5 140664.98 2.55 0.73 BG87P-z0 95645.46 2.45 BG87P-22 136523.4 2.12 0.71 BG87P-m2 202202.02 2.51 BG87P-m7 134492.69 2.43 0.70 BG87P-Bz7 154833.51 2.54 BG87P-27 131381.01 2 0.68 BG87P-Bz5 140664.98 2.55 BG87P-Bz8 130650.51 1.71 0.68 BG87P-Bz3 107318.29 2.63 BG87P-m5 126460 4.19 0.66 BG87P-Bz2 84347.71 2.65 BG87P-m6 118322.89 2.34 0.62 chBG87P 192332.83 2.72 BG87P-Bz4 109529.57 1.88 0.57 BG87P-Bz6 143672.43 2.79 BG87P-Bz3 107318.29 2.63 0.56 BG87P-21 149494.66 3.07 BG87P-Bz0 106974.58 2.1 0.56 BG87P-m1 206987.02 3.56 BG87P-Bz1 99692.6 2.19 0.52 BG87P-m5 126460 4.19 BG87P-z0 95645.46 2.45 0.50 BG87P-m4 143182.68 11.76 BG87P-Bz2 84347.71 2.65 0.44 BG87P-m8 54013.49 48.74 BG87P-m8 54013.49 48.74 0.28 Antibody Name Highest MFI average Ec50 (nM) Antibody Name Highest MFI average Ec50 (nM) Ratio of Emax to BG87P Table 8. Cell binding activity of the second round of humanized variants with PTM removed from the PA-1 cancer cell line Cancer cell line: PA-1 Ec50 Ranking E Maximum Ranking Antibody Name Highest MFI average EC50 (nM) Antibody Name Highest MFI average Ec50 (nM) Ratio of Emax to BG87P BG87P-27 66672.07 1.68 BG87P 124836.78 8.33 1.00 BG87P-24 62436.19 2.27 BG87P-21 76460.34 2.8. 0.61 BG87P-26 7BG143P0.80 2.30 BG87P-25 73677.44 2.34 0.59 BG87P-25 73677.44 2.34 BG87P-23 72521.43 2.49 0.58 BG87P-23 72521.43 2.49 BG87P-26 68012.43 2.52 0.54 BG87P-21 74335.52 2.62 BG87P-22 67778.95 3.01 0.54 BG87P-22 76120.75 3.00 BG87P-27 66672.07 1.68 0.53 BG87P-z0 53183.52 3.25 BG87P-24 62436.19 2.27 0.50 chBG87P 124836.78 8.33 BG87P-z0 53183.52 3.25 0.43 Combination with PTM removal sites to measure cell binding activity of the second round of key reversion mutations

After comprehensive analysis of EC50 and Emax of the first round of humanization cell binding data (Table 7), four key reversion mutation sites VH: V2I, VH: T28S, VH: I69L, VH: Y91F were identified and combined with the PTM removal sites for second round validation and final humanization candidate determination. The PTM removal mutation VH: V65G (a site with a higher prevalence of G in the human germline (G62%; V<1%), indicating potential benefits for antibody framework stability) involved variant BG87P-m3, which showed improved Emax and EC50 compared to chBG87P in Figure 1F and Table 7, which was included in the second round combination for further validation. The VH and VL sequences of the resulting second round humanized variants BG87P-21, BG87P-22, BG87P-23, BG87P-24, BG87P-25, BG87P-26 and BG87P-27 are given in Table 1.

After characterizing the cell binding activity of the humanized combinatorial variants, BG87P-21 was selected as the best humanized candidate for further consideration (VH and VL amino acid sequences are SEQ ID NOs: 24 and 12, respectively), as shown in Figures 1B and 1C for HEK293T/human CLDN6 cells and in Figures 1D and 1E for PA-1 cells. BG87P-21 includes the key reverting mutation site VH: T28S and the PTM site VH: V65G, which revealed comparable cell binding affinity compared to chBG87P. E in HEK293T/human CLDN6 was reduced by 22% at most, and E in PA-1 was reduced by 40% at most (Tables 7 and 8). Developability Assessment of Humanized Anti- CLDN6 Antibodies

Biophysical properties were analyzed to identify the best humanized anti-CLDN6 antibody. The data showed that BG87P-21 showed moderate to high risk of hydrophobicity, followed by the risk of self-interaction in PBS buffer as shown by AC-SINS, B22KD and CIC readings. (Table 9-Table 11). Table 9. Analysis of biophysical properties of BG87P-21 and chBG87P Sample Name BG87P-21 chBG87P Threshold Verification HIC (min) 21.9 25 21.1 CIC (min) 15.5 14.7 14 KD in PBS (mL/g) -20.1 -15.6 ＞-10 B22 in PBS -3.10E-05 -2.20E-05 >0 AC-SINS in PBS Δλ=14.5nm Δλ=6.0nm ＜ 10nm Tm1 (℃) 69.26 70.17 65 Tagg (℃) 69.35 72.33 65 SEC-HPLC (%) 98.42 92.52 0.95

For hydrophobicity assessment, 50 μg of 1 mg/ml sample was diluted with mobile phase A solution (1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0) to obtain a final ammonium sulfate concentration of approximately 1 M before analysis. The MABPac HIC-10 column was used with a linear gradient of mobile phase A and mobile phase B solution (50 mM sodium phosphate, pH 7.0) over 29 minutes at a flow rate of 0.5 ml/min. Peak retention time was monitored at A280 absorbance. As indicated in Table 9, both chBG87P and BG87P-21 showed higher hydrophobicity performance and exceeded the internal standard by 21.1 min in IgG format.

For thermal stability assessment, the thermal stability of BG87P-related engineered variants was described by the thermal defolding transition midpoint Tm (°C), which was measured by external fluorescence. Tm was determined using the QuantStudioTM 6 Flex system from Applied Biosystems. 20 μL of 1 mg/ml sample was mixed with 20 μL of 40XSYPRO Orange. The plate was scanned from 25°C to 95°C at a rate of 0.9°C/min. Tm was assigned by the first derivative of the raw data using the QuantStudioTM 6 Flex system analysis software. The results are summarized in Table 9, which shows that both chBG87P and the humanized variant BG87P-21 showed good thermal stability.

To determine the aggregation tendency of BG87P-related engineered variants, the static light scattering intensity was measured using the Uncle system (Unchained Labs). During the measurement, approximately 8.8 μL of 1 mg/ml protein sample was placed in a cuvette; the sample was incubated at 25°C for 120 s and then heated to 95°C at a rate of 0.3°C/min. Scattering data were collected at an angle of 90° using a 266 nm laser wavelength. Tagg (aggregation temperature) was analyzed and calculated by Uncle analysis software. The results are summarized in Table 9. Both chBG87P and humanized variants showed acceptable Tagg.

CIC is a technique for identifying candidate antibodies with poor solubility or non-specific binding tendencies. IgG or other ligands from human serum are chemically coupled to an NHS-activated chromatography resin. The retention time of the protein on this resin is tested using HPLC to assess protein solubility. After column coupling with IgG in human serum, the antibody sample and sample buffer are diluted to 0.1 mg/mL with the mobile phase (PBS). The diluted sample and buffer are transferred to HPLC vials for LC-MS analysis. The results summarized in Table 9 show that both chBG87P and BG87P-21 show acceptable non-specific interactions with human IgG.

General description and intended use of the B22 and KD assay. This method is used to study weak protein-protein interactions to predict aggregation trends, reveal the effects of formulation components on molecular interactions, and support formulation buffer selection. Antibody and buffer exchange samples were diluted to 1 mg/mL and centrifuged at 14,000 rpm for 30 min, followed by Tm, Tagg, and DLS. Samples were loaded onto the Uni. 9 μL/well. One replicate well was set for each sample. Following Uncle's instructions to set the instrument parameters and run the experiment. In this experiment, we used the B22 & Kd mode. Run Information: Temperature (°C): 25. Incubation Time (sec): 120. Number of Acquisitions: 4. Acquisition Time (sec): 5. Attenuator control: automatic. Laser. Control: automatic run. For kd: diffusion interaction parameter, if the interaction of the proteins increases with increasing concentration (mutual attraction), then the protein behaves as if it is larger and the diffusion coefficient (KD) decreases (negative slope). For B22: second virial coefficient, if the interaction of the proteins increases with increasing concentration (mutual attraction), then the protein behaves as if it is larger and 1/R90 decreases (negative slope). The data show that in PBS, both chBG87P and the humanized variants are attracted to each other and tend to aggregate under these conditions (Table 10).

AC-SINS is an assay that obtains sample self-interactions to predict aggregation potential. It is based on concentrating antibodies in a dilute solution around AuNPs pre-coated with multiple strains of capture agents. The interaction between the immobilized antibodies leads to a decrease in the interparticle distance and an increase in the plasmon wavelength (wavelength of maximum absorbance), which can be easily measured by optical means. The antibodies were diluted to 0.05 mg/mL with the provided buffer. After AuNP preparation, the AuNP solution was mixed with the coating solution using a 9:1 volume ratio. After incubation at room temperature for 1 hour, thiolated PEG (final concentration 0.1 uM) was used to block the vacant sites in the AuNPs. This was followed by another incubation at room temperature for 1 hour. The particle solution was then centrifuged at 15,000 rpm for 6 minutes. The supernatant solution was discarded. The particles were re-dissolved using 1/10 of the starting volume of storage buffer. 10 μL of concentrated coated particles were incubated with 100 μL of test antibody solution in a polypropylene dish at room temperature for 2 hours, and then 90 μL of the resulting solution was transferred to a polystyrene UV transparent dish. The data showed that both chBG87P and BG87P-21 showed suboptimal self-interaction tendencies (Table 9). Table 10. Self-interaction risk assessment of second - round humanized variants and chBG87P Sample kD (mL/g) B₂₂ (mol*mL/g²) k D Fit Quality B₂₂ fit quality BG87P-21 -17.2 -1.1E-05 0.97 0.96 BG87P-22 -22.7 -2.5E-05 0.96 0.97 BG87P-24 -21.1 -1.7E-05 0.95 0.94 BG87P-26 -17.5 -2.0E-05 0.95 0.96 chBG87P -18.5 2.1E-05 0.98 0.97 Table 11. The four humanized antibodies all showed lower hydrophobicity than chimeric antibodies, but the hydrophobicity risk was also relatively higher. Sample Name Retention time (min) BG87P-21 21.48 BG87P-22 21.51 BG87P-24 21.75 BG87P-26 21.83 chBG87P 24.80

The hydrophobic patch resulted in HIC retention times of more than 25 minutes for chBG87P and 21.9 minutes for humanized BG87P-21, both above the acceptable threshold of 21.1 minutes in the IgG format. The underlying cause is the hydrophobic patch in HCDR3, specifically the I97-Y98-Y100-V100a portion of the light chain FR2 (framework region 2) and LCDR2 edges, as well as Y49-W50 (mainly W50) (Figure 2A). Schrodinger's autoantibody pipeline analysis of BG87P aggregation also showed a higher aggregation risk (Table 12). Table 12. Autoantibody pipeline analysis of aggregation chBG87P Global Aggregate Score 164.53 CDR aggregation score 104.38 CDRH1 aggregation score 0 CDRH2 aggregation score 1.11 CDRH3 aggregation score 89.7 CDRL1 aggregation score 0 CDRL2 aggregation score 13.53 CDRL3 aggregation score 0 Example 7. Solubility Engineering of Humanized Anti- CLDN6 Antibody Overall Strategy for Solubility Engineering of BG87P-21

In the foregoing description, chBG87P has been engineered into a humanized antibody, and we have identified BG87P-21 as the final best pure line. However, the potential developability risk of the hydrophobic patch driven by the HCDR3 of chBG87P has not been resolved (Figure 2). Considering that chBG87P showed promising binding activity and excellent CLDN6 selectivity (Figure 1A, Figure 1B, Figure 1C, Figure 1D, Figure 1E, Figure 1F, Figure 1G, and Figure 1H), additional engineering of BG87P-21 has been completed to remove the hydrophobic patch, thereby achieving optimal manufacturability and reducing potential ADA risks.

Two main strategies were used to address the solubility problem of BG87P-21: single-site mutation and framework swapping. Table 13. Overall overview of the solubility engineering of BG87P-21 Design principles Verification Filter Criteria Best variant Comments Round 1 : Structure-based mutation induction: 1: Reduce exposed hydrophilicity 2: Mutation to human germline residues 3: Mutation to residues that are more common in the human antibody library 4: Improve hydrophobic core filling 5: Saturation mutation at key hydrophobic sites FACS HIC-RT ( without AC-SINS ) Improved HIC-RT or Improved Yield or Improved FACS Ec50 or Improved FACS Emax or Improved AC-SINS (only in the last round, as this assay was not discussed previously) VK: Y49K/R VH: Y56H VH: W50Y/N VH: V5Q-V11T-P41S VK-Y49K was later abandoned because it degraded AC-SINS Round 2 : Extending structure-based design 1: Mutating residues near the hydrophobic patch (because the results of round 1 were lower than expected) VK: V29A/G VK: T31S/H VH: Y59K/E/Q/H/D/N VH: Y59K reduces production Combinations of optimal point mutations for rounds 1 and 2 1: Improved HIC-RT 2: Or improved FACS Emax 3: Or improved FACS Ec50 4: Or improved yield or purity BG87P-31 BG87P-32 Both reduced the yield of BG87P-21; BG87P-31 reduced HIC-RT more than BG87P-32, but the AC-SINS value was worse. Round 3 : Framework swapping to improve yield and self-interactions 1: Current human frameworks are IGHV1-3 and IGKV1-5 2: Additional frameworks tested for IGHV3-23 and IGKV1-39 3: Sweet spot mutations on BG87P-21 were incorporated 4: Rare amino acids were also mutated to common amino acids 5: Effect of charge distribution on self-interactions verified. FACS HIC-RT AC-SINS BG87P-33 BG87P-34 The new framework will carry more reversion mutations because it has less homology to the original chimeric sequence

The design of a large number of single-point mutations is based on two basic principles: one basic principle is to replace hydrophobic amino acids with more hydrophilic amino acids; and the other basic principle is to mutate rare amino acids at the same Kabat position in the human antibody library to more common amino acids. 57 variants in the first round of screening and 104 variants in the second round of screening were performed, and seven positions were found to be substituted by other more hydrophilic amino acids with binding affinity comparable to that of the parent BG87P-21 and slightly improved hydrophilicity (Table 14). The best selected mutations in the first and second rounds of screening were combined to generate 56 variants for further validation. Combinatorial variants BG87P-31 and BG87P-32 (VH and VL amino acid sequences are SEQ ID NO: 46 and 42, respectively) were selected as the best candidates, which had comparable binding affinity to BG87P-21 and improved HIC retention, 17.4 minutes for BG87P-31 and 18.49 minutes for BG87P-32, both better than the parent BG87P-21 of 22.3 minutes (Table 14 and Figure 3). Table 14. Characterization of chBG87P solubility engineered variants Antibody Name chBG87P BG87P-21 BG87P-32 BG87P-31 BG87P-33 BG87P-34 Cell binding activity FACS - Ec50 ratio with BG87P (HEK293-hCLDN6) 1 0.91 1.31 1.6 2.45 1.17 FACS E maximum ratio with BG87P (HEK293T-hCLDN6) 1 0.66 0.63 0.76 0.91 1.03 E c 50 (nM) with HEK293T_cyno -CLDN6 7.2 8.9 No data No data 25.8 8.8 FACS E maximum ratio with BG87P (HEK293T-cyno-CLDN6) 1 0.8 No data No data 0.75 1 Biophysical properties Tm1 (℃) 69.8 69.3 69.4 70.5 68.8 69.9 Tagg (℃) 65.6 65.4 69.86 69.16 62.3 65.2 AC-SINS DI (nm) 12.85 21.95 10.09 22.2 7.7/11.4 8.05 HIC-RT (min) 23.54 20.33 18.49 17.41 16.87 17.85 Purity (%) 95.44 97.99 99.18 99.46 94.96 93.14 Yield (mg/L) 242.87 119.57 30.06 34.02 111.65 221.07 Net charge ( at pH 7.4 ) 2.9 8.8 9.3 9.59 5.8. 4.8 frame Chimeric Antibodies IGHV1-3-01 and IGKV1-5 IGHV1-3-01 and IGKV1-5 IGHV1-3-01 and IGKV1-5 IGHV-3-23 and IGKV1-5 IGHV3-23 and IGKV1-39

However, despite the reduction of hydrophobicity in the single-point mutation approach of solubility engineering, the self-association risk measured by AC-SICNS still appears to be moderate to high. The reason why this problem has not been solved is that the hydrophobic risk mainly reflects the amount of hydrophobic patches actually displayed on the antibody surface, while the causes of self-interaction also involve isoelectric point issues, uniform charge distribution, and even some unknown specific interactions. (Doi.org/10.1021/mp200566k). Therefore, the self-interaction risk caused by the above reasons cannot be reduced by simply replacing hydrophobic residues with hydrophilic residues. In addition, we found that the parent chBG87P had a higher HIC retention time of 25 minutes and showed a lower self-interaction tendency, with an AC-SINS value of about 12.85 nm in PBS buffer (Table 14). Another phenomenon found was that the calculated net charge of chBG87P was much less than that of BG87P-21, 2.9 vs 8.8. Therefore, we hypothesized that the framework or net charge may have an effect on the self-association effect.

We tested additional frameworks of IGHV3-23 and IGKV1-39, and selected optimal point mutations based on the reversion mutations of the new paired framework and the solubility engineering of BG87P-21 were incorporated (Table 13). The final optimal candidate BG87P-34 showed no danger signals in all conventional biophysical properties (Table 14; VH and VL amino acid sequences are SEQ ID NO: 43 and 44, respectively).

In addition, the Emax of BG87P-21 lost in the original humanization process was restored after framework swapping to IGHV3-23 and IGKV1-39 (Figure 1G). The key reverting mutation identification principle used in these two humanization processes was the same, thus excluding the possibility that any reverting mutations were missed in the previous round of humanization responsible for Emax. One explanation for the restoration of Emax in cell binding by framework swapping is that the VH-VL angles of different paired frameworks may be different, and specific VH-VL angles may help maintain Emax in cell binding. The final lead clone BG87P-34 showed good cross-reactivity in different species (Figures 1I and 1J). Using HEK293T/human CLDN9 for non-specific binding to human CLDN9, the data showed that BG87P-34 had good selectivity for human CLDN6 relative to CLDN9 (Figure 1H). Example 8. Humanization and scFv engineering of anti-human CD3 antibody sp34

The widely reported mouse clone sp34 (Blumberg 1990 PNAS 87 (18):7220–24) is the best clone for the development of anti-CD3 based therapeutics due to its cross-reactivity with cynomolgus macaque CD3. For humanization of sp34, human germline IgG genes were searched by blast analysis of the human immunoglobulin gene databases at the IMGT (http://www(dot)imgt(dot)org/IMGT_vquest/share/textes/index(dot)html) and NCBI (http://www(dot)ncbi(dot)nlm(dot)nih(dot)gov/igblast/) websites for sequences with high homology to the protein sequence of the sp34 variable region (SEQ ID NO: 48-57). Human IGVH and IGVK genes, which are present at high frequency in human antibody libraries (Glanville 2009 PNAS 106:20216-20221) and are homologous to sp34, were selected as templates for humanization.

Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and the humanized antibody (hu-sp34) was engineered into a human IgG1 format using an in-house developed expression vector. In the initial round of humanization, mutations from mouse to human amino acid residues in the framework regions were guided by modeled 3D structures, and mouse framework residues that were structurally important for maintaining the CDR canonical structure were retained in the first version of the humanized antibody sp34. Specifically, the CDRs of sp34 VL (SEQ ID NOs: 51-53) were transplanted into the framework of the human germline variable gene IGVκ3-15, and several mouse framework residues (Q1, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, I85, and F87) were retained. The CDRs of sp34 VH (SEQ ID NOs: 48-50) were transplanted into the framework of the human germline variable gene IGVH3-7, and several mouse framework residues (D73, S76, M89, V93) were retained.

Humanized sp34 (hu-sp34) and chimeric sp34 (ch-sp34) were constructed in human full-length antibody format using in-house developed expression vectors containing the constant regions of human IgG1 and κ chains, respectively, with readily adaptable secondary cloned sites. Expression and preparation of humanized sp34 and chimeric sp34 antibodies were achieved by co-transfection of heavy chain and corresponding light chain constructs into 293G cells (in-house developed) and purification using a protein A column. Purified antibodies were concentrated to 0.5-5 mg/mL in PBS and stored in aliquots in a -80°C freezer for the following assays.

For affinity determination, antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. Binding activity of humanized sp34 to native CD3 on live cells was assessed using HuT78 cells in a FACS-based assay. Live HuT78 cells were seeded in 96-well plates and incubated with a series of dilutions of chimeric or humanized sp34. Mouse anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC50 values for dose-dependent binding to human native CD3 were determined by fitting the dose-response data to a four-parameter logical model in GraphPad Prism. Humanized sp34 BG53P (SEQ ID NO: 48-53 and 58-61) showed comparable binding affinity to ch-sp34 in both SPR and FACS assays (Table 15 and FIG. 4A ). Table 15. Comparison of binding affinity of hu-sp34 and ch-sp34 to CD3 by SPR and FACS antibody SPR binding affinity to CD3ε ​ SPR binding affinity to CD3 ε / δ HuT78 combined with FACS k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) KD (nM ₎ k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) KD (nM ₎ EC50 (nM) Ch-sp34 2.16E+05 4.96E-04 2.30E-09 1.09E+05 2.96E-03 2.71E-08 1.9 BG53P 1.98E+05 5.25E-04 2.65E-09 9.93E+04 2.52E-03 2.54E-08 1.8

Based on the humanized sp34 BG53P template, we performed several single mutations to convert the retained mouse residues in the framework regions to the corresponding human germline residues, including four retained mouse residues in VH (D73, S76, M89, V93) and fifteen retained mouse residues in VL (Q1, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, I85 and F87). All humanizing mutations were performed using primers containing mutations at specific positions and a site-directed mutagenesis induction kit (Catalog No. FM111-02, TransGen, Beijing, China). The desired mutations were verified by sequencing analysis. These hu-sp34 variant antibodies were tested in the binding assay as described above. V36Y, G46L and G49Y (Kabat numbering) mutations on VK significantly impaired the binding affinity of the humanized variants compared to hu-sp34-1A-1f, while the remaining forms of the hu-sp34 humanized variants had comparable binding activity to hu-sp34-1A-1f. D73N in VH significantly reduced the expression level (data not shown).

In summary, a well-engineered version of the humanized monoclonal antibody BG56P (SEQ ID NOs: 70-77 and 72-86) was derived from the mutagenesis process described above and was characterized in detail (Table 16 and Figure 4B). Table 16. Comparison of binding affinity of humanized sp34 to CD3 antibody SPR binding affinity to CD3ε ​ SPR binding affinity to CD3 ε / δ HuT78 combined with FACS k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) K _D (M) k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) K _D (M) Ec50 (nM) Ch-sp34 2.16E+05 4.96E-04 2.30E-09 1.09E+05 2.96E-03 2.71E-08 2.49 BG53P 1.06E+05 4.84E-04 4.58E-09 1.09E+05 1.05E-03 9.62E-09 1.72 BG56P 4.60E+04 3.25E-04 7.06E-09 6.16E+04 1.12E-03 1.82E-08 4.69 Example 9. Engineering of humanized sp34 ScFv

To generate a plug-and-play bispecific format and avoid light-heavy chain mispairing, we reformatted the BG56P antibody into a single-chain fragment variable (scFv) format with a 3xG4S linker between VH and VƘ. The reformatted scFv was fused to the N-terminus of the human IgG1 Fc region into a scFv-Fc format using an in-house developed expression vector with easily adaptable subcloning sites. Expression and preparation of parental and reengineered hu-sp34 scFv-Fc were achieved by transfecting the scFv-Fc constructs into 293G cells (developed in-house) and purifying using a protein A column. Purified scFv-Fc format antibodies were concentrated to 0.5-5 mg/mL in PBS and stored in aliquots in a -80°C freezer for the following assays. scFvized BG56P (referred to as BG561P, SEQ ID NOs: 48-53 and 62-65) showed binding affinity comparable to that of the antibody format of BG56P in SPR and FACS (Table 17 and Figure 5). Table 17. Comparison of humanized sp34 and scFv humanized sp34 binding affinity to CD3 by SPR and FACS antibody SPR binding affinity to CD3ε ​ SPR binding affinity to CD3 ε / δ HuT78 combined with FACS k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) KD (nM ₎ k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) KD (nM ₎ EC50 (nM) BG56P 4.60E+04 3.25E-04 7.06E-09 6.16E+04 1.12E-03 1.82E-08 4.69 BG561P 4.42E+04 4.01E-04 9.08E-09 7.25E+04 1.88E-03 2.59E-08 4.35

Based on BG561P, we made several mutations in the framework and CDRs to remove potential PTM sites and improve thermal stability and colloidal stability for human therapeutic use. Mutation of L4V in VL (the resulting humanized scFv designated as BG562P, SEQ ID NOs: 48-53 and 69-70) showed an increase in aggregation temperature (Tagg) by 5 degrees. The combination of L4V in VL with A49G and D65G in VH (the resulting humanized scFv designated as BG563P) (SEQ ID NOs: 48, 71, 50, 51-53, 73 and 74) showed improved thermal stability and colloidal stability compared to BG561P, and slightly improved binding affinity to human CD3 in FACS assays. Potential PTM sites include potential deamidation sites N30 (NT) (Kabat CDR definition) in the joining region of FR1 and HCDR1 and N100 (NS) in HCDR3. Each N was mutated to S to remove potential deamidation sites. All mutations were performed using primers containing mutations at specific positions and a site-directed mutagenesis induction kit (Catalog No. FM111-02, TransGen, Beijing, China). In summary, a well-engineered version of humanized scFv BG564P (SEQ ID NO: 48, 71, 75, 51 - 53, 77 and 78) was derived from the above mutagenesis process and was characterized in detail. The results showed that compared with humanized scFv BG561P, humanized scFv BG564P retained binding affinity to CD3 (Tables 18 - 20 and Figure 6) and improved biophysical stability (Table 20). Table 18. Comparison of binding affinity of different forms of humanized sp34 scFv-Fc to CD3 by SPR antibody SPR binding affinity to CD3ε ​ k _total (M ^-1 s ^-1 ) k _ion (s ^-1 ) KD (nM ₎ BG561P 7.22E+04 3.60E-04 4.98E-09 BG562P 1.16E+05 3.67E-04 3.16E-09 BG563P 4.93E+05 7.72E-04 1.57E-09 BG564P 4.14E+05 5.76E-04 1.39E-09 Table 19. Comparison of binding affinity of humanized sp34 scFv-Fc to CD3 by FACS antibody HuT78 combined with FACS Test 1 Ec50 (nM) Test 2 Ec50 (nM) Test 3 Ec50 (nM) BG561P 2.46 BG562P 2.85 2.85 BG563P 0.94 0.94 BG564P 1.23 Table 20. Comparison of thermal stability and colloidal stability of humanized sp34 scFv-Fc scFv -Fc Tm (℃) Tagg (℃) BG561P 58.1 45.5 BG562P 59.0 50.8 BG563P 59.9 50.9 BG564P 61.6 51.2

Melting temperatures (Tm) were determined using a high-throughput MicroCal™ VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermograms were obtained for each protein (at 0.5 mg/mL, 350 μL) from 20°C to 100°C using a scan rate of 90°C/h. Thermograms of buffer alone were subtracted from each protein sample. The results obtained show the midpoint values of the transition temperatures (Tm) and calorimetric enthalpy (ΔH) of the samples, which indicate that the Tm of BG564P is improved compared to BG561P (Table 20).

The aggregation temperature Tagg (°C) represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation using UNCLE™ (Unchained lab, Pleasanton, CA) by SLS266. The sample was loaded into the Uni and the temperature was raised uniformly from 15°C to 95°C. Back reflection optics cannot detect near-UV light scattering by protein aggregates, and therefore only non-scattered light reaches the detector. Therefore, the reduction in back reflection light is a direct measure of aggregation in the sample, which shows that the Tagg of BG564P is improved compared to BG561P (Table 20). Example 10. Generation of CLDN6×CD3 BsAb BG143P

Agonistic anti-CD3 antibodies have shown toxicity in the clinical setting, which may indicate that systemic Fc R cross-linking is not ideal for CD3 activation. The goal is to achieve effective CD3 stimulation at the tumor site without the need for systemic CD3 activation in multiple cancers. R cross-linking dependence, we generated CLDN6 × CD3 BsAb BG143P with the following characteristics, as shown in Figure 7. This specific construct BG143P includes an IgG fusion-like multispecific antibody format with a module ratio of 1:1, a well-engineered Fab fragment BG87P-34 that binds to CLDN6 and a scFv of BG564P that binds to the CD3 fusion at the N-terminus of CH2, and an Fc empty version of huIgG1 that does not have FcγR binding but retains FcRn binding. A knob-in-hole (KIH) was also introduced into the Fc to increase heterodimerization. The sequence information of BG143P is listed in SEQ ID NO: 79-84. Example 11. Target binding activity of CLDN6×CD3 BsAb BG143P

The binding kinetics of CLDN6×CD3 BsAb BG143P were measured using SPR. SPR was used to measure the association rate constant ( _ka ) and dissociation rate constant ( _kd ) of the CDεγ recombinant protein antibody, and then the affinity constant ( _KD ) was determined. The results showed that CLDN6 × CD3 BsAb has a strong binding affinity to human CDεγ, as shown in Table 21. Table 21. Amino acid and DNA sequence of CLDN6×CD3 BsAb BG143P antibody antigen ka (1/Ms) kd (1/s) K _D (M) BG143P Human CDεγ 2.17E+06 1.81E-02 8.31E-09 Crab-eating macaque CDεγ 1.32E+06 5.99E-03 4.55E-09

FACS results further confirmed the binding activity of BG143P to CD3 and CLDN6. The BsAb showed strong binding activity to Jurkat expressing CD3 in a dose-responsive manner with an EC ₅₀ of 6.98 nM (Figure 8A). Similarly, BG143P showed strong binding activity to PA-1 expressing CLDN6 in a dose-responsive manner with an EC50 of 81.26 nM (Figure 8B). Example 12. In vitro functional activity of CLDN6×CD3 antibodies [ targeting T cells to redirect cytotoxicity and interleukin release

T cell redirected cytotoxicity of BG143P against PA-1 (cancer cell line with high CLDN6 expression), Hutu80 (cancer cell line with moderate CLDN6 expression), AGS (cancer cell line with low and heterogeneous CLDN6 expression) and NCI-H1299 (cancer cell line with negative CLDN6 expression) was evaluated using human PBMCs as effector cells. To measure cytotoxicity, the target cancer cell lines were engineered to express Nano-luciferase. Approximately 10,000 target cells and 25,000 human PBMCs (E/T=2.5) were seeded into each well of a 96-well U-bottom plate and incubated with various concentrations of antibodies at 37°C and 5% _CO2 for 48 hours. The supernatant was collected for interleukin detection. Target cell killing was measured by Nano-Glo detection kit (Promega). The cytotoxic activity (%) of the antibody was calculated using the following formula. Cytotoxic activity (%) = (AB) / (AC) * 100%. "A" represents the average luminescence signal of the wells with only untreated target cells, "B" represents the average luminescence signal of the wells with antibodies and PBMCs, and "C" represents the average luminescence signal of the wells with target cells completely lysed with Triton-X100. IFN-γ and IL-2 in the supernatant were detected by HTRF kit (Cisbio).

As shown in Figure 9, BG143P showed potent T cell redirected killing and interleukin release inducing efficacy at pM EC50 level in a dose-dependent manner. Functional specificity against human CLDN6 and CLDN9

The amino acid sequences of human CLDN6 and CLDN9 are highly conserved, with only three amino acid differences in the extracellular domain. CLDN9 is widely expressed in normal human tissues, so the binding specificity between CLDN6 and CLDN9 is important and was examined by FACS analysis.

Expression vectors of human CLDN6 and CLDN9 were established by inserting the corresponding sequences of synthetic coding cDNA into mammalian expression vectors. NCI-H1299 stable cells expressing human CLDN6 and CLDN9 were generated by transfecting the corresponding plasmids. The cells were suspended in FACS buffer (2% FBS, 1× PBS) at a concentration of 1×10 ⁶ cells, and the cell suspension was dispensed into a U-bottom 96-well plate (100 μL/well). Antibodies were added thereto at a final maximum concentration of 100 nM and 2× diluted 11 dilutions, then mixed with the cells and incubated at 4°C for 1 hour. After centrifugation, the reaction solution was removed and the cells were washed twice with 200 μL/well of FACS buffer. Then, APC-anti-human Fcγ was diluted 500-fold with FACS buffer and added to the cells as a secondary antibody. The cells were incubated at 4°C for 30 minutes, then washed twice as described above, and suspended in 100 μL FACS buffer. The cell suspension was subjected to flow cytometry.

Killing of BG143P on NCI-H1299-CLDN6/CLDN9 was assessed by Nano-Glo assay using human PBMCs. Approximately 10,000 target cells and 25,000 human PBMCs (E/T=2.5) were seeded into each well of a 96-well U-bottom plate and incubated with various concentrations of antibodies at 37°C and 5% _CO2 for 48 hours. The supernatant was collected for interleukin detection. Killing of target cells was measured by Nano-Glo detection kit (Promega). Cytotoxic activity (%) of the antibody was calculated using the following formula. Cytotoxic activity (%) = (AB)/(AC)*100%. "A" represents the average luminescence signal of wells with untreated target cells only, "B" represents the average luminescence signal of wells with antibodies and PBMCs, and "C" represents the average luminescence signal of wells with target cells completely lysed with Triton-X100. IFN-γ was detected by HTRF kit (Cisbio).

As shown in FIG. 10 , BG143P is an antibody that has specific binding ( FIG. 10A ), cell killing (e.g., lysis) ( FIG. 10B ), and IFN-γ inducing activity ( FIG. 10B ) against human CLDN6 but not human CLDN9. Example 13. In vivo efficacy of CLDN6×CD3 BsAb BG143P in the OV90 xenograft model

The in vivo antitumor efficacy of CLDN6 × CD3 BsAb BG143P was evaluated in a PBMC humanized mouse xenograft model. NCG (NOD/ShiLtJGpt-Prkdc ^em26Cd52 Il2rg ^em26Cd22 /Gpt) mice were subcutaneously inoculated with human ovarian cancer cell line OV-90 (ATCC) expressing human CLDN6, and the mice were injected intravenously with human PBMCs the next day. When the tumor volume reached approximately 200 ^mm3 , tumor-bearing mice were randomized into treatment groups to receive antibody or vehicle (PBS) as a control. Antibody/vehicle was administered once a week. The length (L) and width (W) of the tumor mass and body weight of each mouse were measured three times a week. The tumor volume (TV) was calculated as follows: TV = (L × W ² ) / 2. FIG11A shows the in vivo antitumor efficacy of BG143P, which showed stronger efficacy at 0.03 mg/kg and 0.1 mg/kg, with TGI% (tumor growth inhibition ratio, %) of 115.43% and 125.92%.

The reconstitution of mouse hPBMCs was examined at 2, 3, and 4 weeks after PBMC injection. hCD45+ cells among viable cells in peripheral blood were 20% at 2 weeks and increased to 60% at 4 weeks. Figure 11B shows hPBMC reconstitution. Example 14. In vivo efficacy of CLDN6×CD3 BsAb BG143P in the B16F10-/hCLDN6 syngeneic model

Another efficacy model was used to evaluate the in vivo efficacy of CLDN6×CD3 BsAb BG143P. Human CLDN6 expression plasmids were constructed and stably transfected in the B16F10 cell line, and the resulting B16F10/human CLDN6 cells were shown to be able to grow in human CD3EDG transgenic mice and retain hCLDN6 expression after tumor formation. To establish this model, B16F10/human CLDN6 cells were subcutaneously inoculated into hCD3EDG transgenic mice, in which the mouse CD3 gene was replaced by the human counterpart. Mice were randomized after tumors reached approximately 100 ^mm3 in size. Mice were injected intraperitoneally with test article or PBS weekly. The length (L) and width (W) of the tumor mass and body weight of each mouse were measured three times a week. The tumor volume (TV) was calculated as follows: TV = (L × W2) / 2. BG143P showed a strong efficacy at 0.1 mg/kg, with a TGI% of 93.54%, as shown in Figure 12A. As shown in Figure 12B, no significant weight loss was observed in the study .

without

Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I and 1J show the cell binding activity of chBG87P engineered variants. Figure 1A shows the binding activity of the first round BG87P humanized revertant variants (BG87P-z0, BG87P-Bz0, BG87P-Bz1, BG87P-Bz2, BG87P-Bz3, BG87P-Bz4, BG87P-Bz5, BG87P-Bz6, BG87P-Bz7 and BG87P-Bz8) against HEK293T/human CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P). Figure 1B shows the cell binding activity of the combined humanized variants (BG87P-21, BG87P-22, BG87P-23 and BG87P-24) against HEK293T/human CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P). Figure 1C shows the cell binding activity of the combined humanized variants (BG87P-25, BG87P-26 and BG87P-27) against HEK293T/human CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P). Figure 1D shows the cell binding activity of the combined humanized variants (BG87P-21, BG87P-22, BG87P-23 and BG87P-24) against the cancer cell line PA-1 compared to the anti-CLDN6 chimeric BG87P (chBG87P). Figure 1E shows the cell binding activity of the combined humanized variants (BG87P-25, BG87P-26 and BG87P-27) against the cancer cell line PA-1 compared to the anti-CLDN6 chimeric BG87P (chBG87P). Figure 1F shows the cell binding activity of post-translational modification (PTM) removal engineered variants (BG87P-m1, BG87P-m2, BG87P-m3, BG87P-m4, BG87P-m5, BG87P-m6, BG87P-m7 and BG87P-m8) against HEK293T/human CLDN6 compared to anti-CLDN6 chimeric BG87P (chBG87P) and BG87P-Bz0. Figure 1G shows the cell binding activity of BG87P solubility engineered variants (BG87P-21, BG87P-34 and BG87P-33) against HEK293T/human CLDN6 compared to anti-CLDN6 chimeric BG87P (chBG87P). Figure 1H shows the non-specific binding activity of solubility engineered variants (BG87P-21, BG87P-34 and BG87P-33) against HEK293T-human CLDN9 compared to anti-CLDN6 chimeric BG87P (chBG87P). Figure 1I shows the cross-reactivity of humanized variants (BG87P-21, BG87P-34 and BG87P-33) against CHOK1-cynomolgus macaque CLDN6 compared to anti-CLDN6 chimeric BG87P (chBG87P). Figure 1J shows the cross-reactivity of humanized variants (BG87P-21, BG87P-34 and BG87P-33) against CHOK1-mouse CLDN6 compared to anti-CLDN6 chimeric BG87P (chBG87P). Figure 2 depicts the predicted hydrophobic patch in Schroedinger's chimeric BG87P homology model. I97-Y98-Y100-V100a of HCDR3 and Y49-W50 of HCDR2 are predicted to form an exposed hydrophobic patch together (Y49 is the last residue of the light chain variable region FR2, and W50 is the first residue of LCDR2). Figure 3 shows the hydrophobicity of selected humanized BG87P variants (BG87-33, BG87-34, BG87P-21) after engineering, as determined by HIC-HPLC. Figures 4A and 4B show a comparison of binding activity between chimeric and humanized sp34s in Hut78 cells. Figure 4A shows a comparison of binding affinity between chimeric sp34 (ch-sp34) and humanized sp34 BG53P (BG53P) measured by melt flow index (MFI) in Hut78 cells. Figure 4B shows a comparison of binding affinity between chimeric sp34 (ch-sp34), humanized sp34 BG53P (BG53P) and BG56P. Figure 5 shows a comparison of binding activity between humanized sp34 BG56P (BG56P) and humanized sp34 scFv BG561p (BG561P). Figures 6A, 6B, and 6C show a comparison of binding affinity between humanized sp34 scFvs in Hut78 cells. Figure 6A shows a comparison of binding affinity between humanized sp34 scFv BG561p (BG561P) and humanized scFv BG562P (BG562P) in Hut78 cells. Figure 6B shows a comparison of binding affinity between humanized scFv BG562P (BG562P) and humanized scFv BG563P (BG563P) in Hut78 cells. Figure 6C shows a comparison of binding affinity between humanized scFv BG563P (BG563P) and humanized scFv BG564P (BG564P) in Hut78 cells. Figure 7 shows a schematic diagram of CLDN6×CD3 BsAb BG143P. Figures 8A and 8B show the target binding activity of CLDN6×CD3 BsAb BG143P. Figure 8A shows the CD3 binding activity of BG143P in Jurkat cells expressing CD3. Figure 8B shows the CLDN6 binding activity of BG143P in PA-1 cells expressing CLDN6. Figures 9A, 9B and 9C show the targeting functional activity of CLDN6×CD3 BsAb BG143P in tumor cell lines with different CLDN6 expression. Figure 9A shows the redirected T cell cytotoxicity of BG143P in PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells expressing CLDN6 according to the cell lysis assay. Figure 9B shows the IFN-γ inducing activity of BG143P in PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells expressing CLDN6. Figure 9C shows the IL-2 inducing activity of BG143P in PA-1 cells, Hutu80 cells, AGS cells, and NCI-H1299 cells expressing CLDN6. Figures 10A, 10B and 10C show the functional specificity of CLDN6 × CD3 BsAb BG143P against human CLDN6 and CLDN9. Figure 10A shows the binding specificity of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells. Figure 10B shows the killing specificity (cytolytic activity) of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells. Figure 10C shows the IFN-γ induction of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells. Figures 11A and 11B show the in vivo efficacy of CLDN6×CD3 BsAb BG143P in the OV-90 xenograft model in PBMC humanized mice. Figure 11A shows the change in tumor volume over time. Mice were untreated (no PBMC), treated with PBS (PBS ip QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), treated with 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, ip). Treatments were once a week and are represented by triangles on the X-axis. Figure 11B shows the percentage of hCD45+ cells in peripheral blood of mice untreated (no PBMC), treated with PBS (PBS ip QW), treated with 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), treated with 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), or treated with 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, ip) at days 13, 21, and 27 after PBMC injection, indicating human PBMC reconstitution. Figures 12A and 12B show the in vivo efficacy of CLDN6 × CD3 BsAb BG143P in the B16F10/human CLDN6 syngeneic model of hCD3EDG transgenic mice. Figure 12A shows the changes in tumor volume over time. Mice were treated with PBS (PBS ip QW), 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), or 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, ip). Treatments were given once a week and are represented by triangles on the X-axis. Figure 12B shows the body weight of mice treated with PBS (PBS ip QW), 0.01 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), 0.03 mg/kg of BG143P (BG143P -0.01 mg/kg, ip), or 0.1 mg/kg of BG143P (BG143P -0.1 mg/kg, ip) during days 11 to 27 after inoculation as an indicator of mouse tolerance to antibodies.

TW202436354A_113107891_SEQL.xml

Claims (34)

An antibody or an antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to human claudin 6 (CLDN6). The antibody or antigen-binding fragment of claim 1, wherein the antigen-binding domain does not bind to other members of the claudin (CLDN) protein family. The antibody or antigen-binding fragment of claim 2, wherein the antigen-binding domain does not bind to human claudin 9 (CLDN9). The antibody or antigen-binding fragment of claim 3, wherein the antigen-binding domain is highly selective for human CLDN6 over human CLDN9. An antibody or antigen-binding fragment as claimed in any of the preceding claims, wherein the antigen-binding domain that specifically binds to human CLDN6 comprises: (a) a heavy chain variable region comprising (i) a heavy chain complementation determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 1, (ii) a HCDR2 having an amino acid sequence of SEQ ID NO: 39, (iii) a HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (iv) a light chain complementation determining region (LCDR) 1 having an amino acid sequence of SEQ ID NO: 40, (v) a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and (vi) a LCDR3 having an amino acid sequence of SEQ ID NO: 6; (b) a heavy chain variable region comprising: (i) a heavy chain complementation determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having an amino acid sequence of SEQ ID NO: 2, (iii) HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 having an amino acid sequence of SEQ ID NO: 4, (v) LCDR2 having an amino acid sequence of SEQ ID NO: 5, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO: 6; (c) a heavy chain variable region comprising: (i) HCDR1 having an amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having an amino acid sequence of SEQ ID NO: 23, (iii) HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 having an amino acid sequence of SEQ ID NO: 4, (v) LCDR2 having an amino acid sequence of SEQ ID NO: 5, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO: 6; 5, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6; or (d) a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 45, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 40, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 5, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 6. An antibody or antigen-binding fragment as claimed in any of the preceding claims, wherein the antigen-binding domain comprises: (a) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44; (b) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8 having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24; (c) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12; (d) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 42 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46; (e) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 47; or (f) a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46, and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 42Amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. The antibody or antigen-binding fragment of any of the preceding claims, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46 or 47 have been inserted, deleted or substituted. An antibody or antigen-binding fragment as claimed in any of the preceding claims, wherein the antigen-binding domain comprises: (a) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 44; (b) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 8; (c) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 12; (d) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 42; (e) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 47; or (f) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 42. The antibody or antigen-binding fragment of any of the preceding claims, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody is a multispecific antibody. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody is a bispecific antibody. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or is hypofucosylated. The antibody or antigen-binding fragment of any of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an increased bisecting GlcNac structure. An antibody or antigen-binding fragment as claimed in any preceding claim, wherein the Fc domain is IgG1. The antibody or antigen-binding fragment of any of the preceding claims, wherein the Fc domain is IgG1 with reduced effector function. The antibody or antigen-binding fragment of any of the preceding claims, wherein the Fc domain is IgG4. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 17, further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 18, further comprising histidine/histidine HCl, trehalose dihydrate and/or polysorbate 20. A method for treating cancer, comprising administering an effective amount of the antibody or antigen-binding fragment of claims 1 to 17 to a patient in need thereof. The method of claim 20, wherein the cancer is a solid cancer. The method of claim 20, wherein the cancer is selected from gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, brain cancer, colorectal cancer, prostate cancer, cervical cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor and/or neuroglioma. The method of claim 22, wherein the antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agents. The method of claim 23, wherein the one or more therapeutic agents are selected from paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine. The method of claim 24, wherein the one or more therapeutic agents are paclitaxel, lelizumab, or 5-azacytidine. The method of claim 23, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody. The method of claim 25, wherein the anti-PD1 antibody is Tislelizumab. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 17. A vector comprising the nucleic acid of claim 28. A host cell comprising the nucleic acid of claim 28 or the vector of claim 29. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell of claim 30 and recovering the antibody or antigen-binding fragment from the culture. An antibody or antigen-binding fragment of any one of claims 1 to 17, or a pharmaceutical composition of claim 18 or 19, for use in medicine or therapy. The antibody or antigen-binding fragment of any one of claims 1 to 17, or the pharmaceutical composition of claim 18 or 19, for use in a method for treating cancer. A use of the antibody or antigen-binding fragment of any one of claims 1 to 17 for the manufacture of a medicament for treating cancer.