Anti-CLDN6 and anti-CD3 multispecific antibodies and methods of use thereof

Abstract

The present disclosure provides multispecific antibodies and antigen-binding fragments thereof that bind to human CLDN6 and CD3, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and the use of the multispecific antibodies or antigen-binding fragments thereof or the compositions for the treatment of diseases such as cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to International Application PCT / CN2023 / 079805, filed March 6, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] Disclosed herein are multispecific antibodies or antigen-binding fragments thereof that bind to human Claudin 6 (CLDN6) and human Cluster of Differentiation 3 (CD3) and methods of producing the same. In particular, the present disclosure provides, inter alia, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and methods of treating cancer. BACKGROUND

[0003] The following description of background art may be aimed to assist in understanding the present technology and is not admitted to be prior art to the present technology.

[0004] The Claudin (CLDN) gene family encodes integral membrane proteins that serve as key structural and functional components of tight junctions (TJs). CLDNs display a four-pass 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 that are expressed in a tissue-specific manner in epithelial and endothelial cells (Günzel et al., Physiol Rev. 2013). Through interactions with one another in cis (intracellular) and trans (intercellular) interactions, CLDNs play important roles in regulating paracellular permeability and maintaining cell polarity (Tsukita et al., Trends in Biochemical Sciences. 2019). Additionally, CLDNs can serve as protein scaffolds that assemble complexes at cell junctions and transmit signals to the interior of the cell to regulate gene expression and cellular behavior (Matter et al., Nat Rev Mol Cell Biol. 2003; Singh et al., Pflugers Arch. 2017).

[0005] CLDN6 was first identified and characterized in 2001 (Turksen et al., Developmental Dynamics. 2001). Expression of CLDN6 is dynamically regulated by a variety of factors and mechanisms (Du et al., Mol Med Rep. 2021). CLDN6 is one of the first proteins expressed in embryonic stem cells committed to an epithelial cell fate and is a cell surface specific 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, while transcriptionally silenced 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, and others (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 and membrane localization of CLDN6 between cancer and normal tissues make it an attractive target for cancer immunotherapy.

[0006] CD3 bispecific antibodies (BsAbs) are an emerging therapeutic modality in the field of cancer immunotherapy. CD3-BsAbs act by simultaneously binding to a tumor-associated antigen expressed on tumor cells and CD3 on T cells. CD3-BsAbs tightly link these two cell types, allowing the formation of an MHC-independent immunological synapse and leading to T cell activation and subsequent anti-tumor immune response (Kamakura et al., Pharmaceuticals (Basel). 2021). Currently, CD3-BsAbs show great potential for hematological malignancies, and early encouraging clinical data against solid tumors have been reported. A key obstacle in developing CD3-BsAbs for solid tumors is to identify cell surface targets with high cancer-specific expression to allow efficient tumor eradication and low risk of off-tumor toxicity (Middelburg et al., Cancers (Basel). 2021; Singh et al., Br J Cancer. 2021; Baeuerle et al., Current Opinion in Oncology. 2022). Based on the highly cancer-specific expression pattern, CLDN6 can be used as a promising target for CD3-BsAb development (Stadler et al., Oncoimmunology. 2016; Stadler et al., Nat Med. 2017).

[0007] An important consideration for targeting CLDN6 is that many members of the CLDN family have high sequence identity, with Claudin 9 (CLDN9) having the highest similarity to CLDN6. Only 3 of the 76 residues in the extracellular loop of CLDN6 and CLDN9 are different. Given that CLDN9 is highly expressed in certain normal tissues, achieving high selectivity for CLDN6 over CLDN9 is critical for any CLDN6-targeting antibody-based therapeutic. SUMMARY

[0008] The present disclosure provides multispecific anti-CLDN6xCD3 antibodies and antigen-binding fragments thereof. The present disclosure encompasses the following embodiments.

[0009] In some aspects, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human Claudin 6 (CLDN6) and a second antigen-binding domain that specifically binds to human Cluster of Differentiation 3 (CD3).

[0010] In some embodiments, wherein the first antigen-binding domain does not bind to other Claudin (CLDN) family members.

[0011] In some embodiments, the first antigen binding domain does not bind to human Claudin 9 (CLDN9).

[0012] In some embodiments, the first antigen binding domain has high selectivity for human CLDN6 over human CLDN9.

[0013] In some embodiments, the first antigen binding domain that specifically binds to human CLDN6 comprises: (a) a heavy chain variable region comprising: (i) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 2, (iii) a HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (iv) a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 4, (v) a LCDR2 of SEQ ID NO: 5, and (vi) a LCDR3 of SEQ ID NO: 6; (b) a heavy chain variable region comprising: (i) a HCDR1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 23, (iii) a HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (iv) a LCDR1 of SEQ ID NO: 4, (v) a LCDR2 of SEQ ID NO: 5, and (vi) a LCDR3 of SEQ ID NO: 6; (c) a heavy chain variable region comprising: (i) a HCDR1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 39, (iii) a HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (iv) a LCDR1 of SEQ ID NO: 40, (v) a LCDR2 of SEQ ID NO: 5, and (vi) a LCDR3 of SEQ ID NO: 6; or (d) a heavy chain variable region comprising: (i) a HCDR1 of SEQ ID NO: 1, (ii) a HCDR2 of SEQ ID NO: 45, (iii) a HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (iv) a LCDR1 of SEQ ID NO: 40, (v) a LCDR2 of SEQ ID NO: 5, and (vi) a LCDR3 of SEQ ID NO: 6.

[0014] In some embodiments, the first 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; (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; (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.

[0015] In some embodiments, one, two, three, four, five, six, seven, eight, nine, or ten amino acids of SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 have been inserted, deleted, or substituted.

[0016] In some embodiments, the first antigen binding domain comprises: (a) 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; (b) 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; (c) 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; (d) 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; (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.

[0017] In some embodiments, the second antigen binding domain that specifically binds to human CD3 comprises: (a) a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 49, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising: (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 53; (b) a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising: (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 53; or (c) a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 75, and a light chain variable region comprising: (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 53.

[0018] In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region 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: 54, and a light chain variable region 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: 55; (b) a heavy chain variable region 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: 58, and a light chain variable region 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: 59; (c) a heavy chain variable region 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: 62, and a light chain variable region 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: 63; (d) a heavy chain variable region 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: 62, and a light chain variable region 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: 68; (e) a heavy chain variable region 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: 72, and a light chain variable region 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: 68.or (f) a heavy chain variable region 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: 76, and a light chain variable region 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: 68.

[0019] In some embodiments, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids within SEQ ID NO: 54, 55, 58, 59, 62, 68, 72, or 76 have been inserted, deleted, or substituted.

[0020] In some embodiments, wherein the second antigen binding domain comprises: (a) a heavy chain variable region comprising SEQ ID NO: 54 and a light chain variable region comprising SEQ ID NO: 55; (b) a heavy chain variable region comprising SEQ ID NO: 58 and a light chain variable region comprising SEQ ID NO: 59; (c) a heavy chain variable region comprising SEQ ID NO: 62 and a light chain variable region comprising SEQ ID NO: 63; (d) a heavy chain variable region comprising SEQ ID NO: 62 and a light chain variable region comprising SEQ ID NO: 68; (e) a heavy chain variable region comprising SEQ ID NO: 72 and a light chain variable region comprising SEQ ID NO: 68; or (f) a heavy chain variable region comprising SEQ ID NO: 76 and a light chain variable region comprising SEQ ID NO: 68.

[0021] In some embodiments, wherein the second antigen binding domain comprises: (a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; (b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; (c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or (e) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0022] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region comprising (i) HCDR1 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) LCDR1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vii) LCDR3 of SEQ ID NO: 6; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region comprising (i) HCDR1 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) LCDR1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53;or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region comprising (i) HCDR1 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) LCDR1 of SEQ ID NO: 4, (v) LCDR2 of SEQ ID NO: 5, and (vi) LCDR3 of SEQ ID NO: 6; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0023] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 49, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53;or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0024] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 49, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53;or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0025] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0026] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 49, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53;or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0027] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 of SEQ ID NO: 48, (ii) HCDR2 of SEQ ID NO: 71, (iii) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 of SEQ ID NO: 51, (v) LCDR2 of SEQ ID NO: 52, and (vi) LCDR3 of SEQ ID NO: 53.

[0028] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 2, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 4, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 23, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 4, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 39, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 40, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; or (iv) the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 45, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 40, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; and (b) the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55, (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63;(i) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0029] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 39, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 40, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; or / and (b) the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0030] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 23, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 4, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; or (ii) a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 39, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 40, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; or (iii) the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region comprising (1) HCDR1 of SEQ ID NO: 1, (2) HCDR2 of SEQ ID NO: 45, (3) HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising: (4) LCDR1 of SEQ ID NO: 40, (5) LCDR2 of SEQ ID NO: 5, and (6) LCDR3 of SEQ ID NO: 6; and (b) the second antigen binding domain that specifically binds to human CD3 comprises: (i) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; (ii) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; (iii) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or (iv) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0031] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42, and (ii) the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0032] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (b) the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0033] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (b) the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0034] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0035] In some embodiments, wherein the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0036] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0037] In some embodiments, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (b) the second antigen binding domain that specifically binds to human CD3 comprises: (i) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; (ii) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; (iii) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or (iv) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0038] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: (a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; (b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; (c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or (d) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0039] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: a single chain variable fragment (scFv) comprising SEQ ID NO: 66.

[0040] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 69.

[0041] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 73.

[0042] In some embodiments, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0043] In some embodiments, the antibody 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.

[0044] In some embodiments, wherein the multispecific antibody is a bispecific antibody.

[0045] In some embodiments, wherein the multispecific antibody is BG143P and has a sequence comprising SEQ ID NO: 80, SEQ ID NO: 82, or SEQ ID NO: 84.

[0046] In some embodiments, wherein the antibody or antigen binding fragment thereof has antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).

[0047] In some embodiments, wherein the antibody or antigen binding fragment thereof has reduced glycosylation or no glycosylation or low fucosylation.

[0048] In some embodiments, wherein the antibody or antigen binding fragment thereof comprises increased bisecting GlcNac structures.

[0049] In some embodiments, the Fc domain is an IgGl with reduced effector function.

[0050] In some embodiments, the Fc domain is an IgG4.

[0051] In some aspects, the disclosure provides a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment as disclosed herein In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0052] In some embodiments, the pharmaceutical composition comprises histidine / histidine HC1, trehalose dihydrate, and / or polysorbate 20.

[0053] In some aspects, the disclosure provides a method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of a multispecific antibody or antigen-binding fragment as disclosed herein.

[0054] In some embodiments, the cancer is a solid tumor.

[0055] In some embodiments, the cancer is selected from gastric cancer, colon 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 glioma.

[0056] In some embodiments, the multispecific antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agents.

[0057] 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.

[0058] In some embodiments, the one or more therapeutic agents is a paclitaxel agent, lenalidomide, or 5-azacytidine.

[0059] In some embodiments, at least one of the one or more therapeutic agents is an anti-PDl or anti-PDLl antibody.

[0060] In some embodiments, the anti-PD1 antibody is tislelizumab.

[0061] In some embodiments, the present disclosure provides an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment as disclosed herein.

[0062] In some embodiments, the present disclosure provides a vector comprising a nucleic acid as disclosed herein.

[0063] In some embodiments, the present disclosure provides a host cell comprising a nucleic acid or vector as disclosed herein.

[0064] In some aspects, the present disclosure provides a process for producing a multispecific antibody or antigen-binding fragment as disclosed herein, the process comprising culturing a host cell as disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

[0065] In some aspects, the present disclosure provides a multispecific antibody or antigen-binding fragment as disclosed herein for use in a method of treating cancer.

[0066] In some aspects, the present disclosure provides a multispecific antibody or antigen-binding fragment as disclosed herein for use in the manufacture of a medicament for treating cancer.

[0067] In some aspects, the present disclosure provides a pharmaceutical composition for use in a method of treating cancer.

[0068] A multispecific antibody or antigen-binding fragment thereof comprising a first antigen binding domain that specifically binds to human CLDN6 and a second antigen binding domain that specifically binds to human CD3.

[0069] The multispecific antibody or antigen-binding fragment, wherein the first antigen binding domain does not bind to other CLDN family members.

[0070] The multispecific antibody or antigen-binding fragment, wherein the first antigen binding domain does not bind to human CLDN9.

[0071] The multispecific antibody or antigen-binding fragment, wherein the first antigen binding domain has high selectivity over human CLDN9.

[0072] The multispecific antibody or antigen-binding fragment, wherein the first 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; (iii). 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; or (iv). 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.

[0073] the multispecific antibody or antigen binding fragment, wherein the first 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; (iii). 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: 41, 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; (iv). 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: 43, 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: 44; (v). 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: 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.

[0074] the multispecific antibody or antigen-binding fragment, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids within SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 have been inserted, deleted, or substituted.

[0075] the multispecific antibody or antigen-binding fragment, wherein the first antigen binding domain comprises: (i). a heavy chain variable region (VH) comprising SEQ ID NO: 7, and a light chain variable region (VL) comprising SEQ ID NO: 8; (ii). a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; (iii). a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; (iv). a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; (v). a heavy chain variable region (VH) 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.

[0076] the multispecific antibody or antigen-binding fragment, wherein the second antigen binding domain that specifically binds to human CD3 comprises: (i). a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (ii). a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; or (iii). a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0077] the multispecific antibody or antigen binding fragment, wherein the second antigen binding domain comprises: 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: 54, 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: 55; 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: 58, 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: 59; 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: 62, 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: 63; 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: 62, 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: 68; 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: 72, 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: 68; or 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: 76, 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: 68.

[0078] the multispecific antibody or antigen binding fragment, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids within SEQ ID NO: 58, 59, 62, or 63 have been inserted, deleted, or substituted.

[0079] the multispecific antibody or antigen binding fragment, wherein the second antigen binding domain comprises: (i). a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; (ii). a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii). a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; (iv). a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v). a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or (vi). a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0080] the multispecific antibody or antigen binding fragment, wherein the second antigen binding domain comprises: (a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; (b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; (c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or (d) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0081] The multispecific antibody or antigen binding fragment, wherein: (i) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (ii) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53;(iii) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and (iv) the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0082] the multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (ii). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; or (iii). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0083] the multispecific antibody or antigen binding fragment, wherein: (i). the multispecific antibody or antigen binding fragment, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (ii). the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; or (iii). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0084] The multispecific antibody or antigen binding fragment, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0085] The multispecific antibody or antigen binding fragment, wherein: (i). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; (ii). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; or (iii). The first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0086] The multispecific antibody or antigen binding fragment, wherein:

[0087] The multispecific antibody or antigen binding fragment, wherein: (i). The first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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; b) 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; c) 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; or d) a first antigen binding domain that specifically binds to human CLDN6 comprises 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; and (ii). a second antigen binding domain that specifically binds to human CD3 comprises: a) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; b) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53; or c) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0088] the multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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; b) 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; c) 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; or d) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and (ii). the second antigen binding domain that specifically binds to human CD3 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; b) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; c) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; d) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0089] The multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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; or and (ii). the second antigen binding domain that specifically binds to human CD3 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; b) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; c) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; d) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0090] The multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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; or b) 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; or c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and (ii). the second antigen binding domain that specifically binds to human CD3 comprises: a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or d) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0091] the multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; b) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; c) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; d) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or e) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (ii). the second antigen binding domain that specifically binds to human CD3 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; b) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; c) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; d) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0092] the multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; b) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; c) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; d) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or e) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (ii). the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0093] the multispecific antibody or antigen binding fragment, wherein: (i). the first antigen binding domain that specifically binds to human CLDN6 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; b) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; c) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; d) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or e) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42; and (ii). the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0094] The multispecific antibody or antigen-binding fragment, wherein: a) a heavy chain variable region (VH) comprising SEQ ID NO: 54, and a light chain variable region (VL) comprising SEQ ID NO: 55; b) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; c) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 63; d) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 72, and a light chain variable region (VL) comprising SEQ ID NO: 68; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 76, and a light chain variable region (VL) comprising SEQ ID NO: 68.

[0095] The multispecific antibody or antigen-binding fragment, wherein:

[0096] The multispecific antibody or antigen-binding fragment, wherein:

[0097] The multispecific antibody or antigen-binding fragment, wherein: (i). The first antigen binding domain that specifically binds to human CLDN6 comprises: a) a heavy chain variable region (VH) comprising SEQ ID NO: 24, and a light chain variable region (VL) comprising SEQ ID NO: 12; b) a heavy chain variable region (VH) comprising SEQ ID NO: 41, and a light chain variable region (VL) comprising SEQ ID NO: 42; c) a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; d) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 47; or e) a heavy chain variable region (VH) comprising SEQ ID NO: 46, and a light chain variable region (VL) comprising SEQ ID NO: 42.

[0098] and (ii). The second antigen binding domain that specifically binds to human CD3 comprises: a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or d) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0099] The multispecific antibody or antigen binding fragment, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: a) a single chain variable fragment (scFv) comprising SEQ ID NO: 66; b) a single chain variable fragment (scFv) comprising SEQ ID NO: 69; c) a single chain variable fragment (scFv) comprising SEQ ID NO: 73; or d) a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0100] The multispecific antibody or antigen-binding fragment, wherein: the first antigen-binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen-binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 66.

[0101] The multispecific antibody or antigen-binding fragment, wherein: the first antigen-binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen-binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 69.

[0102] The multispecific antibody or antigen-binding fragment, wherein: the first antigen-binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen-binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 73.

[0103] The multispecific antibody or antigen-binding fragment, wherein: the first antigen-binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising SEQ ID NO: 43, and a light chain variable region (VL) comprising SEQ ID NO: 44; and the second antigen-binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) comprising SEQ ID NO: 77.

[0104] The multispecific antibody or antigen-binding fragment, 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.

[0105] The multispecific antibody, wherein the multispecific antibody is a bispecific antibody.

[0106] The multispecific antibody, wherein the multispecific antibody is BG143P (SEQ ID NO: 80, SEQ ID NO: 82, and SEQ ID NO: 84).

[0107] The multispecific antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0108] The multispecific antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or low fucosylation.

[0109] The multispecific antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof comprises increased bisected GlcNac structures.

[0110] The multispecific antibody or antigen-binding fragment, wherein the Fc domain is an IgG1 with reduced effector function.

[0111] The multispecific antibody or antigen-binding fragment, wherein the Fc domain is an IgG4.

[0112] A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof, further comprising a pharmaceutically acceptable carrier.

[0113] The pharmaceutical composition, further comprising histidine / histidine HC1, trehalose dihydrate, and polysorbate 20.

[0114] A method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of the multispecific antibody or antigen-binding fragment.

[0115] The method, wherein the cancer is gastric cancer, breast cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, testicular cancer, endometrial cancer, bladder cancer, rhabdoid tumor, and glioma.

[0116] The method, wherein the multispecific antibody or antigen-binding fragment is administered in combination with another therapeutic agent.

[0117] The method, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacitidine.

[0118] The method, wherein the therapeutic agent is a paclitaxel agent, lenalidomide, or 5-azacitidine.

[0119] The method, wherein the therapeutic agent is an anti-PD1 or anti-PDL1 antibody.

[0120] The method, wherein the anti-PD1 antibody is tiragulimab.

[0121] An isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment.

[0122] A vector comprising the nucleic acid.

[0123] A host cell comprising the nucleic acid or the vector.

[0124] A process for producing a multispecific antibody or antigen binding fragment thereof, the process comprising culturing the host cell and recovering the antibody or antigen binding fragment from the culture.

[0125] In one embodiment, the multispecific antibody or antigen binding fragment thereof comprises one or more complementarity 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: 23, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53.

[0126] In another embodiment, the multispecific antibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising one or more complementarity 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, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 71, SEQ ID NO: 75; and / or (b) a light chain variable region comprising one or more complementarity 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, SEQ ID NO: 40, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53.

[0127] In another embodiment, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs) that are a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs) that are a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0128] In another embodiment, the multispecific antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs) that are a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; or a HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs) that 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.

[0129] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0130] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:49, (c) HCDR3 of SEQ ID NO:50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0131] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:49, (c) HCDR3 of SEQ ID NO:50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0132] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 49, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0133] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0134] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:71, (c) HCDR3 of SEQ ID NO:50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0135] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:71, (c) HCDR3 of SEQ ID NO:50, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0136] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:71, (c) HCDR3 of SEQ ID NO:75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0137] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:48, (b) HCDR2 of SEQ ID NO:71, (c) HCDR3 of SEQ ID NO:75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:51, (e) LCDR2 of SEQ ID NO:52, and (f) LCDR3 of SEQ ID NO:53.

[0138] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0139] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0140] In another embodiment, the multispecific antibody or antigen-binding fragment comprises: a first 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; and a second antigen binding domain comprising: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 48, (b) HCDR2 of SEQ ID NO: 71, (c) HCDR3 of SEQ ID NO: 75, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO: 51, (e) LCDR2 of SEQ ID NO: 52, and (f) LCDR3 of SEQ ID NO: 53.

[0141] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region having an amino acid sequence of a HCDR or VH listed in Table 1 and Table 2; and / or (b) a light chain variable region comprising an amino acid sequence of a LCDR or VL listed in Table 1 and Table 2.

[0142] In another embodiment, the multispecific antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) an amino acid sequence comprising one, two, or three amino acid substitutions in an amino acid sequence of a HCDR or VH listed in Table 1 and Table 2; and / or (b) a light chain variable region comprising an amino acid sequence comprising one, two, three, four, or five amino acid substitutions in an amino acid sequence of a LCDR or VL listed in Table 1 and Table 2. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0143] In one embodiment, the multispecific antibody of the present disclosure is an IgGl, IgG2, IgG3, or IgG4 isotype. In a more specific embodiment, the antibody of the present disclosure comprises the Fc domain of wild-type human IgGl (also referred to as human IgGl wt or huIgGl) or IgG2.

[0144] In one embodiment, the multispecific antibody of the present disclosure binds to CLDN6 with a binding affinity (K -6 M to 1 x 10 -10 M. In another embodiment, the antibody of the present disclosure binds to CLDN6 with a binding affinity (K D ) of about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, or about 1 x 10 -10 M. In another embodiment, the antibody of the present disclosure binds to CLDN6 with a binding affinity (K D ) of about 1 x 10

[0145] In another embodiment, the anti-human CLDN6 multispecific antibody of the present disclosure exhibits cross-species binding activity against cynomolgus monkey CLDN6.

[0146] In one embodiment, the antibody of the present disclosure has strong Fc-mediated effector functions. The antibody mediates antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing CLDN6.

[0147] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a CLDN6xCD3 multispecific antibody or antigen binding fragment thereof, and optionally a pharmaceutically acceptable excipient.

[0148] In yet another aspect, the present disclosure relates to a method of treating a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a CLDN6xCD3 multispecific antibody or antigen binding fragment thereof, or a CLDN6xCD3 multispecific antibody pharmaceutical composition. In another embodiment, the disease treated by the antibody or antigen binding fragment is cancer.

[0149] The present disclosure relates to the use of a CLDN6xCD3 multispecific antibody or antigen binding fragment thereof, or a CLDN6xCD3 multispecific antibody pharmaceutical composition for treating a disease such as cancer. BRIEF DESCRIPTION OF DRAWINGS

[0150] Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1D 、 Figure 1E 、 Figure 1F 、 Figure 1G 、 Figure 1H 、 Figure 1I and Figure 1J Cellular binding activity of chBG87P engineered variants is shown. Figure 1A Cellular binding activity of 1st round BG87P humanization back-mutation 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 anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1B Cellular binding activity of combined humanization variants (BG87P-21, BG87P-22, BG87P-23, and BG87P-24) against HEK293T / human CLDN6 compared to anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1CCellular binding activity of the combination humanized variants (BG87P-21, BG87P-22, BG87P-23, and BG87P-24) against HEK293T / human CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1D Cellular binding activity of the combination 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) is shown. Figure 1E Cellular binding activity of the combination humanized variants (BG87P-25, BG87P-26, and BG87P-27) against the cancer cell line PA-1 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1F Cellular binding activity of the 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 the anti-CLDN6 chimeric BG87P (chBG87P) and BG87P-Bz0 is shown. Figure 1G Cellular binding activity of the BG87P soluble engineered variants (BG87P-21, BG87P-34, and BG87P-33) against HEK293T / human CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1H Non-specific binding activity of the soluble engineered variants (BG87P-21, BG87P-34, and BG87P-33) against HEK293T-human CLDN9 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1I Cross-reactivity of the humanized variants (BG87P-21, BG87P-34, and BG87P-33) against CHOK1-cynomolgus CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown. Figure 1J Cross-reactivity of the humanized variants (BG87P-21, BG87P-34, and BG87P-33) against CHOK1-mouse CLDN6 compared to the anti-CLDN6 chimeric BG87P (chBG87P) is shown.

[0151] Figure 2Hydrophobic patch regions in the chimeric BG87P homology model predicted by Schroedinger are delineated. I97-Y98-Y100-V100a of HCDR3 is predicted to form an exposed hydrophobic patch region together with Y49-W50 of HCDR2 (Y49 is the last residue of FR2 of the light chain variable region and W50 is the first residue of LCDR2).

[0152] Figure 3 Hydrophobicity of selected humanized BG87P variants (BG87-33, BG87-34, BG87P-21) after engineering as determined by HIC-HPLC is shown.

[0153] Figure 4A and Figure 4B Comparison of binding activity between chimeric sp34 and humanized sp34 BG53P is shown in Hut78 cells. Figure 4A Comparison of binding affinity between chimeric sp34 (ch-sp34) and humanized sp34 BG53P (BG53P) is shown in Hut78 cells as measured by melt flow index (MFI). Figure 4B Comparison of binding affinity between chimeric sp34 (ch-sp34), humanized sp34 BG53P (BG53P), and BG56P is shown.

[0154] Figure 5 Comparison of binding activity between humanized sp34 BG56P (BG56P) and humanized sp34 scFv BG561p (BG561P) is shown.

[0155] Figure 6A , Figure 6B and Figure 6C Comparison of binding affinity of humanized sp34 scFv is shown in Hut78 cells. Figure 6A Comparison of binding affinity between humanized sp34 scFv BG561p (BG561P) and humanized scFv BG562P (BG562P) is shown in Hut78 cells. Figure 6B Comparison of binding affinity between humanized scFv BG562P (BG562P) and humanized scFv BG563P (BG563P) is shown in Hut78 cells. Figure 6C Comparison of binding affinity between humanized scFv BG563P (BG563P) and humanized scFv BG564P (BG564P) is shown in Hut78 cells.

[0156] Figure 7A schematic of CLDN6xCD3 BsAb BG143P is shown.

[0157] Figure 8A and Figure 8B Target binding activity of CLDN6xCD3 BsAb BG143P is shown. Figure 8A CD3 binding activity of BG143P in CD3 expressing Jurkat cells is shown. Figure 8B CLDN6 binding activity of BG143P in CLDN6 expressing PA-1 cells is shown.

[0158] Figure 9A , Figure 9B and Figure 9C Targeting functional activity of CLDN6xCD3 BsAb BG143P in tumor cell lines with different CLDN6 expression is shown. Figure 9A Redirected T cell cytotoxicity of BG143P in CLDN6 expressing PA-1 cells, Hutu80 cells, AGS cells and NCI-H1299 cells by cell lysis assay is shown. Figure 9B IFN-γ induction activity of BG143P in CLDN6 expressing PA-1 cells, Hutu80 cells, AGS cells and NCI-H1299 cells is shown. Figure 9C IL-2 induction activity of BG143P in CLDN6 expressing PA-1 cells, Hutu80 cells, AGS cells and NCI-H1299 cells is shown.

[0159] Figure 10A , Figure 10B and Figure 10C Functional specificity of CLDN6xCD3 BsAb BG143P against human CLDN6 and CLDN9 is shown. Figure 10A Binding specificity of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells is shown. Figure 10B Killing specificity (cell lysis activity) of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells is shown. Figure 10C Cytokine (IFN-γ) induction of BG143P against human CLDN6 (left panel) and CLDN9 (right panel) in NCI-H1299 cells is shown.

[0160] Figure 11A and Figure 11B In vivo efficacy of CLDN6xCD3 BsAb BG143P in OV-90 xenograft model in PBMC humanized mice is shown.Figure 11A Tumor volume over time is shown. Mice were untreated (no PBMC), treated with PBS (PBS i.p QW), treated with 0.01 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), treated with 0.03 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), or treated with 0.1 mg / kg BG143P (BG143P - 0.1 mg / kg, i.p). Treatments were given once per week and are indicated on the X-axis with triangles. Figure 11B The percentage of hCD45+ cells in the peripheral blood of the following mice is shown: untreated (no PBMC), treated with PBS (PBS i.p QW), treated with 0.01 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), treated with 0.03 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), or treated with 0.1 mg / kg BG143P (BG143P - 0.1 mg / kg, i.p), at day 13, day 21, and day 27 post-PBMC injection, indicating human PBMC reconstitution.

[0161] Figure 12A and Figure 12B In vivo efficacy of CLDN6xCD3 BsAb BG143P in the aB16F10 / human CLDN6 syngeneic model in hCD3EDG transgenic mice is shown. Figure 12A Tumor volume over time is shown. Mice were treated with PBS (PBS i.p QW), treated with 0.01 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), treated with 0.03 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), or treated with 0.1 mg / kg BG143P (BG143P - 0.1 mg / kg, i.p). Treatments were given once per week and are indicated on the X-axis with triangles. Figure 12B Body weight of the following mice is shown: treated with PBS (PBS i.p QW), treated with 0.01 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), treated with 0.03 mg / kg BG143P (BG143P - 0.01 mg / kg, i.p), or treated with 0.1 mg / kg BG143P (BG143P - 0.1 mg / kg, i.p), over the course of days 11-27 post-inoculation, as an indicator of mouse tolerance to the antibody.

[0162] Definitions Unless otherwise defined herein, all other technical and scientific terms used have the meaning commonly understood by one of ordinary skill in the art.

[0163] As used herein, including the appended claims, the singular forms “a,” “an,” and “the” include their corresponding plural references unless the context clearly dictates otherwise.

[0164] As used herein, unless the context clearly dictates otherwise, the term “or” is used to mean “and / or” and is equivalent to the term “and / or” as used in the art. As also used herein, the term “and / or” means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when the alternative option (“or”) is interpreted.

[0165] As used herein, “about” when used in reference to a numerical value means the numerical value recited and the numerical value plus or minus 10%. For example, “about 10” is to be understood as “10” and “9-11”.

[0166] As used herein, the phrase in the alternative “A / B” or “A and / or B” means (A), (B), or (A and B); the phrase 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).

[0167] The term “anti-cancer agent” as used herein refers to any agent useful in the treatment of a cell proliferative disorder such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0168] The term “claudin 6” or “CLDN6” refers to a member of the CLDN family. CLDN6 has a molecular weight of 23 kDa. CLDN6 has four transmembrane domains and a PDZ binding region at the carboxy terminus in the cytoplasm. The amino acid sequence of human CLDN6 can be found at UniPort ID P56747. An exemplary human CLDN6 sequence is SEQ ID NO: 87.

[0169] The term “claudin 9” or “CLDN9” refers to another member of the CLDN family. CLDN9 has a molecular weight of 23 kDa and its amino acid sequence can be found at UniPort ID O95484. An exemplary human CLDN9 sequence is SEQ ID NO: 88.

[0170] The term "cluster of differentiation 3" or "CD3," as used herein, 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, CD3 epsilon, CD3 gamma, CD3 alpha, and CD3 beta chains. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3 epsilon or CD3 gamma), as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, a human CD3 epsilon protein of 207 amino acids in length (NCBI RefSeq No. NP_000724), and a human CD3 gamma protein of 182 amino acids in length (NCBI RefSeq No. NP_000064).

[0171] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, the terms “administration,” “administering,” “treating,” and “treatment” as used herein mean contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. Cellular treatment encompasses contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. The terms “administration” and “treatment” also mean, for example, in vitro and in vitro treatment of cells by means of a reagent, diagnostic agent, conjugated compound, or by means of another cell. The term “subject” as used herein includes any organism. Non-limiting examples include animals. In any embodiment, an 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, a mammal is a human. In any embodiment, a subject is a patient who has or is at risk of having the condition described herein. In any implementation, treating any disease or condition means improving the disease or condition (i.e., slowing down, stopping, or reducing the development of the disease or at least one of its clinical symptoms). In another aspect, “treat,” “treating,” or “treatment” means reducing or improving at least one bodily parameter, including those that the patient may not be able to discern. In yet another aspect, “treat,” “treating,” or “treatment” means regulating a disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of bodily parameters), or both. In yet another aspect, “treat,” “treating,” or “treatment” means preventing or delaying the onset, development, or progression of a disease or condition. In one aspect, the terms “prevent,” “preventing,” or “prevention,” as used herein with respect to cancer, mean eliminating or reducing the risk of developing cancer. Prevention can also refer to preventing recurrence or secondary cancer after the initial cancer has been treated or cured.

[0172] 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 a specific implementation, the subject, individual, or patient is a human.

[0173] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within an antigen, the variable region of an antibody interacts with the antigen at many sites through noncovalent forces. Generally, the more interactions, the stronger the affinity.

[0174] The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that can noncovalently, reversibly, and in a specific manner bind to a corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer that includes at least two heavy (H) chains and two light (L) chains, which are inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL or VK) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four framework regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin 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.

[0175] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. An antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2).

[0176] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the rest of the heavy and / or light chain is derived from a different source or species.

[0177] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain Fc regions.

[0178] In some embodiments, an anti-CLDN6 antibody comprises at least one antigen binding site, at least one variable region. In some embodiments, an anti-CLDN6 antibody comprises an antigen binding fragment from a CLDN6 antibody described herein. In some embodiments, an anti-CLDN6 antibody is isolated or recombinant.

[0179] In some embodiments, the anti-CD3 antibody comprises at least one antigen binding site, at least one variable region. In some embodiments, the anti-CD3 antibody comprises an antigen binding fragment from a CD3 antibody described herein. In some embodiments, the anti-CD3 antibody is isolated or recombinant.

[0180] The term "monoclonal antibody" or "mAb" or "Mab" means here a population of essentially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that can be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their complementarity determining regions (CDRs), which generally are specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of essentially homogeneous antibodies, and it is not to be construed as requiring production of the antibody 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. Pat. 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 can be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclass thereof, such as IgGl, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained by in vivo production, in which cells from an individual hybridoma are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of isotype IgM or IgG can be purified from such ascites fluid or from culture supernatants using column chromatography methods well known to those skilled in the art.

[0181] Generally, the basic antibody structural unit includes a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (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 primarily responsible for antigen recognition. The carboxy-terminal portion of each heavy chain can define a constant region primarily responsible for effector functions. Generally, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as alpha, delta, epsilon, gamma, or mu, and define the class of an antibody as IgA, IgD, IgE, IgG, and IgM, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain further including a "D" region of about 10 or more amino acids.

[0182] The variable regions of each light / heavy (VL / VH) pair contribute to the formation of the antibody binding site. Thus, generally, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are generally identical in primary sequence.

[0183] Generally, the variable domains of both heavy and light chains comprise three hypervariable regions, also called "complementarity determining regions" or "CDRs", located between relatively conserved framework regions (FRs). The CDRs are typically aligned with the framework regions such that the combination of CDRs and framework regions can bind to a particular epitope. Generally, both the light chain variable domain and the heavy chain variable domain comprise, from N-terminus to C-terminus, 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 location of CDRs and framework regions can be determined using various well-known definitions 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 combining 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, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in Kabat; and the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in Kabat. The CDR amino acid residues in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3) in Chothia; and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in Chothia. By combining the CDR definitions of both 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. The CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3) and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) in IMGT (numbered according to Kabat). According to IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0184] The term "hypervariable region" means amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "CDR" (e.g., LCDR1, LCDR2, and LCDR3 in a light chain variable domain and HCDR1, HCDR2, and HCDR3 in a 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 as defined herein.

[0185] Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., a fragment of an antibody that retains the ability to specifically bind to the 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, e.g., single-chain Fv (ScFv); nanobodies formed of antibody fragments, and multispecific antibodies.

[0186] 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, although such specificity need not be absolute. Antibody "specific binding" or "selective binding" is used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, refers to a binding reaction that is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological agents (e.g., in a biological sample, blood, serum, plasma, or tissue sample). Thus, under designated immunoassay conditions, the antibodies or antigen-binding fragments thereof specifically bind to a particular antigen at least two-fold over background levels and do not substantially bind in a specific manner to other antigens present in the sample. In one aspect, under designated immunoassay conditions, the antibodies or antigen-binding fragments thereof specifically bind to a particular antigen at least ten (10) fold over background levels of binding and do not substantially bind in a specific manner to other antigens present in the sample.

[0187] An "antigen binding domain" as used herein comprises at least three CDRs and specifically binds to an epitope. An "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 also comprises at least three CDRs and specifically binds to a second epitope. A multispecific antibody can be bispecific, trispecific, tetraspecific, etc., with an antigen binding domain for each particular epitope. A multispecific antibody can 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.

[0188] The term "human antibody" means an antibody comprising only human immunoglobulin sequences. A human antibody can contain murine carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or "rat antibody" means an antibody comprising only mouse or rat immunoglobulin sequences, respectively.

[0189] The term "humanized" or "humanized antibody" means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. 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. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. When it is desirable to distinguish humanized antibodies from the parent rodent antibody, the prefix "hum," "hu," "Hu," or "h" is added to the antibody clone designation. The humanized form of a rodent antibody generally will comprise the same CDR sequences as the parent rodent antibody, but can include certain amino acid substitutions to increase affinity, increase stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0190] 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 by, e.g., crystallography. Hydroxyl radical protein footprinting and alanine scanning mutagenesis methods, among others, can also be used, but can provide lower resolution.

[0191] The term“monospecific antibody” refers to an antibody that specifically binds to only one antigen. A monospecific antibody can bind to only one epitope of an antigen, or can 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.

[0192] The term“multispecific antibody” or“multi-specific antibody” refers to an antibody that specifically binds to two or more antigens (e.g., a bispecific antibody, a trispecific antibody, 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), where the VH / VL unit has multi-epitope specificity; antibodies having two or more VL and VH domains, where each VH / VL unit binds to a different epitope; antibodies having two or more single variable domains, where each single variable domain binds to a different epitope; diabodies, triabodies, etc.; and full-length antibodies and / or antibody fragments linked covalently or noncovalently.

[0193] The terms“multi-epitope antibody” and“antibody having multi-epitope specificity” are used interchangeably herein to refer to an antibody that binds to two or more epitopes on the same or different antigens.

[0194] The term“Fc region” is used herein to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy-terminus. The C-terminal lysine (residue 447 according to EU numbering system) of the Fc region can be removed, e.g., during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies with all Lys447 residues removed, a population of antibodies with Lys447 residues not removed, and a population of antibodies with a mixture of antibodies having and not having Lys447 residues.

[0195] A "functional Fc region" has an effector function of a native sequence Fc region. Exemplary effector functions include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of B cell receptor (BCR); and the like. Such effector functions generally require the Fc region to be combined 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 can have substantially similar, decreased, or enhanced effector function as compared to a wild-type IgG. For antibodies comprising a human Fc region, comparisons are generally made with wild-type human IgGl.

[0196] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non-A and A allotypes); native sequence human lgG2 Fc region; native sequence human lgG3 Fc region; and native sequence human lgG4 Fc region, as well as naturally occurring variants thereof.

[0197] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, for example, from about one to about ten amino acid modifications, and in some embodiments, from about one to about five amino acid modifications, preferably one or more amino acid substitutions. A variant Fc region herein will preferably possess at least about 80% identity with a native sequence Fc region, preferably at least about 90% identity therewith, or preferably at least about 95% identity therewith. In some embodiments, a variant Fc region can have decreased or enhanced effector function as compared to a wild-type IgG. For antibodies comprising a human Fc region, comparisons are generally made with wild-type human IgGl.

[0198] The term "Fc component" as used herein refers to the hinge region, CH2 domain, or CH3 domain of an Fc region.

[0199] The term "hinge region" is defined generally as extending from about residues 216-230 of IgG (Eu numbering), from about residues 226-243 of IgG (Kabat numbering), or from about residues 1-15 of IgG (IMGT unique numbering).

[0200] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the 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 fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), triabody, tetrabody, single-chain antibody, scFv, scFv dimer, single domain antibody, single-domain antibody, and multivalent domain antibody. Generally, the binding fragment competes with the intact antibody from which it was derived for specific binding. Binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0201] The term "Fab" refers to an antibody moiety consisting of a single light chain (both variable and constant regions) bound via disulfide bonds to a variable region and a first constant region of a single heavy chain.

[0202] The term "Fab'" refers to a Fab fragment that includes a portion of the hinge region.

[0203] The term "F(ab')2" refers to a dimer of Fab'. F(ab')2 antibody fragments are initially generated as a pair of Fab' fragments with a hinge cysteine between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0204] The term "Fv" refers to the minimum fragment of an antibody that carries the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0205] 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, but not all, cases, the linker can be a peptide. The length of the linker varies depending on the type of single-chain antibody. Covalent or non-covalent linking of two or more single-chain antibodies together produces higher order forms. Single-chain antibodies and higher order forms thereof can include, but are not limited to, single domain antibodies, multivalent domain antibodies, single-chain variable fragment (scFv), di-valent scFv (di-scFv), tri-valent scFv (tri-scFv), tetra-valent scFv (tetra-scFv), diabodies, and triabodies and tetrabodies.

[0206] The terms "single-chain Fv antibody" and "scFv" are used interchangeably herein to refer to a single-chain antibody consisting of a heavy variable region and a light variable region linked by a linker. In most cases (but not all), the linker may be a peptide. The linker peptide is preferably about 5 to 30 amino acids long, or about 10 to 25 amino acids long. Typically, the linker allows for the stabilization of the variable domain without interfering with the proper folding and creation of the active binding site. In a preferred embodiment, the linker peptide is rich in glycine as well as serine or threonine. Covalently or nonvalently linking two or more scFvs together produces higher-order forms such as di-scFv, tri-scFv, tetra-scFv, etc. The antigen-binding site of each scFv in the higher-order forms can target the same or different antigens or epitopes.

[0207] The term "single-chain Fv-Fc antibody" or "scFv-Fc" refers to a full-length antibody composed of scFv linked to the Fc region.

[0208] "Dib antibodies" are a more advanced variant of single-chain antibodies, consisting of two single-chain antibodies. For each single-chain antibody, the linker used is too short to allow pairing between two domains on the same chain, thus forcing said domain to pair with a complementary domain of the other chain, resulting in two antigen-binding sites. In most cases (but not all), the linker may be a peptide. The antigen-binding sites can target the same or different antigens or epitopes. Similarly, triantibodies (three single-chain antibodies assembling to form three antigen-binding sites), tetraantibodies (four single-chain antibodies assembling to form four antigen-binding sites), and more advanced variants can be generated. See, for example, Holliger P. et al. Proc Natl Acad Sci USA . Jul 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161.

[0209] "Single-domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more V... H Domains are covalently linked via peptide linkers to create multivalent domain antibodies. Multivalent domain antibodies contain two or more V... H Domains can target the same or different antigens or epitopes.

[0210] The term "heavy chain antibody" refers to an antibody composed of two heavy chains. Heavy chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNARs from cartilaginous fish. See, for example, Riechmann L. and Muyldermans S. J Immunol Methods.December 10; 231(1-2): 25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; or U.S. Patent No. 6,005,079. Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Despite the lack of light chains, camelized antibodies still have authentic antigen binding profiles (Hamers-Casterman C. et al, Nature. June 3; 363(6428):446-8 (1993); Nguyen V. K. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics April; 54(1):39-47 (2002); Nguyen V. K. et al., Immunology May; 109(1):93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen binding unit produced by the adaptive immune response (Koch-Nolte F. et al, FASEB J. November; 21(13):3490-8. Epub 2007 Jun 15 (2007).

[0211] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity to 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 can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. The corresponding human germline sequence can be a framework region only, a complementarity determining region only, a framework region and a complementarity determining region, a variable segment (as defined above), or other combination of sequences or subsequence comprising a variable region. Sequence identity can be determined using methods described herein, e.g., using BLAST, ALIGN, or another alignment algorithm known in the art to align two sequences. A 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 to a reference variable region nucleic acid or amino acid sequence. Additionally, if the antibody contains a constant region, the constant region is also derived from such human sequences, e.g., a human germline sequence or a mutated version of a human germline sequence or an antibody containing a consensus framework sequence derived from an analysis of human framework sequences, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0212] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time1) divided by the association rate constant (ka, time1M1). The equilibrium dissociation constant can be measured using any known method in the art. Antibodies of the present disclosure will generally have an equilibrium dissociation constant of less than about 10"7or 10"8M, e.g., less than about 10"9M or 10"10M, in some aspects, less than about 10"11M, 10"12M, or 10"13M.

[0213] The terms "cancer" and "tumor" have the broadest meaning as understood in the art and refer to a physiological condition in mammals generally characterized by unregulated cell growth. In the context of the present disclosure, cancer is not limited to a certain type or location.

[0214] In the context of the present disclosure, the term "conservative substitution" when referring to an amino acid sequence means the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to CLDN6 or to CD3. In particular, common conservative substitutions of amino acids are well known in the art The term "knob-into-hole" technology as used herein refers to amino acids that direct two polypeptides to pair together in vitro or in vivo by introducing a steric bulge (knob) into one polypeptide and a hole or cavity (hole) into the other polypeptide at the interface of the two polypeptide interactions. For example, knob-into-hole has been introduced in the Fc:Fc binding interface, the CL:CHI interface, or the VH / VL interface of an antibody (see, e.g., US 2011 / 0287009, US 2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knob-into-hole ensures the correct pairing of two different heavy chains during the manufacture of a multispecific antibody. For example, a multispecific antibody having knob-into-hole amino acids in the Fc region can also 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. Knob-into-hole technology can also be used in the VH or VL region, thereby also ensuring the correct pairing.

[0215] The term "knob" as used herein in the context of "knob-into-hole" technology refers to an amino acid change that introduces a bulge (knob) into a polypeptide at the interface of the polypeptide interaction with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.

[0216] The term "hole" as used herein in the context of "knob-into-hole" refers to an amino acid change that introduces a hole or cavity (hole) into a polypeptide at the interface of the polypeptide interaction with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.

[0217] An example of an algorithm that is 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 short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching 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 are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring 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 as defaults a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLAST program uses as defaults a word length of 3, and an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation value (E) of 10, M=5, N=-4, and a comparison of both strands.

[0218] The BLAST algorithm also performs a 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 by which an 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 the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0219] 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 a PAM120 weight 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 Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970) (an algorithm that has been incorporated into the GAP program in the GCG software package), using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6.

[0220] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modifications or that are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties to reference nucleic acids and are metabolized in a similar manner to reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, phosphoramidites, chiral phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0221] 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 the functional relationship of a transcriptional regulatory sequence to the sequence being transcribed. For example, a promoter or enhancer sequence is operably linked to a coding sequence if the promoter or enhancer sequence stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence that is operably linked to the sequence being transcribed is physically contiguous with the transcriptional sequence, i.e., they are in cis-acting relationship. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequence whose transcription they enhance.

[0222] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-CLDN6 x CD3 multispecific antibody as described herein formulated together with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The excipients can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

[0223] The compositions disclosed herein can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable 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.

[0224] The term "therapeutically effective amount" as used herein refers to the amount of an antibody that, when administered to a subject for treating a disease, or at least one clinical symptom of a disease or condition, is sufficient to effect such treatment for the disease, condition, or symptom. The "therapeutically effective amount" can vary with the antibody, the disease, condition, and / or symptoms of the disease or condition, the severity of the disease, condition, and / or symptoms of the disease or condition, the age of the subject to be treated, and / or the body weight of the subject to be treated. In any given case, the appropriate amount can be apparent to those skilled in the art or can be determined using routine experimentation. In the case of combination therapy, "therapeutically effective amount" refers to the total amount of the combination that is effective for the treatment of the disease, condition, or disorder.

[0225] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule, powder, and liquid. Powders and / or liquids can be reconstituted or diluted to the desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide combined levels of the drugs in the body that will have a therapeutic effect.

[0226] As used herein, the phrase "in combination with" means that the anti-CLDN6xCD3 multispecific antibody is administered to a subject at the same time as, immediately before, or immediately after administration of the additional therapeutic agent. In certain embodiments, the anti-CLDN6xCD3 multispecific antibody is administered as a co-formulation with the additional therapeutic agent. DETAILED DESCRIPTION

[0227] The present disclosure provides antibodies, antigen-binding fragments, and anti-CLDN6xCD3 multispecific antibodies. In addition, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable attributes, and thus 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 making and using such pharmaceutical compositions for the prevention and treatment of cancer and related disorders.

[0228] Anti-CLDN6 antibodies The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CLDN6. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof produced as described below.

[0229] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence set forth in Table 1. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein the antibody or antigen-binding fragment comprises a HCDR having an amino acid sequence of any one of the HCDRs set forth in Table 1. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein the antibody comprises one, two, three, or more HCDRs (or alternatively, consists of) having an amino acid sequence of any one of the HCDRs set forth in Table 1.

[0230] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein the antibody or antigen-binding fragment comprises a VL domain having an amino acid sequence set forth in Table 1. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CLDN6, wherein the antibody or antigen-binding fragment comprises a LCDR having an amino acid sequence of any one of the LCDRs set forth in Table 1. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CLDN6, comprising one, two, three or more LCDRs (or alternatively consisting of) having an amino acid sequence of any one of the LCDRs set forth in Table 1.

[0231] Other antibodies or antigen-binding fragments thereof of the present disclosure include those in which amino acids or nucleic acids encoding amino acids have been altered, but which are at least 60%, 70%, 80%, 90%, 95%, or 99% identical 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, this includes changes in the amino acid sequence wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR regions have been altered when compared to the CDR regions depicted in the sequences described in Table 1.

[0232] Other antibodies of the present disclosure include those in which amino acids or nucleic acids encoding amino acids have been altered; but which are at least 60%, 70%, 80%, 90%, 95%, or 99% identical 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, this includes changes in the amino acid sequence wherein no more than 1, 2, 3, 4, or 5 amino acids in the variable regions have been altered when compared to the variable regions depicted in the sequences described in Table 1, while retaining substantially the same therapeutic activity.

[0233] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full length heavy chain, and full length light chain of the antibodies that specifically bind to CLDN6. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0234] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CLDN6. In certain aspects, the antibodies and antigen-binding fragments can bind to the same epitope of CLDN6.

[0235] 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. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in a binding assay. Demonstration of a test antibody’s ability 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 one theory, such an antibody can bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on CLDN6 as the antibody or antigen-binding fragment thereof with which it competes. In certain aspects, an antibody that binds to the same epitope on CLDN6 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0236] Anti-CD3 antibodies The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof produced as described below.

[0237] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD3, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence set forth in Table 2. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CD3, wherein the antibody or antigen-binding fragment comprises a HCDR having an amino acid sequence of any one of the HCDRs set forth in Table 2. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD3, wherein the antibody comprises one, two, three, or more HCDRs (or, alternatively, consists of one, two, three, or more HCDRs) having an amino acid sequence of any one of the HCDRs set forth in Table 2.

[0238] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acids that have been altered, but are at least 60%, 70%, 80%, 90%, 95%, or 99% identical to the CDR regions disclosed in Table 2, in the CDR regions. 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, it includes amino acid changes, wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR regions have been altered when compared to the CDR regions depicted in the sequences described in Table 2.

[0239] Other antibodies of the present disclosure include those in which an amino acid or nucleic acid encoding an amino acid has been altered; but which have at least 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequences described in Table 2. 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, it includes variations in the amino acid sequence in which no more than 1, 2, 3, 4, or 5 amino acids in the variable region have been altered when compared to the variable region depicted in the sequences described in Table 2, while retaining substantially the same therapeutic activity.

[0240] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full length heavy chain, and full length light chain of the antibodies that specifically bind to CD3. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0241] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CD3. In certain aspects, the antibodies and antigen-binding fragments can bind to the same epitope of CD3.

[0242] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CD3 antibodies described in Table 2. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in a binding assay. The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CD3 demonstrates that the test antibody can compete with the antibody or antigen-binding fragment thereof for binding to CD3. Without being bound by any one theory, such an antibody can bind to the same or related (e.g., structurally similar or spatially proximal) epitope on CD3 as the antibody or antigen-binding fragment thereof with which it competes. In certain aspects, the antibody that binds to the same epitope on CD3 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0243] Anti-CLDN6xCD3 multispecific antibodies In one embodiment, an anti-CLDN6 and anti-CD3 antibody as disclosed herein can be incorporated into an anti-CLDN6xCD3 multispecific antibody. The antibody molecule is a multispecific antibody molecule, e.g., comprising multiple antigen binding domains, wherein at least one antigen binding domain sequence specifically binds CLDN6 as a first epitope, and a second antigen binding domain specifically binds CD3 as 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 instance, the multispecific antibody comprises at least one anti-CLDN6 antigen binding domain and at least one anti-CD3 antigen binding domain.

[0244] In one embodiment, the multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. The bispecific antibody comprises a first antigen binding domain that specifically binds CLDN6 and a second antigen binding domain that specifically binds CD3. This includes bispecific antibodies comprising a heavy chain variable domain and a light chain variable domain that specifically bind CLDN6 as a first epitope and a heavy chain variable domain that specifically binds CD3 as a second epitope. In another embodiment, the bispecific antibody comprises an antigen binding fragment of an antibody that specifically binds CLDN6 and an antigen binding fragment that specifically binds CD3. The bispecific antibody comprises an antigen binding fragment that can be a Fab, F(ab')2, Fv, or single chain Fv (ScFv) or scFv.

[0245] Previous experiments (Coloma and Morrison Nature Biotech. 15: 159-163 (1997)) describe tetravalent bispecific antibodies engineered by fusing DNA encoding single chain anti-dansyl antibody Fv (scFv) either at the C-terminus (CH3-scFv) or at the hinge of IgG3 anti-dansyl antibody (hinge-scFv). 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 antibody polypeptide chains. The multivalent antibodies herein comprise three to eight, but preferably four, antigen binding domains that specifically bind at least two antigens.

[0246] Linker It is also to be 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 by linker regions, i.e. CL, CH1, hinge, CH2, CH3 or the entire Fc region. For example, VL1-CL-(linker) VH2-CH1, VH-linker-VL. Such linker regions can comprise randomly assorted amino acids or a restricted set of amino acids. Such linker regions can be flexible or rigid (see US 2009 / 0155275).

[0247] Multispecific antibodies have been constructed by genetically fusing two single chain Fv (scFv) or Fab fragments, with or without flexible linkers (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 devices such as leucine zippers (Kostelny et al., J. Immunol. 1992 148:1547-53; de Kruif et al J. Biol. Chem. 1996 271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 422:259-64); by diabodies (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 fusions (Schoonjans et al., J. Immunol. 2000 165:7050-7); and minibody formats (Packet et al., Biochemistry 1992.31:1579-84; Packet et al., Bio / Technology 1993 11:1271-7).

[0248] The bispecific tetravalent antibody as disclosed herein comprises a linker region of 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 its antigen binding domains, CL domains, CH1 domains, hinge regions, CH2 domains, CH3 domains, or Fc regions. In some embodiments, the amino acids glycine and serine make up the amino acids within the linker region. In another embodiment, the linker can be GS, GGS, GSG, SGG, GGG, GGGS, SGGG, GGGGS, GGGGSGS, 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 WO 2007 / 024715).

[0249] 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 "knob-in-hole" interaction and increases assembly of the correct multivalent antibody. The dimerization-specific amino acids can be within 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 CL domains with other CL domains (CL-CL) and can be found at least in the disclosure of WO2014082179, WO2015181805 series and WO2017059551. Dimerization-specific amino acids can also be within the Fc domain and can be combined with dimerization-specific amino acids within the CH1 or CL domains. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one pair of dimerization-specific amino acids.

[0250] Further alterations of the framework of the Fc region In other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand, but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. Such an approach is described in, for example, both U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0251] In another aspect, one or more amino acid residues are replaced with one or more different amino acid residues such that the antibody has altered Clq binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). Such an approach is described in, for example, U.S. Patent No. 6,194,551 to Idusogie et al.

[0252] In yet another aspect, one or more amino acid residues are altered to change the ability of the antibody to fix complement. Such an approach is described in, for example, publication WO 94 / 29351 to Bodmer et al. In a specific aspect, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgGl subclass and kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl, IgG2, and IgG3 subclasses and the constant region of the light chain of the kappa isotype as described in Jefferis et al., MAbs. 1 :332-338 (2009).

[0253] In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of an antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the affinity of the antibody for an Fc gamma receptor. Such an approach is described in, e.g., the publication WO 00 / 42072 by Presta. In addition, the binding sites on human IgGl for FcyRI, FcyRII, FcyRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0254] In yet another aspect, the glycosylation of the multispecific antibody is modified. For example, an aglycosylated antibody can be made (i.e., an antibody that lacks glycosylation or has reduced glycosylation). Glycosylation can be altered, e.g., to increase the affinity of the antibody for "antigen." Such carbohydrate modifications can be accomplished by, e.g., altering one or more of the glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0255] Additionally or alternatively, antibodies with altered types of glycosylation can be produced, such as aglycosylated antibodies with reduced amounts of fucose residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by expressing the antibody in a host cell with altered glycosylation pathway. Cells with altered glycosylation pathway have been described in the art and can be used as host cells in which to express recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, Hang et al. EP 1,176,195 describes cell lines with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such cell lines exhibit reduced fucosylation. Presta's publication WO 03 / 035835 describes a variant CHO cell line, Lecl3 cells, which have a reduced ability to attach fucose to the 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. WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0256] In another aspect, if reduced ADCC is desired, a number of previous reports show that the human antibody subclass IgG4 has only modest ADCC and little CDC effector function (Moore G L et al., 2010 MAbs, 2: 181-189). However, native IgG4 was found to be less stable under stress conditions, such as in acidic buffer or at increased 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). Reduced ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with altered combinations that reduce FcyR binding or Clq binding activity, thereby reducing or eliminating ADCC and CDC effector function. One of the less desirable intrinsic properties of IgG4, given the physicochemical properties of antibodies as biopharmaceuticals, 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). Mutation of the serine at position 228 (EU numbering system) to proline appears to be inhibitory for IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al., 2002 Immunol, 105:9-19). Some amino acid residues in the hinge and gamma Fc regions have been reported to have an impact on antibody interaction with Fcy receptors (Chappel S M et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9: 115-119; Armour, K. L. et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, R. A. et al., 2000 Nature Medicine, 6:443-446; Arnold J. N., 2007 Annu Rev immunol, 25:21-50).In addition, some rare IgG4 isotypes in the human population can cause 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 with good stability, the hinge region and Fc region of human IgG4 can be modified and a number of changes can be introduced. These modified IgG4 Fc molecules can be found in U.S. Patent No. 8,735,553 to Li et al., which is incorporated by reference herein.

[0257] Antibody production Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, e.g., mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0258] The present disclosure also provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding a heavy chain or light chain variable region or segment comprising a complementarity determining region as described herein. In some aspects, a polynucleotide encoding a heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to 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, a polynucleotide encoding a light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 10, 57, 61, or 65.

[0259] The polynucleotides of the present disclosure can encode variable region sequences of anti-CLDN6 x CD3 antibodies. They can also encode both variable and constant regions of the antibodies. Some polynucleotide sequences encode polypeptides comprising the variable regions of both the heavy and light chains of the exemplified anti-CLDN6 x CD3 antibodies.

[0260] The present disclosure also provides expression vectors and host cells for producing anti- CLDN6xCD3 antibodies. The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Generally, the expression vector will contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-CLDN6xCD3 antibody chains or antigen-binding fragments. In some aspects, an inducible promoter is employed to prevent expression of the inserted sequences except under conditions of induction. Inducible promoters include, for example, the arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be expanded under non-inducing conditions without deviating from the population of coding sequences whose expression products are better tolerated by the host cell. In addition to the promoter, other control elements can also be required or desired for efficient expression of the anti-CLDN6xCD3 antibody or antigen-binding fragment. These elements generally include an ATG start codon and adjacent ribosome binding site or other sequences. Additionally, expression efficiency can be enhanced by inclusion of enhancers appropriate for the cellular system used (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 CMV enhancer can be used to increase expression in mammalian host cells.

[0261] Host cells used to carry and express the anti-CLDN6 x CD3 antibody chains can be prokaryotic or eukaryotic. Escherichia coli (E. coli) is one prokaryotic host that can be used to clone and express the polynucleotides of the present disclosure. Other microbial hosts that are suitable for use 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 made that typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of various well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from bacteriophage lambda. The promoters generally optionally control expression with operator sequences and have ribosome binding site sequences, etc., for the initiation and termination of transcription and translation. Other microorganisms, such as yeast, can also be used as hosts. Insect cells in combination with baculovirus vectors can also be used. In other aspects, mammalian host cells are used to express and produce the anti-CLDN6 x CD3 antibodies of the present disclosure. For example, they can be hybridoma cell lines that express endogenous immunoglobulin genes or mammalian cell lines that carry exogenous expression vectors. These cells include any normal mortal or normal or abnormal immortal animal or human cell. For example, several suitable host cell lines have been developed that are capable of secreting intact immunoglobulins, including CHO cell lines, various COS cell lines, HEK 293 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, N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors will typically contain a promoter derived from a mammalian gene or a mammalian virus. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or inducible or controllable.Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoter, dexamethasone inducible MMTV promoter, SV40 promoter, MRP pol III promoter, constitutive MPSV promoter, tetracycline inducible CMV promoter (such as the human immediate early CMV promoter), constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0262] Generation of bispecific antibodies The current standard for engineered heterodimeric antibody Fc domains is the knob-in-hole (KiH) design, which introduces mutations at the core CH3 domain interface. The resulting heterodimer has a reduced CH3 melting temperature (69°C or less). In contrast, the ZW heterodimeric Fc design has a thermal stability of 81.5°C, which is comparable to the wild-type CH3 domain.

[0263] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure 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. The term "detecting" as used herein includes quantitative or qualitative detection. In certain aspects, the biological sample comprises a cell or tissue. In other aspects, such tissue includes normal and / or cancerous tissue that expresses CLDN6 at higher levels relative to other tissues.

[0264] In one aspect, the present disclosure provides a method of detecting the presence of CLDN6 in a biological sample. In certain aspects, the method comprises contacting a biological sample with an anti-CLDN6xCD3 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 can include, but is not limited to, a urine, tissue, saliva, or blood sample.

[0265] Also included is a method of diagnosing a disorder associated with CLDN6 expression. In certain aspects, the method comprises contacting a test cell with an anti-CLDN6xCD3 antibody; determining the expression level (quantitative or qualitative) of CLDN6 expressed by the test cell by detecting the binding of the anti-CLDN6xCD3 antibody to the CLDN6 polypeptide; and comparing the expression level of the test cell to the expression level of CLDN6 in a control cell (e.g., a normal cell or a non-CLDN6 expressing cell having the same tissue origin as the test cell), wherein a higher expression level of CLDN6 in the test cell compared to the control cell is indicative of the presence of a disorder associated with CLDN6 expression.

[0266] Therapeutic methods The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods for treating a CLDN6-associated disorder or disease. In one aspect, the CLDN6-associated disorder or disease is cancer.

[0267] In one aspect, the present disclosure provides a method of treating cancer. In certain aspects, the method comprises administering to a patient in need thereof an effective amount of an anti-CLDN6 x CD3 antibody or antigen-binding fragment. In some embodiments, the cancer is a solid tumor. The cancer can include, but is not limited to, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, renal 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, rhabdoid tumor, and / or glioma.

[0268] The antibodies or antigen-binding fragments as disclosed herein can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if local treatment is desired, intralesionally. 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, depending partly on whether the administration is short term or long term. Contemplated herein are various dosing schedules including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0269] The antibodies or antigen-binding fragments of the present disclosure can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular 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 scheduling of administration, and other factors known to medical practitioners. The antibody need not, but optionally, be formulated with one or more agents currently used in preventing or treating 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 other factors discussed above. These are generally used in the same dosages and with administration routes and in the same manner as the antibodies described herein, or about from 1 to 99% of the dosages described herein.

[0270] For the prevention or treatment of disease, the appropriate dosage of an antibody or antigen binding fragment of the disclosure 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 for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the treating physician. The antibody is suitably administered 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 antibody can be an initial candidate dosage, whether by injection or continuous infusion. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations up to about 25% of the dose can be acceptable. Generally, the treatment is continued until the disease symptoms are brought back under control. Such doses can be given intermittently, e.g. every week or every three weeks (e.g. so that the patient receives about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by a conventional techniques and assays.

[0271] Combination therapy In one aspect, the anti-CLDN6xCD3 antibodies of the present disclosure can be used in combination with other therapeutic agents.Other therapeutic agents that can be used with the anti-CLDN6xCD3 antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents; (e.g., Abraxane®), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacitidine, ifosfamide, oxaliplatin, pemetrexed 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), multi-kinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeting agents (e.g., rituximab, ofatumumab, RO 5072759, LFB-R603), CD52 targeting agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, 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).

[0272] The anti-CLDN6xCD3 antibodies of the present disclosure can be used in combination with other therapeutic agents, such as immune checkpoint antibodies. Such immune checkpoint antibodies can include anti-PD1 antibodies. Anti-PD1 antibodies can 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 lgG4-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 indications for 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 lgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in US 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.

[0273] Other immune checkpoint antibodies in combination with anti-CLDN6xCD3 antibodies can include anti-TIGIT antibodies. Such anti-TIGIT antibodies can include, but are not limited to, anti-TIGIT antibodies as disclosed in WO2019 / 129261.

[0274] Other immune checkpoint antibodies in combination with anti-CLDN6xCD3 antibodies can include anti-OX40 antibodies. Such anti-OX40 antibodies can include, but are not limited to, anti-OX40 antibodies as disclosed in WO2019 / 223733.

[0275] Other immune checkpoint antibodies in combination with anti-CLDN6xCD3 antibodies can include anti-TIM3 antibodies. Such anti-TIM3 antibodies can include, but are not limited to, anti-TIM3 antibodies as disclosed in WO2018 / 036561.

[0276] Pharmaceutical compositions and formulations Also provided are compositions, including pharmaceutical formulations, comprising an anti-CLDN6 x CD3 antibody or antigen-binding fragment thereof or a polynucleotide comprising a sequence encoding an anti-CLDN6 x CD3 antibody or antigen-binding fragment. In certain embodiments, a composition comprises one or more anti-CLDN6 x CD3 antibodies or antigen-binding fragments, or one or more polynucleotides comprising a sequence encoding one or more anti-CLDN6 x CD3 antibodies or antigen-binding fragments. These compositions can also comprise a suitable carrier, such as a pharmaceutically acceptable excipient, including buffers well known in the art.

[0277] Pharmaceutical formulations of the anti-CLDN6 x CD3 antibodies or antigen binding fragments as described herein are prepared by mixing such antibody or antigen binding fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, in either lyophilized or aqueous solution form, (Remington's Pharmaceutical Sciences 16thEdition, Osol, A. Ed. (1980)), in solid, semisolid, or liquid state, as appropriate. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl 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, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include an interstitial drug dispersion agent such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in US Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0278] In one embodiment, the formulation comprises L-histidine / L-histidine hydrochloride monohydrate, trehalose, and polysorbate 20. In another embodiment, the anti-CLDN6xCD3 antibody drug product, after reconstitution with sterile water for injection, is an isotonic solution comprising 10 mg / mL anti-CLDN6xCD3 antibody, 20 mM histidine / histidine HCl, 240 mM trehalose dihydrate, and 0.02% polysorbate 20, with a pH of approximately 5.5.

[0279] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter of which comprises histidine-acetate buffer.

[0280] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a membrane or microcapsule.

[0281] Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane.

[0282] sequence list The sequence listings of this disclosure are provided in Tables 1 through 3 below.

[0283] Table 1. Sequence List (Kabat Numbers)

[0284] Table 2. Sequence List (Kabat Numbers)

[0285] Table 3. Sequences (Kabat numbering)

[0286] Examples Example 1. Generation of mouse anti-CLDN6 antibodies To generate antibodies against CLDN6, groups of 20-25 inbred mice of BALB / C, SJL strain were immunized with human CLDN6 overexpressing cells (L929 / human CLDN6, in-house preparation), each group subjected to a unique combination of CLDN6 antigen, dose, route of injection, adjuvant and time of immunization. A total of 5 animals in 4-5 groups were immunized. Animals received immunizations at different time periods between day 0 and day 56. To monitor the immune response, titers of sera were screened by FACS after 21-56 days from 2-4 immunizations. Antibodies in the screened sera that bound to CLDN6 overexpressing cells CHOK1 / human CLDN6 were selected. CLDN6 specific antibody responses in each animal were measured and animals with sufficient anti-CLDN6 Ig titers were selected for a 4-day final boost.

[0287] Lymphoid organs, including spleen and lymph nodes, were isolated from mice immunized as described above. Hybridomas were generated by PEG-based fusion with immortalized mouse myeloma cells derived from SP2 / 0. Resulting cells were plated in 96-well cell culture plates in regular 1640 medium supplemented with HAT for selection of hybridomas.

[0288] Example 2. Screening and selection of anti-CLDN6 antibodies Hybridomas were generated as described in Example 1. After 10-13 days of culture and media replacement, hybridoma culture supernatants were collected from each well 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 (in-house prepared). Antibody binding on overexpressing cell lines was measured by FACS. CLDN6 antibody screening was performed on supernatants from over approximately 20,000 culture wells from 4 hybridoma fusions. Briefly, 100 μL of hybridoma culture supernatant was co-incubated with a CLDN6-expressing cancer cell line (such as PA-1 or CHOK1 / human CLDN6 stable cell line) or a control cell (such as parental CHOK1) for 30-60 minutes, washed, and incubated with an anti-mouse IgG Fc secondary antibody conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0289] Hybridomas from positive wells were transferred to 24-well plates with fresh media and grown for 2-3 days, then re-screened by flow cytometry to confirm antibody binding to cynomolgus CLDN6 overexpressing cell lines and human CLDN6 positive cancer cell lines (PA-1). Antibody binding to cynomolgus CLDN6 overexpressing cell lines and human CLDN6 positive cancer cell lines (PA-1) was measured by flow cytometry. Briefly, 100 μL of hybridoma culture supernatant was co-incubated with a CLDN-expressing cancer cell line (such as PA-1 or CHOK1 / human CLDN6 and CHOK1 / human CLDN9 stable cell lines) or a control cell (such as parental CHOK1) for 30-60 minutes, washed, and incubated with an anti-mouse IgG Fc secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0290] Example 3. Subcloning of selected CLDN6 Ab secreting hybridomas Selected anti-CLDN6 Ab secreting hybridomas were subcloned once or twice to ensure monoclonality. Briefly, approximately 80-100 live hybridoma cells were plated in 3 mL of semi-solid methylcellulose media (Stem Cell Technologies) in a 6-well plate. After 7-10 days, hybridoma colonies that arose from single cells as visible colonies were picked into 96-well plates and further cultured in fresh media for 2-4 days. Culture supernatants were screened by ELISA and flow cytometry as previously described to confirm human and cynomolgus CLDN6 binding. In vitro culture stable hybridoma subclones were generated for cell cryopreservation, antibody production, and antibody VH and VL gene cloning and sequencing.

[0291] Example 4. Determination of CLDN6 binding EC50values for mouse anti-CLDN6 antibodies Selected anti-CLND6 antibodies were subcloned and the subcloned selected anti-CLDN6 antibody secreting hybridomas were plated in T75 flasks containing 40 ml of fresh 1640 media supplemented with 2% FBS to produce antibodies. After 7-10 days of culture, the hybridoma supernatants were harvested for antibody purification using a protein A column. The mouse anti-CLDN6 antibodies were then characterized for their binding activity to CLDN6 positive cells using flow cytometry. The EC50values for clone BG87P are presented in Tables 4-6. The data indicate that clone BG87P binds to human CLDN6, but not to human CLDN9. In addition, BG87P binds to mouse CLDN6 and cynomolgus CLDN6.

[0292] Table 4. Binding activity of BG87P to CHOK1 stable cells expressing human CLDN6 and human CLDN9

[0293] Table 5. Cross-species binding activity of BG87P to mouse CLDN6 and cynomolgus CLDN6

[0294] Table 6. Binding activity of BG87P to cancer cell line PA-1 with endogenous CLDN6 expression

[0295] Example 5. Antibody VH and VL gene cloning and sequencing After removal of the supernatant, the subcloned selected CLDN6 antibody secreting hybridomas were lysed in a 96-well round bottom plate by addition of 100 mL RLT buffer. 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 was prepared using a total RNA isolation kit according to the manufacturer’s instructions. cDNA was generated by reverse transcription of mRNA using a Super Script III First-Strand Synthesis SuperMix (Invitrogen®) according to the manufacturer’s instructions. The nucleic acid and amino acid sequences for BG87P are shown in Table 1 (SEQ ID NOs: 1-10).

[0296] Generation of chimeric BG87P antibody (chBG87P) The ChBG87P antibody was generated by subcloning the variable regions of mouse BG87P (SEQ ID NOS: 7 and 8) into an in-house developed expression vector containing the constant regions of human wild-type IgG1 and kappa chains. The antibody was expressed by co-transfecting the two constructs described above into HEK293T cells and purified using a protein A column (cat. 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 at -80 °C freezer.

[0297] Example 6. Humanization of anti-CLDN6 chimeric BG87P antibody (chBG87P) Humanization approach For humanization of chBG87P, sequences in human germline IgG genes that share high homology to the protein sequences of chBG87P variable regions were searched by sequence comparison against the human immunoglobulin gene database in IMGT. Human IGHV and IGKV genes that exist at high frequency in human antibody repertoire and are highly homologous to chBG87P were selected as templates for humanization.

[0298] Design of humanized variants Humanization was performed by CDR grafting followed by incorporation of key back mutations. Humanized antibodies were engineered into human IgGl wild type format by using in-house developed expression vectors. In the initial first round of humanization, mutations from murine variable regions to human amino acid residues in the framework regions were guided by 3D structure analysis and murine framework residues that were structurally important for maintaining canonical structure of CDRs were preserved in the first round of humanization design. Five back mutations on the heavy chain and three mutations on the light chain were selected and single point mutations were made to explore key back 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: 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 the variant incorporating all the theoretical back mutations and the binding capacity of BG87P-Bz0 should be comparable to the parent chBG87P. Comparison of the binding data revealed which back mutations significantly impact binding. Specifically, the LCDRs (SEQ ID NO: 4-6) of chBG87P were grafted into the framework of human germline variable genes IGKV1-5 and 01-IGKJ4 with A43S, L78V, and Y87F murine framework residues (resulting in SEQ ID NO: 14). The HCDRs (SEQ ID NO: 1-3) of chBG87P were grafted into the framework of human germline variable genes IGHV1-3 and 01-JH6c with V2I, T28S, I69L, R71V, and Y91F murine 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 grafting but without any back mutations from the murine VH and VL framework. 01. Comparison of the binding data revealed which back mutations significantly impact binding. Specifically, the LCDRs (SEQ ID NO: 4-6) of chBG87P were grafted into the framework of human germline variable genes IGKV1-5 and 01-IGKJ4 with A43S, L78V, and Y87F murine framework residues (resulting in SEQ ID NO: 14). The HCDRs (SEQ ID NO: 1-3) of chBG87P were grafted into the framework of human germline variable genes IGHV1-3 and 01-JH6c with V2I, T28S, I69L, R71V, and Y91F murine 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 grafting but without any back mutations from the murine VH and VL framework.

[0299] 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 constant regions of human wild-type IgG1 and kappa chains, respectively, with easily adaptable subcloning sites. All humanized variants were expressed by co-transfecting the above two constructs into HEK293T cells and purified using a protein A column (cat# 17-5438-02, GE Life Sciences). Purified antibodies were concentrated to 0.5-10 mg / ml in PBS and stored at -80 °C freezer in aliquots.

[0300] Determination of cell binding activity of 1st round humanized BG87P variants (hBG87P) and PTM removal variants To determine affinities, HEK293T cells overexpressing CLDN6 and cancer cell line PA-1 expressing high levels of human CLDN6 were used to evaluate 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 secondary antibody to detect the binding of antibodies to cell surface. EC50 values for dose-dependent binding to CLDN6 expressing cell lines were determined by fitting the dose-response data to a four-parameter logistic model with GraphPad Prism. The cell binding activity of first round BG87P humanized variants against HEK293T / human CLDN6 was compared to chBG87P and shown in Figure 1A

[0301] ​With chBG87P antibody and BG87P-Bz0, several additional amino acid changes were made in the CDR regions of both VH and VL to further improve biophysical properties in human therapeutic use. Considerations included removal of post-translational modifications (PTM), increase in 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 chBG87P and BG87P-Bz0 related PTM removal variants were compared against chBG87P and BG87-Bz0, respectively, for cell binding activity against HEK293T / human CLDN6 Figure 1F ). Cell binding affinities (EC50) and Emax (MFI) were normalized to chBG87P for direct comparison and ranking (Table 7). The results demonstrated that, with the exception of H33A mutation resulting in BG87P-m4 and BG87P-m8, other substitutions of potentially deleterious residues all maintained the binding capacity of their respective parents.

[0302] Table 7. Summary of cell binding activity of humanized and PTM removal variants against CLDN6 overexpressing HEK293T

[0303] Table 8. Cell binding activity of 2nd round PTM removal humanized variants against cancer cell line PA-1

[0304] Determination of cell binding activity of 2nd round key back mutations in combination with PTM removal sites Following a comprehensive analysis of the EC50and Emaxof the 1stround humanization cell binding data (Table 7), four key back mutation sites were identified, VH: V2I; VH: T28S; VH: I69L; VH: Y91F, and combined with PTM removal sites for a 2ndround validation and to determine the final humanization candidates. PTM removal mutation VH: V65G, which has a higher G prevalence in the human germline (G 62%; V < 1%), indicating a potential benefit in antibody framework stability, was associated with variant BG87P-m3, which Figure 1F and showed improved Emaxand EC50compared to chBG87P in Table 7, and were incorporated into the 2ndround combination for further validation. The resulting VH and VL sequences of the 2ndround humanization variants BG87P-21, BG87P-22, BG87P-23, BG87P-24, BG87P-25, BG87P-26, and BG87P-27 are given in Table 1.

[0305] The humanization combination variants were identified for cell binding activity in HEK293T / human CLDN6 cells (as shown in Figure 1B and Figure 1C ) and cell binding activity in PA-1 cells (as shown in Figure 1D and Figure 1E ), BG87P-21 was selected as the top humanization candidate for further consideration (VH and VL amino acid sequences are SEQ ID NO: 24 and 12, respectively). BG87P-21 includes the key back mutation site VH: T28S and the PTM site VH: V65G, which revealed comparable cell binding affinities compared to chBG87P. Emaxwas reduced by 22% in HEK293T / human CLDN6 and by 40% in PA-1 (Table 7 and Table 8).

[0306] Developability assessment of humanized anti-CLDN6 antibodies Biophysical properties were profiled to identify the top humanized anti-CLDN6 antibodies. The data indicated that BG87P-21 showed moderate to high hydrophobic risk, followed by a risk of self-interaction in PBS buffer, which was demonstrated by the AC-SINS, B22KD, and CIC readouts. (Table 9 to Table 11).

[0307] Table 9. Biophysical property analysis of BG87P-21 and chBG87P

[0308] To assess hydrophobicity, 50 μg samples at 1 mg / ml were diluted with mobile phase A solution (1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0) to achieve a final ammonium sulfate concentration of approximately 1 M prior to analysis. A linear gradient was used with MABPac HIC-10 columns, mobile phase A and mobile phase B solution (50 mM sodium phosphate, pH 7.0) at a flow rate of 0.5 ml / min over 29 min. Peak retention times were monitored at A280 absorbance. As indicated in Table 9, both chBG87P and BG87P-21 showed higher hydrophobicity performance and exceeded the internal standard 21.1 min in IgG format.

[0309] For thermal stability evaluation, the thermal stability of BG87P-related engineered variants was described by the thermal unfolding transition midpoint Tm (°C) measured by exogenous fluorescence. Tm was determined using a QuantStudio™ 6 Flex system from Applied Biosystems. 20 μL of 1 mg / ml sample was mixed with 20 μL of 40X SYPRO Orange. The plate was scanned from 25 °C to 95 °C at a rate of 0.9 °C / min. The first derivative of the raw data from the QuantStudio™ 6 Flex system analysis software was used to assign Tm. Results are summarized in Table 9, indicating that both chBG87P and humanized variant BG87P-21 showed good thermal stability.

[0310] To determine the aggregation propensity of BG87P-related engineered variants, 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 loaded into a cuvette; the sample was held at 25 °C for 120 seconds, then ramped to 95 °C at a rate of 0.3 °C / min. Scattering data were collected at an angle of 90°, with a laser wavelength of 266 nm. Tagg (aggregation temperature) was analyzed and calculated by the Uncle analysis software. Results are summarized in Table 9. Both chBG87P and humanized variants showed acceptable Tagg.

[0311] CIC is a technique to discriminate antibody candidates with lower solubility or non-specific binding propensity. IgG from human serum or other ligands is chemically coupled to NHS activated chromatography resin. The retention time of proteins on this resin is tested using HPLC to evaluate the solubility of the proteins. After column coupling with IgG from human serum, antibody samples and sample buffer are diluted to 0.1 mg / mL with mobile phase (PBS). The diluted samples and buffer are transferred to HPLC vials for LC-MS analysis. The results summarized in Table 9 indicate that both chBG87P and BG87P-21 show acceptable non-specific interaction with human IgG.

[0312] General description and intended use of B22 and KD test method. The method is used to investigate weak protein-protein interactions to predict aggregation tendency, reveal the impact of formulation ingredients on intermolecular interactions and support formulation buffer selection. Antibodies are diluted to 1 mg / mL with buffer exchange samples and centrifuged at 14000 rpm for 30 min, then Tm, Tagg and DLS are checked. Samples are loaded to the Uncle at 9 pL / well. Duplicate experimental wells are set up for each sample. Equipment parameters are set and experiments are run according to Uncle guidelines. In this experiment we use B22 and 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 parameters, if proteins interact with an increase in concentration (mutual attraction), proteins behave as if they become larger and diffusion coefficient (KD) decreases (negative slope). For B22: Second virial coefficient, if proteins interact with an increase in concentration (mutual attraction), proteins behave as if they are larger and 1 / R90 decreases (negative slope). Data indicate that both chBG87P and humanized variants attract each other in PBS, tending to aggregate under this condition (Table 10).

[0313] The AC-SINS is an assay that obtains the self-interaction of the sample to predict the aggregation potential. The assay is based on concentrating antibodies from a dilute solution around gold nanoparticles pre-coated with a polyclonal capture agent. Interactions between the immobilized antibodies result in a decrease in interparticle distance and an increase in plasmon wavelength (wavelength of maximum absorbance), which can be easily measured optically. Antibodies were diluted to 0.05 mg / mL each by providing the buffer. After gold nanoparticles were prepared, the gold nanoparticle solution was mixed with the coating solution using a volume ratio of 9: 1. After incubation at room temperature for 1 hour, the voids in the AuNPs were blocked using thiolated PEG (final concentration 0.1 uM). Then incubate for another 1 hour at room temperature. The particle solution was then centrifuged at 15000 rpm for 6 minutes. Discard the supernatant. Resuspend the particles using 1 / 10 of the starting volume of storage buffer. Incubate 10 pL of concentrated coated particles with 100 pL of test antibody solution in a polypropylene plate for 2 hours at room temperature, then transfer 90 pL of the resulting solution to a polystyrene UV transparent plate. The data indicated that both chBG87P and BG87P-21 showed suboptimal self-interaction propensity (Table 9).

[0314] Table 10. Self-interaction risk determination of Round 2 humanized variants and chBG87P

[0315] Table 11. All four humanized antibodies show lower hydrophobicity than the chimeric antibody, however, the hydrophobic risk is also relatively high

[0316] The hydrophobic patch region results in a HIC retention time of chBG87P over 25 minutes and a HIC retention time of humanized BG87P-21 of 21.9 minutes, both of which are above the acceptable threshold of 21.1 minutes for IgG format. The root cause is the hydrophobic patch region in HCDR3, especially the I97-Y98-Y100-V100a portion at the edge of FR2 (framework region 2) and LCDR2 of the light chain and Y49-W50 (mainly W50) of the heavy chain Figure 2 A). The automatic antibody pipeline analysis by Schrodinger also shows a higher aggregation risk for BG87P (Table 12).

[0317] Table 12. Automatic antibody pipeline analysis for aggregation

[0318] Example 7. Solubility engineering of humanized anti-CLDN6 antibodies General strategy for soluble engineering of BG87P-21 In the above description, chBG87P has been engineered as a humanized antibody, and we have identified BG87P-21 as the final top clone. However, the potential developability risk driven by the hydrophobic patch of HCDR3 of chBG87P has not been addressed Figure 2 ). Considering that chBG87P shows promising binding activity and superior CLDN6 selectivity Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1D 、 Figure 1E 、 Figure 1F 、 Figure 1G and Figure 1H , BG87P-21 has been subjected to additional engineering to remove the hydrophobic patch, to optimize manufacturability and reduce potential ADA risk.

[0319] The solubility issue of BG87P-21 was engineered using two major strategies: single point mutation and framework exchange.

[0320] Table 13. Overall summary of solubility engineering of BG87P-21

[0321] A large number of single point mutations were designed based on two principles: one is to substitute the hydrophobic amino acid to a more hydrophilic one; and the other is to mutate the rare amino acid at the same Kabat position in the human antibody library to a more common one. Seven positions were found to be replaceable by other more hydrophilic amino acids with comparable binding affinity to the parent BG87P-21 and slightly improved hydrophilicity in the 1st round of screening using 57 variants and the 2nd round of screening using 104 variants (Table 14). The selected top mutations from the 1st and 2nd rounds of screening were combined to generate 56 variants for further validation. The combined variants BG87P-31 and BG87P-32 (VH and VL amino acid sequences are SEQ ID NO: 46 and 42, respectively) were selected as top candidates with comparable binding affinity to BG87P-21 and improved HIC retention, 17.4 min for BG87P-31 and 18.49 min for BG87P-32, both better than the parent BG87P-21 22.3 min (Table 14 and Figure 3 ).

[0322] Table 14. Characterization of chBG87P solubility engineered variants

[0323] However, even with the reduction in hydrophobicity during the soluble engineered single point mutation approach, the self-association risk determined by AC-SICNS still showed moderate to high. The reason for this unresolved issue is that the hydrophobic risk is mainly manifested in the amount of hydrophobic patch actually presented on the antibody surface, while the cause of inducing self-interaction also involves isoelectric point issues, uniform distribution of charge, and even some unknown specific interactions. (Doi.org / 10.1021 / mp200566k). Therefore, merely replacing hydrophobic residues with hydrophilic residues does not reduce the self-interaction risk triggered by the above reasons. In addition, we have found that the parental chBG87P has a higher HIC retention time of 25 min, while exhibiting a lower self-interaction tendency in PBS buffer with an AC-SINS value of about 12.85 nm (Table 14). Another finding is that the calculated net charge of chBG87P is much less than that of BG87P-21, 2.9 vs. 8.8. Therefore, we hypothesize that the framework or net charge can have an impact on the self-association effect.

[0324] We tested additional frameworks for IGHV3-23 and IGKV1-39, both the backmutations based on the new pairing frameworks and the selected top mutations from the soluble engineering of BG87P-21 were incorporated (Table 13). The final top candidate, BG87P-34, showed no dangerous signals in all the routine biophysical properties (Table 14; VH and VL amino acid sequences are SEQ ID NOs: 43 and 44, respectively).

[0325] In addition, after the framework exchange to IGHV3-23 and IGKV1-39, the Emax that BG87P-21 lost in the original humanization program has been recovered ( Figure 1G ). The principle of identifying the key backmutations used in both humanization programs is the same, thus ruling out the possibility of missing any backmutations in the previous round of humanization due to Emax responsibility. One explanation for this recovery of cell binding Emax by framework exchange is that the VH-VL angle can be different for different pairing frameworks, and a specific VH-VL angle can help maintain the Emax of cell binding. The final lead clone, BG87P-34, showed good cross-reactivity in different species ( Figure 1I and Figure 1J ). By using HEK293T / human CLDN9 for non-specific binding to human CLDN9, the data indicated that BG87P-34 has good selectivity for human CLDN6 over CLDN9 ( Figure 1H ).

[0326] 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) became the best clone for the development of anti-CD3 based therapeutics due to its cross-reactivity with cynomolgus CD3. For the humanization of sp34, sequences sharing high homology with the protein sequences of the variable regions of sp34 (SEQ ID NO: 48 - 57) were searched in human germline IgG genes by querying human immunoglobulin gene databases in the IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) websites. Human IGVH and IGVK genes that are present at high frequency in the human antibody repertoire (Glanville 2009 PNAS 106:20216-20221) and share homology with sp34 were selected as templates for humanization.

[0327] Humanization was performed by CDR grafting (Methods in Molecular Biology, Volume 248: Antibody Engineering, Methods and Protocols, Humana Press) and the humanized antibody (hu-sp34) was engineered into human IgGl format using in-house developed expression vectors. In the initial round of humanization, mutations from murine to human amino acid residues in the framework regions were guided by modeling the 3D structure and murine framework residues that are structurally important for maintaining the canonical structure of the CDRs were retained in the 1stversion of the humanized antibody sp34. Specifically, the CDRs of sp34 VL (SEQ ID NO: 51 - 53) were grafted into the framework of the human germline variable gene IGVK3-15 and several murine 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 NO: 48 - 50) were grafted into the framework of the human germline variable gene IGVH3-7 and several murine framework residues (D73, S76, M89, V93) were retained.

[0328] Humanized sp34 (hu-sp34) and chimeric sp34 (ch-sp34) were constructed as human full length antibody formats using in-house developed expression vectors containing constant regions of human IgGl and kappa chain, respectively, and with easily adaptable subcloning sites. Expression and production of humanized sp34 and chimeric sp34 antibodies were achieved by co-transfecting heavy and corresponding light chain constructs into 293G cells (in-house developed) and purification using protein A columns. Purified antibodies were concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80 °C freezer for use in the assays below.

[0329] To determine affinity, antibodies were captured with anti-human Fc surface and used in an affinity assay based on surface plasmon resonance (SPR) technology. HuT78 cells were used in a FACS-based assay to evaluate the binding activity of humanized sp34 to native CD3 on live cells. Live HuT78 cells were seeded in 96-well plates and incubated with a range of dilutions of chimeric or humanized sp34. Mouse anti-human IgG was used as 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 logistic model with 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 Figure 4A ).

[0330] Table 15. Comparison of hu-sp34 and ch-sp34 binding affinity to CD3 by SPR and FACS

[0331] Based on the humanized sp34 BG53P template, we made several single mutations to convert the remaining murine residues in the framework regions to the corresponding human germline residues, including the four remaining murine residues in VH (D73, S76, M89, V93) and the fifteen remaining murine residues in VL (Q1, A2, V4, V36, E38, L43, F44, T45, G46, G49, L66, D69, A71, I85, and F87). All humanization mutations were made using primers containing the mutation at the specific position and a site-directed mutagenesis 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 previously described. Mutations of V36Y, G46L, and G49Y (Kabat numbering) on VK significantly impaired the binding affinity of the humanized variant compared to hu-sp34-1A-1f, while the rest 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).

[0332] In summary, the excellent engineered version of the humanized monoclonal antibody, BG56P (SEQ ID NO: 70-77 and 72-86), was derived from the mutation process as described above, and was characterized in detail (Table 16 and Figure 4B ).

[0333] Table 16. Comparison of the binding affinity of humanized sp34 to CD3

[0334] Example 9. ScFv engineering of humanized sp34 To generate a ready-to-use bispecific format and avoid light chain-heavy chain mispairing, we re-arranged the BG56P antibody into a single chain fragment variable (scFv) format with a 3xG4S linker between VH and VK. Using an in-house developed expression vector with easily adaptable subcloning sites, the re-arranged scFv was fused to the N-terminus of the human IgGl Fc region into a scFv-Fc format. Expression and production of the parental and re-engineered hu-sp34 scFv-Fc was achieved by transfection of the scFv-Fc construct into 293G cells (in-house developed) and purification using a protein A column. The purified scFv-Fc format of the antibodies was concentrated to 0.5-5 mg / mL in PBS and aliquoted and stored at -80°C freezer for use in the assays below. The scFv-ized BG56P (referred to as BG561P, SEQ ID NO: 48 - 53 and 62 - 65) showed comparable binding affinities to the antibody format of BG56P in SPR and FACS (Table 17 and Figure 5 ).

[0335] Table 17. Comparison of affinity of humanized sp34 and scFv humanized sp34 to CD3 by SPR and FACS

[0336] Based on BG561P, we made several mutations in the framework and CDRs to remove potential PTM sites and improve thermal and colloidal stability for therapeutic use in humans. The L4V mutation in the VL (resulting humanized scFv designated BG562P, SEQ ID NO: 48 - 53 and 69 - 70) showed an increase in aggregation temperature (Tagg) of 5 degrees. The combination of L4V in the VL and A49G and D65G in the VH (resulting humanized scFv designated BG563P) (SEQ ID NO: 48, 71, 50, 51-53, 73, and 74) showed improved thermal and colloidal stability compared to BG561P, while showing a slight improvement in binding affinity to human CD3 in the FACS assay. Potential PTM sites include potential deamidation sites N30 (NT) (Kabat CDR definition) in the FR1 and HCDR1 junction and N100 (NS) in the HCDR3. Each N was mutated to S to remove the potential deamidation sites. All mutations were performed using primers containing the mutations at the specified positions and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). In summary, the top engineered version of the humanized scFv, BG564P (SEQ ID NO: 48, 71, 75, 51-53, 77, and 78) was derived from the mutagenesis process as described above and was characterized in detail. The results showed that humanized scFv BG564P retained binding affinity to CD3 (Table 18 - Table 20 and Figure 6) and improved biophysical stability (Table 20) compared to humanized scFv BG561P.

[0337] Table 18. Comparison of binding affinity of different versions of humanized sp34 scFv-Fc to CD3 by SPR

[0338] Table 19. Comparison of binding affinity of humanized sp34 scFv-Fc to CD3 by FACS

[0339] Table 20. Comparison of thermal and colloidal stability of humanized sp34 scFv-Fc

[0340] Thermal melting temperatures (Tm) were determined using high-throughput MicroCal™ VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermal profiles were acquired for each protein (350 μL, 0.5 mg / mL) from 20 °C to 100 °C using a scan rate of 90 °C / hour. Thermal profiles of buffer alone were subtracted from each protein sample. The resulting results show values for the midpoint of the transition temperature (Tm) and the enthalpy of the reaction (ΔH) for the samples, which indicates that the Tm of BG564P is increased compared to BG561P (Table 20).

[0341] Aggregation temperature Tagg (°C) represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation using SLS266 with UNCLE™ (Unchained lab, Pleasanton, CA). The sample was loaded into the UNCLE and the temperature was ramped from 15 °C to 95 °C. Backscattered optics cannot detect near-UV light scattering of protein aggregates, so only the non-scattered light can reach the detector. Therefore, a decrease in backscattered light is a direct measure of aggregation in the sample, which indicates that the Tagg of BG564P is increased compared to BG561P (Table 20).

[0342] Example 10. Generation of CLDN6 x CD3 BsAb BG143P Agonistic anti-CD3 antibodies have demonstrated toxicity in the clinical setting, which can indicate that systemic FcyR cross-linking is not ideal for CD3 activation. The goal is to achieve potent CD3 stimulation specifically at the tumor site for a broad range of cancers without systemic CD3 activation. To overcome the dependency on FcyR cross-linking, we generated a CLDN6 x CD3 BsAb BG143P with the following features as shown in Table 1. Figure 7 This specific construct, BG143P, includes a multispecific antibody format of an IgG-like fusion (module ratio of 1:1), a superbly 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, as well as an Fc-less version of huIgG1 that does not bind to FcyR but retains FcRn binding. A knob-in-hole (KIH) was also introduced into the Fc to increase heterodimerization. Sequence information for BG143P is listed in SEQ ID NOs: 79-84.

[0343] Example 11. Target binding activity of CLDN6 x CD3 BsAb BG143P Binding kinetics of CLDN6xCD3 BsAb BG143P were measured using SPR. The on-rate constant (k a ) and off-rate constant (k d ) of the antibody to CDεγ recombinant protein were measured using SPR, followed by determination of the affinity constant (K D ). The results showed that the CLDN6xCD3 BsAb has strong binding affinity to human CDεγ, as shown in Table 21.

[0344] Table 21. Amino acid and DNA sequences of CLDN6xCD3 BsAb BG143P

[0345] 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- response 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- response manner, with an EC 50 of 81.26 nM Figure 8B .

[0346] Example 12. In vitro functional activity of CLDN6xCD3 antibody Targeting T cell redirected cytotoxicity and cytokine 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 negative for CLDN6 expression) was evaluated using human PBMCs as effector cells. To measure cytotoxicity, the target cancer cell lines were engineered to express NanoLuc luciferase. About 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 cytokine detection. Target cell killing was measured by Nano-Glo detection kit (Promega). The cytotoxic activity (%) of the antibodies was calculated using the following formula. Cytotoxic activity (%) = (A-B) / (A-C) 100%. "A" represents the average luminescence signal of the well containing only untreated target cells, "B" represents the average luminescence signal of the well containing antibody and PBMC, and "C" represents the average luminescence signal of the well containing target cells completely lysed with Triton-X100. IFN-g and IL-2 in the supernatant were detected by HTRF kit (Cisbio).

[0347] As shown in Figure 9, BG143P showed strong T cell redirected killing and cytokine release induction potency in a dose dependent manner at pM EC50 level.

[0348] Functional specificity against human CLDN6 and CLDN9 The amino acid sequence between human CLDN6 and CLDN9 is highly conserved with only 3 amino acid differences in the extracellular domain. CLDN9 is widely expressed in human normal tissues, so the binding specificity between CLDN6 and CLDN9 is important and checked by FACS analysis.

[0349] Expression vectors of human CLDN6 and CLDN9 were established by inserting synthetic cDNA encoding the corresponding sequence into a mammalian expression vector. NCI-H1299 stable cells expressing human CLDN6 and CLDN9 were produced by transfecting the corresponding plasmid. Cells were suspended in FACS buffer (2% FBS, 1x PBS) at a concentration of 1x10 6 Cells were suspended in FACS buffer (2% FBS, 1x PBS) at a concentration of 1x10

[0350] Human PBMCs were used to evaluate BG143P killing of NCI-H1299-CLDN6 / CLDN9 by Nano-Glo assay. About 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. Supernatants were collected for cytokine detection. Target cell killing was measured by Nano-Glo detection kit (Promega). Cytotoxic activity (%) of antibodies was calculated using the following formula. Cytotoxic activity (%) = (A-B) / (A-C) 100%. “A” represents the average luminescence signal of the wells containing only untreated target cells, “B” represents the average luminescence signal of the wells containing antibodies and PBMCs, and “C” represents the average luminescence signal of the wells containing target cells completely lysed with Triton-X100. IFN-γ was detected by HTRF kit (Cisbio).

[0351] As shown in Figure 10, BG143P is an antibody with specific binding ( Figure 10A ), cell killing (e.g., lysis) Figure 10B ), and IFN-γ induction activity Figure 10B ) against human CLDN6 but not human CLDN9.

[0352] Example 13. In vivo efficacy of CLDN6xCD3 BsAb BG143P in OV90 xenograft model In vivo anti-tumor efficacy of CLDN6xCD3 BsAb BG143P was evaluated in a xenograft model of PBMC humanized mice. Human ovarian cancer cell line OV-90 (ATCC) expressing human CLDN6 was subcutaneously inoculated into NCG (NOD / ShiLtJ Gpt-Prkdc em26Cd52 Il2rg em26Cd22 / Gpt) mice, and the next day, human PBMCs were intravenously injected into the mice. When the tumor volume reached about 200 mm 3 , the tumor-bearing mice were randomly divided into treatment groups to receive administration of antibodies or vehicle (PBS) control. Antibodies / vehicle were administered once a week. The length (L) and width (W) of the tumor mass and the body weight of each mouse were measured three times a week. And the tumor volume (TV) was calculated as follows: TV = (L x W 2 ) / 2. Figure 11A In vivo anti-tumor efficacy of BG143P was shown, BG143P showed strong efficacy at 0.03 mg / kg and 0.1 mg / kg with TGI% (tumor growth inhibition ratio, %) of 115.43% and 125.92%, respectively.

[0353] hPBMC reconstitution in vivo was examined at weeks 2, 3, and 4 after injection of PBMCs. hCD45+ cells in viable cells in peripheral blood were 20% at week 2 and increased to 60% at week 4. Figure 11B hPBMC reconstitution was demonstrated.

[0354] Example 14. In vivo efficacy of CLDN6xCD3 BsAb BG143P in B16F10- / hCLDN6 syngeneic model Another type of efficacy model was used to evaluate the in vivo efficacy of CLDN6xCD3 BsAb BG143P. A human CLDN6 expression plasmid was constructed and stably transfected in B16F10 cell line, and the resulting B16F10 / human CLDN6 cell line was confirmed to be able to grow in human CD3EDG transgenic mice, retaining 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 with the human counterpart. When the tumor volume reached about 100 mm3, the mice were randomly divided into groups. The test article or PBS was intraperitoneally injected into the mice every week. The length (L) and width (W) of the tumor mass and the body weight of each mouse were measured three times a week. And the tumor volume (TV) was calculated as follows: TV = (L x W2) / 2. BG143P at 0.1 mg / kg showed strong efficacy with TGI% of 93.54%, as shown in 3 Figure 12A No significant body weight loss was observed in the study, as illustrated in Figure 12B

[0355] Literature ​​

Claims

1. A multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human Claudin 6 (CLDN6) and a second antigen-binding domain that specifically binds to human Cluster of Differentiation 3 (CD3).

2. The multispecific antibody or antigen-binding fragment of claim 1, wherein the first antigen-binding domain does not bind to other Claudin (CLDN) family members.

3. The multispecific antibody or antigen-binding fragment of claim 2, wherein the first antigen-binding domain does not bind to human Claudin 9 (CLDN9).

4. The multispecific antibody or antigen-binding fragment of claim 3, wherein the first antigen-binding domain has high selectivity for human CLDN6 over human CLDN9.

5. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the first antigen-binding domain that specifically binds to human CLDN6 comprises: (a) a heavy chain variable region comprising: (i) a heavy chain complementarity 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, and (iii) a HCDR3 having an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising: (iv) a light chain complementarity determining region (LCDR)1 having the amino acid sequence of SEQ ID NO:40, (v) a LCDR2 having the amino acid sequence of SEQ ID NO:5, and (vi) a LCDR3 having the amino acid sequence of SEQ ID NO:6; (b) a heavy chain variable region comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) a HCDR2 having the amino acid sequence of SEQ ID NO:2, and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising: (iv) a LCDR1 having the amino acid sequence of SEQ ID NO:4, (v) a LCDR2 having the amino acid sequence of SEQ ID NO:5, and (vi) a LCDR3 having the amino acid sequence of SEQ ID NO:6; (c) a heavy chain variable region comprising: (i) a HCDR1 having the amino acid sequence of SEQ ID NO: 1, (ii) a HCDR2 having the amino acid sequence of SEQ ID NO:23, and (iii) a HCDR3 having the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising: (iv) a LCDR1 having the amino acid sequence of SEQ ID NO:4, (v) a LCDR2 having the amino acid sequence of SEQ ID NO:5, and (vi) a LCDR3 having the amino acid sequence of SEQ ID NO:6; or (d) 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: 45, and (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: 40, (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.

6. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the first 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; (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; (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:

42.

7. The multispecific antibody or antigen-binding fragment of claim 6, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids of SEQ ID NO: 7, 8, 12, 24, 41, 42, 43, 44, 46, or 47 have been inserted, deleted, or substituted.

8. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the first 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.

9. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the second antigen binding domain that specifically binds to human CD3 comprises: (a) a heavy chain variable region comprising (i) HCDR1 having an amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having an amino acid sequence of SEQ ID NO:71, (iii) HCDR3 having an amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising: (iv) LCDR1 having an amino acid sequence of SEQ ID NO:51, (v) LCDR2 having an amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO:53; (b) a heavy chain variable region comprising (i) HCDR1 having an amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having an amino acid sequence of SEQ ID NO:49, and (iii) HCDR3 having an amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising: (iv) LCDR1 having an amino acid sequence of SEQ ID NO:51, (v) LCDR2 having an amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO:53; or (c) a heavy chain variable region comprising (i) HCDR1 having an amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having an amino acid sequence of SEQ ID NO:71, (iii) HCDR3 having an amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising: (iv) LCDR1 having an amino acid sequence of SEQ ID NO:51, (v) LCDR2 having an amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO:

53.

10. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the second 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:76, 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:68; (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: 54, 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: 55; (v) 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: 58, 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: 59; (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: 62, 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: 63; (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: 62, 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: 68; 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: 72, 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:

68.

11. The multispecific antibody or antigen-binding fragment of claim 10, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids of SEQ ID NO: 54, 55, 58, 59, 62, 68, 72, or 76 have been inserted, deleted, or substituted.

12. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the second antigen binding domain comprises: (a) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 76 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 68; (b) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 54 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 55; (c) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 58 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 59; (d) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 62 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 63; (e) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 62 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 68; or (f) a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 72 and a light chain variable region having an amino acid sequence comprising SEQ ID NO:

68.

13. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the second antigen binding domain comprises: (a) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 77; (b) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 66; (c) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 69; or (d) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

73.

14. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:2, and (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:4, (v) LCDR2 having the amino acid sequence of SEQ ID NO:5, and (vii) LCDR3 having the amino acid sequence of SEQ ID NO:6; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:49, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:2, and (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:4, (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID N6O:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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: 2, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having an amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having an amino acid sequence of SEQ ID NO: 71, (iii) HCDR3 having an amino acid sequence of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 having an amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having an amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO:

53.

15. The multispecific antibody or antigen binding fragment of any one of the preceding claims, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:23, and (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:4, (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:49, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:23, and (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:4, (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having an amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having an amino acid sequence of SEQ ID NO: 71, and (iii) HCDR3 having an amino acid sequence of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 having an amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having an amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having an amino acid sequence of SEQ ID NO:

53.

16. The multispecific antibody or antigen binding fragment of any one of the preceding claims, wherein: (a) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:39, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:49, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:39, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (f) LCDR3 having the amino acid sequence of SEQ ID NO:53; or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:39, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:49, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:

53. (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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:39, and (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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, and (iii) HCDR3 having the amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:

53.

17. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises 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:39, (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 (f) LCDR3 having the amino acid sequence of SEQ ID NO:6; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:

53.

18. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein: (a) the first antigen-binding domain that specifically binds to human CLDN6 comprises 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 (ivd) 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; and the second antigen-binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 49, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 53; (b) the first antigen-binding domain that specifically binds to human CLDN6 comprises 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 (ivd) 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; and the second antigen-binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 49, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO: 53; (b) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO:50, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:53; or (c) the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO:48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO:71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO:51, (v) LCDR2 having the amino acid sequence of SEQ ID NO:52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:

53.

19. The multispecific antibody or antigen-binding fragment of any of the preceding claims, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises 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; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region comprising (i) HCDR1 having the amino acid sequence of SEQ ID NO: 48, (ii) HCDR2 having the amino acid sequence of SEQ ID NO: 71, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 75, and a light chain variable region comprising (iv) LCDR1 having the amino acid sequence of SEQ ID NO: 51, (v) LCDR2 having the amino acid sequence of SEQ ID NO: 52, and (vi) LCDR3 having the amino acid sequence of SEQ ID NO:

53.

20. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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: 2, (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: 4, (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; b) 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; 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: 39, (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: 40, (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; or d) the first antigen binding domain that specifically binds to human CLDN6 comprises: 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: 45, (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: 40, (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; and the second antigen binding domain that specifically binds to human CD3 comprises: a) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 54, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 55; b) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 58, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 59; c) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 63; d) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 63; e) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; or f) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68.

21. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: a) 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: 39, (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: 40, (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; or and the second antigen binding domain that specifically binds to human CD3 comprises: b) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 54, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 55; c) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 58, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 59; d) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 63; e) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; f) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; or g) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 76, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68.

22. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: (a) 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: 23, (iii) HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: (ivd) LCDR1 having the amino acid sequence of SEQ ID NO: 4, (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; or (b) 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: 39, (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; or (c) 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; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 66; (ii) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 69; (iii) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 73; or (iv) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

77.

23. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 54, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 58, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 76, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68.

24. The multispecific antibody or antigen binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 54, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 55; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 58, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 63; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 62, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 76, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68. (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68.

25. The multispecific antibody or antigen binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 24, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 41, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; (iii) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 44; (iv) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 47; or (v) a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 46, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO: 42; and the second antigen binding domain that specifically binds to human CD3 comprises: a heavy chain variable region (VH) that has an amino acid sequence comprising SEQ ID NO: 72, and a light chain variable region (VL) that has an amino acid sequence comprising SEQ ID NO:

68. The second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 76, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

68.

26. The multispecific antibody or antigen-binding fragment of claim 1, wherein: The first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 54, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 55; (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 62, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 63; (iv) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 62, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 72, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 68; or (vi) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 76, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

68.

27. The multispecific antibody or antigen-binding fragment of claim 1, wherein: The first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 43, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 72, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

68.

28. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:44; and the second antigen binding domain that specifically binds to human CD3 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:76, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:

68.

29. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises: (i) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:24, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 12; (ii) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:41, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:42; (iii) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:44; (iv) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:46, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:47; or (v) a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:46, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:42; and the second antigen binding domain that specifically binds to human CD3 comprises: (i) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:66; (ii) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:69; (iii) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:73; or (iv) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

77.

30. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO:43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO:44; and the second antigen binding domain that specifically binds to human CD3 comprises: (a) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 66; (b) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 69; (c) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO: 73; or (d) a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

77.

31. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

66.

32. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

69.

33. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

73.

34. The multispecific antibody or antigen-binding fragment of claim 1, wherein: the first antigen binding domain that specifically binds to human CLDN6 comprises a heavy chain variable region (VH) having an amino acid sequence comprising SEQ ID NO: 43, and a light chain variable region (VL) having an amino acid sequence comprising SEQ ID NO: 44; and the second antigen binding domain that specifically binds to human CD3 comprises a single chain variable fragment (scFv) having an amino acid sequence comprising SEQ ID NO:

77.

35. The multispecific 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.

36. The multispecific antibody or antigen-binding fragment of any of the preceding claims, wherein the multispecific antibody is a bispecific antibody.

37. The multispecific antibody or antigen-binding fragment of claim 36, wherein the multispecific antibody is BG143P.

38. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

39. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or low fucosylation.

40. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises increased bisecting GlcNac structures.

41. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the Fc domain is an IgGl with reduced effector function.

42. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the Fc domain is an IgG4.

43. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 42, further comprising a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43, further comprising histidine / histidine HC1, trehalose dihydrate, and / or polysorbate 20.

45. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of the multispecific antibody or antigen-binding fragment of claims 1 to 42.

46. The method of claim 45, wherein the cancer is a solid tumor.

47. The method of claim 45, wherein the cancer is selected from gastric cancer, colon 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 glioma.

48. The method of claim 45, wherein the multispecific antibody or antigen-binding fragment is administered in combination with one or more additional therapeutic agents.

49. The method of claim 48, wherein the one or more therapeutic agents are selected from paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacitidine.

50. The method of claim 49, wherein the one or more therapeutic agents are a paclitaxel agent, lenalidomide, or 5-azacitidine.

51. The method of claim 48, wherein at least one of the one or more therapeutic agents is an anti-PDl or anti-PDLl antibody.

52. The method of claim 51, wherein the anti-PDl antibody is tiragulimab.

53. An isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment of any one of claims 1 to 42.

54. A vector comprising the nucleic acid of claim 53.

55. A host cell comprising the nucleic acid of claim 53 or the vector of claim 54.

56. A method for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 55 and recovering the antibody or antigen-binding fragment from the culture.

57. The multispecific antibody or antigen-binding fragment of any one of claims 1 to 42, or the pharmaceutical composition of claim 43 or 44, for use in medicine or therapy.

58. The multispecific antibody or antigen-binding fragment of any one of claims 1 to 42, or the pharmaceutical composition of claim 43 or 44, for use in a method of treating cancer.

59. Use of the multispecific antibody or antigen-binding fragment of any one of claims 1 to 42 in the manufacture of a medicament for treating cancer.

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