CLDN6 / 9 antibodies and conjugates and uses thereof

Abstract

The present disclosure provides anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same and the uses thereof. The present disclosure also provides ADCs comprising the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising the ADCs and the uses thereof.

Description

Attorney Docket No.: 091787-8001W001CLDN6 / 9 ANTIBODIES AND CONJUGATES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 756,793, filed February 10, 2025, the disclosure of which is incorporated herein by reference.SEQUENCE LISTING

[0002] The sequence listing that is contained in the file named “091787-8001 WOOl.xml”, which is 115,291 bytes and was created on January 8, 2026, is filed herewith by electronic submission and is incorporated by reference herein.FIELD OF THE APPLICATION

[0003] This application pertains to antibodies that specifically Claudin-6 (CLDN6) and Claudin-9 (CLDN9), immunoconjugates and methods of manufacture and uses thereof, including methods of treating cancer.BACKGROUND OF THE APPLICATION

[0004] Claudin-6 (CLDN6) belongs to the claudin family of membrane proteins, which includes 27 distinct proteins, each characterized by four transmembrane domains and two extracellular loops. These claudins are primarily found at tight junctions between epithelial cells, where they play a vital role in maintaining barrier functions. Recently, CLDN6 has been linked to key intracellular signaling pathways, such as the YAP1–snail1 axis (Yu et al., 2019), the ASKl-p38 / JNK MAPK secretory signaling pathway (Lin et al, 2017), and it has been shown to activate ERa transcriptional activity in a ligand-independent manner, contributing to tumor progression in endometrial cancer (Kojima et al, 2021).

[0005] CLDN6 is notably expressed in undifferentiated stem cells, suggesting its role in the tumorigenic potential of cultures containing human pluripotent stem cells (Wang et al., 2012). There is significant sequence homology between CLDN6 and its family members; for instance, CLDN6 and CLDN9 differ by only three amino acids in their extracellular domains. CLDN3 and CLDN4 are also closely related and are highly expressed in various normal tissues, including the small intestine, colon, salivary glands, thyroid, kidneys, adrenal glands, and pancreas (Hewitt et al., 2006).

[0006] While CLDN6 is widely expressed during early embryonic and fetal development (Qu et al., 2021), its expression in adults is mainly confined to malignant tissues, where it has been associatedAttorney Docket No.: 091787-8001W001 with the initiation, progression, and metastasis of several cancers (Tsukita et al., 2019). Notably, it has been found to be aberrantly expressed in ovarian, lung, and endometrial cancers (Wang et al., 2013; Micke et al., 2014; Kojima et al., 2020). High levels of CLDN6 expression in malignant tissues of ovarian and endometrial cancer patients serve as an independent prognostic marker for poorer progression-free and overall survival (Kojima et al., 2020; Gao et al., 2021). In addition to CLDN6, CLDN9 is also highly expressed in certain cancers and is associated with cancer development, enhanced metastasis, and poor prognosis (Zhuang et al., 2023; Endo et al., 2022; Sharma et al., 2016). Given the pronounced expression profile and important roles of CLDN6 and CLDN9 in cancerous tissues, they represent promising targets for the development of antibodybased therapeutics.

[0007] Thus, there remains a need in the art for therapeutic antibodies and antibody-drug conjugates that demonstrate their potential as a treatment for patients whose tumors are CLDN6 and / or CLDN9-positive.

[0008] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.SUMMARY OF THE DISCLOSURE

[0009] Provided herein are antibody specifically binding to CLDN6 and CLDN9 (anti-CLDN6 / 9 antibody) or antigen-binding fragments thereof, and the conjugates comprising the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, and the uses of the same.

[0010] In one aspect, the present disclosure provides an anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, comprising a heavy chain complementary determining region 1 (HCDR1), a HCDR2 and a HCDR3 respectively having an at least 70% sequence identity of the HCDR1, HCDR2 and HCDR3 comprised in the heavy chain variable region as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21; and / or, a light chain complementary determining region 1 (LCDR1), a LCDR2 and a LCDR3 respectively having an at least 70% sequence identity of the LCDR1, LCDR2 and LCDR3 comprised in the light chain variable region as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

[0011] In a further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigenbinding fragments thereof described herein are determined according to the Kabat numberingAttorney Docket No.: 091787-8001W001 scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are determined according to the IMGT numbering scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are determined according to the IMGT numbering scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are determined according to the Chothia numbering scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are determined according to the AbM numbering scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are determined according to the Aho numbering scheme. In another further embodiment, the CDR sequences of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein are further according to the Contact numbering scheme.

[0012] In some embodiments, the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof described herein comprises clone-paired HCDR1-3 and LCDR1-3 as listed in Table 14.

[0013] In some embodiments, the HCDR1 comprises an amino acid sequence of SYGMS (SEQ ID NO: 27) or a homologous sequence having 1-3 amino acid residue mutations therein; the HCDR2 comprises an amino acid sequence of SINSNGGRTYYPDSEKG (SEQ ID NO: 28) or a homologous sequence having 1-3 amino acid residue mutations therein; and the HCDR3 comprises an amino acid sequence of WGGQYVMDY (SEQ ID NO: 29) or a homologous sequence having 1-3 amino acid residue mutations therein.

[0014] In some embodiments, the LCDR1 comprises an amino acid sequence of TADSSVTSSYLH (SEQ ID NO: 30) or a homologous sequence having 1-3 amino acid residue mutations therein; the LCDR2 comprises an amino acid sequence of ATSNLAS (SEQ ID NO: 31) or a homologous sequence having 1-3 amino acid residue mutations therein; and the LCDR3 comprises an amino acid sequence of HQSHRSPPT (SEQ ID NO: 32) or a homologous sequence having 1-3 amino acid residue mutations therein.

[0015] In another embodiment, the present disclosure provides an anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, which comprises a heavy chain and / or a light chain; wherein the heavy chain comprises a heavy chain variable region (VH) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQAttorney Docket No.: 091787-8001W001 ID NO: 21, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55; and, wherein the light chain comprises a light chain variable region (VL) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

[0016] In some further embodiments, the present disclosure provides a chimeric anti-CLDN6 / 9 antibody or antigen-binding fragments thereof disclosed herein. In some embodiments, the chimeric anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises human constant region. In some further embodiments, the human constant region is human IgGl constant region. In some embodiments, the chimeric anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises a heavy chain constant region having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 23 or SEQ ID NO: 25, and / or, a light chain constant region having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 24 or SEQ ID NO: 26.

[0017] In some further embodiments, the present disclosure provides a humanized anti-CLDN6 / 9 antibody or antigen-binding fragments thereof disclosed herein. In some embodiments the humanized anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises a heavy chain and / or a light chain; wherein the heavy chain comprises a heavy chain variable region (VH) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55; and, wherein the light chain comprises a light chain variable region (VL) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

[0018] In some embodiments, the present disclosure provides an anti-CLDN6 / 9 antibody or antigenbinding fragments thereof, comprising a heavy chain having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 57 or SEQ ID NO: 59; and / or, a light chain having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 58 or SEQ ID NO: 60.

[0019] In some embodiments, wherein the sequence identity further is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some furtherAttorney Docket No.: 091787-8001W001 embodiments, wherein the sequence identity further is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0020] In another aspect, the present disclosure provides a nucleic acid molecule encoding the antibody or antigen-binding fragments thereof disclosed herein. In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule disclosed herein. In one aspect, the present disclosure provides host cell comprising the vector disclosed herein.

[0021] In another aspect, the present disclosure provides anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, comprising a fusion moiety. In some further embodiments, the fusion moiety is selected from the group consisting of: an antibody heavy chain constant region, an anti-human serum albumin (aHSA), and other kind of moieties to improve functions of the antibody or antigenbinding fragment thereof. In some further embodiments, the heavy chain constant region is a fragment crystallizable (Fc) region derived from human Immunoglobulin (IgG) (e.g., IgGl, IgG2, IgG3 or IgG4), optionally, the heavy chain constant region is an IgG isotype to provide for altered (e.g., reduced) effector functions (e.g., ADCC or CDC), more optionally, the heavy chain constant region is an IgGl isotype which comprises one or more amino acid substitution(s) selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof.

[0022] In another aspect, the present disclosure provides anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, comprising a signal peptide, optionally the signal peptide is at the N-terminal of the heavy chain variable region.

[0023] In another aspect, the present disclosure provides anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, which is a monovalent antibody, a bivalent antibody, a monoclonal antibody, a bispecific antibody, a multi-specific antibody, a recombinant antibody, a labeled antibody, a fusion protein, or a single domain antibody.

[0024] In another aspect, the present disclosure provides anti-CLDN6 / 9 antibody or antigen-binding fragments thereof, comprising one or more amino acid residue substitutions or modifications yet retains binding specificity to CLDN6 / 9, optionally human CLDN6 / 9, cynomolgus CLDN6 / 9 or mouse CLDN6 / 9.

[0025] In some embodiments, the present disclosure provides anti-CLDN6 / 9 antibody or antigenbinding fragments thereof which binds to extracellular loop-1 (ECL-1) or ECL-2 of the extracellular domain (ECD) of CLDN6 / 9.Attorney Docket No.: 091787-8001W001

[0026] In some embodiments, the present disclosure provides anti-CLDN6 / 9 antibody or antigenbinding fragments thereof which is linked to one or more conjugate moieties. In some further embodiments, the conjugate moiety is selected from a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide, protein, therapeutic agent, or a diagnostic agent.

[0027] In another aspect, the present disclosure provides a conjugate comprising the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof disclosed herein.

[0028] In some embodiments, the present disclosure provides a conjugate of Formula (A):Ab-[Lk-T]r(A),or a pharmaceutically acceptable salt thereof,wherein,Ab is the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof;T is a therapeutic agent moiety;Lk is a linker; andr is about 1 to about 12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0029] In some embodiments, wherein Lk is represented by the following Formula (B):-Lkl-(Lk2)z-Lk3- (B),wherein,Lkl is a linking moiety to Ab formed by a reaction between a reactive group of Lk and a reactive residue of Ab;each Lk2 is independently a linking unit selected from the group consisting of -O-, -S-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-, -S(=O)-, -S(=O)2-, -NHC(=O)-, -C(=O)NH-, -SC(=O)-, -C(=O)S-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=NH)NH-, -NHS(=O)2-, -S(=O)2NH-, -C(=NH)-, -C(=N-NH2)-, -S(=NH)-, -S(=O)(=NH)-, -CH2-, -CH2CH2-, -CH=CH-, -C=C-, -CH2CH2O-, -OCH2CH2-, -CH2CH2OCH2-, -CH2OCH2CH2-, -S-S-, -NHCH2CH2O-, -OCH2CH2NH-, -CH2CH2NH-, -NHCH2CH2-, -CH2CH2S-, -SCH2CH2-, alanine residue, arginine residue, asparagine residue, aspartic acid residue, cysteine residue, glutamine residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, phenylalanine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, valine residue, selenocysteine residue, pyrrolysine residue, P-alanine residue, y-aminobutyric acid residue, 5-aminolevulinic residue, 4-aminobenzoic acid residue, cystine residue,Attorney Docket No.: 091787-8001W001 cystathionine residue, lanthionine residue, djenkolic acid residue, diaminopimelic acid residue, norvaline residue, norleucine residue, alloisoleucine residue, t-leucine residue, a-amino-n-heptanoic acid residue, pipecolic acid residue, a, P-diaminopr opionic acid residue, a,y-diaminobutyric acid residue, ornithine residue, allothreonine residue, homocysteine residue, homoserine residue, P-amino-n-butyric acid residue, P-aminoisobutyric acid residue, y-aminobutyric acid residue, a-aminoisobutyric acid residue, isovaline residue, sarcosine residue, N-ethylglycine residue, N-propylglycine residue, N-isopropylglycine residue, N-methylalanine residue, N-ethylalanine residue, N-methyl-P-alanine residue, N-ethyl-P-alanine residue, isoserine residue, a-hydroxy-y-aminobutyric acid residue, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each Lk2 is independently optionally substituted with one or more RLk;each RLkis independently selected from the group selected from halogen, oxo, cyano, nitro, -ORLkl, -OC(=O)RLkl, -OC(=O)ORLkl, -OC(=O)N(RLk2)2, -SRLkl, -S(=O)RLkl, -S(=O)2RLkl, -S(=O)2N(RLk2)2, -S(=O)(=NRLk2)RLkl, -N(RLk2)2, -NRLk2C(=O)N(RLk2)2, -NRLk2C(=O)RLkl, -NRLk2C(=O)ORLkl, -NR^S^O)^1, -N=S(=O)(RLkl)2, -C(=O)RLkl, -C(=O)ORLk2, -C(=O)N(RLk2)2, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, cycloalkyl or heterocyclyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl and heterocyclyl are optionally substituted with one or more RLk3;each RLklis independently hydrogen, alkyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl, each optionally substituted with one or more RLk3; each RLk2is independently hydrogen, alkyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -alkyl-cycloalkyl, or -alkyl-heterocyclyl, optionally substituted with one or more RLk3;or two RLk2on the same atom are taken together with the atom to which they are attached to form a heterocyclyl optionally substituted with one or more RLk3;each RLk3is independently halogen, cyano, hydroxy, oxo, -SF5, -SH, -S(=O)-alkyl, -S(=O)2-alkyl, -S(=O)2NH2, -S(=O)2NH-alkyl, -S(=O)2N(alkyl)2, -S(=O)(=N-alkyl)(alkyl), -NH2, -NH-alkyl, -N(alkyl)2, -N=S(=O)(alkyl)2, -C(=O)-alkyl, -C(=O)OH, -C(=O)O-alkyl, -C(=O)NH2, -C(=O)NH-alkyl, -C(=O)N(alkyl)2, -P(=O)(alkyl)2, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, aminoalkyl, heteroalkyl or cycloalkyl;z is any integer of 1-20; andAttorney Docket No.: 091787-8001W001 Lk3 is a linking moiety to T formed by a reaction between a reactive group of Lk and a reactive residue of T.

[0030] In some embodiments, wherein the reactive residue of Ab is a cysteine residue.

[0031] In some embodiments, wherein Lkl is covalently linked to the S atom of the cysteine residue.

[0032] In some embodiments, wherein -Lk-T is represented by the following Formula (A-I-l), (A-I- 2) or (A-I-3):M / ^O\ x^N-(CH2)n3(CH2O)n4(CH2)n5^<Un1 LTZn2 [I W-E-TO (A-I-l),o on1 i_i 'Zn2 [| u E~ TO (A-I-2),H O WNx(CH2)n3(CH2O)n4(CH2)n5^E-T (A-I-3),wherein,X’ is the linking moiety connecting to Ab;E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC);T is the therapeutic agent moiety;W is a polypeptide moiety;nl is any integer of 0-10;n2 is any integer of 0-10;n3 is any integer of 0-10;n4 is any integer of 0-10; andn5 is any integer of 0-10.

[0033] In some embodiments, wherein -Lk-T is selected from the group consisting of:-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NHCH2CH2-C(=O)-E-T; -X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NHCH2CH2CH2-C(=O)-E- T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NHCH2-O-CH2-C(=O)-E- T;Attorney Docket No.: 091787-8001W001 -X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2- C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T.

[0034] In some embodiments, wherein -Lk-T is selected from the group consisting of:-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;Attorney Docket No.: 091787-8001W001 -X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NHCH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NHCH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NHCH2-O- CH2-C(=O)-E-T; and-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T.

[0035] In some embodiments, wherein -Lk-T is represented by the following Formula (A-II):wherein,X’ is the linking moiety connecting to Ab;L1is an amino acid side chain residue;L2is a spacer moiety;Y is a hydrophilic end group;E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC);W is a polypeptide moiety;T is the therapeutic agent moiety;n is an integer from 1 to 20;m is an integer from 0 to 5;p is an integer from 1 to 24;r is an integer from 1 to 10;a is 0 or 1; andAttorney Docket No.: 091787-8001W001 b is 0 or 1.

[0036] In some embodiments, wherein Lk-T is represented by the following Formula (A-II-1), (A- II-2), or (A-II-3):

[0037] In some embodiments, X’ is maleimide, ring opening maleimide, NHS ester chemistry, or enzyme mediated conjugation.

[0038] In some embodiments, X’ is, or, wherein q is an integer from 1 to 10, and ** end indicates the attachment point of X’ to Ab.

[0039] In some embodiments, wherein W is selected from a peptide composed of 2-5 amino acids.

[0040] In some embodiments, wherein W is selected from Gly-Ala, Gly-Gly, Ala-Gly, Glu-Gly, Glu-Ala, Gly-Glu, Asp-Gly, Asp-Ala, Gly-Asp, Cit-Val, Ala-Val, Phe-Lys, Lys-Val, Val-Ala, Val-Cit, Phe-Gly, Ala-Ala, Val-Cys, Val-Gly, Val-Thr, Val-Val, Val-Leu, Val-Ile, Val-Asn or Val-Lys.Attorney Docket No.: 091787-8001W001

[0041] In some embodiments, wherein W is selected from Glu-Ala-Gly, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Glu-Ala, Asp-Ala-Gly, Asp-Gly-Gly, Gly-Asp-Gly, Gly-Asp-Ala, Gly-Gly-Ala, Gly-Gly-Arg, Gly-Ala-Gly, Gly-F-Gly, Ala-Ala-Gly, Ala-Ala-Ala, Ala-Ala-Asn, Val-Ala-Gly, Val-Cys-Gly, or Val-Lys-Gly.

[0042] In some embodiments, wherein W is selected from Gly-Gly-Phe-Gly, Gly-Gly-Ala-Gly, Gly-Gly-Gly-Gly, Gly-Glu-Gly-Gly, Gly-Glu-Ala-Gly, Gly-Asp-Gly-Gly, Gly-Asp-Ala-Gly, Ala- Ala-Ala-Gly or Glu-Ala-Gly-Gly.

[0043] In some embodiments, wherein W is Val-Ala, Val-Cit, or Gly-Gly-Phe-Gly.HN

[0044] In some embodiments, wherein L1is selected from, wherein the * end indicates the attachment point of L1to L2.

[0045] In some embodiments, wherein L2is selected fromor O wherein the * end indicates the attachment point of L2to L1.N

[0046] In some embodiments, wherein L1is, and L2isN HN

[0047] In some embodiments, wherein L1is, and L2is O

[0048] In some embodiments, wherein Y is selected from -COOH, -OH, -OCH3, or -CONH2.

[0049] In some embodiments, wherein n is an integer from 1 to 12.

[0050] In some embodiments, wherein p is an integer from 1 to 16.

[0051] In some embodiments, wherein m is 0, 1 or 2.Attorney Docket No.: 091787-8001W001

[0052] In some embodiments, wherein -Lk-T is selected from the group consisting of:Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001oAttorney Docket No.: 091787-8001W001 ooAttorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001HNoo

[0053] In some embodiments, wherein T is selected from a cytotoxic agent, a detection reagent, a diagnostic reagent, a nucleic acid, a radionuclide, a hormone, an immunomodulatory agent, an enzyme, an antibody, a fusion protein, a metal ion or a combination thereof.

[0054] In some embodiments, wherein the cytotoxic agent is an antitubulin agent or a topoisomerase inhibitor.

[0055] In some embodiments, wherein T is of Formula (T-1), Formula (T-2) or Formula (T-3):whereinR1is hydrogen, halogen, alkyl, haloalkyl, -OR1a, -SR1a, -S(O)R1a, or -S(O)2R1a;R2is hydrogen, halogen, alkyl or haloalkyl;each of R3, R6, R7and R10is hydrogen or alkyl;each of R4, R5and R8is independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, -alkyl-cycloalkyl, -alkyl-aryl, or -alkyl-heterocyclyl;Attorney Docket No.: 091787-8001W001 each R9is independently H, -OH, alkyl, alkoxyl, or cycloalkyl;R11is -[C(Rlla)2]2-cycloalkyl, -[C(Rlla)2]2-heterocyclyl, or -[C(Rlla)2]2-aryl;R12is heterocyclyl or aryl;Z is -O-, -S-, -NH-, or -N(alkyl)-;R13is hydrogen, alkyl, heterocyclyl, or aryl;R1ais hydrogen or alkyl; andeach Rllais independently selected from hydrogen, hydroxyl, alkyl, alkoxyl, or cycloalkyl.

[0056] In some embodiments, wherein T is selected from:

[0057] In some embodiments, wherein Lk-T is selected from the group consisting of:Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001 o

[0058] In some embodiments, selected from the group consisting ofoo0Attorney Docket No.: 091787-8001W001

[0059] In another aspect, the present disclosure provides a pharmaceutical composition, comprising: (i) the anti-CLDN6 / 9 antibody or an antigen-binding fragment thereof disclosed herein, or the nucleic acid molecule encoding the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof disclosed herein, or the conjugate disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0060] In another aspect, the present disclosure provides a method of expressing the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof disclosed herein, comprising culturing the host cell of the present disclosure under the condition at which the vector of the present disclosure is expressed.

[0061] In another aspect, the present disclosure provides a method of treating, preventing or alleviating a CLDN6 / 9-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof provided herein, or the nucleic acid molecule encoding the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof provided herein, the conjugate disclosed herein, and / or the pharmaceutical composition disclosed herein.

[0062] In some embodiments, the CLDN6 / 9-related disease or disorder is a tumor, wherein the cells of the tumor express claudin 6 (CLDN6) and / or claudin 9 (CLDN9).

[0063] In another aspect, the present disclosure provides a method of detecting the presence or amount of CLDN6 and / or CLDN9 in a sample, comprising contacting the sample with the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof disclosed herein, and determining the presence or the amount of CLDN6 and / or CLDN9 in the sample.DESCRIPTION OF DRAWINGS

[0064] Fig. 1A-1C. Binding and internalization of mouse hybridoma antibodies to CLDN6+ cell line. Fig. 1A, Flow binding (MFI values) of mouse hybridoma antibodies (1 ug / ml) to PA-1 cell line. Fig. 1B, Flow internalization (%) of mouse hybridoma antibodies (0.4 ug / ml) in PA-1 cell line.Fig. 1C, Internalization of mouse hybridoma antibodies (0.1 ug / ml) labeled with anti-mouse IgGFc specific antibody conjugated to monomethyl auristatin E (MMAE) with a cleavable linker (Fab(aMuFc)-MMAE). Cell viability (%) of PA-1 has been shown.Attorney Docket No.: 091787-8001W001

[0065] Fig.2A-2G. Binding of chimeric antibodies to cell lines. Flow binding of chimeric antibodies to PA-1 cell line (Fig. 2A), ES2 cells overexpressing huCLDN6 (Fig.2B and Fig. 2C), huCLDN9 (Fig. 2D), huCLDN3 (Fig.2E), huCLDN4 (Fig. 2F), and muCLDN6 (Fig. 2G). MFI values are shown.

[0066] Fig.3A-3B. Internalization of chimeric antibodies in CLDN6+ cell line. Fig.3A and Fig. 3B, Flow internalization of chimeric antibodies (0.2 ug / ml) in PA-1 cell line. MFI values are shown.

[0067] Fig.4A-4B. Binding of chimeric ADCs to CLDN6+ and CLDN9+ cell lines. Flow binding of chimeric ADCs to PA-1 cell line (Fig. 4A) and ES2 cells overexpressing huCLDN9 (Fig. 4B). MFI values are shown.

[0068] Fig. 5A-5B. Internalization of chimeric ADCs in CLDN6+ and CLDN9+ cell lines. Flow internalization of chimeric ADCs (0.1 ug / ml) in PA-1 cell line (Fig. 5A) and ES2 cells overexpressing huCLDN9 (Fig. 5B). MFI values are shown.

[0069] Fig 6A-6B. In vitro potency of chimeric ADCs. Cell viability assays with PA-1 cell line (Fig.6A) and JEG-3 cell line (Fig. 6B) following treatment with a range of concentrations of chimeric ADCs (0-25 nmol / L).

[0070] Fig. 7. In vivo efficacy of chimeric ADCs. Growth of PA-1 tumor xenografts in BALB / c nude mice after two doses (2 mg / kg, intraperitoneally on day 0 and 7) of non-binding control ADC, 7C10 chimera ADC, 2D5-A chimera ADC, and 2D5-S chimera ADC. Data points show mean tumor volumes ± SEM (n = 5).

[0071] Fig. 8A-8D. Assessment of humanized antibodies and ADCs. Fig. 8A, Flow binding of humanized antibodies (1 ug / ml) to ES2 cells overexpressing huCLDN6. Fig. 8B, Flow binding of humanized ADCs to PA-1 cell line. Fig. 8C, Internalization of humanized ADCs in PA-1 cells. The ADC internalization was measured using the Incucyte live-cell analysis system. Fig. 8D, Cell viability assay with PA-1 cell line following treatment with a range of concentrations of humanized ADCs (0-25 nmol / L).

[0072] Fig.9A-9D. Characterization of hu2D5-S_seq6_WT and LALA antibodies. Fig. 9A, HIC profile of hu2D5-S_seq6_WT antibody. Fig. 9B, Sec profiles of hu2D5-S_seq6_WT antibody (left panel) and hu2D5-S_seq6_LALA antibody (right panel). Fig. 9C, SPR binding to measure the affinity to human CLDN6-VLP. SPR sensorgrams of hu2D5-S_seq6_WT antibody (left panel) and hu2D5-S_seq6_LALA antibody (right panel) are shown. Fig.9D, SPR binding to measure the affinity to human CLDN9-VLP. SPR sensorgrams of hu2D5-S_seq6_WT antibody (left panel) and hu2D5-S_seq6_LALA antibody (right panel) are shown.Attorney Docket No.: 091787-8001W001

[0073] Fig. 10A-10F. Binding of humanized 2D5-S_seq6_WT and LALA antibodies to cell lines. Flow binding of hu2D-S_seq6_WT and LALA antibodies to PA-1 cell line (Fig. 10A), ES2 cells overexpressing huCLDN6 (Fig. 10B), huCLDN9 (Fig. 10C), muCLDN6 (Fig. 10D), huCLDN3 (Fig. 10E), and huCLDN6 (Fig. 10F). MFI values are shown.

[0074] Fig. 11A-11B. Internalization of humanized 2D5-S_seq6_WT and LALA antibodies in CLDN6+ and CLDN9+ cell lines. The internalizations of humanized 2D5-S_seq6_WT and LALA antibodies in PA-1 cell line (Fig. 11A) and ES2 cell line overexpressing huCLDN9 (Fig. 11B) were measured using the Incucyte live-cell analysis system.

[0075] Fig. 12A-12C. Characterization of hu2D5-S_seq6_WT ADCs. Fig. 12A, HIC profile of hu2D5-S_seq6_WT-BL001-Ex ADC. Fig 12B, Sec profile of hu2D5-S_seq6_WT-BL001-Ex ADC.Fig 12C, HIC profile of hu2D5-S_seq6-WT-VC-PABC-MMAE ADC.

[0076] Fig. 13A-13F. Binding of humanized 2D5-S_seq6 ADCs to cell lines. Flow binding of hu2D-S_seq6_WT-BL001-Ex and hu2D-S_seq6_LALA-BL001-Ex ADCs to PA-1 cell line (Fig. 13A), ES2 cells overexpressing huCLDN6 (Fig. 13B), huCLDN9 (Fig. 13C), muCLDN6 (Fig. 13D), huCLDN3 (Fig. 13E), and huCLDN6 (Fig. 13F). MFI values are shown.

[0077] Fig. 14A-14B. Internalization of humanized 2D5-S_seq6 ADCs in CLDN6+ and CLDN9+ cell lines. The internalizations of hu2D-S_seq6_WT-BL001-Ex and hu2D-S_seq6_LALA-BL001-Ex ADCs in PA-1 cell line (Fig. 14A) and ES2 cell line overexpressing huCLDN9 (Fig. 14B) were measured using the Incucyte live-cell analysis system.

[0078] Fig. 15A-15E. In vitro potency of humanized 2D5-S_seq6 ADCs. Cell viability assays with PA-1 cell line (Fig. 15A), JEG-3 cell line (Fig. 15B), and ES2 cells overexpressing huCLDN6 (Fig.15C) and huCLDN9 (Fig. 15D) following treatment with a range of concentrations of hu2D-S_seq6_WT-BL001-Ex or hu2D-S_seq6_LALA-BL001-Ex ADCs (0-100 nmol / L). Fig. 15E, in vitro potency of hu2D-S_seq6_WT-BL003-Ex and hu2D-S_seq6_WT-VC-MMAE in PA-1 cell line.

[0079] Fig. 16A-16C. ADCC, CDC, and ADCP activities of hu2D5-S_seq6_WT Ab and ADC. (Fig. 16A) ADCC activity. PA-1 cells were plated, and IL-2-activated human PBMCs were added at different E: T ratios to detect the ADCC. (Fig. 16B) CDC activity. Human serum was added to PA-1 cells in the presence of hu2D5-S_seq6_WT Ab and ADC. (Fig. 16C) ADCP activity. THP-1 cells were cultured for 1 hour with PA-1 cells and treated with isotype control or hu2D5-S_seq6_WT Ab and ADC. ADCP was determined by flow cytometric analysis as % of CFSE+ THP-1 cells.Attorney Docket No.: 091787-8001W001

[0080] Fig. 17A-17B. Bystander killing effect of humanized 2D5-S_seq6 ADCs. Fig. 17A, The CLDN6-positive PA-1 cells and the CLDN6-negative ES2 cells labeled with NucLight Red were mixed and cultured at 2: 1 ratio overnight. The cells were treated with ADC (2 nM) for 2 days, and then the number of ES2 cells labeled with NucLight Red was determined using the Incucyte livecell analysis system. Fig. 17B, CLDN6-negative ES2 cells labeled with NucLight Red were treated with ADC (2 nM) for 2 days, and then the number of ES2 cells was determined. Each bar represents the mean and ± SEM (n=2).

[0081] Fig. 18A-18C. In vivo efficacy of humanized 2D5-S_seq6 ADCs. Growths of (Fig. 18A) OVACR3, (Fig. 18B) PA-1, and (Fig. 18C) ES2-huCLDN9 tumor xenografts in BALB / c nude mice after a single injection of vehicle control, non-binding control ADC, hu2D5-S_seq6_WT-BL001-Ex ADC. Data points show mean tumor volumes ± SEM (n = 5).

[0082] Fig. 19A-19B. In vivo efficacy of humanized 2D5-S_seq6 ADC in patient derived xenograft tumor model. Growths of tumor LD1-0032-370726 (Fig. 19A) and tumor LD 1-0032-410821 (Fig.19B) in C-NKG mice after a single injection of vehicle control, hu2D5-S_seq6_WT-BL001-Ex ADC, or 3-7-MMAE. Data points show mean tumor volumes ± SEM (n = 6).

[0083] Fig.20A. Epitope mapping at single amino acid resolution. Critical residues for antibody binding were identified by Shotgun Mutagenesis alanine scanning. Antibody binding was assessed by flow cytometry of each antibody against each of 9 individual mutations expressed in ES2 cells. A residue was considered critical if it resulted in <50% of binding signal relative to wild-type CLDN6.

[0084] Fig.20B. Intracellular staining of CLDN6 in engineered ES2 cells. Mean fluorescence intensity (MFI) was determined by flow cytometry to evaluate CLDN6 expression using an antibody (Abeam, ab314134) specific to the CLDN6 intracellular domain. The analysis included three cell types: parental ES2 cells, ES2 cells expressing wild-type human CLDN6 (ES2-huCLDN6-WT), and ES2 cells expressing the N38A mutant (ES2-huCLDN6_N38A).DETAILED DESCRIPTION

[0085] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of theAttorney Docket No.: 091787-8001W001 present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0086] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination.

[0087] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural forms of the same unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0088] It also be noted that, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0089] Definitions

[0090] The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (VH) and a first, second, third, and optionally fourth constant region (CHI, CH2, CH3, CH4 respectively); mammalian light chains are classified as X or K, while each light chain consists of a variable region (VL) and a constant region. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chainAttorney Docket No.: 091787-8001W001 CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3).

[0091] An anti-CLDN6 / 9 antibodies of the present disclosure may be described or specified in terms of the particular CDRs they comprise. The precise amino acid sequence boundaries of a given CDR or FR for the antibodies and antigen-binding fragments disclosed herein may be determined or identified by any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest, ” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography, ” J. Mol. Biol. 262, 732-745. ” (“Contact” numbering scheme); LefrancM P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, ” Dev Comp Immunol, 2003 January; 27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, ” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm, ” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, a “CDR” or “complementarity determining region,” or individual specified CDRs (e.g., HCDR1, HCDR2, HCDR3), of a given antibody or region thereof (e.g., variable region thereof) should be understood to encompass a (or the specific) CDR as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a HCDR3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., HCDR3) within the variable region, as defined by any of the aforementioned schemes. The scheme for identification of a particular CDR or CDRs may be specified, such as the CDR as defined by the Kabat, Chothia, AbM, IMGT, Aho or Contact method.

[0092] The three CDRs are interposed between flanking stretches known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, heavy chain FRs including HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than the CDRs and form a scaffold to support the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions. Antibodies are assigned toAttorney Docket No.: 091787-8001W001 classes based on the amino acid sequences of the constant regions of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (gamma 1 heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgAl (alphal heavy chain), or IgA2 (alpha2 heavy chain).

[0093] In certain embodiments, the antibody provided herein encompasses any antigen-binding fragments thereof. The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, an Fv fragment, a bispecific antibody, a multispecific antibody, a single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.

[0094] ‘ ‘Fc” with regard to an antibody (e.g., of IgG, IgA, or IgD isotype) refers to that portion of the antibody consisting of the second and third constant domains of a first heavy chain bound to the second and third constant domains of a second heavy chain via disulfide bonding. Fc with regard to antibody of IgM and IgE isotype further comprises a fourth constant domain. The Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC), but does not function in antigen binding.

[0095] ‘ ‘Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. In some embodiments, the Fv fragment consists of a heavy chain variable region.

[0096] A “single domain antibody”, “camelized single domain antibody,” “heavy chain antibody,” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1 -2):25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4): 277-302 (2001); WO94 / 04678; WO94 / 25591; U. S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas). Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428): 446- 8 (1993); Nguyen VK. etal.Immunogenetics. Apr;54(l):39-47 (2002); Nguyen VK. etal. Immunology. May; 109(l):93-101Attorney Docket No.: 091787-8001W001 (2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. el al., FASEB J. Nov; 21 (13): 3490-8. Epub 2007 Jun 15 (2007)).

[0097] As used herein, a “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens.

[0098] The term “chimeric” as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and / or light chain derived from one species, and the rest of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

[0099] The term “humanized” as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.

[0100] The term “affinity” as used herein refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e., antibody) or fragment thereof and an antigen.

[0101] The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. Specific binding can be characterized in binding affinity, for example, represented by KD value, i.e., the ratio of dissociation rate to association rate (koff / kon) when the binding between the antigen and antigen-binding molecule reaches equilibrium. KD may be determined by using any conventional method known in the art, including but are not limited to, surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. A KD value of <10’6M (e.g. <5x1 O'7M, <2x1 O'7M, <10’7M, <5x10’8M, <2x10’8M, <10’8M, <5x1 O'9M, <4X10'9M, <3X10'9M, <2x1 O'9M, or <10'9M) can indicate specific binding between an antibody or antigen binding fragments thereof and CLDN6 and / or CLDN9 (e.g. human CLDN6).

[0102] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. An epitope can be linear or conformational (i.e., including amino acid residues spaced apart).Attorney Docket No.: 091787-8001W001

[0103] The term “amino acid” as used herein refers to an organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.

[0104] The term “natural amino acid” as used herein refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins, the names of which are also represented as standard single letter or three-letter codes in the present disclosure, which are summarized as follows.Name of Amino Acid Three-letter Code Single-letter CodeAlanine Ala AArginine Arg RAsparagine Asn NAspartic acid Asp DCysteine Cys CGlutamic acid Glu EGlutamine Gin QGlycine Gly GHistidine His HIsoleucine Ile ILeucine Leu LLysine Lys KMethionine Met MPhenylalanine Phe FProline Pro PSerine Ser SThreonine Thr TTryptophan Trp WTyrosine Tyr YValine Val V

[0105] The term “non-natural amino acid” as used herein refers to any amino acid which is not a natural amino acid. This includes, for example, amino acids that comprise a-, P-, co-, D-, L-amino acyl residues. More generally, the non-natural amino acid comprises a residue of the generalformula, wherein the side chain R is other than the amino acid side chains occurring in nature. Exemplary unnatural amino acids, include, but are not limited to, sarcosine (N-methylglycine), citrulline (cit), homocitrulline, P-ureidoalanine, thiocitrulline, hydroxyproline, allothreonine, pipecolic acid (homoproline), a-aminoisobutyric acid, tert-butylglycine, tertbutylalanine, allo-isoleucine, norleucine, a-methyl leucine, cyclohexylglycine, P-cyclohexylalanine,Attorney Docket No.: 091787-8001W001 P-cyclopentylalanine, a-methylproline, phenylglycine, a-methylphenylalanine and homophenylalanine.

[0106] A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Vai, Leu, and He), among amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gin), among amino acid residues with acidic side chains (e.g., Asp, Glu), among amino acid residues with basic side chains (e.g., His, Lys, and Arg), or among amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

[0107] The term “homologous” as used herein refers to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optimally aligned.

[0108] ‘ ‘Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical relative to the reference sequence to which it is being compared by the total number of the amino acid residues (or bases) in the candidate sequence or in the reference sequence, whichever is shorter. Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLAS TN, BLASTp (available on the website of U. S. National Center for Biotechnology Information (NCBI), see also, Altschul S. F. etal., J. Mol. Biol., 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)), ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G et al., Methods in Enzymology, 266:383-402 (1996); Larkin M. A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. A person skilled in the art may use the defaultAttorney Docket No.: 091787-8001W001 parameters provided by the tool or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

[0109] ‘ ‘Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as Cl complex and Fc receptor. Exemplary effector functions include complement dependent cytotoxicity (CDC) mediated by interaction of antibodies and Clq on the Cl complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis. Effector functions can be evaluated using various assays such as Fc receptor binding assay, Clq binding assay, and cell lysis assay.

[0110] An “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an “isolated antibody or an antigen-binding fragment thereof’ refers to the antibody or antigen-binding fragments thereof having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC).

[0111] The term “nucleic acid molecule” as used herein refers to a polymer of DNA or RNA, or modified forms thereof, which can be single- stranded or double- stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non- natural or altered nucleotides, and which can contain a natural, non-natural or altered inter-nucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. The nucleic acid can comprise any nucleotide sequence which encodes any of the antigen-binding proteins of the present disclosure.

[0112] The term “vector” as used herein refers to a vehicle into which a genetic element may be operably inserted so as to bring about the expression of that genetic element, such as to produce the protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of theAttorney Docket No.: 091787-8001W001 genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or an antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0113] The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and / or a vector can be or has been introduced.

[0114] The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.

[0115] “Treating” or “treatment” of a disease, disorder or condition as used herein includes preventing or alleviating a disease, disorder or condition, slowing the onset or rate of development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or ending symptoms associated with a disease, disorder or condition, generating a complete or partial regression of a disease, disorder or condition, curing a disease, disorder or condition, or some combination thereof.

[0116] The term “diagnosis” “diagnoses” or “diagnosing” refers to the identification of a pathological state, disease or condition, such as identification of a CLDN6 / 9 related disease, or refer to identification of a subject with a CLDN6 / 9 related disease who may benefit from a particular treatment regimen. Wherein, the CLDN6 / 9 related disease refers to a disease or disorder that is responsive to CLDN6 / 9 inhibition. In some embodiments, diagnosis contains the identification of abnormal amount or activity of CLDN6 / 9. In some embodiments, diagnosis refers to the identification of an inflammatory disease, an autoimmune disease, or a cancer in a subject.Attorney Docket No.: 091787-8001W001

[0117] As used herein, the term “sample” or “biological sample” refers to a biological composition that is obtained or derived from a subject of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example based on physical, biochemical, chemical and / or physiological characteristics. A biological sample includes, but is not limited to, cells, tissues, organs and / or biological fluids of a subject, obtained by any method known by those of skill in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, blood serum, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, chest fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites or pericardial fluid. In some embodiments, the biological sample is a tissue or cell obtained from heart, liver, spleen, lung, kidney, skin or blood vessels of the subject.

[0118] ‘ ‘CLDN6” as used herein, refers to an important member of claudin family, which was firstly identified from a differential display analysis of differentiating embryoid bodies in 2001, although its cDNA was isolated from an ectoderm-specific library in 1995 and was amplified by RT-PCR from mouse kidney in 1999 (Harrison SM, etal. Development, 1995, 121, 2479-89; Morita K, et al. Proc. Natl. Acad. Sci. USA, 1999, 96, 511-6). CLDN6 gene is located on chromosome 16p13.3 and contains three exons, which encodes CLDN6 protein with 220 amino acids that has a Mw of 23 kDa (SEQ ID NO: 1) (Singh AB, et al. J oncol, 2010, 541957).

[0119] CLDN6 has close overall sequence identity and ECL pairwise identity with its homologues, CLDN9 (71.8% and 96.0%) (SEQ ID NO: 2), CLDN3 (59.1% and 81.6%) (SEQ ID NO: 3), and CLDN4 (57.1% and 78.4%) (SEQ ID NO: 4) (Gunzel D, Yu ADL. Physiol Rev, 2013, 93, 525-69).

[0120] Amino acid sequence of the human CLDN6 protein (SEQ ID NO: 61):

[0121] MASAGMQILGVVLTLLGWVNGLVSCALPMWKVTAFIGNSIWAQVVWEGLWMS CVVQSTGQMQCKVYDSLLALPQDLQAARALCVIALLVALFGLLVYLAGAKCTTCVEEKDS KARLVLTSGIVFVISGVLTLIPVCWTAHAIIRDFYNPLVAEAQKRELGASLYLGWAASGLLL LGGGLLCCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV

[0122] Amino acid sequence of the human CLDN9 protein (SEQ ID NO: 62):

[0123] MASTGLELLGMTLAVLGWLGTLVSCALPLWKVTAFIGNSIWAQWWEGLWMSC WQSTGQMQCKVYDSLLALPQDLQAARALCVIALLLALLGLLVAITGAQCTTCVEDEGAK ARIVLTAGVILLLAGILVLIPVCWTAHAIIQDFYNPLVAEALKRELGASLYLGWAAAALLML GGGLLCCTCPPPQVERPRGPRLGYSIPSRSGASGLDKRDYV

[0124] Amino acid sequence of the human CLDN3 protein (SEQ ID NO: 63):Attorney Docket No.: 091787-8001W001

[0125] MSMGLEITGTALAVLGWLGTIVCCALPMWRVSAFIGSNIITSQNIWEGLWMNCW QSTGQMQCKVYDSLLALPQDLQAARALIVVAILLAAFGLLVALVGAQCTNCVQDDTAKA KITIVAGVLFLLAALLTLVPVSWSANTIIRDFYNPVVPEAQKREMGAGLYVGWAAAALQLL GGALLCCSCPPREKKYTATKWYSAPRSTGPGASLGTGYDRKDYV

[0126] Amino acid sequence of the human CLDN4 protein (SEQ ID NO: 64):

[0127] MASMGLQVMGIALAVLGWLAVMLCCALPMWRVTAFIGSNIVTSQTIWEGLWMN CVVQSTGQMQCKVYDSLLALPQDLQAARALVIISHVAALGVLLSWGGKCTNCLEDESAK AKTMIVAGWFLLAGLMVIVPVSWTAHNIIQDFYNPLVASGQKREMGASLYVGWAASGLL LLGGGLLCCNCPPRTDKPYSAKYSAARSAAASNYV

[0128] It is worth noting that CLDN6 is nearly identical to the widely expressed CLDN9, with only 3 extracellular amino acids different (M29L in ECL1, as well as R145Q and Q156L in ECL2). Therefore, achieving therapeutic CLDN6 specific monoclonal antibody is extremely challenging.

[0129] CLDN6 is highly associated with tumor initiation and metastasis in specific types of cancers. Pan-cancer analysis using TCGA tumors with the data of the GTEx database as controls indicated that CLDN6 was found significant upregulated in about 20 types of cancers, especially in testicular germ cell tumors (TGCT), ovarian cancer (OV), lung cancers (LU AD and LUSC), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC), et al., while it is downregulated in glioblastoma multiforme, kidney chromophobe, kidney renal clear cell carcinoma, acute myeloid leukemia, and brain lower-grade glioma, et al (Zhang C, et al. Front Cell Dev Biol, 2021, 9, 726656). CLDN6 was found most highly expressed in testicular germ cell tumors (TGCT) (28 / 28, 100%), and second highly expressed in ovarian cancers (34 / 62, 54.8%) (Ushiku T, Histopathology, 2012, 61, 1043-56; Qu H, et al. Int J Mol Sci, 2021, 22, 13416). Bioinformatic analysis also indicated that CLDN6 differential expression was correlated with different molecular subtypes and immune subtypes in UCEC, BRCA, ESCA, LUSC, HNSC, OV, and STAD, et al. For instance, in UCEC, CLDN6 was identified to express more in the molecular subtype of CN HIGH than other molecular subtypes; in BRCA, CLDN6 was significantly upregulated in the molecular subtype of basal. Survival prognosis analysis by Kaplan-Meier plots indicated that CLDN6 expression is notably correlated with the overall survival (OS), disease specific survival (DSS), and progression-free interval (PFI) of UCEC, adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), and STAD. For UCEC, CLDN6 expression was found significantly related to tumor stage, age, weight, histological type and grade, and menopause status of UCEC (Zhang C, et al. Front Cell Dev Biol, 2021, 9, 726656).Attorney Docket No.: 091787-8001W001

[0130] The term “anti-CLDN6 / 9 antibody” refers to an antibody that is capable of specifically binding to CLDN6 and / or CLDN9 (e.g., human CLDN6 / 9). The term “anti-human CLDN6 / 9 antibody” refers to an antibody that is capable of specifically binding to human CLDN6 / 9.

[0131] A “CLDN6 / 9-related” disease, disorder or condition refers to any disease or condition linked to increased or decreased expression or activities of CLDN6 / 9.

[0132] In some embodiments, the CLDN6 / 9-related disease, disorder or condition is associated with dysregulation of CLDN6 / 9 mediated signaling compared to the control level. In some embodiments, the dysregulation of CLDN6 / 9 mediated signaling includes dysregulation of CLDN6 / 9-expressing cell proliferation. In some embodiments, the control level is the level in a healthy individual.

[0133] In some embodiments, the CLDN6 / 9-related disease, disorder or condition is a disorder related to excessive cell proliferation, such as, for example, cancer. In certain embodiments, the CLDN6 / 9-related disease or condition is characterized in expressing or over-expressing of CLDN6 / 9 gene. In certain embodiments, the CLDN6 / 9-related disease or condition is characterized in over-expression of CLDN6 / 9 and / or dysregulation of CLDN6 / 9 mediated signaling.

[0134] The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0135] The term “CLDN6 positive” cell as used herein refer to a cell which shows an abnormal expression level of CLDN6 relative to a control cell. The abnormal expression level can be up-regulated or down-regulated relative to the level of the control cell and can be associated with dysregulation of CLDN6 mediated signaling. The control cell can be a normal or healthy counterpart cell, which may or may not express CLDN6. In case the control cell expresses CLDN6, the abnormal expression level of the CLDN6 positive cell can be up-regulated or down-regulated. In case the control cell does not express CLDN6, the abnormal expression level of the CLDN6-positive cell can be up-regulated.

[0136] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; SmithAttorney Docket No.: 091787-8001W001 and March March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0137] At various places in the present disclosure, linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl”, then it is understood that the “alkyl” represents a linking alkylene group.

[0138] When any variable (e.g., R1) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R1moieties, then the group may optionally be substituted with up to two R1moieties and R1at each occurrence is selected independently from the definition of R1. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0139] As used herein, a dash at the front or end of a chemical group is used, a matter of convenience, to indicate a point of attachment for a substituent. For example, -OH is attached through the carbon atom; chemical groups may be depicted with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates a point of attachment of a group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which a chemical group is written or named. As used herein, a solid line coming out of the center of a ring indicates that the point of attachment for a substituent on the ring can be at any ring atom. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0140] When any variable (e.g., R1) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R1moieties, then the group may optionally be substituted with up to two R1moieties and R1at each occurrence is selected independently from the definition of R1. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.Attorney Docket No.: 091787-8001W001

[0141] The term “about”, when used in connection with a numerical value, means that a collection or range of values is included. For example, “about X” includes a range of values that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value. In one embodiment, the term “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 2% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 1% more or less than the specified value.

[0142] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. For example, the expressions “n is an integer between 1 and 6” and “n being an integer of 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”.

[0143] As used herein, the term “Ci-j” indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e., i and j) and each integer point in between, and wherein j is greater than i. For examples, Ci-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms. In some embodiments, the term “C1-12” indicates 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms. In similar manner, the term “m-n membered” ring, wherein m and n are integers and n is greater than m, refers to a ring containing m to n atoms.

[0144] As used herein, the term “aliphatic” includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties.

[0145] As used herein, the term “alkyl”, whether as part of another term or used independently, refers to a saturated linear or branched- chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term “Ci-j alkyl” refers to a linear or branched- chain alkyl having i to j carbon atoms. For example, alkyl groups contain 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of “C1-6 alkyl” include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-Attorney Docket No.: 091787-8001W001 butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3 -methyl- 1 -butyl, 2-methyl-1 -butyl, 2-ethyl-l -butyl, 1 -hexyl, 2-hexyl, 3 -hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3 -methyl-3 -pentyl, 2-methyl-3 -pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0146] As used herein, the term “alkenyl”, whether as part of another term or used independently, refers to linear or branched-chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted independently with one or more substituents described herein, and includes radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. In some embodiments, alkenyl groups contain 2 to 12 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms. In some embodiments, alkenyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkenyl groups contain 2 carbon atoms. Examples of alkenyl group include, but are not limited to, ethylenyl (or vinyl), propenyl (allyl), butenyl, pentenyl, l-methyl-2 buten-l-yl, 5-hexenyl, and the like.

[0147] As used herein, the term “alkynyl”, whether as part of another term or used independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, which may be optionally substituted independently with one or more substituents described herein. In some embodiments, alkynyl groups contain 2 to 12 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms. In some embodiments, alkynyl groups contain 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkynyl groups contain 2 carbon atoms. Examples of alkynyl group include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0148] As used herein, the term “alkoxy!” or “alkoxy”, whether as part of another term or used independently, refers to a radical of the formula -ORawhere Rais an alkyl radical as defined herein. Whenever it appears herein, a numerical range such as “Ci-Ce alkoxy” or “Ci-ealkoxy”, means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although the present definition also covers the occurrence of the term “alkoxy” where no numerical range is designated. In some embodiments, the alkoxy is a Ci-io alkoxy. In some embodiments, the alkoxy is a Ci-6 alkoxy. In some embodiments, the alkoxy is a Ci-5 alkoxy. In some embodiments, the alkoxy is a Ci-4 alkoxy. In some embodiments, the alkyl is aAttorney Docket No.: 091787-8001W001 Ci-3 alkoxy. In some embodiments, the alkyl is a C1-2 alkoxy. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, carboxyl, carboxylate, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, heteroaryl, and the like.

[0149] As used herein, the term “amino” refers to the group -NRaRb, wherein Raand Rbare independently selected from groups consisting of hydrogen, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl and each of which may be optionally substituted.

[0150] As used herein, the term “aryl”, whether as part of another term or used independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring system, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0151] As used herein, the term “cycloalkyl”, whether as part of another term or used independently, refers to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring system, in which all the ring atoms are carbon, and which contains at least three ring forming carbon atoms. In some embodiments, the cycloalkyl group may contain 3 to 12 ring forming carbon atoms, 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms, 3 to 8 ring forming carbon atoms, 3 to 7 ring forming carbon atoms, 3 to 6 ring forming carbon atoms, 3 to 5 ring forming carbon atoms, 4 to 12 ring forming carbon atoms, 4 to 10 ring forming carbon atoms, 4 to 9 ring forming carbon atoms, 4 to 8 ring forming carbon atoms, 4 to 7 ring forming carbon atoms, 4 to 6 ring forming carbon atoms, 4 to 5 ring forming carbon atoms. The cycloalkyl group may be saturated or partially unsaturated. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system.

[0152] In some embodiments, the cycloalkyl group may be saturated or partially unsaturated monocyclic carbocyclic ring system, examples of which include, but are not limited to, cyclopropyl,Attorney Docket No.: 091787-8001W001 cyclobutyl, cyclopentyl, 1 -cyclopent- 1-enyl, l-cyclopent-2-enyl, 1 -cyclopent-3 -enyl, cyclohexyl, 1-cyclohex-l-enyl, 1 -cyclohex-2-enyl, 1 -cyclohex-3 -enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.

[0153] In some embodiments, the cycloalkyl group may be saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring system, which can be arranged as a fused, spiro or bridged ring system. As used herein, the term “fused ring” refers to a ring system having two rings sharing two adjacent atoms, the term “spiro ring” refers to a ring systems having two rings connected through one single common atom, and the term “bridged ring” refers to a ring system with two rings sharing three or more atoms. Examples of fused cycloalkyl include, but are not limited to, naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl and the like. Examples of spiro cycloalkyl include, but are not limited to, spiro[5.5]undecanyl, spiro-pentadienyl, spiro[3.6]-decanyl, and the like. Examples of bridged cycloalkyl include, but are not limited to bicyclo[l,l,l]pentenyl, bicyclo[2,2,l]heptenyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, bicyclo[3.3.1]nonanyl, bicyclo[3.3.3]undecanyl, and the like.

[0154] As used herein, the term “halo” or “halogen” refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo) and iodine (or iodo).

[0155] As used herein, the term “haloalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, tri chloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0156] As used herein, the term “heteroatom” refers to nitrogen, oxygen, sulfur or phosphorus, and includes any oxidized form of nitrogen, sulfur or phosphorus, and any quaternized form of a basic nitrogen.

[0157] As used herein, the term “heteroaliphatic” refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched or linear unbranched. As will be appreciated by one of ordinary skill in the art, “heteroaliphatic” is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, heteroalkynyl moieties. In certain embodiments, heteroaliphatic moieties are substituted (“substituted heteroaliphatic”) by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylaryl;Attorney Docket No.: 091787-8001W001 alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; - or -GRG1, wherein G is -O-, -S-, -NRG2-, -C(=O)-, -S(=O)-> -SO2-, -C(=O)O-, -C(=O)NRG2-, -OC(=O)-, -NRG2C(=O)-, -OC(=O)O-, -OC(=O)NRG2-, -NRG2C(=O)O-, -NRG2C(=O)NRG2-, -C(=S)-> -C(=S)S-> -SC(=S)-> -SC(=S)S-> -C(=NRG2)-, -C(=NRG2)O-, -C(=NRG2)NRG3-, -OC(=NRG2)-, -NRG2C(=NRG3)-, -NRG2SO2-, -NRG2SO2NRG3-, or -SO2NRG2-, wherein each occurrence of RG1, RG2and RG3independently includes, but is not limited to, hydrogen, halogen, or an optionally substituted aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

[0158] As used herein, the term “heteroalkyl” refers to an alkyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein. The term “heteroalkyl” encompasses alkoxyl and heteroalkoxy radicals.

[0159] As used herein, the term “heteroalkenyl” refers to an alkenyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkenyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein.

[0160] As used herein, the term “heteroalkynyl” refers to an alkynyl, at least one of the carbon atoms of which is replaced with a heteroatom selected from N, O, or S. The heteroalkynyl may be a carbon radical or heteroatom radical (i.e., the heteroatom may appear in the middle or at the end of the radical), and may be optionally substituted independently with one or more substituents described herein.

[0161] As used herein, the term “heteroaryl”, whether as part of another term or used independently, refers to an aryl group having, in addition to carbon atoms, one or more heteroatoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The heteroaryl group alsoAttorney Docket No.: 091787-8001W001 includes polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, heteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryl include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[l,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0162] As used herein, the term “heterocycloalkyl” refers to a saturated or partially unsaturated cycloalkyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, the remaining ring atoms being carbon, wherein one or more ring atoms may be optionally substituted independently with one or more substituents. In some embodiments, the heterocycloalkyl is a saturated heterocycloalkyl. In some embodiments, the heterocycloalkyl is a partially unsaturated heterocycloalkyl having one or more double bonds in its ring system. In some embodiments, the heterocycloalkyl may contains any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of a basic nitrogen. The heterocycloalkyl radical may be carbon linked or nitrogen linked where such is possible. In some embodiments, the heterocycle is carbon linked. In some embodiments, the heterocycle is nitrogen linked. For example, a group derived from pyrrole may be pyrrol- 1-yl (nitrogen linked) or pyrrol-3-yl (carbon linked). Further, a group derived from imidazole may be imidazol-l-yl (nitrogen linked) or imidazol-3-yl (carbon linked).

[0163] Heterocycloalkyl group may be monocyclic. Examples of monocyclic heterocycloalkyl include, but are not limited to oxetanyl, 1, 1 -dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like.

[0164] Heterocycloalkyl group may be polycyclic, including the fused, spiro and bridged ring systems. The fused heterocycloalkyl group includes radicals wherein the heterocycloalkyl radicals are fused with a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocycloalkyl include, but are not limited to, phenyl fused ring or pyridinyl fused ring, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinolizinyl,Attorney Docket No.: 091787-8001W001 quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[l,2-a]pyridinyl, furo[3,4-d]pyrimidinyl, pyrrolo[3,4-d]pyrimidinyl, dihydrofuro[3,4-b]pyridinyl groups, and the like.Examples of spiro heterocycloalkyl include, but are not limited to, spiropyranyl, spirooxazinyl, 5-aza-spiro[2.4]heptanyl, 6-aza-spiro[2.5]octanyl, 6-aza-spiro[3.4]octanyl, 2-oxa-6-aza-spiro[3.3]heptanyl, 2-oxa-6-aza-spiro[3.4]octanyl, 6-aza-spiro[3.5]nonanyl, 7-aza-spiro[3.5]nonanyl, l-oxa-7-aza-spiro[3.5]nonanyl, 3,8-dioxa-l-azaspiro[4.5]dec-l-enyl and the like. Examples of bridged heterocycloalkyl include, but are not limited to, 3-aza-bicyclo[3.1.0]hexanyl, 8-aza-bicyclo[3.2.1]octanyl, l-aza-bicyclo[2.2.2]octanyl, 2-aza-bicyclo[2.2. l]heptanyl, 1,4-diazabicyclo[2.2.2]octanyl, and the like.

[0165] As used herein, the term “hydroxyl” refers to -OH.

[0166] As used herein, the term “leaving group” refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to halides such as Cl', Br, and E, sulfonate esters, such as para-toluenesulfonate (“tosylate”, TsO'), and RC(O)O' in which R is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0167] As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the said event or circumstance occurs and instances in which it does not.

[0168] As used herein, the term “partially unsaturated” refers to a radical that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0169] As used herein, the term “protecting group” means that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In some embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions. The protecting group must be selectively removed in good yield by readily available, preferably nontoxic reagents that do not attack the other functional groups. The protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers). The protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailedAttorney Docket No.: 091787-8001W001 herein, oxygen, sulfur, nitrogen and carbon protecting groups may be utilized. For example, in some embodiments, certain exemplary oxygen protecting groups may be utilized. These oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM (p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, and TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, and dichloroacetate), carbonates, cyclic acetals and ketals. In some other embodiments, nitrogen protecting groups are utilized. Nitrogen protecting groups, as well as protection and deprotection methods are known in the art. Nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives. In yet other embodiments, certain exemplary sulphur protecting groups may be utilized. The sulfur protecting groups include, but are not limited to those oxygen protecting group describe above as well as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinyl sulfonyl, and optionally substituted maleic acid. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in “Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. and Wuts, P. G, Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.

[0170] As used herein, the term “substituted”, whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. The substituents may include, but not limited to, alkyl, alkenyl, alkynyl,Attorney Docket No.: 091787-8001W001 alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or combinations thereof. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted”, references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

[0171] As used herein, the term “biocompatible” as used herein is intended to describe compounds that exert minimal destructive or host response effects while in contact with body fluids or living cells or tissues. Thus, a biocompatible group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety, which falls within the definition of the term biocompatible, as defined above and herein. The term “biocompatibility” as used herein, is also taken to mean that the compounds exhibit minimal interactions with recognition proteins, e.g., naturally occurring antibodies, cell proteins, cells and other components of biological systems, unless such interactions are specifically desirable. Thus, substances and functional groups specifically intended to cause the above minimal interactions, e.g., drugs and prodrugs, are considered to be biocompatible. In some embodiments, compounds are “biocompatible” if their addition to normal cells in vitro, at concentrations similar to the intended systemic in vivo concentrations, results in less than or equal to 1% cell death during the time equivalent to the halflife of the compound in vivo (e.g., the period of time required for 50% of the compound administered in vivo to be eliminated / cleared), and their administration in vivo induces minimal and medically acceptable inflammation, foreign body reaction, immunotoxicity, chemical toxicity and / or other such adverse effects. As used herein, the term “normal cells” refers to cells that are not intended to be destroyed or otherwise significantly affected by the compound being tested.

[0172] As used herein, “biodegradable” polymers are polymers that are susceptible to biological processing in vivo. As used herein, “biodegradable” compounds or moieties are those that, when taken up by cells, can be broken down by the lysosomal or other chemical machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells. The term “biocleavable” as used herein has the same meaning of “biodegradable”. The degradation fragments preferably induce little or no organ or cell overload or pathological processes caused by such overload or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction.Attorney Docket No.: 091787-8001W001 Suitable conditions for non-enzymatic hydrolysis of the biodegradable protein-polymer-drug conjugates (or their components, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or the drug molecule) described herein, for example, include exposure of the biodegradable conjugates to water at a temperature and a pH of lysosomal intracellular compartment. Biodegradation of some protein-polymer-drug conjugates (or their components, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or the drug molecule), can also be enhanced extracellularly, e.g., in low pH regions of the animal body, e.g. an inflamed area, in the close vicinity of activated macrophages or other cells releasing degradation facilitating factors. In certain embodiments, the effective size of the polymer carrier at pH~7.5 does not detectably change over 1 to 7 days and remains within 50% of the original polymer size for at least several weeks. At pH~5, on the other hand, the polymer carrier preferably detectably degrades over 1 to 5 days and is completely transformed into low molecular weight fragments within a two-week to several-month time frame. Polymer integrity in such tests can be measured, for example, by size exclusion HPLC. Although faster degradation may be in some cases preferable, in general it may be more desirable that the polymer degrades in cells with the rate that does not exceed the rate of metabolization or excretion of polymer fragments by the cells. In certain embodiments, the polymers and polymer biodegradation byproducts are biocompatible.

[0173] As used herein, the term “bioavailability” refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug or compound administered to a subject.Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug or compound that reaches the general circulation from an administered dosage form.

[0174] As used herein, the term “drug release mechanism” refers to a linking moiety that is biocleavable / biodegradable under intracellular conditions, such that the cleavage of the linking moiety release the drug in the intracellular environment. In some embodiments, the linking moiety is hydrolytically labile in water or in aqueous solutions including for example, body fluid such as blood, i.e., sensitive to hydrolysis at certain pHs. In some embodiments, the linking moiety is enzymatically labile, i.e., degradable by one or more enzymes. In some embodiments, the linking moiety is photo labile and is useful at the body surface and in many body cavities that are accessible to light. In some embodiments, the linking moiety is biocleavable under reducing conditions under which the activity of drug is not affected.Attorney Docket No.: 091787-8001W001

[0175] As used herein, the term “therapeutic agent” or “drug” refers to a compound, prodrug or payload which is biologically active and provides a desired physiological effectresponse following administration to a subject in need thereof (e.g., an active pharmaceutical ingredient) biological entity. Exemplary biological responses include, without limitation, increase or decrease in DNA or protein synthesis, upregulation or down-regulation of signalling pathways, and increase or decrease in cell proliferation, and the like. In some embodiments, the therapeutic agent is small molecule drug.

[0176] The term “pharmaceutically acceptable salt” refers to a salt of a compound described in the present invention, which is safe and effective when used in a biological entity and has the expected biological activity. The pharmaceutically acceptable salt includes a salt with base or acid. Non-restrictive examples of pharmaceutically acceptable salts include sodium salts, potassium salts, calcium salts, magnesium salts, hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and the like.

[0177] As used herein, the term “small molecule” refers to molecules, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, such as mammals, for example humans. In certain embodiments, the small molecule is a drug, and the small molecule is referred to as “drug molecule” or “drug” or “therapeutic agent”. In certain embodiments, the drug molecule has Mw less than or equal to about 5 kDa. In other embodiments, the drug molecule has Mw less than or equal to about 1.5 kDa.

[0178] Classes of drug molecules that can be used in the present disclosure include, but are not limited to, anti-cancer substances, radionuclides, vitamins, anti- AIDS substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, anti-parasite and / or anti-protozoal compounds, modulators of cell- extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and / or antithrombotic agents, local anesthetics,Attorney Docket No.: 091787-8001W001 ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, imaging agents.

[0179] In certain embodiments, large molecules can be used as the therapeutic agents in the present disclosure. Examples of suitable large molecules include, but are not limited to, amino acid-based molecules, such as peptides, polypeptides, enzymes, antibodies, immunoglobulins, or functional fragments thereof, among others.

[0180] In some embodiments, the therapeutic agent used in the present disclosure is a therapeutic agent that has antiproliferative (cytostatic and / or cytotoxic) activity against a target cell or pathway. The drug may have a chemically reactive group such as, for example, -COOH, primary amine, secondary amine -NHR, -OH, -SH, -C(O)H, -C(O)R, -C(O)NHR’, -C(S)OH, -S(O)2OR’, -P(O)2OR’, -CN, -NC or -ONO, in which R is aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moiety and R’ is a hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl moiety.

[0181] As used herein the term “cytotoxic” means toxic to cells or a selected cell population (e.g., cancer cells). The toxic effect may result in cell death and / or lysis. In certain instances, the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing or arresting cell growth. In order to achieve a cytotoxic effect, the drug or prodrug may be selected from a group consisting of a DNA damaging agent, a microtubule disrupting agent, or a cytotoxic protein or polypeptide, amongst others.

[0182] As used herein, the term “specific binding” or “specifically binds” refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to a target antigen with a binding affinity (KD) of about 0.01 nM to about 100 nM, about 0.1 nM to about 100 nM, 0.01 nM to about 10 nM, about 0.1 nM to about 10 nM, 0.01 nM to about 1 nM, about 0.1 nM to about 1 nM or about 0.01 nM to about 0.1 nM) at pH 7.4. KD as used herein refers to the ratio of the dissociation rate to the association rate (koff / kon), may be determined using surface plasmon resonance methods for example using instrument such as Biacore.

[0183] As used herein, the term “effective amount” refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc. For example, the effective amount of microparticles containing an antigen to be delivered toAttorney Docket No.: 091787-8001W001 immunize an individual is the amount that results in an immune response sufficient to prevent infection with an organism having the administered antigen.

[0184] The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include13C and14C.

[0185] The present invention is intended to include all isomers of the compound, which refers to and includes, optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in a mixture with one or more other isomers.

[0186] Anti-CLDN6 / 9 Antibodies

[0187] The present disclosure provides anti-CLDN6 / 9 antibodies and antigen-binding fragments thereof. The anti-CLDN6 / 9 antibodies and antigen-binding fragments provided herein are capable of specific binding to CLDN6 / 9.

[0188] In certain embodiments, the antibodies and the antigen-binding fragments thereof provided herein specifically bind to human CLDN6 (i.e., long-form CLDN6) at a KD value of no more than 10-6M, no more than 10-7M, no more than 8×10-8M, no more than 5×10-8M, no more than 2×10-8M, no more than 8×10-9M, no more than 5×10-9M, no more than 2×10-9M, no more than 10-9M, no more than 8×10-10M, no more than 7×10-10M, or no more than 6×10-10M by Surface Plasmon Resonance (SPR) assay, see, for example, Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006.

[0189] Binding of the antibodies or the antigen-binding fragments thereof provided herein to human CLDN6 can also be represented by “half maximal effective concentration” (ECso) value. As used herein, the term “half maximal effective concentration” or “ECso” refers to the concentration of an antibody where 50% of its maximal binding is observed. The ECso value can be measured by binding assays known in the art, for example, direct or indirect binding assay such as enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS) assay, and other binding assays.

[0190] Preparation of antibody

[0191] Antibodies and antigen-binding fragments thereof can be prepared by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g.,Attorney Docket No.: 091787-8001W001 hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0192] The antigen-binding fragment of the present invention can be obtained by hydrolyzing an intact antibody molecule (see, Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992), and Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab') 2 fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

[0193] In certain embodiments, the present invention provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or light chain variable region thereof. In some preferred embodiments, the isolated nucleic acid molecule encodes the antibody or antigenbinding fragment thereof of the present invention, or a heavy chain variable region and / or light chain variable region thereof.

[0194] In some embodiments, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present invention. In some preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like. In some preferred embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, for example, a human).

[0195] In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. Such host cell includes, but is not limited to, prokaryotic cell such as E. coli cell, eukaryotic cell such as yeast cell, insect cell, plant cell, and animal cell (e.g., mammalian cell, such as mouse cell, human cell, etc.). In some preferred embodiments, the host cell of the present invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DG44).Attorney Docket No.: 091787-8001W001

[0196] In some embodiments, provided is a method for producing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under a condition that allows the expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cell.

[0197] Antibody Variants

[0198] The anti-CLDN6 / 9 antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the anti-CLDN6 / 9 antibody sequences provided herein.

[0199] In certain embodiments, the antibody variants comprise one or more modifications or substitutions in one or more of the CDR sequences as provided herein, one or more of the non-CDR sequences of the heavy and / or light chain variable region provided herein, and / or the constant region (e.g., Fc region). Such variants retain binding specificity to CLDN6 / 9 of their parent antibodies but have one or more desirable properties conferred by the modification(s) or substitution(s). For example, the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility to conjugation (e.g., one or more introduced cysteine residues).

[0200] The parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for the interested property. If substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substituting with a different type of residue (e.g., cysteine residue, positively charged residue, etc.).

[0201] Affinity Variants

[0202] Affinity variants of antibodies may contain modifications or substitutions in one or more CDR sequences as provided herein, or the heavy and / or light chain variable region sequences provided herein. FR sequences can be readily identified by a person skilled in the art based on the CDR sequences and variable region sequences provided herein, as it is well-known in the art that aAttorney Docket No.: 091787-8001W001 CDR region is flanked by two FR regions in the variable region. The affinity variants retain specific binding affinity to CLDN6 / 9 of the parent antibody, or even have improved CLDN6 / 9 specific binding affinity over the parent antibody. In certain embodiments, at least one (or all) of the substitution(s) in the CDR sequences, FR sequences, or variable region sequences comprises a conservative substitution.

[0203] A person skilled in the art will understand that in the CDR sequences and variable region sequences provided herein, one or more amino acid residues may be substituted yet the resulting antibody or antigen-binding fragment still retain the binding affinity or binding capacity to CLDN6 / 9, or even have an improved binding affinity or capacity. Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human CLDN6. For another example, computer software can be used to virtually simulate the binding of the antibodies to human CLDN6 and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity or targeted for substitution to provide for a stronger binding.

[0204] In certain embodiments, the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid residue substitutions in one or more of the CDR sequences, and / or one or more of the FR sequences. In certain embodiments, an affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution(s) in the CDR sequences and / or FR sequences in total.

[0205] In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) provided herein yet retaining the specific binding affinity to CLDN6 at a level similar to or even higher than its parent antibody.

[0206] In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) provided herein yet retaining the specific binding affinity to CLDN6 at a level similar to or even higher than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted,Attorney Docket No.: 091787-8001W001 inserted, or deleted in a variable region sequence provided herein. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs).

[0207] Glycosylation Variants

[0208] The anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants, which can be obtained to either increase or decrease the extent of glycosylation of the antibodies or antigen binding fragments thereof.

[0209] The antibodies or antigen binding fragments thereof may comprise one or more modifications that introduce or remove a glycosylation site. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.

[0210] In certain embodiments, the anti-CLDN6 / 9 antibodies and antigen-binding fragments provided herein comprise a mutation at N297 (e.g., N297A, N297Q, or N297G) to remove the glycosylation site.

[0211] Cysteine-engineered Variants

[0212] The anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein also encompass cysteine-engineered variants, which comprise one or more introduced free cysteine amino acid residues.

[0213] A free cysteine residue is one which is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and / or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine residues are known in the art, see, for example, W02006 / 034488.

[0214] Fc VariantsAttorney Docket No.: 091787-8001W001

[0215] The anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants, which comprise one or more amino acid residue modifications or substitutions at the Fc region and / or hinge region, for example, to provide for altered effector functions such as ADCC and / or CDC. Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol Chem. 2001. 276(9): 6591-604; IdusogieEE. et aL, J Immunol. 2000.164(8):4178-84; SteurerW. et al., J Immunol. 1995, 155(3): 1165- 74; IdusogieEE. et al., J Immunol. 2001, 166(4): 2571-5; Lazar GA. et aL, PNAS, 2006, 103(11): 4005-4010; RyanMC. etal., Mol. Cancer Ther., 2007, 6: 3009-3018; Richards JO,. etal., Mol Cancer Ther. 2008, 7(8): 2517-27; Shields R. L. et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. etal., J. Biol. Chem, 2003, 278: 3466-3473.

[0216] CDC activity of the antibodies or antigen-binding fragments provided herein can also be altered, for example, by improving or diminishing Clq binding and / or CDC (see, for example, WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U. S. Pat. No. 5,648,260; U. S. Pat. No.5,624,821; and WO94 / 29351 concerning other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331 and 322 of the Fc region can be replaced with a different amino acid residue to alter Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC) (see, U. S. Pat. No. 6,194,551 by Idusogie et aL). One or more amino acid substitution(s) can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94 / 29351 by Bodmer etaL).

[0217] In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein has reduced effector functions, and comprise one or more amino acid substitution(s) in IgGl at a position selected from the group consisting of: 234, 235, 237, and 238, 268, 297, 309, 330, and 331. In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein is of IgGl isotype and comprise one or more amino acid substitution(s) selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof. In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein is of IgG2 isotype, and comprises one or more amino acid substitution(s) selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein is of IgG4 isotype, and comprises one or more amino acid substitution(s) selected from the group consisting of: N297A, N297Q,Attorney Docket No.: 091787-8001W001 N297G, L235E, L234A, L235A, M252Y / S254T / T256E, T307Q / N434 and any combination thereof. In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein is of IgG2 / IgG4 cross isotype. Examples of IgG2 / IgG4 cross isotype is described in RotherRP et al., Nat Biotechnol 25:1256-1264 (2007).

[0218] In certain embodiments, the anti-CLDN6 / 9 antibodies and antigen-binding fragments provided herein is of IgG4 isotype and comprises the amino acid substitutions of S228P / F234A / L235A (PAA). In certain embodiments, the anti-CLDN6 / 9 antibodies and antigenbinding fragments provided herein is of IgG4 isotype and comprises the amino acid substitutions of M252Y / S254T / T256 (YTE). In certain embodiments, the anti-CLDN6 / 9 antibodies and antigenbinding fragments provided herein is of IgG4 isotype and comprises the amino acid deletion of K447del.

[0219] In certain embodiments, the anti-CLDN6 / 9 antibodies and antigen-binding fragments provided herein is of IgG4 isotype and comprises one or more amino acid substitution(s), for example at the point of 228. In certain embodiments, the anti-CLDN6 / 9 antibodies and antigenbinding fragments provided herein is of IgG4 isotype and comprises one or more mutations selected from the group consisting of S228P, F234A, L235A, M252Y, S254T, T256E and K447del in the Fc region, or any combination thereof.

[0220] In certain embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof comprise one or more amino acid substitution(s) that improves pH-dependent binding to neonatal Fc receptor (FcRn). Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody or antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al., Structure, 6(1): 63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. etal., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. etal., J. Immunology, 176:346-356 (2006).

[0221] In certain embodiments, anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof comprise one or more amino acid substitution(s) in the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to formAttorney Docket No.: 091787-8001W001 a heterodimer or a complex. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U. S. Pat. No. 5,731,168.

[0222] Bispecific or Multi-specific Molecules

[0223] The antibody or antigen-binding fragment thereof of the present invention can be used to form a bispecific or multispecific molecule. The antibody or antigen-binding fragment thereof of the present invention may be part of a bispecific or multispecific molecule, and the bispecific or multispecific molecule comprises a second functional module (e.g., a second antibody) that is different from the antibody or antigen-binding fragment thereof of the present invention in binding specificity, thereby being able to bind to at least two different binding sites and / or target molecules. For example, the antibody or antigen-binding fragment thereof of the present invention may be linked to a second antibody or antigen-binding fragment thereof that is capable of specifically binding to any protein that may be used as a potential target for combination therapy. To generate the bispecific or multispecific molecule, the antibody or antigen-binding fragment thereof of the present invention may be linked (e.g., by chemical conjugation, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules (e.g., additional antibody, antibody fragment, peptide or binding mimetic).

[0224] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen other than CLDN6, or a second epitope on CLDN6. In some embodiments, the second antigen other than CLDN6 is selected from the group consisting of CD3, 4-1BB, CD28, CD16A, FRa, CLDN18.2, PD-L1, HER2, EGFR, MSLN, CD47, VEGFR, Trop2, MUC1, MUC16 or the like.

[0225] In some embodiments, the present invention provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof of the present invention.

[0226] In some embodiments, the bispecific or multispecific molecule specifically binds to CLDN6 and additionally specifically binds to one or more other targets.

[0227] In some embodiments, the bispecific or multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

[0228] Chimeric Antigen Receptor

[0229] The antibody or antigen-binding fragment thereof of the present invention can be used to construct a chimeric antigen receptor (CAR), which comprises an extracellular antigen-binding domain (e.g., scFv) that is capable of specific binding to CLDN6, linking to transmembrane domain, and linking to one or more intracellular T cell signaling domain. The intracellular T cell signalingAttorney Docket No.: 091787-8001W001 domain can comprise, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or a combination thereof.

[0230] Features of the CAR of the present invention comprises an ability to direct T-cell specificity and reactivity toward a cell expressing CLDN6(e.g., a tumor cell) in a non-MHC-restricted manner. The non-MHC-restricted CLDN6 recognition ability endows a T cell expressing the CAR of the present invention with the ability to recognize antigens that is independent to antigen processing.

[0231] In certain embodiments, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain of the antibody or antigen-binding fragment thereof of the present invention.

[0232] In some embodiments, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof of the present invention.

[0233] In some embodiments, the antigen-binding domain is a scFv.

[0234] In some embodiments, the chimeric antigen receptor comprises the antigen-binding fragment (e.g., scFv) of the antibody of the present invention.

[0235] In some embodiments, the chimeric antigen receptor is expressed by an immune effector cell (e.g., a T cell).

[0236] In some embodiments, a spacer domain comprising a polypeptide sequence may exist between the antigen-binding domain and the transmembrane domain of the CAR. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In some embodiments, the spacer domain may comprise an immunoglobulin domain, for example, a human immunoglobulin sequence. In certain exemplary embodiments, the immunoglobulin domain comprises immunoglobulin CH2 and CH3 domain sequences.

[0237] In some embodiments, the transmembrane domain may be derived from a natural or synthetic source. In such embodiments, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains useful in the CAR of the present invention may comprise at least transmembrane regions of α, β or ζ chain of a T cell receptor, and the T cell receptor may be selected from the group consisting of CD28, CD3s, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,

[0238] In certain exemplary embodiments, the transmembrane domain comprises a transmembrane domain of a T cell receptor, for example, a CD 8 transmembrane domain.Attorney Docket No.: 091787-8001W001

[0239] In some embodiments, the transmembrane domain comprises a transmembrane domain of a T cell co-stimulatory molecule (e.g., CD 137 or CD28).

[0240] In some embodiments, examples of intracellular T cell domains useful in the CAR include cytoplasmic sequences and co-stimulatory molecules of a T cell receptor (TCR), wherein the T cell receptor (TCR) cytoplasmic sequences and co-stimulatory molecules cooperate to initiate signal transduction following antigen receptor engagement, and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0241] In some embodiments, the intracellular region of the CAR may comprise a primary cytoplasmic signal sequence acting in a stimulatory manner, which may comprise what is known as an immunoreceptor tyrosine-based activation motif or IT AM signal motif. Examples of ITAM comprising a primary cytoplasmic signal sequence that can be included in the CAR include those from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d proteins.

[0242] In some embodiments, the intracellular region of the CAR may comprise an ITAM comprising a primary cytoplasmic signaling domain (e.g., CD3ζ itself or in combination with any other desired cytoplasmic domain that can be used in the context of the CAR. For example, the cytoplasmic domain of the CAR comprises a CD3ζ chain portion and an intracellular co-stimulatory signaling domain. The co-stimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of the co-stimulatory molecule.

[0243] In some embodiments, the CAR may comprise a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a CD137 signaling domain, or any combination thereof. The order of the one or more T cell signaling domains on the CAR can be changed by those skilled in the art as needed.

[0244] Methods of Generating Chimeric Antigen Receptors

[0245] T cells comprising such receptors, and their uses (e.g., uses for the treatment of cancers) are known in the art, and described in detail, for example, in Brentjens et al., 2010, Molecular Therapy, 18: 4,666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pp. 1-9; Till etal., 2008, Blood, 112: 2261-2271; Park etal., Trends Biotechnol., 29: 550-557,2011;Grupp et al., NEnglJMed., 368: 1509-1518, 2013; Han et al., J. Hematol Oncol., 6: 47, 2013; P

[0246] In certain embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the present invention. In some preferred embodiments, the isolated nucleic acid molecule encodes the chimeric antigen receptor of the present invention.Attorney Docket No.: 091787-8001W001

[0247] In some embodiments, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In some preferred embodiments, the vector of the present invention is, for example, a plasmid.

[0248] In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule or the vector as described above. In some preferred embodiments, the host cell is a T cell. In some preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0249] Polynucleotides and Recombinant Methods

[0250] The present disclosure provides isolated polynucleotides that encode the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof provided herein. The term “nucleic acid” or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer etal., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

[0251] DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0252] The isolated polynucleotide that encodes the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0253] The present disclosure provides vectors comprising the isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes the antibodies or antigen-binding fragments thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linkedAttorney Docket No.: 091787-8001W001 to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g. herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (e.g. SV40), lambda phage, andM13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, pl5TV-L, pProl8, pTD, pRSlO, pLexA, pACT2.2, pCMV-SCRIPT. RTM., pCDM8, pCDNAl.l / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXTl, pCDEF3, pSVSPORT, pEF-Bos etc.

[0254] Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0255] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CLDN6 / 9 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe,' Kluyveromyces hosts such as, e.g. K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus,' yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida,' Trichoderma reesia (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalism and filamentous fungi such as, e.g. Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0256] Suitable host cells for the expression of glycosylated antibodies or antigen-fragment thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),Attorney Docket No.: 091787-8001W001 Aedes albopictus (mosquito), Drosophila melanogaster (fruiffly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[0257] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and their derivatives.

[0258] Host cells are transformed with the above-described expression or cloning vectors for anti-CLDN6 / 9 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the antibody may be produced by homologous recombination known in the art. In certain embodiments, the host cell is capable of producing the antibody or antigen-binding fragment thereof provided herein.

[0259] The present disclosure also provides a method of expressing the antibody or an antigenbinding fragment thereof provided herein, comprising culturing the host cell provided herein under the condition at which the vector of the present disclosure is expressed. The host cells used to produce the antibodies or antigen-binding fragments thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's MediumAttorney Docket No.: 091787-8001W001 (DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U. S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U. S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to a person skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to a person skilled in the art.

[0260] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. colt. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[0261] The anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0262] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is presentAttorney Docket No.: 091787-8001W001 in the antibody. Protein A can be used to purify antibodies that are based on human gammal, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N. J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0263] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[0264] Conjugate

[0265] In some embodiments, the anti-CLDN6 / 9 antibodies and antigen-binding fragments thereof provided herein is linked to one or more conjugate moieties. As used herein, the term “conjugate moiety” refers to any molecule (chemical or biochemical, naturally-occurring or non-coded) which is different from the anti-CLDN6 / 9 antibodies and antigen-binding fragments thereof provided herein. In some embodiments, the conjugate moiety is selected from a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide, protein, therapeutic agent, a diagnostic agent, or a toxin (e.g., a chemotherapeutic agent).

[0266] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.

[0267] In some embodiments, the conjugate moiety is a polymer. The polymer can be branched or unbranched. The polymer can be of any molecular weight, such as an average molecular weight of between about 2 kDa to about 100 kDa, between about 5 kDa and about 50 kDa, between about 12Attorney Docket No.: 091787-8001W001 kDa to about 40 kDa or between about 20 kDa to about 35 kDa. Exemplary polymers include but are not limited to polyamides, polycarbonates, polyalkylenes and derivatives thereof including, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic and methacrylic esters, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers including polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl acetate), and polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses including alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt, polypropylene, polyethylenes including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene.

[0268] In some embodiments, the conjugate moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.

[0269] In some embodiments, the conjugate moiety is a lipid. Exemplary lipids include but are not limited to a fatty acid, eicosanoid, prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g., mono-, di-, tri- substituted glycerols), glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, a phospholipid.

[0270] In some embodiments, the conjugate moiety is a therapeutic agent. The therapeutic agent can be any of those known in the art. Examples of therapeutic agents that are contemplated herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobins, beta-adrenergic blocking agents, calcium channel blockers,Attorney Docket No.: 091787-8001W001 coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomemetics, angiotensin converting enzyme (ACE) inhibitors, diuretics, inotropes, cholesterol and triglyceride reducers, bile acid sequestrants, fibrates, 3-hydroxy-3- methylgluteryl (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agents, alpha- adrenergic blocking agents, centrally acting antiadrenergic agents, vasodilators, potassium- sparing agents, thiazides and related agents, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulins and analogs, alpha- glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thizaolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators, antithyroid agents, thyroid hormones, bulk forming agents, laxatives, antiperistaltics, flora modifiers, intestinal adsorbents, intestinal anti-infectives, antianorexic, anticachexic, antibulimics, appetite suppressants, antiobesity agents, antacids, upper gastrointestinal tract agents, anticholinergic agents, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotectives, histamine H2-receptor antagonists, mucosal protective agent, proton pump inhibitors, H. pylori eradication therapy, erythropoieses stimulants, hematopoietic agents, anemia agents, heparins, antifibrinolytics, hemostatics, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors, thromboxane- A2inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralcorticoids, corticosteroids, selective immunosuppressive agents, antifungals, drugs involved in prophylactic therapy, AIDS-associated infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptse inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopoptides, lincosamides, macrolies, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amebicies, biguanides, cinchona alkaloids, folic acid antagonists, quinoline derivatives, Pneumocystis carinii therapy, hydrazides, imidazoles, triazoles, nitroimidzaoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapy, barbiturates and derivatives, benzodiazepines, gamma aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigrane preparations, biological response modifiers, carbamic acid eaters, tricyclic derivatives, depolarizing agents, nondepolarizing agents,Attorney Docket No.: 091787-8001W001 neuromuscular paralytic agents, CNS stimulants, dopaminergic reagents, monoamine oxidase inhibitors, COMT inhibitors, alkyl sulphonates, ethylenimines, imidazotetrazines, nitrogen mustard analogs, nitrosoureas, platinum-containing compounds, antimetabolites, purine analogs, pyrimidine analogs, urea derivatives, antracyclines, actinomycinds, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitor antineoplastics, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamind reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisooxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergenic extracts, nonsteroidal agents, leukotriene receptor antagonists, xanthines, endothelin receptor antagonist, prostaglandins, lung surfactants, mucolytics, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetic agents, halogenated hydrocarbons, esters of amino benzoic acid, amides (e.g. lidocaine, arti caine hydrochloride, bupivacaine hydrochloride), antipyretics, hynotics and sedatives, cyclopyrrolones, pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitor, heparin antagonists, adsorbents, emetics, opoid antagonists, cholinesterase reactivators, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, and the like.

[0271] In some embodiments, the conjugate moiety is a toxin. The toxin can be any of those known in the art. Examples of toxin that are contemplated herein include, but are not limited to, taxol, deruxtecan, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), mertansine, emtansine, DM1, maytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin di one, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mer captopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibioticsAttorney Docket No.: 091787-8001W001 (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), anti-mitotic agents (e.g., vincristine and vinblastine), a topoisomerase inhibitor, and a tubulin-binders.

[0272] In some embodiments, the anti-CLDN6 / 9 antibodies and antigen-binding fragments thereof provided herein is linked to one or more conjugate moieties via a linker. The linker refers to the direct or indirect connection between the antibody and the drug. The linker can be connected to the antibody in many ways, such as through surface lysine, reductive coupling to oxidized carbohydrates, and through cysteine residues released by reducing interchain disulfide bonds. Many ADC connection systems are known in the art, including connections based on hydrazones, disulfides, and peptides. The linker can be any of those known in the art.

[0273] Synthetic Methods

[0274] The therapeutic agent used for the conjugate provided herein are commercially available or can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, by employing standard synthetic methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith, M. B., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, incorporated by reference herein, are useful and recognized reference textbooks of organic synthesis known to those in the art.

[0275] Any available techniques can be used to make the conjugates provided herein or compositions including them, and intermediates and components (e.g., carriers and modifiers) useful for making them.

[0276] The conjugate described herein can be synthesized by coupling the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof to the therapeutic agent via one or more cysteine or lysine residues of the antibody. For example, the conjugates of Formula (A) provided herein can prepared by subjecting the antibody to a reducing agent, for example, dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, for example, by gel filtration, and subsequently treating the antibody with a linker-payload containing a suitable reactive moiety, forAttorney Docket No.: 091787-8001W001 example, a maleimido group. Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO. Conjugates can be purified using known protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration / diafiltration.

[0277] Conjugates provided herein, once produced, can be characterized using a variety of assays known to those skilled in the art to determine whether the conjugates have biological activity. For example, the conjugates can be characterized by conventional assays, including but not limited to those assays described below, to determine the binding activity, binding specificity, cytotoxicity, stability etc.

[0278] Pharmaceutical Composition

[0279] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid molecule encoding the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers. Antibodies provided herein can also be produced in vivo by delivery of the nucleic acid molecule encoding the antibodies or antigenbinding fragments thereof provided herein, such as, for example, in-vitro-transcribed mRNA, or expression vectors. Methods are known in the art for polynucleotide delivery for antibody expression in vivo, see, for example, Rybakova, Y. et al, Molecular Therapy, vol. 27 (8), pp. 1415-1423 (2019); Deal, C. E. etal, Vaccines, 2021, 9, 108.

[0280] The present disclosure further provides pharmaceutical compositions comprising an expression vector comprising the nucleic acid molecule encoding the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers.

[0281] In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, without limitation, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, and adenovirus vector. Examples of non-viral vectors include, without limitation, naked DNA, plasmid, exosome, mRNA, and so on. In certain embodiments, the expression vector is suitable for gene therapy in human. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV), or adenovirus vector. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carriers are polymeric excipients, such as without limitation, microspheres, microcapsules, polymeric micelles and dendrimers. The polynucleotides, or polynucleotide vectors of the present disclosure may be encapsulated, adhered to, or coated on the polymer-based components by methods known in the art (see for example, W. Heiser, NonviralAttorney Docket No.: 091787-8001W001 gene transfer techniques, published by Humana Press, 2004; U. S. patent 6025337; Advanced Drug Delivery Reviews, 57(15): 2177-2202 (2005)).

[0282] In a further aspect, the present disclosure provides a pharmaceutical composition comprising the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof or the conjugate as disclosed herein. For the purposes of administration, in some embodiments, the anti-CLDN6 / 9 antibodies or conjugates provided herein are administered as a raw chemical or are formulated as pharmaceutical compositions.

[0283] In another aspect, the present disclosure provides a pharmaceutical composition comprising the anti-CLDN6 / 9 antibodies or conjugates as disclosed herein and an acceptable carrier. Examples of suitable carriers include, but are not limited to, buffers for maintenance of proper composition pH (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants.

[0284] The administration of the pharmaceutical composition provided herein may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), intratracheal, intranasal, epidermal, transdermal, oral or parenteral administration including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion or intracranial.

[0285] The pharmaceutical composition provided herein can be formulated into various suitable dosage forms depending on administration routes, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, subdermals, aerosols, powders, and sprays.

[0286] The pharmaceutical composition provided herein comprises pharmaceutically effective amount of the anti-CLDN6 / 9 antibodies or conjugates as disclosed herein. As used herein, the term “pharmaceutically effective amount” refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Pharmaceutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.Attorney Docket No.: 091787-8001W001

[0287] The pharmaceutical composition provided herein may be administered at a dosage level between about 0.001 mg / kg to about 200 mg / kg of the subject's body weight, between about 0.001 mg / kg to about 150 mg / kg of the subject's body weight, between about 0.001 mg / kg to about 100 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 90 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 80 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 70 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 60 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 50 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 40 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 30 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 20 mg / kg of the subject's body weight, between about 0.01 mg / kg to about 15 mg / kg of the subject's body weight, between about 0.1 mg / kg and about 15 mg / kg of the subject's body weight, between about 0.1 mg / kg and about 20 mg / kg of the subject's body weight, between about 0.1 mg / kg to about 5 mg / kg, between about 0.1 mg / kg to about 4 mg / kg, between about 0.1 mg / kg to about 3 mg / kg, between about 0.1 mg / kg to about 2 mg / kg or about 0.1 mg / kg to about 1 mg / kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg / kg to about 10 mg / kg of the subject's body weight.

[0288] In some embodiments, the therapeutically effective amount of the anti-CLDN6 / 9 antibodies or conjugates in the pharmaceutical composition provided herein may be administered on a regular schedule, i.e., daily, weekly, monthly, or yearly basis or on an irregular schedule with varying administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In some embodiments, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In some embodiments, the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The number and frequency of dosages corresponding to a completed course of therapy will be determined according to the recommendations of the relevant regulatory bodies and judgment of a health-care practitioner. The therapeutically effective amounts described herein refer to total amounts administered for a givenAttorney Docket No.: 091787-8001W001 time period; that is, if more than one different antibody or conjugate described herein is administered, the therapeutically effective amounts correspond to the total amount administered. It is understood that the specific dose level for a particular subject depends upon a variety of factors including the activity of the specific antibody or conjugate, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, combination with other active agents, and the severity of the particular disease undergoing therapy.

[0289] The anti-CLDN6 / 9 antibodies, conjugates and pharmaceutical composition provided herein can also be administered in combination with one or more additional therapeutic agents to increase the overall therapeutic effect. The one or more additional therapeutic agents can be administered prior to, concurrent with, or after the administration of the anti-CLDN6 / 9 antibodies, conjugates or pharmaceutical compositions described herein. The additional therapeutic agents may be an anticancer agent, an immunosuppressant agent, and an anti-infectious agent. Examples of anticancer agent include but are not limited to methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. Examples of immunosuppressant agents include but are not limited to cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate cyclophosphamide, prednisorie, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, and infliximab. Examples of anti-infectious agents include but are not limited to β-lactam antibiotics (such as penicillin G, penicillin V, cloxacilliin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacarnpicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, and ticarcillin), aminoglycosides (such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin), macrolides (such as azithromycin, clarithromycin, erythromycin, lincomycin, and clindamycin), tetracyclines (such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline), quinolones (such as cinoxacin and nalidixic acid), fluoroquinolones (such as ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, and trovafloxicin), and sulfonamides (such as sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, and sulfacetamide).Attorney Docket No.: 091787-8001W001

[0290] Kits

[0291] The present disclosure also provides pharmaceutical kits comprising one or more containers filled with one or more of the anti-CLDN6 / 9 antibodies or conjugates and / or compositions of the present disclosure, optionally together with additional therapeutical agents, a device(s) for administering the anti-CLDN6 / 9 antibodies, conjugates and / or compositions, and written instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products. The anti-CLDN6 / 9 antibodies, conjugates or compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration. For continuous administration, a package or kit can include the anti-CLDN6 / 9 antibodies, conjugates, or compositions in each dosage unit (e.g., solution or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses daily, weekly, or monthly, for a predetermined length of time or as prescribed. If varying dosing level of the anti-CLDN6 / 9 antibodies, conjugates or compositions over time is desired, a package or kit may contain a sequence of dosage units which provide the desired variability.

[0292] Use

[0293] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a CLDN6-related disease or disorder in a subject in need thereof. In another aspect, methods are provided to treat a disease, disorder or condition that is responsive to CLDN6 inhibition in a subject in need thereof.

[0294] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof provided herein, the nucleic acid molecule encoding the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof, the conjugates and / or the pharmaceutical composition provided herein. In certain embodiments, the subject is human.

[0295] In some embodiments, the subject to be treated has been identified as having a CLDN6-related disease or disorder. In some embodiments, the CLDN6 related disease, disorder or condition is responsive to CLDN6 inhibition. In some embodiments, the CLDN6 related disease, disorder or condition is associated with dysregulation of CLDN6 mediated signaling, or more specifically, associated with up-regulated CLDN6 signaling.

[0296] In some embodiments, the disease or disorder is associated with cells dysregulation of CLDN6 mediated signaling. In some embodiments, the dysregulation of CLDN6 mediated signalingAttorney Docket No.: 091787-8001W001 includes dysregulation of CLDN6 -expressing cell proliferation, compared with control level (e.g., the level in a healthy subject).

[0297] In some embodiments, the disease or disorder is a cancer.

[0298] In certain embodiments, the cancer is selected from: ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, cancer of the urinary bladder, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small bowel cancer, including cancer of the ileum, in particular small bowel adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, a germ cell tumor such as a teratocarcinoma or an embryonal carcinoma, in particular a germ cell tumor of the testis, and the metastatic forms thereof.

[0299] In certain embodiments, the disease is selected from the group consisting of hepatocellular carcinoma (HCC), esophageal adenocarcinoma (EAC), gastric cancer (GC), testicular germ cell tumors (TGCT), ovarian cancer (OV), lung cancers (LU D and LUSC), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC).

[0300] In certain embodiments, the anti-CLDN6 / 9 antibody or antigen-binding fragment or the conjugates provided herein may be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 2 mg / kg or about 0.1 mg / kg to about 1 mg / kg of the subject's body weight. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.

[0301] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.Attorney Docket No.: 091787-8001W001

[0302] The anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof or the conjugates provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0303] In some embodiments, the anti-CLDN6 / 9 antibodies or antigen-binding fragments thereof or the conjugates provided herein may be administered alone or in combination a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof or the antibody drug conjugates disclosed herein may be administered in combination with a second therapeutic agent.

[0304] In some embodiments, the second therapeutic agent is selected from a chemotherapeutic agent, radiation, or an agent that modulates, e.g., enhances or inhibits, the expression or activity of an Fc receptor, e.g. an Fc-gamma receptor, such as a cytokine. Typical cytokines for administration during treatment include granulocyte colony-stimulating factor (G- CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-y (IFN-y), and tumor necrosis factor (TNF).Typical therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin, cisplatin, taxotere, 5- fluoruracil, methotrexat, gemzitabin and cyclophosphamide.

[0305] In certain of these embodiments, the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof or the conjugates provided herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment thereof or the antibody drug conjugates and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof or the antibody drug conjugates administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof or the antibody drug conjugates administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment or the antibody drug conjugates and the second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments thereof or the antibody drug conjugates disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to theAttorney Docket No.: 091787-8001W001 Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)) or protocols well known in the art.

[0306] In another aspect, the present disclosure provides methods of detecting the presence or amount of CLDN6 in a sample.

[0307] The presence and / or amount of CLDN6 in an interested biological sample can be indicative of whether the subject from whom the biological sample is derived could likely respond to an anti-CLDN6 antibody. Various methods can be used to determine the presence and / or amount of CLDN6 in a test biological sample from the subject. For example, the test biological sample can be exposed to anti-CLDN6 / 9 antibody or antigen-binding fragment thereof, which binds to and detects the expressed CLDN6 protein. Alternatively, CLDN6 can also be detected at nucleic acid expression level, using methods such as qPCR, reverse transcriptase PCR, microarray, serial analysis of gene expression (SAGE), fluorescence in situ hybridization (FISH), and the like. In some embodiments, the method provided herein comprises contacting a test sample with the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof provided herein, and determining the presence or the amount of CLDN6 in the test sample. In some embodiments, the test sample is derived from an epithelial tissue. In certain embodiments, presence or up-regulated level of the CLDN6 in the test biological sample indicates likelihood of responsiveness. The term “up-regulated” as used herein, refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or greater, in the expression level of CLDN6 in the test sample, as compared to the CLDN6 expression level in a reference sample as detected using the same method. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from whom the test sample is obtained.

[0308] In another aspect, the present disclosure provides a method of diagnosing a CLDN6 related disease, disorder or condition in a subject, comprising a) contacting a sample obtained from the subject with the antibody or an antigen-binding fragment thereof or the antibody drug conjugates provided herein; b) determining the presence or amount of CLDN6 in the sample; and c) correlating the presence or the amount of CLDN6 to existence or status of the CLDN6 related disease, disorder or condition in the subject.

[0309] In another aspect, the present disclosure also provides use of the antibody or antigenbinding fragment thereof or the conjugates provided herein in the manufacture of a medicament forAttorney Docket No.: 091787-8001W001 treating, preventing or alleviating a CLDN6 related disease, disorder or condition in a subject, in the manufacture of a diagnostic reagent for diagnosing a CLDN6 related disease, disorder or condition.

[0310] Some of the Examples provided herein describe the synthesis of the compounds and conjugates disclosed herein as well as intermediates used to prepare the compounds and conjugates. However, it is to be understood that these examples do not limit the present disclosure and are only meant to suggest a method of practicing the present disclosure. Persons skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds or conjugates of the present disclosure, and alternative methods for preparing the compounds and conjugates of the present disclosure are deemed to be within the scope of the present disclosure. Besides, persons skilled in the art will also understand that individual steps described herein or in the separate batches of a compound may be combined. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure.EXAMPLES

[0311] Different features and variations of the disclosure are demonstrated in the following examples, which serve as illustrations rather than limitations. Experts in the field will recognize that these specific examples are merely representative of the invention as detailed in the subsequent claims. Every embodiment and feature outlined in this application should be viewed as interchangeable and combinable with all other embodiments included.Example 1: Generation of Anti-Hu-CLDN6 Antibodies Using Hybrid oma Methods, Screening and Characterization

[0312] This example illustrates the methods using mouse hybridoma technology to generate anti-hu-CLDN6 antibodies, and methods to screen and select antibodies for further characterization.

[0313] Hybridoma generation: Mouse spleens and lymph nodes were collected after immunization with human CLDN6 and processed following standard protocols. Mouse B cells were isolated using the EasySep Mouse B Cell Isolation Kit (StemCell, Cat# 19854A). These B cells were then fused with SP2 / 0-Agl4 myeloma cells (ATCC, CRL 1581) using PEG. According to standard procedures, the fused cells were plated in six-well plates with semi-solid ClonalCell-HY Cloning-Medium D (StemCell, Cat# 03804). Monoclonal hybridoma clones were selected using the Clone Pix 2 Machine (Molecular Devices) and transferred into 96-well plates for culture in low-Ig HT medium.

[0314] Flow Cytometry assays: After 10-14 days of culture, supernatants were collected for primary screening using Flow Cytometry. Cell lines expressing the cell surface CLDN6 proteins,Attorney Docket No.: 091787-8001W001 such as PA-1 cells (ATCC, Cat# CRL-1572), were resuspended in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at pH 7.4 (Flow Cytometry buffer). A total of 50,000 cells per well in 50 pL of Flow Cytometry buffer were distributed into 96- well round-bottom plates (Corning, Cat# 3799). Subsequently, 50 pL of culture supernatant from each hybridoma clone (or purified antibodies at the specified concentration) was added to the wells and incubated at 4°C for 30 minutes. The plates were then washed twice with Flow Cytometry buffer, and 50 pL of goat antimouse antibody-PE (BioLegend, Cat# 405307) diluted 1:200 was added to each well. After another 30-minute incubation at 4°C and two additional washes with Flow Cytometry buffer, the samples were resuspended in 50 pL of Flow Cytometry buffer. The plates were analyzed using a CytoFlex LX (Beckman Coulter) or an iQue3 flow cytometry (Sartorius).

[0315] The parental hybridoma candidates identified from the primary screen were expanded into 48 or 24-well plates, and a confirmatory Flow Cytometry was conducted following the primary screening protocol. This step aimed to further verify and screen for anti-human or anti-mouse CLDN6 and CLDN9 binders.

[0316] Internalization assay of hybridoma hits: The supernatants from the hybridoma candidates identified in the primary screen were also assessed for their ability to internalize in cells expressing cell surface CLDN6 proteins. Culture supernatants from individual hybridoma clones (or purified antibodies at the specified concentration) were labeled with a mouse antibody internalization reagent (Sartorius, Cat# 90564) at 37°C for 15 minutes. PA-1 cells (50,000 cells in 40 pL per well) were then treated with the labeled antibody in 96-well round-bottom plates at 36°C for 1 hour. The plates were analyzed using a CytoFlex LX or an iQue3 flow cytometry.

[0317] Purification of hybridoma antibodies: Hybridoma clones were verified using a primary screening Flow cytometry assay. Positive clones were expanded to 30 mL cultures in serum-free medium, and the antibodies were purified as follows: The supernatant media were clarified by centrifugation at 300g for 10 minutes to remove cells and then filtered through a 0.22 micron filter. The clarified supernatant was mixed with protein A resin (Thermo Fisher Scientific, Cat# A26458), equilibrated with PBS buffer, and incubated with gentle rotation for 1.5 hours at room temperature. After incubation, the mixture was loaded into a column, and the resin was washed with ten column volumes of PBS buffer containing 0.5M NaCl, followed by elution with 0. IM glycine-HCl at pH 2.8. The eluent was quickly neutralized with IM Tris-HCl (pH 8.5) and then exchanged to PBS. The binding and blocking capabilities of the purified hybridoma antibodies were further validated using the aforementioned protocol.Attorney Docket No.: 091787-8001W001

[0318] The purified hybridoma antibodies were tested for their binding activities to PA-1 cells in flow cytometry assay. As shown in Fig. 1A, the hybridoma antibodies, 2D5, 2D11, 8C11, 10C1, and 7C10, displayed binding activities to PA-1 cells at 1 ug / ml. Antibody binding is represented as median fluorescence intensity (MFI)

[0319] The purified hybridoma antibodies (0.4 ug / ml) were then assessed in the flow cytometrybased internalization assay. The internalization activities of 2D11, 7C10, and 10C1 antibodies in PA-1 cell line have been shown in Fig. IB

[0320] The internalization activities of the purified hybridoma antibodies were further investigated using secondary ADC in a cell-based cytotoxicity assay. 0.1 ug / ml of the hybridoma antibody was labeled with 0.2 ug / ml of a Fab fragment of an anti-mouse IgG Fc specific antibody conjugated to monomethyl auristatin E (MMAE) with a cleavable linker (Fab(aMuFc)-MMAE) (Moradec, Cat# AM-202-AE) at 37C for 15 minutes. PA-1 cells (2,000 cell / well) were seeded in 96-well flat-bottom plate (Corning, Cat# 3595), and the cells were incubated with the labeled antibodies in an incubator with a 5% CO2 atmosphere at 37°C for 4 days. The cell viability was measured using the CellTiter-Glo reagent (Promega, Cat# G9241). As illustrated in Fig. 1C, the hybridoma antibodies, 7C10, 2D11, and 2D5, internalized in PA-1 cells along with the Fab(aMuFc)-MMAE and displayed cytotoxicity at 0.1 ug / ml of antibodies.

[0321] Sequencing and amplification of hybridoma antibody clones

[0322] RNA Extraction. Monoclonal anti-human CLDN6 hybridoma hits were cultured to a density of 1-3 x 105cells in standard hybridoma medium (DMEM / F12, 10% FBS, 1% Glutamax, 1% penicillin / streptomycin) for 7-10 days in a T75 flask, maintaining over 80% viability.Approximately 1-3 million cells were pelleted in a 15 mL Falcon tube by centrifugation at 300g for 5 minutes. The pelleted cells were washed by resuspending them in 5 mL of ice-cold PBS. After removing the PBS, the cells were resuspended in 600 pL of Buffer RLT Plus (Qiagen, Cat# 74134). Total RNA was then isolated from the lysate according to the manufacturer's protocol (Qiagen, cat # 74134).

[0323] PCR amplification to generate cDNA, The synthesis of cDNA utilized specific reverse PCR primers along with switch oligos for heavy and kappa chains. One microgram of RNA was employed as a template for reverse transcription using the SMART Scribe Reverse Transcriptase kit from TAKARA (Cat# 639537). The reaction included 10 pM primers (Integrated DNA Technologies), a 10 mM deoxynucleotide triphosphate mix (New England Biolab, Cat# N0447S), H2O, and an 80 U / pL RNase inhibitor (Invitrogen, Cat# 10000840).Attorney Docket No.: 091787-8001W001

[0324] For the 5 ’-RACE PCR reactions, constant region-specific reverse primers were paired with a universal forward primer. The PCR products were gel-purified, cloned into the TOPO TA vector (Thermo Fisher, Cat# 451641), and transformed into competent cells (Thermo Fisher, Cat# 451641). After transformation and blue / white screening, white colonies were selected and allowed to grow overnight in LB broth containing carbenicillin. Miniprep-purified plasmids were sequenced using Ml 3 forward and T7 forward primers. The variable domain sequences of the anti-human CLDN6 hybridomas are summarized in Table 1 and included in the attached Sequence Listing. The CDR sequences of the antibody variable sequences (VH and VL) listed in Table 1 are exemplarily identified according to the Kabat numbering scheme and are set forth in Table 14. The initial identified 2D5 clone contains a cysteine residue (91, Kabat numbering) in CDR-L3 (HQCHRSPPT; SEQ ID NO: 70). To facilitate the generation of ADC, this cysteine residue was replaced with serine (the variant was referred to as 2D5-S) or alanine (the variant was referred to as 2D5-A).

[0325] Table 1. Anti-human CLDN6 hybridoma antibody variable sequencesAntibodies variable amino acid sequence2D5-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVASINSNGG RTYYPDSEKGRFTMSRDNAKNTLYLQMSSLKSEDTAMYYCARWGGQYVMDYWG HGTSVTVSS (SEQ ID NO: 1)2D5-VL QIVLTQSPAIMSASLGERVTMTCTADSSVTSSYLHWYQQKPGSSPKLWIFATSNLAS GVPARFTGSGSGTSYSLTISSMEAEDAATYYCHQCHRSPPTFGAGTKLELK (SEQ ID NO: 2)2D5-A-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVASINSNGG RTYYPDSEKGRFTMSRDNAKNTLYLQMSSLKSEDTAMYYCARWGGQYVMDYWG HGTSVTVSS (SEQ ID NO: 3)2D5-A-VL QIVLTQSPAIMSASLGERVTMTCTADSSVTSSYLHWYQQKPGSSPKLWIFATSNLAS GVPARFTGSGSGTSYSLTISSMEAEDAATYYCHQAHRSPPTFGAGTKLELK (SEQ ID NO: 4)2D5-S-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVASINSNGG RTYYPDSEKGRFTMSRDNAKNTLYLQMSSLKSEDTAMYYCARWGGQYVMDYWG HGTSVTVSS (SEQ ID NO: 5)2D5-S-VL QIVLTQSPAIMSASLGERVTMTCTADSSVTSSYLHWYQQKPGSSPKLWIFATSNLAS GVPARFTGSGSGTSYSLTISSMEAEDAATYYCHQSHRSPPTFGAGTKLELK (SEQ ID NO: 6)2D11-VH EVQLQESGPGLAKPSQTLSLTCSVTGYSITSDYWNWIRKFPGNKLEYMGYISYSGST YYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCATLLSGSSPWFAYWGQGTL VTVSA (SEQ ID NO: 7)2D11-VL DIVMTQAAFSNPVTLGTSASISCRSSKSLLHYNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVETEDVGVYYCAQNLELPYTFGGGTKLEIK(SEQ ID NO: 8)Attorney Docket No.: 091787-8001W001 3D6-VH QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGS TDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCASPDGYYVYYAMDYWG QGTSVTVSS (SEQ ID NO: 9)3D6-VL QIVLTQSPAIMCSSPGEKVTMTCSASSSVRYMYWYQQKPGSSPRLLIYDTSKLASGV PVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELK (SEQ ID NO: 10)4B1-VH EIQLQQTGPELVKPGASVKISCKTSDYSFTDYIMLWVKQSHGKSLEWIGNINPYYGS TSYNLNFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYNGKPFYAMDYWGQ GTSVTVSS (SEQ IDNO: 11)4B1-VL DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANSLVNG VPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 12)6H6-VH EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMHWVRQAPGKGLEWVARIRGKS SNYATYYADSVKDRFTISSDDSQSMLYLQMNNLKTEDTAMYHCVRGVHNSNSWF AYWGQGTLVTVSA (SEQ ID NO: 13)6H6-VL DIVMTQSQKFMSTSVGDRVAITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRH TGVPDRFTGSGYGTDFTLTISNVKSEDLADYFCLQHWNYPLTFGAGTKVELK (SEQ ID NO: 14)6H11-VH EVQLQESGPGLAKPSQTLSLTCSVTGYSITSDYWNWIRKFPGNKLEYMGYISYSGST YYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCATLLSGSSPWFAFWGQGTL VTVSA (SEQ IDNO: 15)6H11-VL DIVMTQAAFSNPVTLGTSASISCRSSKSLLHYNGITYLYWYLQKPGQSPQLLIYQMS NLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPYTFGGGTKLEIK (SEQ ID NO: 16)7C10-VH EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMHWVRQAPGKGLEWVARIRSKS GNFATFYADSVKDRFTISRDDSQSMLYLQMSNLKIEDTAIYYCVRGVHNSNSWFAY WGQGTL VTVSA (SEQ ID NO: 17)7C10-VL DFVMTQSQKFMSTSVGDRVSITCKASQNVRTAVAWFQQKPGQSPKALIYLASNRHT GVPDRFTGSGFGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAGTKVELK (SEQ ID NO: 18)8C11-VH QVQLQQSGTELVRPGTSVKVSCKASGYAFTNYLIQWIKQRPGQGLEWIGVINPGSG GTNYNEKFKGKATLTADKSSNTAYMQLGSLTSEDSAVYFCARLYDGYYEDYYAM DYWGQGTSVTVSS (SEQ ID NO: 19)8C11-VL QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLAS GVPPRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPWTFGGGTKLEIK (SEQ ID NO: 20)10C1-VH QVQLQQSGAELARPGASVKMSCKASGYTFTNYTIHWVKQRPGQGLDWIGYINPSS GYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASNLAWFAYWGQG TLVTVSA (SEQ ID NO: 21)10C1-VL DIQMTQSPSSLSASLGQRVSLTCRASQEISGYLSWLQQKPDGTIIPLIYAASTLYSGVP RRFSGSRSGSDYSLTISSLESEDFADYYCLQYASHPLTFGAGTKLDLK (SEQ ID NO:22)Example 2: In Vitro Assays of Anti-CLDN6 / 9 Chimeric Antibodies

[0326] This example illustrates cell-based assays used to characterize the functional activity of the anti-CLDN6 / 9 chimeric antibodies described in the previous Examples.

[0327] Generation of recombinant IgG versions of anti-CLDN6 / 9 antibodiesAttorney Docket No.: 091787-8001W001

[0328] The heavy and light chain variable domain of mouse anti-CLDN6 / 9 antibodies were synthesized with human constant region to make recombinant anti-CLDN6 / 9 chimeric antibody constructs. The expression of recombinant anti-CLDN6 / 9 chimeric antibodies were performed using Expi293 expression system in accordance with the instruction provided. The ratio of the plasmids for the heavy chain and the light chain was kept at 1 to 1 for the transfection reaction and the transfected cells were cultured for 6 days before harvest.

[0329] Recombinant IgG molecules were purified with the following protocols. Supernatant media were clarified by centrifugation at 300 g for 10 min to remove cells and by filtration with 0.22 pm filter. Clarified supernatant media were mixed with MabSelect protein A resin equilibrated with PBS buffer and incubated with gentle rotation for 1.5 h at room temperature. After incubation, the slurry was loaded into a column and the resin was washed with 20 column volumes of PBS buffer containing 0.15M NaCl then eluted with 3 column volumes of 50 mM sodium phosphate, pH 3.0. The pH of the eluent was quickly adjusted to pH 5.2 with 1 M Tris-HCl, pH 9.0 and buffer exchanged into to PBS buffer with PD-10 column.

[0330] Binding to hu-CLDN6 expressed on cells

[0331] To examine the binding of anti-CLDN6 / 9 antibodies to hu-CLDN proteins expressed on cells, we used ES2 cells (ATCC, Cat# CRL-1978) stably expressing hu-CLDN protein family and flow cytometry analysis.

[0332] Hu-CLDN3 (Umprot, 015551), hu-CLDN4 (Umprot, 014493), hu-CLDN6 (Umprot, P56747), hu-CLDN9 (Uniprot, 095484), mu-CLDN6 (Uniprot, Q9Z262) were cloned into lentiviral vector and the virus was packaged according to the instruction of the virus packaging kit (Lenti-X™ Packaging Single Shots, Cat# 631275, Takada). The ES2 cells were transduced and selected with puromycin. The cell line stably expressing CLDN proteins were incubated with anti-CLDN6 / 9 antibodies for 30 minutes in PBS with 0.5% BSA, 1 mM EDTA, and 0.1% sodium azide (flow cytometry buffer) at 4°C. The cells were washed, and then incubated with lOnM phycoerythrin (PE) conjugated anti-Human Fc Ab (Biolegend, Cat# 409304) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX with a 561 nm channel. Data were analyzed with FlowJo software. Antibody binding is represented as MFI.

[0333] As shown by the results in Fig. 2A-2C, the anti-CLDN6 / 9 antibodies, 2D5, 2D5-A, 2D5-S, 2D11, 7C10, and 10C1, bind to ES2 cells expressing Hu-CLDN6 (ES2-huCLDN6) and PA-1 cells dose-dependently.Attorney Docket No.: 091787-8001W001

[0334] Dose-dependent binding activities of the anti-CLDN6 / 9 antibodies, 2D5, 2D5-A, and 2D5-S, to ES2 cells expressing Hu-CLDN9 (ES2-huCLDN9) are shown in Fig. 2D.

[0335] As shown in Fig. 2E-2G, the anti-CLDN6 / 9 antibodies, 2D5, 2D11, 7C10, and 10C1, do not bind to ES2 cells expressing Hu-CLDN3 (ES2-huCLDN3) and ES2 cells expressing Hu-CLDN4 (ES2-huCLDN4), but bind to ES2 cells expressing Mu-CLDN6 (ES2-muCLDN6).

[0336] The flow cytometry determined binding affinity EC50 values are summarized in Table 2.

[0337] Table 2. Binding activity of anti-CLDN6 / 9 IgGs to hu-CLDN6 and hu-CLDN9 expressed on cells.EC50 (nM)CloneES2-huCLDN6 ES2-huCLDN92D5 0.865 0.8692D5-A 0.818 0.4222D5-S 0.793 0.4302D11 2.2857C10 64.27510C1 3.914

[0338] Internalization assay of anti-CLDN6 / 9 chimeric antibodies: The anti-CLDN6 / 9 antibodies were also assessed for their ability to internalize in PA-1 cells. 0.1 ug / ml of the anti-CLDN6 / 9 antibodies, 2D5, 2D5-A, 2D5-S, 2D11, 7C10, and 10C1, were labeled with a human antibody internalization reagent (Sartorius, Cat# 4722) at 37°C for 15 minutes. PA-1 cells (50,000 cells in 40 pL per well) were then treated with the labeled antibody in 96-well round-bottom plates in an incubator with a 5% CO2 atmosphere at 37°C for 3 hours. The plates were analyzed using a CytoFlex LX, and antibody internalization is represented as MFI. As shown in Fig. 3A-3B, internalization of anti-CLDN6 / 9 chimeric antibodies was confirmed in PA- 1 cell line.Example 3: In vitro and in vivo Assays of Anti-CLDN6 / 9 Chimeric ADCs

[0339] This example illustrates cell-based assays and in vivo study used to characterize the functional activity of the anti-CLDN6 / 9 chimeric antibodies and ADCs described in the previous Examples.

[0340] DAR8 ADC Conjugation with maleimide linker-payload and purification

[0341] Antibody in 1XPBS, pH 7.4 buffer (4 mg / mL) was added with 1 mM EDTA, 14 molar excesses of TCEP (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 150 rpm withAttorney Docket No.: 091787-8001W001 a benchtop rotating shaker (Serial# 300100524). After that, 14 molar excesses of linker-payload, BL003-exatecan (BL003-Ex, see structure below), to antibody were added, and the reaction mixture was left at room temperature for 1 hour to reach complete conjugation. At the end of the reaction, a 10-fold molar excess of N-Acetyl-L-cysteine (CAS Number: 616-91-1) was added to quench the reaction. The conjugation mixture was then buffer exchanged to IxPBS, pH7.4 by Amicon concentrator (Cat# UFC803024, 50 kD cutoff) via concentration and dilution to remove excess small molecules and free drug.A?.,_A 'r°HA <?BL003-Ex

[0342] Binding to hu-CLDN6 expressed on cells

[0343] The PAI cells and ES2-huCLDN6 cells were incubated with anti-CLDN6 / 9 ADCs for 30 minutes in flow cytometry buffer at 4°C. The cells were washed and then incubated with lOnM phycoerythrin (PE) conjugated anti-Human Fc Ab (Biolegend, Cat# 409304) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX with a 561 nm channel. Data were analyzed with FlowJo software. ADC binding is represented as MFI.

[0344] As shown by the results in Fig. 4A and Fig.4B, the anti-CLDN6 / 9 ADCs, 2D5 chimera-BL003-Ex, 2D5-A chimera-BL003-Ex, 2D5-S chimera-BL003-Ex, and 7C10 chimera-BL003-Ex, bind to PA-1 cells and ES2-huCLDN9 cells in a dose-dependent manner.

[0345] Internalization assay of anti-CLDN6 / 9 chimeric ADCs: The anti-CLDN6 / 9 ADCs were also assessed for their ability to internalize in PA-1 cells and ES2-huCLDN9 cells, respectively. 0.1 ug / ml of the anti-CLDN6 / 9 antibodies, 2D5, 2D5-A, and 2D5-S, were labeled with a mouse antibody internalization reagent (Sartorius, Cat# 4722) at 37°C for 15 minutes. The cells (50,000 cells in 40 pL per well) were then treated with the labeled antibody in 96-well round-bottom plates at 36°C for 3 hours. The plates were analyzed using a CytoFlex LX. The MFI values are shown in Fig. 5A-5B.

[0346] In vitro potency of anti-CLDN6 / 9 chimeric ADCs: The anti-CLDN6 / 9 ADCs were examined for their in vitro cytotoxicity. PA-1 and JEG-3 cells (ATCC, Cat# HTB-36) (2,000 cells / well) were seeded in 96-well flat-bottom plates (Corning, Cat# 3595). The cells wereAttorney Docket No.: 091787-8001W001 incubated with the anti-CLDN6 / 9 ADCs in an incubator with a 5% CO2 atmosphere at 37°C for 4 days, and then the cell viability was measured using the CellTiter-Glo reagent (Promega, Cat# G9241). As shown in Fig. 6A-6B, the anti-CLDN6 / 9 ADCs show a dose-dependent cytotoxicity in PA-1 (Fig. 6A) and JEG-3 cells (Fig. 6B).

[0347] The IC50 values of the anti-CLDN6 / 9 ADCs are summarized in Table 3.

[0348] Table 3. In vitro potency of anti-CLDN6 / 9 ADCs.IC50 (nM)ADC PA-1 JEG-32D5 chimera-BL003-Ex 0.037 0.0882D5-A chimera-BL003-Ex 0.317 2.1382D5-S chimera-BL003-Ex 0.163 1.6777C10 chimera-BL003-Ex 0.059

[0349] In vivo efficacy of anti-CLDN6 / 9 chimeric ADCs: The anti-CLDN6 / 9 ADCs were evaluated for their efficacy in CDX model. PA-1 cells (5 million / mouse) were subcutaneously inoculated in BALB / c nude mice. When tumors reached 100mm3, the mice received two injections of each anti-CLDN6 / 9 ADCs (2 mg / kg, intraperitoneally) on day 0 and day 7. As shown by the tumor growth curves in Fig. 7, the anti-CLDN6 / 9 ADCs displayed anti-tumor efficacy in PA-1 tumor model, and the 2D5-S chimera-BL003-Ex ADC showed a stronger efficacy compared to other chimera-BL003-Ex ADCs.Example 4: Assessment of Humanized Versions of 2D5-S

[0350] This example illustrates the preparation of humanized versions of the murine anti-hu-CLDN6 / 9 antibody derived from the hybridoma clone 2D5-S.

[0351] Ab humanization

[0352] The CDR graft technology was used to humanize the variable domain of heavy chain and light chain of 2D5-S. The constant region CHI, CH2, CH3 were further substituted with human IgGl sequences shown in Table 4. The sequences for heavy chain in Kabat numbering CDR1(31-35), CDR2(50-65), CDR3 (95-102) and the light chain in Kabat numbering CDRl(24-34), CDR2(50-56), and CDR3(89-97) were kept unchanged during the humanization process (Table 5).While the framework for heavy chain FRl(l-30), FR2(36-49), FR3(66-94) and FR4(103-l 13) and the light chain FRl(l-23), FR2 (35-49), FR3(57-88) and FR4 (98-107) (Table 6) were humanizedAttorney Docket No.: 091787-8001W001 with mostly closed human germline sequences other than the Vernier zone residues. The human germline was exemplified as listed in Table 7 (Prihoda et al., 2022). The list of variable sequences from humanized variants shown in Table 8.

[0353] Table 4. The human IgGl constant regionHeavy chain constant region Light chain constant region Human ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT RTVAAPSVFIFPPSDEQLKS IgGl VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTASVVCLLNNFYPREAK GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP VQWKVDNALQSGNSQESV APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH TEQDSKDSTYSLSSTLTLS EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS KADYEKHKVYACEVTHQ VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG GLSSPVTKSFNRGEC (SEQ QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV ID NO: 24) EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 23)IgGllala ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT RTVAAPSVFIFPPSDEQLKS VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTASVVCLLNNFYPREAK GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP VQWKVDNALQSGNSQESV APEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSH TEQDSKDSTYSLSSTLTLS EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS KADYEKHKVYACEVTHQ VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG GLSSPVTKSFNRGEC (SEQ QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV ID NO: 26) EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 25)

[0354] Table 5. The variable domain of chimeric 2D5-SName CDR1 Kabat numbering CDR2 Kabat numbering CDR3 Kabat numbering Heavy SYGMS (31-35) (SEQ ID SINSNGGRTYYPDSEKG (SO- WGGQYVMDY (95- chain NO: 27) 65) (SEQ ID NO: 28) 102) (SEQ ID NO: 29) Light TADSSVTSSYLH (24- ATSNLAS (50-56) (SEQ ID HQSHRSPPT (89-97) chain 34) (SEQ ID NO: 30) NO: 31) (SEQ ID NO: 32)

[0355] Table 6 The framework sequence of chimeric 2D5-SFR1 Kabat FR2 Kabat FR3 Kabat numbering FR4 Kabat Name numbering numbering numbering RFTMSRDNAKNTLYLQ WGHGTSVTVS EVQLVESGGGLVQPG WVRQTPDKRLE MSSLKSEDTAMYYCA S (103-113) Heavy GSLKLSCAASGFTFS LVA (36-49) R (66-94) (SEQ ID NO: (SEQ ID NO: 36) chain (1-30) (SEQ ID NO: 33) (SEQ ID NO: 34) 35)QIVLTQSPAIMSASLG WYQQKPGSSPK GVPARFTGSGSGTSYS FGAGTKLELKLight ERVTMTC (1-23) (SEQ LWIF (35-49) LTIS SMEAEDAATYYC (98-107) (SEQ chain ID NO: 37) (SEQ ID NO: 38) (57-88) (SEQ ID NO: 39) ID NO: 40)Attorney Docket No.: 091787-8001W001

[0356] Table 7. Examples of human germline used for humanizationHumanized Human HC Human HC Human LC Human LC joining region variants germline joining region germlinehu2D5S_seql IGHV3-23*04 IGHJ4*01 IGKV3- 11*01 IGKJ2*01 hu2D5S_seq2 IGHV3-23*04 IGHJ4*01 IGKV1-39*O1 IGKJ2*01 hu2D5S_seq3 IGHV3-23*04 IGHJ4*01 IGKV3-7*02 IGKJ2*01 hu2D5S_seq4 IGHV3-64*04 IGHJ4*01 IGKV1-39*O1 IGKJ2*01 hu2D5S_seq5 IGHV3-64*04 IGHJ4*01 IGKV3- 11*01 IGKJ2*01 hu2D5S_seq6 IGHV3-64*04 IGHJ4*01 IGKV3-7*02 IGKJ2*01 hu2D5S_seq7 IGHV3-64*04 IGHJ4*01 IGKV 1-9*01 IGKJ2*01 hu2D5S_seq8 IGHV3-23*04 IGHJ4*01 IGKV 1-9*01 IGKJ2*01

[0357] Table 8. The list of variable sequences from humanized variantsHumanized variable sequence from heavySeq ID chain Humanized variable sequence from light chain EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP EIVLTQSPATLSLSPGERATLSCTADSSVTSSY DSEKGRFTMSRDNAKNTLYLQMNSLRAEDT LHWYQQKPGQAPRLWIFATSNLASGVPARFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYSLTISSLEPEDFAVYYCHQSHRSP _seql (SEQ ID NO: 41) PTFGQGTKLEIK (SEQ ID NO: 42)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP DIQLTQSPS SL S AS VGDRVTITCT ADS S VT S S Y DSEKGRFTMSRDNSKNTLYLQMNSLRAEDT LHWYQQKPGQAPRLWIFATSNLASGVPDRFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYTLTISSLQPEDFATYYCHQSHRSP _seq2 (SEQ ID NO: 43) PTFGQGTKLEIK (SEQ ID NO: 44)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP EIVLTQSPGTLSLSPGERATLSCTADSSVTSSY DSEKGRFTMSRDNSKNTLYLQMNSLRAEDT LHWYQQKPGQAPRLWIFATSNLASGIPARFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYTLTISSLQPEDFAVYYCHQSHRSP _seq3 (SEQ ID NO: 45) PTFGQGTKLEIK (SEQ ID NO: 46)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP DIQLTQSPS SL SAS VGDRVTITCT ADS SVT S S Y DSEKGRFTMSRDNSKNTLYLQMNSLRTEDT LHWYQQKPGQAPRLWIFATSNLASGVPDRFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYTLTISSLQPEDFATYYCHQSHRSP _seq4 (SEQ ID NO: 47) PTFGQGTKLEIK (SEQ ID NO: 48)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP EIVLTQSPATLSLSPGERATLSCTADSSVTSSY DSEKGRFTMSRDNSKNTLYLQMNSLRTEDT LHWYQQKPGQAPRLWIFATSNLASGIPARFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYTLTISSLEPEDFAVYYCHQSHRSP _seq5 (SEQ ID NO: 49) PTFGQGTKLEIK (SEQ ID NO: 50)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP EIVLTQSPGTLSLSPGERATLSCTADSSVTSSY DSEKGRFTMSRDNSKNTLYLQMNSLRTEDT LHWYQQKPGQAPRLWIFATSNLASGIPARFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTDYTLTISSLQPEDFAVYYCHQSHRSP _seq6 (SEQ ID NO: 51) PTFGQGTKLEIK (SEQ ID NO: 52)Attorney Docket No.: 091787-8001W001 EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP DIQLTQSPSFL S AS VGDRVTITCT ADS S VT S S Y DSEKGRFTMSRDNSKNTLYLQMNSLRTEDT LHWYQQKPGQAPRLWIFATSNLASGVPDRFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTEYTLTISSLQPEDFATYYCHQSHRSP _seq7 (SEQ ID NO: 53) PTFGQGTKLEIK (SEQ ID NO: 54)EVQL VESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQAPGKGLELVASINSNGGRTYYP DIQLTQSPSFL SAS VGDRVTITCT ADS SVT S S Y DSEKGRFTMSRDNSKNTLYLQMNSLRAEDT LHWYQQKPGQAPRLWIFATSNLASGVPDRFShu2D5S AVYYCARWGGQYVMDYWGQGTLVTVSS GSGSGTEYTLTISSLQPEDFATYYCHQSHRSP _seq8 (SEQ ID NO: 55) PTFGQGTKLEIK (SEQ ID NO: 56)

[0358] Binding to hu-CLDN6 expressed on cells

[0359] To examine the binding of humanized anti-CLDN6 / 9 antibodies to hu-CLDN6 proteins expressed on cells, we used ES2-huCLDN6 cells and flow cytometry analysis.

[0360] As illustrated in Fig. 8A, the humanized versions of 2D5-S, hu2D5-S_seql - seq8 antibodies, showed comparable ES2-huCLDN6 cell binding activities to the 2D5-S chimeric antibody in flow cytometry analysis. Antibody binding is represented as MFI.

[0361] DAR8 ADC Conjugation with maleimide linker-payload and purification

[0362] Antibody in 1XPBS, pH 7.4 buffer (4 mg / mL) was added with 1 mM EDTA, 14 molar excesses of TCEP (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 150 rpm with a benchtop rotating shaker (Serial# 300100524). After that, 14 molar excesses of linker-payload, BL003-Ex, to antibody were added, and the reaction mixture was left at room temperature for 1 hour to reach complete conjugation. At the end of the reaction, a 10-fold molar excess of N-Acetyl-L-cysteine (CAS Number: 616-91-1) was added to quench the reaction. The conjugation mixture was then buffer exchanged to IxPBS, pH7.4 by Amicon concentrator (Cat# UFC803024, 50 kD cutoff) via concentration and dilution to remove excess small molecules and free drug.

[0363] Binding activity of anti-CLDN6 / 9 humanized ADCs to PA-1 cells

[0364] PA-1 cells were incubated with the humanized ADCs, hu2D5-S_seql, 4, 6, and 7 conjugated with BL003-Ex, for 30 minutes in flow cytometry buffer at 4°C. The cells were washed and then incubated with lOnM PE conjugated anti-Human Fc Ab (Biolegend, Cat# 409304) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX. Data were analyzed with FlowJo software, and ADC binding is represented as MFI. The humanized ADCs, hu2D5-S_seql-8 ADCs conjugated with BL003-Ex, displayed dose-dependent binding activities to PA-1 cells (Figure 8B).

[0365] Internalization of anti-CLDN6 / 9 humanized ADCs in PA-1 cellsAttorney Docket No.: 091787-8001W001

[0366] The humanized ADCs, hu2D5-S_seql, 4, 6, and 7 conjugated with BL003-Ex, were also evaluated for their ability to internalize in PA-1 cells. 0.1 ug / ml of the anti-CLDN6 / 9 ADCs were labeled with a human antibody internalization reagent (Sartorius, Cat# 4722) at 37°C for 15 minutes. PA-1 cells (10,000 cells in 50 gL per well) were then treated with the labeled ADC in 96-well flat-bottom plates. The plates were incubated in an incubator with a 5% CO2 atmosphere at 37°C for 24 hours, and the ADC internalization was measured using the Incucyte live-cell analysis system (Sartorius). As shown in Fig. 8C, the humanized anti-CLDN6 / 9 ADCs show internalization activities in a time-dependent manner in PA-1 cells. In contrast, non-binding ADC did not internalize in PA-1 cells.

[0367] In vitro potency of anti-CLDN6 / 9 humanized ADCs:

[0368] The anti-CLDN6 / 9 ADCs were examined for their in vitro cytotoxicity in PA-1 cells. The cells (2,000 cells / well) were seeded in 96-well flat-bottom plates (Corning, Cat# 3595), and then treated with anti-CLDN6 / 9 ADCs, hu2D5-S_seql, 4, 6, and 7 conjugated with BL003-Ex, and hu2D5-S_seq6 conjugated with VC-MMAE. The plates were incubated in an incubator with a 5% CO2 atmosphere at 37°C for 4 days, and then the cell viability was measured using the CellTiter-Glo reagent (Promega, Cat# G9241). As shown in Fig. 8D, the anti-CLDN6 / 9 humanized ADCs show a dose-dependent cytotoxicity inPA-1 cells.Example 5: In vitro Assays of Humanized 2D5-S_seq6 Antibodies

[0369] This example illustrates the characterization of humanized 2D5-S_seq6 antibodies produced in wild-type IgGl constant region (WT) and Fc-silenced mutant IgGl (L234A / L235A) constant region (LALA). The variable regions of the antibodies are shown in Table 9.

[0370] Table 9. The full humanized variant sequence hu2D5-S_seq6_WT and hu2D-5_S-seq6_LALAhu2D5-S seq6 WT Heavy chain sequence _ EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGRTYYPDSEK GRFTMSRDNSKNTLYLQMNSLRTEDTAVYYCARWGGQYVMDYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 57) _hu2D5-S seq6 WT Light chain sequence _EIVLTQSPGTLSLSPGERATLSCTADSSVTSSYLHWYQQKPGQAPRLWIFATSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAVYYCHQSHRSPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWAttorney Docket No.: 091787-8001W001 CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC (SEQ ID NO: 58) _hu2D5-S seq6 LALA Heavy chain sequence _ EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLELVASINSNGGRTYYPDSEK GRFTMSRDNSKNTLYLQMNSLRTEDTAVYYCARWGGQYVMDYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 59) _hu2D5-S seq6 LALA Light chain sequence _ EIVLTQSPGTLSLSPGERATLSCTADSSVTSSYLHWYQQKPGQAPRLWIFATSNLASGIPARFSGSG SGTDYTLTISSLQPEDFAVYYCHQSHRSPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 60) _

[0371] Analytical HIC

[0372] To determine the solubility of antibody, HIC-HPLC was used. Antibody was filtered by 0.2 gm filter spinning tube (Costar #8160). 14 uL, 1 mg / mL of ADC in IX PBS, pH 7.4 was injected over a TSKgel Butyl-NPR Column, 4.6mmx x 10cm, 2.5pm (TOSOH BIOSCIENCE LLC) running with an Agilent HPLC system (1260 Infinity II Bio Flexible Pump). The ADC was eluted as a reduction of gradient elution concentrations of NH4SO4 in buffer A (25 mM sodium phosphate, 1.5 M NH4SO4, pH 7), to completely buffer B (25mM sodium phosphate; 20% IP A, pH 7) with a flow rate 0.4 mL / min within 20 min. The column was then further washed with buffer B for 20 min and equilibrated with buffer A for continuous running.

[0373] As shown in Fig.9A, the hu2D5-S_seq6_WT antibody was eluted from HIC profile at 8.554 min, which suggests the high solubility of humanized antibody.

[0374] Analytical size exclusion chromatography

[0375] AnSec was utilized to determine potentially high molecular weight and aggregation species of antibody. AnSec was run with buffer that contains 100 mM sodium phosphate, 100 mM NaCl, pH 7.0, 15% isopropanol, with a flow rate 0.4 ml / min, 30 min. The AnSec was run with a column TSKgel G30000SW-XL, 7.8 mm x 30 cm (TOSOH BIOSCIENCE LLC).

[0376] The Sec profile of hu2D5-S_seq6_WT (Fig. 9B, left panel) and hu2D5-S_seq6_LALA (Fig.9B, right panel) showed high purity of target antibody after single column Protein A purification.

[0377] SPR binding determination

[0378] The SPR binding affinity measurement was determined with Biacore 8K (Cytiva, Cat#29327020) by Gene script. The HBS-EP Buffer (lOmM Hepes, 150mM NaCl, 3mM EDTA,Attorney Docket No.: 091787-8001W001 0.005% Tween-20) was used for binding affinity measurement. The immobilization of ligand was performed at 25 °C while HBS-EP+ was used as running buffer. The sensor chip surface of flow cells 1 and 2 were activated by freshly mixed 50 mmol / L N-Hy dr oxy succinimide (NHS) and 200 mmol / L l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Afterwards, ligand in 10 mmol / L NaAC as injected into the flow cell 2 to achieve conjugation of appropriate Resonance Unit respectively, while flow cell 1 was set as blank. After the amine coupling reaction, the remaining active coupling sites on chip surface were blocked with 1 mol / L ethanolamine hydrochloride. The assay was performed at 25 °C and the running buffer was HBS-EP+. Analyte was diluted and injected over the surface of flow cell 1 and 2 as association phase, followed by injecting running buffer as dissociation phase. All the data was processed using the Biacore 8K Evaluation Software version 4.0. Flow cell 1 and blank injections of running buffer in each cycle were used as double reference for Resonance Units subtraction.

[0379] hu2D5-S_seq6_WT (left panel) and hu2D5-S_seq6_LALA (right panel) have high binding affinity with Claudin-6, with a calculated binding affinity with human claudin-6 (VLP) are 2.36 nM and 0.71 nM respectively (Fig.9C and Table 10).

[0380] As shown in Fig.9D and Table 10, hu2D5-S_seq6-WT (left panel) and hu2D5-S_seq6_LALA (right panel) have high binding affinity with Claudin-9, with a calculated binding affinity with human claudin-9 (VLP) are 4.32 nM and 5.22 nM respectively.

[0381] Table 10. Binding affinity of hu2D5-S_seq6 antibodiesLigand Analyte Kon (l / Ms) Koff (l / s) KD (M) Human Claudin-6 (VLP) hu2D5-S_seq6_WT 7.30E+05 1.72E-03 2.36E-09 Human Claudin-6 (VLP) hu2D5-S_seq6_LALA 8.80E+05 6.27E-04 7.13E-10 Human Claudin-9 (VLP) hu2D5-S_seq6_WT 5.29E+05 2.28E-03 4.32E-09 Human Claudin-9 (VLP) hu2D5-S_seq6_Lala 5.65E+05 2.95E-03 5.22E-09

[0382] Binding to CLDN proteins expressed on cells

[0383] The cell line expressing cell surface CLDN proteins were incubated with the hu2D5-S_seq6_WT or hu2D5-S_seq6_LALA antibody for 30 minutes in flow cytometry buffer at 4°C. The cells were washed and then incubated with lOnM PE conjugated anti -Human Fc Ab (Biolegend, Cat# 409304) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX. Data were analyzed with FlowJo software. Antibody binding is represented as MFI.

[0384] As shown in Fig. 10A and Fig. 10B, the humanized 2D5-S_seq6 antibodies bind to PA-1 cells and ES2-huCLDN6 cells dose-dependently.Attorney Docket No.: 091787-8001W001

[0385] Dose-dependent binding activity of the humanized 2D5-S_seq6 antibodies to ES2- huCLDN9 cells is shown in Fig. 10C.

[0386] Fig. 10D shows the cross-reactivity of the humanized 2D5-S_seq6 antibodies with mouse CLDN6 proteins expressed on ES2 cells.

[0387] In Fig. 10E and Fig. 10F, the humanized 2D5-S_seq6 antibodies do not bind to ES2- huCLDN3 and ES-huCLDN4 cells.

[0388] The flow cytometry determined binding affinity EC50 values are summarized in Table 11.

[0389] Table 11. Binding activity of the humanized 2D5-S_seq6 antibodies to CLDN proteins expressed on cells.EC50 (nM)AbPA-1 ES2-huCLDN6 ES2-huCLDN9 ES2-muCLDN6hu2D5-S_seq6-WT 0.963 1.663 0.891 0.948hu2D5-S_seq6-LALA 0.573 0.854 0.359 0.827

[0390] Internalization of humanized 2D5-S seq6 antibodies

[0391] The humanized 2D5-S_seq6 antibodies were evaluated for its ability to internalize in PA-1 and ES2-hCLDN9 cells. 0.1 ug / ml of the antibody was labeled with a human antibody internalization reagent (Sartorius, Cat# 4722) at 37°C for 15 minutes. PA-1 and ES2-hCLDN9 cells (10,000 cells each in 50 pL per well) were then treated with the labeled ADC in 96- well flat-bottom plates. The plates were incubated in an incubator with a 5% CO2 atmosphere at 37°C for 24 hours, and the ADC internalization was measured using the Incucyte live-cell analysis system (Sartorius). As shown in Fig. 11A-11B, the humanized anti-CLDN6 / 9 ADCs, both hu2D5-S_seq6_WT antibody and hu2D5-S_seq6_LALA antibody, show efficient internalization activities in PA-1 (Fig.11 A) and ES2-huCLDN9 cells (Fig. 11B) in a time-dependent manner.Example 6: in vitro assessment of humanized 2D5-S_seq6 ADCs

[0392] This example illustrates in vitro characterizations of humanized 2D5-S ADCs including conjugation methods and in vitro activities.

[0393] DAR8 ADC Conjugation with maleimide linker-payload and purification

[0394] Antibody in 1XPBS, pH 7.4 buffer (4 mg / mL) was added with 1 mM EDTA, 14 molar excesses of TCEP (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 150 rpm with a benchtop rotating shaker (Serial# 300100524). After that, 14 molar excesses of linker-payloadAttorney Docket No.: 091787-8001W001 (BLOOl-Ex (see structure below), BL003-Ex or GGFG-DXd (MedChemExpress, Cat # HY-1363 IE)) to antibody were added, and the reaction mixture was left at room temperature for 1 hour to reach complete conjugation. At the end of the reaction, a 10-fold molar excess of N-Acetyl-L-cysteine (CAS Number: 616-91-1) was added to quench the reaction. The conjugation mixture was then buffer exchanged to IxPBS, pH7.4 by Amicon concentrator (Cat#UFC803024, 50 kD cutoff) via concentration and dilution to remove excess small molecules and free drug.o. 1BLOOl-Ex

[0395] DAR4 ADC Conjugation with maleimide linker-payload and purification

[0396] Antibody in IXPBS, pH 7.4 buffer (4 mg / mL) was added with 1 mM EDTA, 2.6 molar excesses of TCEP (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 150 rpm with a benchtop rotating shaker (Serial# 300100524). After that, 8.3 molar excesses of mc-vc-PAB-MMAE (Cat. No.: HY-15575) to antibody were added, and the reaction mixture was left at room temperature for 1 hour to reach complete conjugation. At the end of the reaction, a 10-fold molar excess of N-Acetyl-L-cysteine (CAS Number: 616-91-1) was added to quench the reaction. The conjugation mixture was then buffer exchanged to IxPBS, pH7.4 by Amicon concentrator (Cat#UFC803024, 50 kD cutoff) via concentration and dilution to remove excess small molecules and free drug. HIC profile of hu2D5-S_seq6-WT-VC-PABC-MMAE is shown in Fig. 12C.

[0397] Engineered cysteine mediated site-specific conjugation

[0398] The antibody (2D5S-seq6-A114C or 2D5S-seq6-A114C / S239C) in IXPBS, 1 mMEDTA, pH 7.4 buffer (4 mg / mL) was fully reduced by adding 14 molar excesses of TCEP (Cat# P1021-10). The mixture was incubated at 37 °C for 2 hours at 150 rpm with a benchtop rotating shaker (Serial# 300100524). After that, the buffer was exchanged to 10 mM histidine, 10% propylene glycol, pH 6 to remove excess TCEP. Then, the reaction was supplemented with 2.5% (v / v) 1 M Tris-HCl, pH 7.6 to raise pH 6.5 followed by adding 20 x of DHAA (L-DEHYDRO ASCORBIC ACID, Cat# AC250932500, Fisher scientific), and incubation at RT for 2 hr. After reoxidation of the disulfide bond, the reaction mixture was supplemented with 10% (v / v) DMA, 5x VC-PABC-MMAE (Medchemexpress, Cat # HY-15575, for DAR2), 8.3 x VC-PABC-MMAE (for DAR4) andAttorney Docket No.: 091787-8001W001 incubated at RT overnight. The buffer was then exchanged to IxPBS, pH 7.4, and the DAR was determined by HIC-HPLC and RP-HPLC.

[0399] DAR determination by HIC

[0400] To determine the drug to antibody ratio (DAR), HIC-HPLC was used. ADC was filtered by 0.2 pm filter spinning tube (Costar #8160). 14 uL, 1 mg / mL of ADC in IX PBS, pH 7.4 was injected over a TSKgel Butyl-NPR Column, 4.6mmx x 10cm, 2.5pm (TOSOH BIOSCIENCE LLC) running with an Agilent HPLC system (1260 Infinity II Bio Flexible Pump). The ADC was eluted as a reduction of gradient elution concentrations of NH4SO4 in buffer A (25 mM sodium phosphate, 1.5 M NH4SO4, pH 7), to completely buffer B (25mM sodium phosphate; 20% IP A, pH 7) with a flow rate 0.4 mL / min within 20 min. The column was then further washed with buffer B for 20 min and equilibrated with buffer A for continuous running. HIC profile of hu2D5-S_seq6_WT-BL001-Ex is shown in Fig. 12A.

[0401] Analytical size exclusion chromatography

[0402] AnSec was utilized to determine potentially high molecular weight and aggregation species of freshly made ADC conjugates and ADC under stressed conditions. AnSec was run with buffer that contains 100 mM sodium phosphate, 100 mM NaCl, pH 7.0, 15% isopropanol, with a flow rate 0.4 ml / min, 30 min. The AnSec was run with a column TSKgel G30000SW-XL, 7.8 mm x 30 cm (TOSOH BIOSCIENCE LLC). Sec profile of hu2D5-S_seq6_WT-BL001-Ex ADC is shown in Fig.12B

[0403] Reverse phase HPLC chromatography

[0404] 50 pg ADC (1 mg / mL) in IxPBS, pH 7.4 was reduced with 50 mM DTT at 37 °C for 1 hr. The sample was filtered and loaded onto a reverse phase column PLRP-S 1000A 8 uM 150 X 4.6 mm. The column was run at 70 °C with mobile phase A (2% Acetonitrile, 0.1% TFA) and mobile phase B (99.9% Acetonitrile, 0.1% TFA). The DAR variants were separated by elution gradient from 25% mobile phase B to 95% mobile phase B.

[0405] Concentration determination

[0406] To determine the concentration, the extinction coefficient of the linker payload at 280 nm was first measured by lambert's law. The total extinction coefficient of ADC was calculated by the sum of antibody extinction coefficients and the total linker payload extinction coefficient at 280 nm. The ADC concentration was calculated by using the reading at 280 nm from UV-vis spectroscopy divide the total ADC extinction coefficient multiply by 10A6 uM.

[0407] Binding to CLDN proteins expressed on cellsAttorney Docket No.: 091787-8001W001

[0408] The cell line expressing cell surface CLDN proteins were incubated with the hu2D5-S_seq6-BL001-Ex ADCs for 30 minutes in flow cytometry buffer at 4°C. The cells were washed and then incubated with lOnM PE conjugated anti-Human Fc Ab (Biolegend, Cat# 409304) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX. Data were analyzed with FlowJo software. ADC binding is represented as MFI.

[0409] As shown in Fig. 13A and Fig. 13B, the hu2D5-S_seq6-BL001-Ex bind to PA-1 cells and ES2-huCLDN6 cells dose-dependently. Dose-dependent binding activity of the hu2D5-S_seq6-BLOOl-Ex to ES2-huCLDN9 cells is shown in Fig. 13C. Fig. 13D shows the cross-reactivity of the hu2D5-S_seq6-BL001-Ex with mouse CLDN6 proteins expressed on ES2 cells. In Fig. 13E and Fig. 13F, the hu2D5-S_seq6-BL001-Ex do not bind to ES2-huCLDN3 and ES-huCLDN4 cells. The flow cytometry determined binding affinity EC50 values are summarized in Table 12.

[0410] Table 12. Binding activity of the 2D5-S antibody to CLDN proteins expressed on cells.EC50 (nM)ADC PA-1 ES2-huCLDN6 ES2-huCLDN9 ES2-muCLDN6 hu2D5-S_seq6_WT-BL001-Ex 2.089 2.312 2.011 1.796hu2D5-S_seq6_LALA-BL001-Ex 2.044 4.797 2.281 1.658

[0411] Internalization of hu2D5-S seq6-BL001 -Ex ADC

[0412] The hu2D5-S_seq6-BL001-Ex ADCs were evaluated for their ability to internalize in PA-1 and ES2-hCLDN9 cells. 0.5 ug / ml of the ADC was labeled with a human antibody internalization reagent (Sartorius, Cat# 4722) at 37°C for 15 minutes. PA-1 and ES2-hCLDN9 cells (10,000 cells each in 50 pL per well) were then treated with the labeled ADC in 96-well flat-bottom plates. The plates were incubated in an incubator with a 5% CO2 atmosphere at 37°C for 24 hours, and the ADC internalization was measured using the Incucyte live-cell analysis system (Sartorius). As shown in Figure 14, both ADCs, hu2D5-S_seq6_WT-BL001-Ex and hu2D5-S_seq6_LALA-BL001-Ex, show internalization activities in PA-1 (A) and ES2-hCLDN9 cells (B) in a time-dependent manner.

[0413] In vitro potency of hu2D5-S seq6-BL001-Ex ADC:

[0414] The hu2D5-S_seq6-BL001-Ex ADCs were examined for their in vitro cytotoxicity. PA-1, JEG-3, ES2-huCLDN6, and ES2-huCLDN9 cells (2,000 cells / well) were seeded in 96-well flatbottom plates (Corning, Cat# 3595). The cells were incubated with the ADC in an incubator with a 5% CO2 atmosphere at 37°C for 4 days, and then the cell viability was measured using the CellTiter-Glo reagent (Promega, Cat# G9241). As shown in Figure 15, both ADCs, hu2D5-Attorney Docket No.: 091787-8001W001 S_seq6_WT-BL001-Ex and hu2D5-S_seq6_LALA-BL001-Ex, show anti-tumor activity in multiple cells expressing CLDN6 or CLDN9 in a dose-dependent manner. The IC50 values of the hu2D5-S_seq6-BL001-Ex ADCs are summarized in Table 13.

[0415] Table 13. In vitro potency of humanized 2D5-S_seq6 ADCs.IC50 (nM)ADC PA-1 JEG-3 ES2-huCLDN6 ES2-huCLDN9 hu2D5-S_seq6_WT-BL001-Ex 0.358 2.216 0.273 0.254hu2D5-S_seq6_LALA-BL001-Ex 0.354 2.172 0.354 0.432

[0416] To further understand the mechanisms underlying hu2D5-S_seq6_WT Ab and ADC, we evaluated ADCC, CDC and ADCP activities. For the ADCC assay, PA-1 cells were seeded in a 96-well plate at the density of 5000 cells / well and then treated with isotype Ab, hu2D5-S_seq6_WT Ab, and hu2D5-S_seq6_BL001-Ex (2ug / ml each) for 30 minutes. Next, the cells were incubated with IL-2-activated human peripheral blood mononuclear cells (PBMCs) (effector cells: target cells ratio 20: 1, 40: 1) for 4 h. For the CDC assay, the serum samples were obtained from Quidel Ortho (Al 13). PA-1 cells were seeded in a 96-well plate at the density of 5000 cells / well and then treated with isotype Ab, hu2D5-S_seq6_WT Ab, and hu2D5-S_seq6_BL001-Ex (2ug / ml each) for 30 minutes. Subsequently, the cells were incubated with the human serum samples (final concentration 20%) for 4h. After incubation, the cells were lysed with lysis buffer for 45min, and the CytoTox 96® Non-Radio Reagent (Promega, G1780) was added to measure the OD490 values, after which the dead rate (%) was calculated. For the ADCP assay, PA-1 cells were labeled with CellTrace Yellow cell proliferation kit (Invitrogen, C34573) and THP-1 cells with CellTrace Violet cell proliferation kit (Invitrogen, C34571). The labeled PA-1 cells were plated in a 96-well round bottom plate together with 2ug / ml of isotype Ab, hu2D5-S_seq6_WT Ab, and hu2D5-S_seq6_BL001-Ex for 30 minutes prior to the addition of effector cells. After 30 minutes, the labeled THP-1 cells were added to each well, and the plate was incubated for 1 hour at 37°C and 5% CO2. After 1 hour, cells were washed with PBS and run on a CytoFlex LX (Beckman Coulter). Data were analyzed using FlowJo analysis software and graphed using GraphPad Prism. ADCP activity was determined as percentage of Yellow / Violet double positive THP-1 cell events.

[0417] We observed hu2D5-S_seq6_WT Ab and ADC-mediated ADCC in PA-1 cells with an increase in the E: T ratio (Fig. 16A). We then conducted a CDC assay using human serum as a compliment. The serum alone induced lysis of 26.9% to 28.5%, whereas in the presence of hu2D5-Attorney Docket No.: 091787-8001W001 S_seq6_WT Ab or ADC, we observed lysis of 71.0% to 72.7% and 65.5% to 66.2% respectively in PA-1 cells (Fig. 16B). As shown in Fig. 16C, the addition of hu2D5-S_seq6_WT Ab or ADC, but not isotype control, enhanced the phagocytosis of PA-1 cells by THP-1 cells.

[0418] Bystander killing effect of hu2D5-S seq6-BL001-Ex ADC:

[0419] In order to confirm whether humanized 2D5-S_seq6_WT-BL001-Ex induced bystander killing, a coculture cell killing assay was carried out. The ES2 cells were transduced with NucLight Red Lentivirus reagent (Sartorius Cat# 4476) and selected with puromycin. The CLDN6-positive PA-1 cells and the CLDN6-negative ES2 cells labeled with NucLight Red were mixed and cultured at 2: 1 ratio overnight. The cells were treated with ADC (2 nM) for 2 days, and then the number of ES2 cells labeled with NucLight Red was determined using the Incucyte live-cell analysis system. As shown in Fig. 17A, hu2D5-S_seq6_WT-BL001-Ex showed a superior bystander killing effect against ES2 cells, compared to hu2D5-S_seq6_WT-GGFG-DXd, and the negative control ADC did not induce ES2 cell killing. Both hu2D5-S_seq6_WT-BL001-Ex and hu2D5-S_seq6_WT-GGFG-DXd ADCs did not show a cytotoxicity against HER2-negative ES2 cells in a monoculture cell killing assay (Fig. 17B).Example 7: In vivo Tumor Models to Evaluate Activity of humanized 2D5-S_seq6_WT-BLOOl-Ex ADC

[0420] This example illustrates in vivo tumor model studies of the functional activity of humanized ADCs.

[0421] The anti-CLDN6 / 9 ADC was evaluated for its efficacy in CDX model. Cancer cells (5 million / mouse) were subcutaneously inoculated in BALB / c nude mice. When tumors reached 100mm3, the mice received i.v. injections of each ADC on day 0. As shown by the tumor growth curves in Fig. 18A-18C, the hu2D5-S_seq6_WT-BL001-Ex ADC displayed anti-tumor efficacy in (Fig. 18A) OVACR3, (Fig. 18B) PA-1, and (Fig. 18C) ES2-huCLDN9 tumor models.

[0422] Tumors coded as LD1-0032-370726 and LD1-0032-410821 were from two patients with Ovarian High-Grade Serous Carcinoma. The tumors were sliced into 3 mmx3 mmx3 mm (about 30-60 mg) fragments and implanted subcutaneously on the right flank of female C-NKG mice (Cyagen Biosciences Inc., Suzhou, China). Tumors were allowed to establish in mice until the mean tumor volume reached 100-150 mm3. Mice were randomized into 3 groups, 6 mice per group, based on the body weight and tumor volume of each mouse. The grouping day was regarded as day 0 which was also the day treatment started. Mouse body weight was monitored twice weekly. TumorAttorney Docket No.: 091787-8001W001 volume was measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3using the formula: V = 0.5a * b2where a and b are the long and short diameters of the tumor, respectively.

[0423] Mice transplanted with LD1 -0032-370726 tumors were treated once with vehicle, 3mg / kg of hu2D5-S_seq6_WT-BL001-Ex, or lOmg / kg of hu2D5-S_seq6_WT-BL001-Ex by intravenous injection. As shown by the tumor growth curves in Fig. 19A, hu2D5-S_seq6_WT-BL001-Ex ADC displayed anti-tumor efficacy in a dose-dependent manner.

[0424] To compare with previous CLDN6 ADC, antibody clone 3-7 (humanized AB3-7) from patent No. AU2020241896A1 was synthesized and conjugated with VC-MMAE at DAR of 4. Mice transplanted with LD1-0032-410821 tumors were treated once with vehicle, lOmg / kg of 3-7-MMAE (DAR4), or lOmg / kg of hu2D5-S_seq6_WT-BL001-Ex by intravenous injection. As shown by the tumor growth curves in Fig. 19B, hu2D5-S_seq6_WT-BL001-Ex ADC displayed anti-tumor efficacy superior to the previous CLDN6 ADC, 3-7-MMAE.Example 8: Epitope mapping identifies amino acid residues essential for 2D5-S_seq6 WT Ab binding

[0425] This example illustrates the epitope mapping that identifies the amino acid residues of CLDN6 essential for 2D5-S_seq6 WT Ab binding.

[0426] To better understand how 2D5-S_seq6 WT Ab specifically recognizes CLDN6 / 9 but not CLDN3 / 4, we mapped the critical amino acid residues involved in antibody binding. Using Shotgun Mutagenesis, we performed a selective scan of certain amino acids in the extracellular domain 1 of CLDN6, substituting wild-type residues with alanine, one mutation at a time.

[0427] Hu-CLDN6 WT (Uniprot, P56747) and the derived mutant CLDN6 sequences were cloned into lentiviral vector and the virus was packaged according to the instruction of the virus packaging kit (Lenti-X™ Packaging Single Shots, Cat# 631275, Takada). The ES2 cells were transduced and selected with puromycin. The cell line stably expressing CLDN6 proteins were incubated with 1 mg / ml of hu2D5-S_seq6_WT antibody for 30 minutes in PBS with 0.5% BSA, 1 mMEDTA, and 0.1% sodium azide (flow cytometry buffer) at 4°C. The cells were washed and then incubated with lOnM phycoerythrin (PE) conjugated anti-Human Fc Ab (Biolegend, cat# 409304) for 20 minutes at 4oC. Cells were washed and then acquired by CytoFlex LX with a 561 nm channel. Data was analyzed with FlowJo software. Antibody binding activity was quantified using MFI.Attorney Docket No.: 091787-8001W001

[0428] Residues were classified as critical if mutation reduced binding to less than 50% of wildtype CLDN6. This approach revealed N38 as a key residue required for 2D5-S_seq6 WT Ab binding (Fig.20A).

[0429] To rule out that any lack of binding was due to mutated CLDN6 not expressing at sufficient level, flow cytometry with another intracellular domain binding CLDN6 antibody was performed to verify normal level of receptor expression in cells (Fig.20B).

[0430] Parental ES2 cells and engineered ES2 cells stably expressing CLDN6 proteins were incubated with 1 mg / ml of antibody (Abeam, ab314134) specific to the CLDN6 intracellular domain for 30 minutes in PBS with 0.5% BSA, 1 mM EDTA, and 0.1% sodium azide (flow cytometry buffer) at 4°C. The cells were washed, and then incubated with lOnM phycoerythrin (PE) conjugated anti-Rabbit Fc Ab (Biolegend, cat# 406421) for 20 minutes at 4°C. Cells were washed and then acquired by CytoFlex LX with a 561 nm channel. Data was analyzed with FlowJo software. Antibody binding activity was quantified using MFI.

[0431] Table 14 The CDR sequences of the antibody variable sequences (by Kabat numbering) Variable CDR1 CDR2 CDR3Chain2D5-S H SYGMS (SEQ ID NO: SINSNGGRTYYPDSEKG WGGQYVMDY (SEQ 27) (SEQ ID NO: 28) ID NO: 29) 2D5-S__L TADSSVTSSYLH (SEQ ATSNLAS (SEQ ID NO: 31) HQSHRSPPT (SEQ ID ID NO: 30) NO: 32)2D5JEI SYGMS (SEQ ID NO: SINSNGGRTYYPDSEKG WGGQYVMDY (SEQ 65) (SEQ ID NO: 66) ID NO: 67) 2D5 L TADSSVTSSYLH (SEQ ATSNLAS (SEQ ID NO: 69) HQCHRSPPT (SEQ ID ID NO: 68) NO: 70)2D5- SYGMS (SEQ ID NO: SINSNGGRTYYPDSEKG WGGQYVMDY (SEQ A II 71) (SEQ ID NO: 72) ID NO: 73)2D5- TADSSVTSSYLH (SEQ ATSNLAS (SEQ ID NO: 75) HQAHRSPPT (SEQ ID A L ID NO: 74) NO: 76)2D11JEI SDYWN (SEQ ID NO: YISYSGSTYYNPSLKS LLSGSSPWFAY (SEQ 77) (SEQ ID NO: 78) ID NO: 79) 2D11J RS SKSLLHYNGITYLY QMSNLAS (SEQ ID NO: 81) AQNLELPYT (SEQ ID (SEQ ID NO: 80) NO: 82)3D6JI NYGVH (SEQ ID NO: V1WSGGSTDYNAAFIS PDGYYVYYAMDY 83) (SEQ ID NO: 84) (SEQ ID NO: 85) 3D6_L SASSSVRYMY (SEQ ID DTSKLAS (SEQ ID NO: 87) QQWSSYPLT (SEQ ID NO: 86) NO: 88)4B1 H DYIML (SEQ ID NO: 89) NINPYYGSTSYNLNFKG YNGKPFYAMDY (SEQ (SEQ ID NO: 90) ID NO: 91)4B1 L KASQDINSYLS (SEQ RANSLVN (SEQ ID NO: 93) LQYDEFPLT (SEQ IDID NO: 92) NO: 94)Attorney Docket No.: 091787-8001W001 6H6. H TYAMH (SEQ ID NO: RIRGKSSNYATYYADS VK GVHNSNSWFAY (SEQ 95) D (SEQ ID NO: 96) ID NO: 97) 6H6 L KASQNVRTAVA (SEQ LASNRHT (SEQ ID NO: 99) LQHNYPLT (SEQ ID ID NO: 98) NO: 100)6H11 H SDYWN (SEQ ID NO: YISYSGSTYYNPSLKS LLSGSSPWFAF (SEQ 101) (SEQ ID NO: 102) ID NO: 103) 6H11 L RS SKSLLI IYNGITYLY QMSNLAS (SEQ ID NO: AQNLELPYT (SEQ ID (SEQ ID NO: 104) 105) NO: 106)7C10 H TYAMH (SEQ ID NO: RIRGKSSNYATYYADSVK GVHNSNSWFAY (SEQ 107) D (SEQ ID NO: 108) ID NO: 109) 7C10JL KASQNVRTAVA (SEQ LASNRHT (SEQ ID NO: LQHNYPLT (SEQ ID ID NO: 110) 111) NO: 112)8C11_H NYLIQ (SEQ ID NO: VINPGSGGTNYNEKFKG LYDGYYEDYYAMDY 113) (SEQ ID NO: 114) (SEQ ID NO: 115) 8C11_L TASSSVSSSYLH (SEQ STSNLAS (SEQ ID NO: HQYHRSPWT (SEQ ID ID NO: 116) 117) NO: 118)10C1-H NYTIH (SEQ ID NO: YINPSSGYTNYNQKFKD NLAWAY (SEQ ID 119) (SEQ ID NO: 120) NO: 121)IOC I L RASQEISGYLS (SEQ ID AASTLYS (SEQ ID NO: LQYASHPLT (SEQ IDNO: 122) J23) _ _ NO: 124)Attorney Docket No.: 091787-8001W001 ReferencesThe complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for example, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PBD, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference, regardless of whether the phrase “incorporated by reference” is or is not used in relation to the particular reference. The following references are listed only as examples.Yu S, Zhang Y, Li Q, Zhang Z, Zhao G, Xu J. CLDN6 promotes tumor progression through the YAP1–snail1 axis in gastric cancer. Cell Death Dis 2019;10:949.Lin D, Guo Y, Li Y, Ruan Y, Zhang M, Jin X, et al. Bioinformatic analysis reveals potential properties of human claudin-6 regulation and functions. Oncol Rep 2017;38:875-85.Kojima M, Sugimoto K, Kobayashi M, Ichikawa-Tomikawa N, Kashiwagi K, Watanabe T, et al. Aberrant claudin-6-adhesion signaling promotes endometrial cancer progression via estrogen receptor a. Mol Cancer Res 2021; 19:1208.Wang L, Xue Y, Shen Y, LiW, Cheng Y, Yan X, et al. Claudin-6: a novel surface marker for characterizing mouse pluripotent stem cells. Cell Res 2012;22:1082-5.Hewitt KJ, Agarwal R, Morin PJ. The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer 2006;6:186.Qu H, Jin Q, Quan C. CLDN6: from traditional barrier function to emerging roles in cancers. Int J Mol Sci 2021;22: 13416.Tsukita S, Tanaka H, Tamura A. The claudins: from tight junctions to biological systems. Trends Biochem Sci 2019;44:141-52Wang L, Jin X, LinD, Liu Z, Zhang X, Lu Y, et al. Clinicopathologic significance of claudin-6, occludin, and matrix metalloproteinases-2 expression in ovarian carcinoma. Diagn Pathol 2013;8:190.Micke P, Mattsson JSM, Edlund K, Lohr M, Jirstr€om K, Berglund A, et al. Aberrantly activated claudin-6 and 18.2 as potential therapy targets in non-small cell lung cancer. Int J Cancer 2014;135:2206-14.Kojima M, Sugimoto K, Tanaka M, Endo Y, Kato H, Honda T, et al. Prognostic significance of aberrant claudin-6 expression in endometrial cancer. Cancers 2020;12:2748.Attorney Docket No.: 091787-8001W001 Gao P, Peng T, Cao C, Lin S, Wu P, Huang X, et al. Association of CLDN6 and CLDN10 with immune microenvironment in ovarian cancer: a study of the claudin family. Front Genet 2021; 12: 595436.Prihoda, D., Maamary, J., Waight, A., Juan, V., Fayadat-Dilman, L., Svozil, D., & Bitton, D. A. (2022). BioPhi: A platform for antibody design, humanization, and humanness evaluation based on natural antibody repertoires and deep learning. mAbs, 14(1). 2.Zhuang X, Martin TA, Ruge F, Zeng JJ, Li XA, Khan E, Dou Q, Davies E, Jiang WG. (2023).Expression of Claudin-9 (CLDN9) in Breast Cancer, the Clinical Significance in Connection with Its Subcoat Anchorage Proteins ZO-1 and ZO-3 and Impact on Drug Resistance. Biomedicines, 11(12):3136Endo Y, Sugimoto K, Kobayashi M, Kobayashi Y, Kojima M, Furukawa S, Soeda S, Watanabe T, Higashi AY, Higashi T, Hashimoto Y, Fujimori K, Chiba H. (2022). Claudin-9 is a novel prognostic biomarker for endometrial cancer. Int J Oncol. 61 (5): 135Sharma RK, Chheda ZS, Das Purkayastha BP, Gomez-Gutierrez JG, Jala VR, Haribabu B. (2016). A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis. 33(3):263- Ill -

Claims

Attorney Docket No.: 091787-8001W001WHAT IS CLAIMED IS:

1. An anti-CLDN6 / 9 antibody or antigen-binding fragment thereof, comprising, (a) a heavy chain complementary determining region 1 (HCDR1), a HCDR2 and a HCDR3 respectively having an at least 70% sequence identity of the HCDR1, HCDR2 and HCDR3 comprised in the heavy chain variable region as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21; and / or,(b) a light chain complementary determining region 1 (LCDR1), a LCDR2 and a LCDR3 respectively having an at least 70% sequence identity of the LCDR1, LCDR2 and LCDR3 comprised in the light chain variable region as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

2. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 1, wherein the complementary determining regions (CDRs) comprised in the heavy or light chain variable region are determined by a numbering scheme.

3. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 2, wherein the numbering scheme is Kabat, Chothia, AbM, IMGT, Aho or Contact.

4. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 1, wherein,the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27 or a homologous sequence having 1-3 amino acid residue mutations therein; the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28 or a homologous sequence having 1-3 amino acid residue mutations therein; and, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 29 or a homologous sequence having 1-3 amino acid residue mutations therein.

5. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 1, wherein,Attorney Docket No.: 091787-8001W001the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 30 or a homologous sequence having 1-3 amino acid residue mutations therein;the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 31 or a homologous sequence having 1-3 amino acid residue mutations therein; and, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 32 or a homologous sequence having 1-3 amino acid residue mutations therein.

6. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises a heavy chain and / or a light chain;wherein the heavy chain comprises a heavy chain variable region (VH) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55;wherein the light chain comprises a light chain variable region (VL) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

7. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of any one of claims 1-6, which is a chimeric anti-CLDN6 / 9 antibody or antigen-binding fragments thereof.

8. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 7, wherein the chimeric anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises a heavy chain constant region having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 23 or SEQ ID NO: 25; and / or, a light chain constant region having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 24.Attorney Docket No.: 091787-8001W0019. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of any one of claims 1-8, which is a humanized anti-CLDN6 / 9 antibody or antigen-binding fragments thereof.

10. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 9, wherein the humanized anti-CLDN6 / 9 antibody or antigen-binding fragments thereof comprises a heavy chain and / or a light chain;wherein the heavy chain comprises a heavy chain variable region (VH) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55; and,wherein the light chain comprises a light chain variable region (VL) having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

11. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-CLDN6 / 9 antibody or antigen-binding fragment thereof comprises:a heavy chain having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 57 or SEQ ID NO: 59; and / or,a light chain having an at least 70% sequence identity of the sequence set forth as SEQ ID NO: 58 or SEQ ID NO: 60.

12. The anti-CLDN6 / 9 antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the sequence identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

13. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of claims 1-6, further comprising a fusion moiety.

14. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of claim 13, wherein the fusion moiety is selected from the group consisting of: an antibodyAttorney Docket No.: 091787-8001W001heavy chain constant region, an anti-human serum albumin (aHSA), and other kind of moieties to improve functions of the antibody or antigen-binding fragment thereof.

15. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of claim 14, wherein the heavy chain constant region is a fragment crystallizable (Fc) region derived from human Immunoglobulin (IgG) (e.g., IgGl, IgG2, IgG3 or IgG4), optionally, the heavy chain constant region is an IgG isotype to provide for altered (e.g., reduced) effector functions (e.g., ADCC or CDC), more optionally, the heavy chain constant region is an IgGl isotype which comprises one or more amino acid substitution(s) selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof.

16. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of the preceding claims, further comprising a signal peptide, optionally the signal peptide is at the N-terminal of the heavy chain variable region.

17. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of the claims, which is a monovalent antibody, a bivalent antibody, a monoclonal antibody, a bispecific antibody, a multi-specific antibody, a recombinant antibody, a labeled antibody, or a fusion protein.

18. The anti-CLDN6 / 9 antibody or an antigen-binding fragments thereof of any one of the preceding claims, which is a single domain antibody.

19. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any of the preceding claims, further comprising one or more amino acid residue substitutions or modifications yet retains binding specificity to CLDN6, optionally human CLDN6, cynomolgus CLDN6 or mouse CLDN6.

20. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any of the preceding claims, which binds to extracellular loop-1 (ECL-1) or ECL-2 of the extracellular domain (ECD) of CLDN6.

21. The anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of the preceding claims, which is linked to one or more conjugate moieties.Attorney Docket No.: 091787-8001W00122. The anti-CLDN6 / 9 antibody or an antigen-binding fragment thereof of claim 21, wherein the conjugate moiety is selected from a polymer, a carbohydrate, a lipid, a nucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide, protein, therapeutic agent, or a diagnostic agent.

23. A nucleic acid molecule encoding the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of claims 1-22.

24. A vector comprising the isolated polynucleotide of claim 23.

25. A host cell comprising the vector of claim 24.

26. A conjugate comprising the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of the preceding claims.

27. The conjugate of claim 26, represented by the following Formula (A):Ab-[Lk-T]r(A),or a pharmaceutically acceptable salt thereof,wherein,Ab is the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof;T is a therapeutic agent moiety;Lk is a linker; andr is about 1 to about 12.

28. The conjugate of claim 26 or 27, wherein Lk is represented by the following Formula (B):-Lkl-(Lk2)z-Lk3- (B),wherein,Lkl is a linking moiety to Ab formed by a reaction between a reactive group of Lk and a reactive residue of Ab;each Lk2 is independently a linking unit selected from the group consisting of -O-, -S-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-, -S(=O)-, -S(=O)2-, -NHC(=O)-, -C(=O)NH-, -SC(=O)-, -C(=O)S-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=NH)NH-, -NHS(=O)2-, -S(=O)2NH-, -C(=NH)-, -C(=N-NH2)-, -S(=NH)-, -S(=O)(=NH)-, -CH2-, -CH2CH2-, -CH=CH-, -C=C-, -CH2CH2O-, -OCH2CH2-, -CH2CH2OCH2-, -CH2OCH2CH2-, -S-S-, -NHCH2CH2O-, -OCH2CH2NH-, -Attorney Docket No.: 091787-8001W001CH2CH2NH-, -NHCH2CH2-, -CH2CH2S-, -SCH2CH2-, alanine residue, arginine residue, asparagine residue, aspartic acid residue, cysteine residue, glutamine residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, phenylalanine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, valine residue, selenocysteine residue, pyrrolysine residue, P-alanine residue, y-aminobutyric acid residue, 6-aminolevulinic residue, 4-aminobenzoic acid residue, cystine residue, cystathionine residue, lanthionine residue, djenkolic acid residue, diaminopimelic acid residue, norvaline residue, norleucine residue, alloisoleucine residue, t-leucine residue, a-amino-n-heptanoic acid residue, pipecolic acid residue, a, P-diaminopropionic acid residue, a,y-diaminobutyric acid residue, ornithine residue, allothreonine residue, homocysteine residue, homoserine residue, P-amino-n-butyric acid residue, P-aminoisobutyric acid residue, y-aminobutyric acid residue, a-aminoisobutyric acid residue, isovaline residue, sarcosine residue, N-ethylglycine residue, N-propylglycine residue, N-isopropylglycine residue, N-methylalanine residue, N-ethylalanine residue, N-methyl-P-alanine residue, N-ethyl-P-alanine residue, isoserine residue, a-hydroxy-y-aminobutyric acid residue, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each Lk2 is independently optionally substituted with one or more RLk;each RLkis independently selected from the group selected from halogen, oxo, cyano, nitro, -ORLkl, -OC(=O)RLkl, -OC(=O)ORLkl, -OC(=O)N(RLk2)2, -SRLkl, -S(=O)RLkl, -S(=O)2RLkl, -S(=O)2N(RLk2)2, -S(=O)(=NRLk2)RLkl, -N(RLk2)2, -NRLk2C(=O)N(RLk2)2, -NRLk2C(=O)RLkl, -NRLk2C(=O)ORLkl, -NRLk2S(=O)2RLkl, -N=S(=O)(RLkl)2, -C(=O)RLkl, -C(=O)ORLk2, -C(=O)N(RLk2)2, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, cycloalkyl or heterocyclyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, cycloalkyl and heterocyclyl are optionally substituted with one or more RLk3;Attorney Docket No.: 091787-8001W001each RLklis independently hydrogen, alkyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl, each optionally substituted with one or more RLk3;each RLk2is independently hydrogen, alkyl, SF5, haloalkyl, hydroxyalkyl, aminoalkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -alkyl-cycloalkyl, or -alkyl-heterocyclyl, optionally substituted with one or more RLk3;or two RLk2on the same atom are taken together with the atom to which they are attached to form a heterocyclyl optionally substituted with one or more RLk3;each RLk3is independently halogen, cyano, hydroxy, oxo, -SF5, -SH, -S(=O)-alkyl, -S(=O)2-alkyl, -S(=O)2NH2, -S(=O)2NH-alkyl, -S(=O)2N(alkyl)2, -S(=O)(=N-alkyl)(alkyl), -NH2, -NH-alkyl, -N(alkyl)2, -N=S(=O)(alkyl)2, -C(=O)-alkyl, -C(=O)OH, -C(=O)O-alkyl, -C(=O)NH2, -C(=O)NH-alkyl, -C(=O)N(alkyl)2, -P(=O)(alkyl)2, alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxyalkyl, aminoalkyl, heteroalkyl or cycloalkyl;z is any integer of 1-20; andLk3 is a linking moiety to T formed by a reaction between a reactive group of Lk and a reactive residue of T.

29. The conjugate of any one of claims 26-28, wherein the reactive residue of Ab is a cysteine residue.

30. The conjugate of any one of claims 26-29, wherein Lkl is covalently linked to the S atom of the cysteine residue.

31. The conjugate of any one of claims 26-29, wherein -Lk-T is represented by the following Formula (A-I-l), (A-I-2) or (A-I-3):OKr(CH2)n3(CH2O)n4(CH2)n5^ H E-T(A-I-2),Attorney Docket No.: 091787-8001W001W'N^CH2)n3(CH2O)n4(CH2)n5^E-T (A-I-3), wherein,X’ is the linking moiety connecting to Ab;E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC);T is the therapeutic agent moiety;W is a polypeptide moiety;nl is any integer of 0-10;n2 is any integer of 0-10;n3 is any integer of 0-10;n4 is any integer of 0-10; andn5 is any integer of 0-10.

32. The conjugate of any one of claims 26-31, wherein -Lk-T is selected from the group consisting of:-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NHCH2CH2- C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W- NHCH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NHCH2-O- CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-W-NH- CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;Attorney Docket No.: 091787-8001W001-X’-CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2CH2-C(=O)-W-NH-CH2CH2-O-CH2-C(=O)-E-T.

33. The conjugate of any one of claims 26-32, wherein -Lk-T is selected from the group consisting of:-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T;-X’-CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T; -X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T;-X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E-T; -X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-T; -X’-CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E- T;-X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-C(=O)-E-T; -X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2CH2-C(=O)-E--X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-C(=O)-E-Attorney Docket No.: 091787-8001W001-X’-CH2CH2CH2CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)- E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NHCH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NHCH2CH2CH2-C(=O)-E-T;-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe- Gly-NHCH2-O-CH2-C(=O)-E-T; and-X’-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-Gly-Gly-Phe-Gly-NH-CH2CH2-O-CH2-C(=O)-E-T.

34. The conjugate of any one of claims 26-33, wherein -Lk-T is represented by the following Formula (A- II):(A-II) wherein,X’ is the linking moiety connecting to Ab;L1is an amino acid side chain residue;L2is a spacer moiety;Y is a hydrophilic end group;E is absent, -NHCH2O- or p-aminobenzyloxycarbonyl (PABC);W is a polypeptide moiety;T is the therapeutic agent moiety;n is an integer from 1 to 20;m is an integer from 0 to 5;p is an integer from 1 to 24;r is an integer from 1 to 10;a is 0 or 1; andb is 0 or 1.Attorney Docket No.: 091787-8001W00135. The conjugate of claims 26-34, wherein Lk-T is represented by the following Formula (A-II-1), (A-II-2), or (A-II-3):wherein q is an integer from 1 to 10, and ** end indicates the attachment point of X’ to Ab.

37. The conjugate of any one of claims 26-36, wherein W is selected from a peptide composed of 2-5 amino acids.

38. The conjugate of any one of claims 26-37, wherein W is selected from Gly-Ala, Gly-Gly, Ala-Gly, Glu-Gly, Glu-Ala, Gly-Glu, Asp-Gly, Asp-Ala, Gly-Asp, Cit-Val, Ala-Val, Phe-Lys, Lys-Val, Val-Ala, Val-Cit, Phe-Gly, Ala-Ala, Val-Cys, Val-Gly, Val-Thr, Val-Val, Val-Leu, Val-Ile, Val-Asn or Val-Lys.

39. The conjugate of any one of claims 26-38, wherein W is selected from Glu-Ala-Gly, Glu-Gly-Gly, Gly-Glu-Gly, Gly-Glu-Ala, Asp-Ala-Gly, Asp-Gly-Gly, Gly-Asp-Attorney Docket No.: 091787-8001W001Gly, Gly-Asp-Ala, Gly-Gly-Ala, Gly-Gly-Arg, Gly-Ala-Gly, Gly-F-Gly, Ala-Ala-Gly, Ala-Ala- Ala, Ala-Ala-Asn, Val-Ala-Gly, Val-Cys-Gly, or Val-Lys-Gly.

40. The conjugate of any one of claims 26-39, wherein W is selected from Gly-Gly-Phe-Gly, Gly-Gly-Ala-Gly, Gly-Gly-Gly-Gly, Gly-Glu-Gly-Gly, Gly-Glu-Ala-Gly, Gly-Asp-Gly-Gly, Gly-Asp-Ala-Gly, Ala-Ala-Ala-Gly or Glu-Ala-Gly-Gly.

41. The conjugate of any one of claims 26-40, wherein W is Val-Ala, Val-Cit, or Gly-Gly-Phe-Gly.

42. The conjugate of any one of claims 26-41, wherein L1is selected from:wherein the * end indicates the attachment point of L1to L2.

43. The conjugate of any one of claims 26-42, wherein L2is selected fromor O, wherein the * end indicates the attachment point of L2to L1.

44. The conjugate of any one of claims 26-43, wherein L1isand L2is NH45. The conjugate of any one of claims 26-44, wherein L1is:O 46. The conjugate of any one of claims 26-45, wherein Y is selected from -COOH, -OH, -OCH3, or -CONH2.

47. The conjugate of any one of claims 26-46, wherein n is an integer from 1 to 12.Attorney Docket No.: 091787-8001W00148. The conjugate of any one of claims 26-47, wherein p is an integer from 1 to 16.

49. The conjugate of any one of claims 26-48, wherein m is 0, 1 or 2.

50. The conjugate of any one of claims 26-49, wherein -Lk-T is selected from the group consisting of:Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001oooAttorney Docket No.: 091787-8001W00151. The conjugate of any one of claims 26-50, wherein T is selected from a cytotoxic agent, a detection reagent, a diagnostic reagent, a nucleic acid, a radionuclide, a hormone, an immunomodulatory agent, an enzyme, an antibody, a fusion protein, a metal ion or a combination thereof.

52. The conjugate of any one of claims 26-51, wherein the cytotoxic agent is an anti tubulin agent or a topoisomerase inhibitor.

53. The conjugate of any one of claims 26-52, wherein T is of Formula (T-1), Formula (T-2) or Formula (T-3):(T-l)Attorney Docket No.: 091787-8001W001whereinR1is hydrogen, halogen, alkyl, haloalkyl, -OR1a, -SR1a, -S(O)R1a, or -S(O)2R1a; R2is hydrogen, halogen, alkyl or haloalkyl;each of R3, R6, R7and R10is hydrogen or alkyl;each of R4, R5and R8is independently selected from hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, -alkyl-cycloalkyl, -alkyl-aryl, or -alkyl-heterocyclyl;each R9is independently H, -OH, alkyl, alkoxyl, or cycloalkyl;R11is -[C(Rlla)2]2-cycloalkyl, -[C(Rlla)2]2-heterocyclyl, or -[C(Rlla)2]2-aryl;R12is heterocyclyl or aryl;Z is -O-, -S-, -NH-, or -N(alkyl)-;R13is hydrogen, alkyl, heterocyclyl, or aryl;R1ais hydrogen or alkyl; andeach Rllais independently selected from hydrogen, hydroxyl, alkyl, alkoxyl, or cycloalkyl.

54. The conjugate of any one of claims 26-53, wherein T is selected from:Attorney Docket No.: 091787-8001W00155. The conjugate of any one of claims 26-54, wherein Lk-T is selected from the group consisting of:Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W001Attorney Docket No.: 091787-8001W00156. The conjugate of any one of claims 26-55, selected from the group consisting ofAttorney Docket No.: 091787-8001W00157. A pharmaceutical composition, comprising:(i) the anti-CLDN6 / 9 antibody or an antigen-binding fragment thereof of any one of claims 1-22, or the nucleic acid molecule of claim 23, or the conjugate of any one of claims 26-56; and(ii) a pharmaceutically acceptable carrier or excipient.

58. The pharmaceutical composition of claim 57, further comprising an additional therapeutic agent.

59. The pharmaceutical composition of claim 58, wherein the additional therapeutic agent is an agent for treating a CLDN6-related and / or CLDN9-related disease or disorder, optionally a cancer.

60. A method of expressing the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of claims 1-22, comprising culturing the host cell of claim 25 under the condition at which the vector of claim 24 is expressed.

61. A method of treating, preventing or alleviating a CLDN6-related and / or a CLDN9-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of claims 1-22, the nucleic acid molecule of claim 23, the conjugate of any one of claims 26-56, and / or the pharmaceutical composition of any one of claim 57-59.

62. The method of claim 61, wherein the CLDN6-related and / or a CLDN9-related disease or disorder is associated with abnormally up-regulated expression of CLDN6 and / or CLDN9 compared to the control level.

63. The method of claim 60 or 61, wherein the CLDN6-related and / or a CLDN9- related disease or disorder is a cancer.

64. The method of claim 63, wherein the cancer is selected from the group consisting of: hepatocellular carcinoma (HCC), esophageal adenocarcinoma (EAC),Attorney Docket No.: 091787-8001W001testicular germ cell tumors (TGCT), ovarian cancer (OV), lung cancers (LU AD and LUSC), stomach adenocarcinoma (STAD), and uterine corpus endometrial carcinoma (UCEC).

65. A method of detecting the presence or amount of CLDN6 and / or CLDN9 in a sample, comprising contacting the sample with the anti-CLDN6 / 9 antibody or antigen-binding fragments thereof of any one of claims 1-22, and determining the presence or the amount of CLDN6 and / or CLDN9 in the sample.

66. The method of claim 65, further comprising a step of determining whether CLDN6 and / or CLDN9 is over-expressed in the cells in the sample.

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