MUC1 Antibodies and Methods of Use

Abstract

MUC1 is aberrantly overexpressed in a variety of common human cancers, including lung, breast, colon, gastric, esophageal, and ovarian cancers. The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human MUC1, as well as pharmaceutical compositions comprising the antibodies. In embodiments, the present disclosure relates to anti-MUC1 antibodies and antigen-binding fragments thereof that specifically bind to cancer cells but not to normal cells. In embodiments, the present disclosure relates to anti-MUC1 antibodies or antigen-binding fragments thereof, including antibodies or binding fragments thereof that specifically bind to the SEA domain of human MUC1 (SEQ ID NO: 1) at amino acids 1036 to 1155 (SEQ ID NO: 2).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to PCT Application No. PCT / CN2023 / 080961, entitled "MUC1 Antibodies and Methods of Use," filed March 12, 2023, PCT Application No. PCT / CN2023 / 079507, entitled "MUC1 and CD16A Antibodies and Methods of Use," filed March 3, 2023, and PCT Application No. PCT / CN2023 / 107724, entitled "MUC1 and CD16A Antibodies and Methods of Use," filed July 17, 2023, which are incorporated by reference herein in their entireties.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a file entitled "01368-0023-00PCT_SL.xml," created on February 27, 2024, and having a size of 121,339 bytes. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human MUC1, and methods of use thereof. [Background technology]

[0004] Mucin 1 (MUC1; also known as CA15-3, EMA, MCD, PEM, PUM, KL-6, MAM6, MCKD, PEMT, CD227, H23AG, MCKD1, ADMCKD, and ADTKD2) is a single-pass transmembrane glycoprotein belonging to the mucin family. MUC1 is highly glycosylated and expressed on the apical surface of epithelial cells lining the mucosal surfaces of various normal tissues, including the lung, breast, stomach, pancreas, and uterus. MUC1 plays an important role in forming a protective mucosal barrier on these epithelial surfaces.

[0005] MUC1 is translated as a single polypeptide, the extracellular portion of which undergoes autocleavage at the SEA (sea urchin sperm protein, enterokinase, and agrin) domain to form two subunits. The cleaved extracellular membrane-distal subunit, which contains 20–125 repeats of a 20-amino acid sequence (variable number tandem repeats, VNTR), forms a heterodimeric complex with the membrane-proximal subunit through strong noncovalent interactions. The membrane-proximal subunit consists of a 58-amino acid (aa) extracellular domain, a 28-aa transmembrane domain, and a 72-aa cytoplasmic tail that intersects with multiple oncogenic signaling pathways.

[0006] MUC1 is aberrantly overexpressed in a variety of common human cancers, including lung, breast, colon, gastric, esophageal, and ovarian cancers. In addition to upregulated expression, hypoglycosylation and altered cellular localization of MUC1 are also associated with cancer. Furthermore, the cleaved membrane-distal subunit tends to be shedding from the surface of cancer cells in association with elevated plasma levels.

[0007] Given its overexpression in a variety of human carcinomas, MUC1 is an attractive tumor-associated antigen. However, previous attempts to target the membrane-distal (MUC1 N-terminal) subunit, such as AS1402 (huHMFG-1) and BrevaRex (AR-20.5), have been unsuccessful. This is due, in part, to a circulating pool of shedding MUC1, which prevents antibody targeting to the surface of MUC1-expressing tumor cells. In contrast, the non-shedding MUC1 membrane-proximal (MUC1 C-terminal) subunit, which functions as an oncoprotein, is an attractive target for the development of antibody-based therapeutics.

[0008] The present disclosure provides anti-MUC1 antibodies that target the MUC1 membrane-proximal region (specifically, the MUC1-SEA domain adjacent to the transmembrane region at the MUC1-C terminus) with minimal interference from shed MUC1. Summary of the Invention

[0009] The present disclosure relates to anti-MUC1 antibodies and antigen-binding fragments thereof.

[0010] In embodiments, the present disclosure relates to anti-MUC1 antibodies and antigen-binding fragments thereof that specifically bind to cancer cells and not to normal cells.

[0011] In embodiments, the present disclosure relates to anti-MUC1 antibodies or antigen-binding fragments thereof, including antibodies or binding fragments thereof that specifically bind to the SEA domain of human MUC1 (SEQ ID NO: 1) at amino acids 1036-1155 (SEQ ID NO: 2).

[0012] In embodiments, the present disclosure relates to anti-MUC1 antibodies or antigen-binding fragments that specifically bind to human MUC1, including: (i) A heavy chain variable region comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 56, and (f) an LCDR3 of SEQ ID NO: 9; (iii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (iv) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 78, (b) an HCDR2 of SEQ ID NO: 79, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (v) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; (vi) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29, or (vii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 74, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29.

[0013] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 20, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 21; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 30, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 31; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 59; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (vi) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 58, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 59; (vii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 58, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (viii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 62; (ix) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 66; (x) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 68; (xi) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 71, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 72; (xii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 75, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 76; or (xiii) A heavy chain variable region (VH) comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 80, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 81.

[0014] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 57, and a light chain variable region (VL) comprising SEQ ID NO: 59; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 57, and a light chain variable region (VL) comprising SEQ ID NO: 60; (vi) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (vii) a heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 60; (viii) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; (ix) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; (x) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; (xi) a heavy chain variable region (VH) comprising SEQ ID NO: 71 and a light chain variable region (VL) comprising SEQ ID NO: 72; (xii) A heavy chain variable region (VH) comprising SEQ ID NO: 75 and a light chain variable region (VL) comprising SEQ ID NO: 76, or (xiii) A heavy chain variable region (VH) comprising SEQ ID NO: 80, and a light chain variable region (VL) comprising SEQ ID NO: 81.

[0015] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inserted, deleted, or substituted within SEQ ID NO: 10, 11, 20, 21, 30, 31, 57, 58, 59, 60, 61, 62, 66, 68, 71, 72, 75, 76, 80, or 81.

[0016] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0017] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, and a VL comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81.

[0018] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, and a VL having the amino acid sequence of SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81.

[0019] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising: (i) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (ii) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 71, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 72; (iii) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 75, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 76; or (iv) A VH comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 80, and a VL comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 81.

[0020] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv comprising: (i) VH having the amino acid sequence of SEQ ID NO: 57, and VL having the amino acid sequence of SEQ ID NO: 60; (ii) VH having the amino acid sequence of SEQ ID NO: 71, and VL having the amino acid sequence of SEQ ID NO: 72; (iii) a VH having the amino acid sequence of SEQ ID NO: 75 and a VL having the amino acid sequence of SEQ ID NO: 76; or (iv) VH having the amino acid sequence of SEQ ID NO: 80, and VL having the amino acid sequence of SEQ ID NO: 81.

[0021] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inserted, deleted, or substituted within SEQ ID NO: 57, 60, 71, 72, 75, 76, 80, or 81.

[0022] In embodiments, the VH and VL of the scFv of the anti-MUC1 antibody or antigen-binding fragment thereof are linked via an amino acid linker. The amino acid linker may have the amino acid sequence of SEQ ID NO: 83 or 84.

[0023] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv having the amino acid sequence of SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:77, or SEQ ID NO:82.

[0024] In embodiments, the antibody or antigen-binding fragment thereof comprises an scFv having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 70, 73, 77, or 82.

[0025] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inserted, deleted, or substituted within SEQ ID NO: 70, 73, 77, or 82.

[0026] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof exhibits antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In embodiments, the ADCC is against MUC1-expressing target cells.

[0027] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof is hypoglycosylated, or aglycosylated, or hypofucosylated.

[0028] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof comprises increased bisecting GlcNAc structures.

[0029] In embodiments, the anti-MUC1 antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype. In embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type. In one embodiment, the antibody or antigen-binding fragment thereof comprises the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2. In one embodiment, the antibody or antigen-binding fragment thereof comprises the Fc domain of IgG1.

[0030] In embodiments, the present disclosure relates to a pharmaceutical composition comprising an anti-MUC1 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0031] In embodiments, the present disclosure relates to an isolated nucleic acid encoding an anti-MUC1 antibody or antigen-binding fragment thereof disclosed herein.

[0032] In embodiments, the present disclosure relates to a vector comprising a nucleic acid disclosed herein.

[0033] In embodiments, the present disclosure relates to a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0034] In embodiments, the present disclosure relates to a process for producing an anti-MUC1 antibody or antigen-binding fragment thereof, comprising culturing a host cell disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

[0035] In one embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:56, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:65.

[0036] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising one or more complementarity determining regions (HCDRs) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:74, SEQ ID NO:78, or SEQ ID NO:79, and / or (b) a light chain variable region comprising one or more complementarity determining regions (LCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:56, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:65.

[0037] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:24, or SEQ ID NO:78, the HCDR2 comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:74, or SEQ ID NO:79, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:16, or SEQ ID NO:26; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:17, or SEQ ID NO:27, the LCDR2 comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:56, or SEQ ID NO:65, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:19, or SEQ ID NO:29.

[0038] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs) that are: HCDR1 comprising the amino acid sequence of SEQ ID NO: 4; HCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or HCDR1 comprising the amino acid sequence of SEQ ID NO: 14; HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, or HCDR1 comprising the amino acid sequence of SEQ ID NO: 24; HCDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or HCDR1 comprising the amino acid sequence of SEQ ID NO: 24; HCDR2 comprising the amino acid sequence of SEQ ID NO: 74, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 26, or HCDR1 comprising the amino acid sequence of SEQ ID NO: 78; HCDR2 comprising the amino acid sequence of SEQ ID NO: 79, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and / or (b) a light chain variable region comprising three complementarity-determining regions (LCDRs) that are: LCDR1 comprising the amino acid sequence of SEQ ID NO: 7; LCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9, or LCDR1 comprising the amino acid sequence of SEQ ID NO: 17; LCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 19, or LCDR1 comprising the amino acid sequence of SEQ ID NO: 27; LCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 29, or LCDR1 comprising the amino acid sequence of SEQ ID NO: 7; LCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 9, or LCDR1 comprising the amino acid sequence of SEQ ID NO: 27; LCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and LCDR3 comprising the amino acid sequence of SEQ ID NO:29.

[0039] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9.

[0040] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 56, and (f) an LCDR3 of SEQ ID NO: 9.

[0041] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19.

[0042] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 78, (b) an HCDR2 of SEQ ID NO: 79, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19.

[0043] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29.

[0044] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29.

[0045] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 74, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29.

[0046] In one embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region having an amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80; and / or or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81, or an amino acid sequence which is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81.

[0047] In another embodiment, the anti-MUC1 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, or the amino acid sequence of SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, with one, two, or three amino acid substitutions; and / or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81, or the amino acid sequence of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81, with one, two, three, four, or five amino acid substitutions. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0048] In one embodiment, the anti-MUC1 antibody is administered at a concentration of 1×10 -6 M to 1 x 10 -10 M, or 1 x 10 -11 Binding affinity (K D In another embodiment, the anti-MUC1 antibody binds to MUC1 at about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, about 1 x 10 -10 M, or approximately 1 x 10 -11 K of M D It binds to MUC1.

[0049] In another embodiment, the anti-human MUC1 antibody or antigen-binding fragment thereof exhibits cross-species binding activity for cynomolgus monkey MUC1.

[0050] In some embodiments, the present disclosure relates to an isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of an anti-MUC1 antibody or antigen-binding fragment. In one embodiment, the isolated nucleic acid comprises the VH nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, or SEQ ID NO: 63, or a nucleotide sequence comprising at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, or SEQ ID NO: 63, and encodes the VH region of an antibody or antigen-binding fragment of the present disclosure. In one embodiment, the isolated nucleic acid comprises the VL nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 64, SEQ ID NO: 67, or SEQ ID NO: 69, or a nucleotide sequence comprising at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 64, SEQ ID NO: 67, or SEQ ID NO: 69, and encodes the VL region of an antibody or antigen-binding fragment of the present disclosure.

[0051] In some embodiments, the present disclosure provides anti-human MUC1 antibodies or antigen-binding fragments thereof that exhibit specific binding and high affinity for human MUC1. [Brief explanation of the drawings]

[0052] [Figure 1] Schematic diagram of MUC1-SEA-mIgG2a (top) and MUC1-SEA-huIgG1 (bottom), where "N" is the N-terminus and "C" is the C-terminus. [Figure 2A] Figures A to F show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figures A and B show the binding affinity of chimeric anti-MUC1 monoclonal antibody BG138P to MUC1-overexpressing human and cynomolgus MUC1 cells. [Figure 2B]Figures A to F show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figures A and B show the binding affinity of chimeric anti-MUC1 monoclonal antibody BG138P to MUC1-overexpressing human and cynomolgus MUC1 cells. [Figure 2C] (A) to (F) show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. (C) and (D) show the binding affinity of chimeric BG346P to human and cynomolgus MUC1-overexpressing cells. [Figure 2D] (A) to (F) show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. (C) and (D) show the binding affinity of chimeric BG346P to human and cynomolgus MUC1-overexpressing cells. [Figure 2E] Figures A to F show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figures E and F show the binding affinity of chimeric BG219P to human and cynomolgus MUC1-overexpressing cells. [Figure 2F] Figures A to F show the binding affinity of purified MUC1 antibodies to human and cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figures E and F show the binding affinity of chimeric BG219P to human and cynomolgus MUC1-overexpressing cells. [Figure 3A] A-C show epitope binning determination by competitive SPR assay in which purified human MUC1-mFc antigen was flown over the chip surface and captured by anti-mouse IgG antibody. [Figure 3B] A-C show epitope binning determination by competitive SPR assay in which purified human MUC1-mFc antigen was flown over the chip surface and captured by anti-mouse IgG antibody. [Figure 3C]A-C show epitope binning determination by competitive SPR assay in which purified human MUC1-mFc antigen was flown over the chip surface and captured by anti-mouse IgG antibody. [Figure 4A] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures A–C show the binding profiles of chBG138P and chBG219P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 4B] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures A–C show the binding profiles of chBG138P and chBG219P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 4C] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures A–C show the binding profiles of chBG138P and chBG219P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 4D] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures D–F show the binding profiles of chBG138P and chBG346P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 4E] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures D–F show the binding profiles of chBG138P and chBG346P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 4F] Figures A–F show the effect of soluble MUC1 on the binding of MUC1 antibodies to MUC1-expressing cells. Figures D–F show the binding profiles of chBG138P and chBG346P at different concentrations (30, 3, and 0.3 μg / ml, respectively) in the presence of soluble MUC1, with HMFG1 as a positive control and mIgG and hIgG1 as negative controls. [Figure 5A] Figures A-C show that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. Figures A-C show that chBG138P binds to the MUC1-expressing tumor cell lines HCC827 (A), H1975 (B), and T-47D (C) in a dose-dependent manner (human IgG1 as a negative control). [Figure 5B] Figures A-C show that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. Figures A-C show that chBG138P binds to the MUC1-expressing tumor cell lines HCC827 (A), H1975 (B), and T-47D (C) in a dose-dependent manner (human IgG1 as a negative control). [Figure 5C] Figures A-C show that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. Figures A-C show that chBG138P binds to the MUC1-expressing tumor cell lines HCC827 (A), H1975 (B), and T-47D (C) in a dose-dependent manner (human IgG1 as a negative control). [Figure 6] Figure 1 shows a schematic of the FACS gating strategy for the T cell binding assay. The dashed box indicates the percentage of T cells that bound the antibody. [Figure 7A] Figures 7A to 7H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7B]A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7C] A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7D] A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7E] A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7F] A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7G]A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 7H] A to H show that chimeric anti-MUC1 monoclonal antibodies chBG138P (A, B, E, and F), chBG219P (E and F), or chBG346P (E and F), which target the membrane-proximal region of MUC1, do not bind to activated T cells, whereas antibodies HMFG1 (C and D) and 16A (G and H), which target the membrane-distal portion of MUC1, can bind to normal T cells. [Figure 8A] A to D show that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 (A and B) do not bind to normal T cells, whereas the antibody HMFG1 (C and D), which targets the membrane-distal portion of MUC1, binds to normal activated T cells. [Figure 8B] A to D show that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 (A and B) do not bind to normal T cells, whereas the antibody HMFG1 (C and D), which targets the membrane-distal portion of MUC1, binds to normal activated T cells. [Figure 8C] A to D show that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 (A and B) do not bind to normal T cells, whereas the antibody HMFG1 (C and D), which targets the membrane-distal portion of MUC1, binds to normal activated T cells. [Figure 8D] A to D show that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 (A and B) do not bind to normal T cells, whereas the antibody HMFG1 (C and D), which targets the membrane-distal portion of MUC1, binds to normal activated T cells. [Figure 9A]1A-C are bar graphs showing chBG138P-induced MUC1 internalization in cancer cell lines. [Figure 9B] 1A-C are bar graphs showing chBG138P-induced MUC1 internalization in cancer cell lines. [Figure 9C] 1A-C are bar graphs showing chBG138P-induced MUC1 internalization in cancer cell lines. [Figure 10A] A and B show FACS binding of chimeric BG138P and its humanized antibodies huBG138P-Hz1 to huBG138P-Hz4 to human and cynomolgus monkey cells overexpressing MUC1. [Figure 10B] A and B show FACS binding of chimeric BG138P and its humanized antibodies huBG138P-Hz1 to huBG138P-Hz4 to human and cynomolgus monkey cells overexpressing MUC1. [Figure 11] 1 shows that the humanized antibodies huBG219P-Bz0, -E39, and -E43 bind to the MUC1-overexpressing cell line ZR-75-1 to a similar extent compared to the chimeric antibody chBG219P. [Figure 12] 1 shows the results of a binding competition assay between scFv1-78P, scFv2 (scFv2-14P, scFv2-57P), and scFv3-20P. DETAILED DESCRIPTION OF THE INVENTION

[0053] definition Unless specifically defined below or elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0054] As used in this specification, including the appended claims, singular words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly indicates otherwise.

[0055] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0056] Unless otherwise specified or clear from the context, as used herein, the term "about" refers to a value or composition that falls within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to practice in the art. "About" can also mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% more or less than the stated value. For example, approximately 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or up to 5 times greater. When a specific value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" should be assumed to be within the tolerance range of that specific value or composition.

[0057] The term "MUC1" or "mucin 1," also known as CA 15-3, EMA, MCD, PEM, PUM, KL-6, MAM6, MCKD, PEMT, CD227, H23AG, MCKD1, ADMCKD, or ADTKD2, is a member of the mucin family. The amino acid sequence of human MUC1 is listed as SEQ ID NO: 1 and can also be found under accession number P15941.

[0058] As used herein, the terms "administration" and "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid when the fluid is in contact with the cell.

[0059] The term "subject" or "patient" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient having or at risk of having a disorder described herein).

[0060] In one embodiment, "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0061] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen through non-covalent forces at multiple sites. Generally, the more interactions, the stronger the affinity.

[0062] As used herein, the term "antibody" ("AB") refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0063] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0064] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies, and human engineered antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0065] The term "chimeric antibody" refers to a molecule created by fusing the variable domains of an antibody from one host species (e.g., mouse, rabbit, llama, etc.) with domains from different species, i.e., the constant domains of an antibody from another species (e.g., human).

[0066] In some embodiments, the anti-MUC1 antibody comprises at least one antigen-binding site. In some embodiments, the anti-MUC1 antibody comprises an antigen-binding fragment derived from a MUC1 antibody described herein. In some embodiments, the anti-MUC1 antibody is isolated or recombinant. In some embodiments, the anti-MUC1 antibody also encompasses multispecific antibodies that target MUC1 as at least one arm and other antigen(s) as another arm(s).

[0067] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, etc., and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0068] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the isotype of the antibody is defined as IgA, IgD, IgE, IgG, and IgM, respectively.

[0069] Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including an additional "D" region of about 10 amino acids.

[0070] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0071] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (FR3), CDR-3 (CDR3), and FR-4 (FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering scheme)).The definition of antigen-binding sites is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0072] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from a "CDR" (e.g., LCDR1, LCDR2, and LCDR3 in the light-chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy-chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (where antibody CDR regions are defined by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (where antibody CDR regions are defined by structure). The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0073] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as single-chain Fv (ScFv), nanobodies (or VHH antibodies), multispecific antibodies formed from antibody fragments, and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0074] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target relative to other proteins, although this specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of an antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0075] As used herein, an "antigen-binding domain" comprises at least six CDRs (or, in the context of a single-domain antibody, three CDRs) and specifically binds to an epitope. The "antigen-binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain that specifically binds to a first epitope and a second antigen-binding domain that specifically binds to a second epitope. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with an antigen-binding domain directed to each specific epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) comprising multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope and two, three, four, or more antigen-binding domains that specifically bind to a second epitope.

[0076] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0077] The terms "humanized" or "humanized antibody" refer to forms of antibodies that contain sequences from non-human (e.g., mouse, rabbit, llama, etc.) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Where it is necessary to distinguish a humanized antibody from the parent (e.g., rodent) antibody, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences of the parent rodent antibody, but can contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0078] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence may also refer to the human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other variable region amino acid sequences evaluated. The corresponding human germline sequence may be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable regions, or other combinations of sequences or subsequences. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0079] The term "equilibrium dissociation constant" or "KD" or "M" refers to the dissociation rate constant (kd, time -1 ) to the association rate constant (ka, time -1 , M -l ) The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10 -9 Less than M or 10-10 M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0080] As used herein, the terms "cancer" or "tumor" have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to a particular type or location.

[0081] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to MUC1. Common conservative changes of amino acids are well known in the art.

[0082] As used herein, the term "knob-into-hole" technology refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide (at the interface where they interact). For example, knobs-into-holes can be used to ... L :C HKnobs-into-holes have been introduced into the VH / VL interface or the VH / VL interface (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, the knobs-into-holes ensure correct pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies with knobs-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The knobs-into-holes technology can also be used with VH or VL regions to ensure correct pairing.

[0083] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value; if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and both strands are compared.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) of 50. Alignment (B), expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0084] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0085] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0086] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0087] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0088] In some aspects, the disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-MUC1 antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all solvents, dispersion media, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0089] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. One suitable mode of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0090] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components.

[0091] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration in multiple or separate containers or formulations (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, "combination therapy" encompasses the use of each type of therapeutic agent in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the conditions or disorders described herein.

[0092] As used herein, the term "in combination with" means that the anti-MUC1 antibody is administered to the subject simultaneously with, immediately before, or immediately after the administration of the additional therapeutic agent. In certain embodiments, the anti-MUC1 antibody is administered as a combination with the additional therapeutic agent.

[0093] Detailed Description The present disclosure provides anti-human MUC1 antibodies and antigen-binding fragments thereof. The present disclosure also provides antibodies with desired binding affinity, desired cellular internalization, and other desired properties. The anti-human MUC1 antibodies can be used to construct multispecific antibodies with additional functions, such as binding to a second human tumor-associated antigen (TAA), immune checkpoint inhibition, or immunostimulation, to construct antibody-drug conjugates (ADCs), or to fuse with other domains to form fusion proteins. Furthermore, the anti-human MUC1 antibodies and their constructs, or pharmaceutical compositions containing them, can be used to treat MUC1-expressing cancers and related disorders.

[0094] Anti-MUC1 antibody The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human MUC1. In embodiments, the antibody or antigen-binding fragment thereof specifically binds to the SEA domain, which is the membrane-proximal portion of the human MUC1 protein. In embodiments, the antibody or antigen-binding fragment thereof has no or significantly reduced interference with soluble MUC1 compared to antibodies targeting the N-terminus of MUC1. Antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof produced by the methods described below.

[0095] In embodiments, the antibodies or antigen-binding fragments disclosed herein specifically bind to MUC1 and comprise a VH domain having the amino acid sequence of SEQ ID NO: 10, 20, 30, 57, 58, 61, 71, 75, or 80. In embodiments, the antibodies or antigen-binding fragments specifically bind to MUC1 and comprise an HCDR having the amino acid sequence of any one of the HCDRs listed below in Tables 2, 7, 14, 16, 22, and 23. In one aspect, the antibodies or antigen-binding fragments specifically bind to MUC1, and the antibodies comprise (or alternatively consist of) one, two, three, or more HCDRs having the amino acid sequence of any of the HCDRs listed in Tables 2, 7, 14, 16, 22, and 23 herein.

[0096] In embodiments, the antibodies or antigen-binding fragments disclosed herein specifically bind to MUC1 and comprise a VL domain having the amino acid sequence of SEQ ID NO: 11, 21, 31, 59, 60, 62, 66, 68, 72, 76, or 81. In embodiments, the antibodies or antigen-binding fragments specifically bind to MUC1 and comprise an LCDR having the amino acid sequence of any one of the LCDRs listed in Tables 2, 7, 14, 16, 22, and 23. In embodiments, the antibodies or antigen-binding fragments specifically bind to MUC1 and comprise (or alternatively consist of) one, two, three, or more LCDRs having the amino acid sequence of any of the LCDRs listed in Tables 2, 7, 14, 16, 22, and 23.

[0097] In embodiments, the antibodies or antigen-binding fragments disclosed herein contain altered amino acids but have at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR regions to the CDR regions disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, this includes amino acid alterations in which no more than 1, 2, 3, 4, or 5 amino acids are changed in the CDR regions when compared to the CDR regions set forth in the sequences of Tables 2, 7, 14, 16, 22, and 23.

[0098] In embodiments, antibodies or antigen-binding fragments disclosed herein include those with altered amino acids or nucleic acids encoding the amino acids, but with at least 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequences disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, this includes altered amino acid sequences with no more than 1, 2, 3, 4, or 5 amino acids changed in the variable regions when compared to the variable regions set forth in the sequences disclosed in Tables 2, 7, 14, 16, 22, and 23, but retaining substantially the same therapeutic activity.

[0099] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds MUC1. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0100] In embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are cross-reactive with both human and cynomolgus MUC1 (cyno MUC1, SEQ ID NO: 3). In embodiments, the antibodies or antigen-binding fragments target the membrane-proximal region of MUC1, where there is minimal interference from shedding MUC1. In embodiments, the antibodies or antigen-binding fragments may be useful for treating MUC1-expressing cancers.

[0101] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-MUC1 antibodies described in Tables 2, 7, 14, 16, 22, and 23. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies in binding assays. The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to MUC1 demonstrates that the test antibody can compete with that antibody or antigen-binding fragment thereof for binding to MUC1. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on MUC1 as the competing antibody or antigen-binding fragment thereof. In certain embodiments, antibodies that bind to the same epitope on MUC1 as an antibody or antigen-binding fragment thereof of the present disclosure are human or humanized monoclonal antibodies. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0102] In one embodiment, the anti-MUC1 antibodies or antigen-binding fragments thereof disclosed herein can be used to construct multispecific antibodies with additional functions, such as binding to a second human tumor-associated antigen (TAA), immune checkpoint inhibition, or immunostimulation.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0104] In one embodiment, the antibody or antigen-binding fragment thereof is in an scFv format comprising, from N-terminal to C-terminal, VH-VL or VL-VH. In some embodiments, the VH and VL are linked via an amino acid linker, e.g., an amino acid linker described herein. In some embodiments, the VH or VL is any one of the VH or VL listed in Tables 2, 7, 14, 16, 22, and 23. Other scFvs of the present disclosure contain altered amino acids in the CDR regions that are at least 60%, 70%, 80%, 90%, 95%, or 99% identical to the CDR regions disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, this includes amino acid alterations that result in no more than 1, 2, 3, 4, or 5 amino acids being changed in the CDR regions when compared to the CDR regions set forth in the sequences of Tables 2, 7, 14, 16, 22, and 23.

[0105] Amino Acid Linker In embodiments, the anti-MUC1 antibodies or antigen-binding fragments thereof disclosed herein are used to construct multispecific antibodies, which may be, for example, bispecific tetravalent antibodies. The domains and / or regions of the polypeptide chains of bispecific tetravalent antibodies can be separated by linker regions of various lengths. In some embodiments, the antigen-binding domains are separated from each other, from the CL, CH1, hinge, CH2, CH3, or the entire Fc region by linker regions. For example, a polypeptide chain can comprise the sequence VL1-CL-(linker)VH2-CH1, VH-linker-VL. Such linker regions can contain a random assortment of amino acids or a limited set of amino acids. Such linker regions can be flexible or rigid (see US 2009 / 0155275).

[0106] Multispecific antibodies have been synthesized via dimerization mechanisms such as leucine zippers (Kostelny et al., J. Immunol. 1992 148:1547-53; de Kruifetal J. Biol. Chem. 1996 271:7630-4) and Ig C / CH1 domains (Muller et al., FEBS Lett. 422:259-64), and have been developed in a variety of formats, including diabodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., Bio / Technology (NY) 1996 14:192-6), Fab-scFv fusions (Schoonjans et al., J. Immunol. 2000 165:7050-7), and miniantibody formats (Pack et al., Biochemistry 1992.31:1579-84; Pack et al., Bio / Technology 1993 11:1271-7) have been constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments with or without a flexible linker (Mallender et al., J. Biol. Chem. 1994 269:199-206; Mack et al., Proc. Natl. Acad. Sci. USA 1995 92:7021-5; Zapata et al., Protein Eng. 1995 8:1057-62).

[0107] Dimerization-specific amino acids In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change may result in "knobs-into-holes" interactions and may increase the likelihood of correct assembly of the desired multivalent antibody. The dimerization-specific amino acid may be in the CH1 domain or the CL domain, or a combination thereof. Suitable dimerization-specific amino acids used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL) can be found in at least WO2014082179, WO2015181805, and WO2017059551. The dimerization-specific amino acid may also be in the Fc domain or may be combined with a dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0108] Further modifications to the Fc region framework In some embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260 (both by Winter et al.).

[0109] In another embodiment, one or more amino acid residues can be substituted with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0110] In yet another embodiment, one or more amino acid residues are altered to thereby modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are substituted with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, those from the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0111] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0112] In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks or has reduced glycosylation). Altering glycosylation can, for example, increase the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for an antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0113] Additionally or alternatively, antibodies can be generated with altered types of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures). Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes cell lines in which the FUT8 gene, encoding fucosyltransferase, has been functionally disrupted, such that antibodies expressed in such cell lines exhibit hypofucosylation. Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell line exhibit an increase in bisecting GlcNac structures, which results in increased ADCC activity of the antibody (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0114] In another aspect, if reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). However, native IgG4 has been found to be less stable under stress conditions, such as in acidic buffers or at elevated temperatures (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably linking the antibody to an IgG4 Fc engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form antibody halves, which generates bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M., et al., 2007 Science, 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) appeared to inhibit IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and gamma Fc region have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev Immunol, 25:21-50). Furthermore, some IgG4 isoforms that occur rarely in the human population can also induce different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To generate antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce several alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 by Li et al.

[0115] antibody generation Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including, but not limited to, recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as a mammalian host cell, a bacterial host cell, a yeast host cell, an insect host cell, etc.

[0116] The present disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein.

[0117] The polynucleotides of the disclosure can encode variable region sequences of anti-MUC1 antibodies. They can also encode both the variable and constant regions of the antibodies. Some of the sequences encode polypeptides containing the variable regions of both the heavy and light chains of the exemplified anti-MUC1 antibodies.

[0118] The present disclosure also provides expression vectors and host cells for producing anti-MUC1 antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. The expression vector may contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-MUC1 antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to a promoter, other regulatory elements can be included for efficient expression of the anti-MUC1 antibody or antigen-binding fragment thereof. These elements may include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0119] Host cells for harboring and expressing anti-MUC1 antibody vectors can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors can also be made in these prokaryotic hosts, typically containing expression control sequences compatible with the host cell (e.g., an origin of replication). Additionally, any number of well-known promoters can be present, such as the lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system, or promoter systems derived from phage lambda. Promoters typically control expression (optionally with operator sequences) and contain ribosome binding site sequences for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express anti-MUC1 antibodies. Insect cells can also be used in conjunction with baculovirus vectors.

[0120] In other embodiments, mammalian host cells are used to express and produce the anti-MUC1 antibodies of the present disclosure. Examples include hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0121] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for detecting MUC1. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of MUC1 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal and / or cancerous tissues that express MUC1 at higher levels than other tissues.

[0122] In one embodiment, the present disclosure provides a method for detecting the presence of MUC1 in a biological sample. In certain embodiments, the method includes contacting the biological sample with an anti-MUC1 antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample can include, but is not limited to, urine, tissue, sputum, or blood.

[0123] Also included are methods for diagnosing disorders associated with MUC1 expression. In certain embodiments, the methods include contacting a test cell with an anti-MUC1 antibody, determining the expression level (either quantitatively or qualitatively) of MUC1 expressed by the test cell by detecting binding of the anti-MUC1 antibody to a MUC1 polypeptide, and comparing the expression level by the test cell to the MUC1 expression level in a control cell (e.g., a normal cell or a non-MUC1-expressing cell of the same tissue origin as the test cell), wherein a higher level of MUC1 expression in the test cell compared to the control cell indicates the presence of a disorder associated with MUC1 expression.

[0124] Pharmaceutical Compositions and Formulations Compositions, such as pharmaceutical formulations, comprising an anti-MUC1 antibody or antigen-binding fragment thereof, or a polynucleotide comprising a sequence encoding the anti-MUC1 antibody or antigen-binding fragment, are also provided. These compositions may further comprise suitable carriers, e.g., pharmaceutically acceptable excipients, including buffers, as are well known in the art.

[0125] Pharmaceutical formulations of the anti-MUC1 antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and may include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0126] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0127] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0128] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0129] equivalent While the anti-human 4Ig-B7H3 antibody and antigen-binding fragments thereof have been described in connection with the detailed description thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0130] It is understood that one, some, any, or all of the features of the various embodiments disclosed herein may be combined to form additional embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. [Example]

[0131] Example 1. Generation of anti-MUC1 monoclonal antibodies targeting the MUC1 membrane-proximal region MUC1 recombinant protein for immunization and binding assays A cDNA encoding full-length human MUC1 (SEQ ID NO: 1) was synthesized by Genewiz (Suzhou, China) based on its Uniprot sequence (UniprotKB: P15941) and purchased from the company. The coding region for the full-length human MUC1 SEA domain (sea urchin sperm protein, enterokinase, and agrin) (SEQ ID NO: 2), consisting of amino acids (AA) 1036–1155 of full-length MUC1, was PCR-amplified and C-terminally fused to either the Fc domain of mouse IgG2a or the Fc domain of human IgG1 heavy chain. The cDNA was then cloned into a pcDNA3.4-based expression vector (Invitrogen, Carlsbad, CA, USA), resulting in two recombinant fusion protein expression plasmids, MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1, respectively. A schematic diagram of the MUC1 fusion proteins is shown in Figure 1. To generate recombinant fusion proteins, the MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 plasmids were transiently transfected into Expi293 cells (Thermo Fisher, Waltham, MA, USA) and cultured for 6 days in a CO2 incubator equipped with a rotary shaker. The supernatant containing the recombinant proteins was collected and clarified by centrifugation. MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 were purified using a Protein A column (Cat. No. 17549852, Cytiva Life Sciences) followed by a HiLoad 16 / 600 Superdex 200pg size-exclusion column (Cat. No. 28989335, Cytiva Life Sciences). Both MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots in a freezer at -80°C. [Table 1-1] [Table 1-2]

[0132] Cell lines stably expressing human MUC1 were generated and validated, including the PT67 / human MUC1 cell line (an in-house generated cell line), the HEK293 / human MUC1 cell line (HEK293 obtained from ATCC, CRL-1573), and the HCT116 / human MUC1 cell line (ATCC CCL-247).

[0133] Cell lines stably expressing cynomolgus MUC1 (SEQ ID NO: 3) were generated and validated, including the HEK293 / cynomolgus MUC1, L929 / cynomolgus MUC1 cell line (L929 was obtained from ATCC, CCL-1), HCT116 / cynomolgus MUC1 cell line, and Daudi / cynomolgus MUC1 cell line (Daudi was obtained from ATCC, CCL-213).

[0134] To generate cell lines stably expressing human or cynomolgus MUC1, an ectotrophic vector was constructed using the retroviral construct PFBneo (STRATAGENE, catalog no. 217561-51). Transfection of the retroviral construct into PLAT-E cells (Cyagen, catalog no. IPMPC-01001) was performed using Lipofectamine 2000 (Invitrogen, reference no. 52758) according to the manufacturer's instructions. Viral supernatants were collected 24, 48, and 72 hours after transfection and filtered (0.45 μm) before use. The above-generated ectotrophic virus was used to transduce the dual-tropic packaging cell line PT67 in the presence of polybrene (final concentration: 8 μg / ml). After three rounds of transduction, PT67 cells were selected in G418 (final concentration: 1 mg / ml) for 7 days. To harvest the dual tropism virus produced from PT67 cells, the medium was replaced with fresh complete DMEM medium without G418 when the cells reached 100% confluence. Virus was collected once daily for three days. The cell lines were infected with viruses containing human or cynomolgus MUC1. After three rounds of transduction, infected cells were selected in G418 (final concentration: 1 mg / ml) for seven days.

[0135] immunization To generate antibodies against MUC1, cohorts of 30 inbred mice (BALB / C, MRL strain) were immunized with different MUC1 antigens, with each cohort employing a unique combination of MUC1 antigen (including the protein and cell line described in Example 1), dose, injection route, adjuvant, and immunization timing. Six cohorts, each consisting of five animals, were immunized. Animals were immunized over various time periods ranging from 0 to 90 days. To monitor immune responses, titrated sera were screened by ELISA and FACS after two to six immunizations, typically over a 30-90 day period. Sera were screened for antibody binding to the MUC1 antigen. MUC1-specific antibody responses were measured in each animal, and animals with sufficient anti-MUC1 Ig titers were selected for a final boost at 4 days.

[0136] Hybridoma fusion and screening Lymphoid organs, including spleens and lymph nodes, were isolated from immunized mice as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2 / 0 using PEG-based fusion. The resulting cells were seeded into 96-well cell culture plates using standard 1640 medium supplemented with HAT for hybridoma selection. After 10–13 days of culture and growth medium changes, hybridoma culture supernatants were collected from individual wells and screened to identify wells that secreted MUC1-specific antibodies. Initially, all supernatants were screened against the recombinant protein huMUC1-SEA-huIgG1 (derived from Example 1). Antibody binding to the recombinant protein huMUC1-SEA-huIgG1 was measured by ELISA. Supernatants from culture wells of three hybridoma fusions were screened for MUC1 antibodies. Briefly, 2 μg / mL huMUC1-SEA-huIgG1 was coated onto a 96-well ELISA plate, co-incubated with 50 μl of hybridoma culture supernatant for 30–60 min, washed, and incubated with an anti-mouse IgG Fc secondary Ab conjugated to HRP. After incubation and washing, the plate was developed with HRP substrate, and the absorbance was measured.

[0137] Hybridomas from positive wells were transferred to 24-well plates with fresh culture medium, grown for 2–3 days, and then rescreened by flow cytometry to confirm antibodies that bound to human MUC1 and cynomolgus MUC1-overexpressing cell lines.

[0138] Antibody (Ab) binding to human MUC1 and cynomolgus MUC1-overexpressing cell lines was measured by FACS. Briefly, 100 μl of hybridoma culture supernatant and human MUC1-overexpressing or cynomolgus MUC1-overexpressing cells were co-incubated for 30–60 min, washed, and then incubated with an anti-mouse IgG Fc secondary Ab conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry.

[0139] Subcloning and sequence analysis Selected anti-MUC1 Ab-secreting hybridomas were subcloned once or twice to ensure monoclonality. Briefly, positive hybridoma clones were subcloned by limiting dilution. After 7–10 days, culture supernatants were screened by ELISA and flow cytometry as described above to confirm human and cynomolgus MUC1 Ab binding. Stable hybridoma subclones were cultured in vitro for cell cryopreservation and gene cloning and sequencing of antibody VH and VL.

[0140] After subcloning, the anti-MUC1 Ab-secreting hybridomas were lysed in lysis buffer. The mRNA-containing lysates were then transferred to a 96-well deep-well plate for mRNA isolation, cDNA synthesis, and DNA sequencing using standard sequencing techniques (Sanger sequencing). Generally, total RNA was prepared from cell lysates, and cDNA was generated by reverse transcription of the mRNA using SuperScript III first-strand synthesis SuperMix (Invitrogen) according to the manufacturer's instructions. The sequence of the BG138P antibody is listed in Table 2.

[0141] Single B Screening Immunized mice were sacrificed and their spleens were harvested. Enriched plasma cells were loaded onto a 14K chip. hMUC1 beads, cynoMUC1 beads, and HEK293-cynoMUC1 cells were used for on-chip screening. Hits were selected and exported into lysis buffer. Single-cell RNA was purified, and Ig sequences were recovered using the BLI cDNA Synthesis Kit (BERKELEY LIGHTS) according to the manufacturer's instructions. The sequences of BG219P and BG346P are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0142] Large-scale expression and purification of chimeras BG219P, BG138P, and BG346P Chimeric antibodies chBG219P, chBG138P, and chBG346P were generated by transient transfection of ExpiCHO-s cells with heavy and light chain-containing plasmids produced in-house. Conditioned medium was collected, and antibodies were purified using a MabSelect SuRe column (Cytiva), followed by a POROS™ 50 HS column (Thermofisher Scientific) and a G-25 desalting column (Cytiva). All purified antibodies were stored in small aliquots in a freezer at -80°C.

[0143] Example 2. Determining binding kinetics and affinity of anti-MUC1 antibodies by SPR The chimeric anti-MUC1 antibody was characterized for its binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, mouse anti-human IgG Fc antibody was immobilized on an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences). Purified chimeric anti-MUC1 antibody was flowed over the chip surface and captured by anti-human IgG antibody. Serial dilutions of human or cynomolgus MUC1-SEA protein were then flowed over the chip surface, and the changes in surface plasmon resonance signal were analyzed. Using a 1:1 Langmuir binding model (BIA Evaluation Software, GE Life Sciences), the association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k onThe binding affinity profiles of the chimeric anti-MUC1 antibodies chBG138P, chBG346P, and chBG219P are shown in Table 3 below. chBG138P, chBG346P, and chBG219P exhibited high affinity for both human and cynomolgus MUC1-SEA. [Table 3]

[0144] Example 3. Determination of binding affinity of anti-MUC1 antibodies to MUC1 expressed in stable cell lines The binding affinity of chimeric anti-MUC1 antibodies to human and cynomolgus MUC1-overexpressing cell lines (HEK293 / human MUC1 and HEK293 / cynomolgus MUC1) was determined by FACS. Briefly, human or cynomolgus MUC1-overexpressing cells were incubated with serially diluted purified antibodies, washed, and then incubated with an anti-human IgG secondary antibody conjugated to APC. After incubation and washing, fluorescence was measured by flow cytometry. The binding affinity profiles of the chimeric anti-MUC1 antibodies are shown in Table 4 below and Figures 2A–2F (hIgG1 was used as a negative control). The results demonstrate that all three chimeric anti-MUC1 antibodies have favorable binding affinity to both human and cynomolgus MUC1 expressed in stable cell lines. [Table 4]

[0145] Example 4. Epitope binning of anti-MUC1 antibodies Epitope binning of chimeric anti-MUC1 antibodies was determined by competitive SPR assay. Briefly, an anti-mouse IgG Fc antibody was immobilized on an activated CM5 biosensor chip. Purified huMUC1-SEA-mIgG2a (human MUC1 linked to mouse IgG2a Fc) antigen was flowed over the chip surface and captured by an anti-mouse IgG antibody. The reference MUC1-SEA Ab 5F3 (Cancer Immunol Immunother. 2020 Jul;69(7):1337-1352) was injected first under saturating antigen binding conditions, followed by injection of chBG138P (Figure 3A), chBG219P (Figure 3B), or chBG346P (Figure 3C). Sensorgrams of epitope binning are shown in Figures 3A-3C. As shown in Table 5 below, the three chimeric MUC1 antibodies were grouped into two epitope bins of the MUC1-SEA domain. ChBG138P and chBG219P bind to the same epitope on MUC1-SEA as 5F3, a different MUC1 epitope from chBG346P. More specifically, chBG138P and chBG219P bind to epitope bin A, while chBG346P binds to bin B. [Table 5]

[0146] Example 5. Chimeric anti-MUC1 antibodies chBG138P, chBG219P, and chBG346P show reduced interference by soluble MUC1. The presence of soluble MUC1 in the specific binding of MUC1 antibody to MUC1-expressing cells was determined by competitive FACS assay. Briefly, human MUC1-expressing cells were incubated with 30, 3, and 0.3 μg / ml of MUC1 antibody in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co., Ltd.). After washing and incubation with anti-human IgG secondary antibody, fluorescence was measured by flow cytometry. The IC of soluble MUC1 blocking MUC1 antibody binding to MUC1-expressing cells was calculated. 50The values ​​are shown in Table 6, and the blocking curves are shown in Figures 4A–4F (where HMFG1 is the positive control, and mIgG and hIgG1 are the negative controls). The profiles indicate that HMFG binding to MUC1-expressing cells can be easily interfered with at high, medium, and low antibody concentrations (i.e., 30, 3, and 0.3 μg / ml), whereas the binding of chBG138P, chBG219P, and chBG346P is only slightly interfered with at low antibody concentrations (i.e., 0.3 μg / ml) (Figures 4A–F). Taken together, the profiles in Figure 4 indicate that the binding of MUC1 antibodies to MUC1-expressing cells is significantly less interfered with by soluble MUC1 compared with HMFG1 (Abcam), which targets the N-terminus of MUC1. [Table 6]

[0147] Example 6. Anti-MUC1 monoclonal antibodies targeting the MUC1 membrane proximal region bind to cancer cell lines but not to normal T cells, whereas antibodies HMFG1 or 16A targeting the N-terminus of MUC1 can bind to normal T cells. To assess whether anti-MUC1 monoclonal antibodies targeting the MUC1 membrane-proximal region can differentially bind to MUC1-expressing tumor cells and MUC1-expressing normal cells, such as activated T cells, a FACS binding assay was performed. For tumor cell line binding experiments, cells were stained with the anti-human MUC1 antibody chBG138P or a control (human IgG1) for 1 hour and then harvested. The cells were then washed twice and subsequently stained with a secondary antibody (Alexa Fluor® 647 anti-human IgG Fc) for 30 minutes. The cells were washed and fixed with 1% paraformaldehyde (PFA) / DPBS before FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.), and the data were analyzed using NovoExpress software. As shown in Figures 5A-5C, the chimeric antibody chBG138P targeting the human MUC1 membrane proximal region binds to the MUC1-expressing tumor cell lines HCC827 (Figure 5A), H1975 (Figure 5B), and T-47D (Figure 5C) in a dose-dependent manner (human IgG1 as a negative control), indicating that antibodies targeting the MUC1 membrane proximal region can be used to target cancer cells and, therefore, can be applied to treat cancers that express or overexpress MUC1.

[0148] To evaluate whether monoclonal antibodies targeting the membrane-proximal region of MUC1 can avoid binding to normal cells expressing MUC1, we performed an activated T cell binding assay. Briefly, human peripheral blood mononuclear cells (PBMCs) from six healthy donors purchased from Allcells or Stemcell were stimulated with 1 μg / ml PHA-L for 3 days. FACS staining was then performed using the stimulated PBMCs. The cell suspension was preincubated with the LIVE / DEAD™ Fixable Dead Cell Stain Kit (Invitrogen, reference number L34964) and Fc receptor blocking solution (FACS buffer containing 100 μg / ml human IgG) before staining with anti-human antibodies. Cells were washed twice and incubated for 1 hour with 10 μg / mL of an anti-MUC1 monoclonal antibody targeting the MUC1 membrane-proximal region, or antibodies HMFG1 (positive control, Abcam, catalog no. ab215670) or 16A (positive control, Biolegend, catalog no. 355608) targeting the N-terminus of MUC1. Cells were then washed and stained for 30 minutes with PE-CY7 anti-human αβ TCR (eBioscience, catalog no. 25-9986-42) or AF647 anti-human IgG Fc (Biolegend, reference no. 409320). Cells were washed and fixed with 1% PFA in DPBS before FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.), and data were analyzed using NovoExpress software. As shown in Figures 6 and 7A-7H, none of the chimeric antibodies chBG138P (Figures 7A, 7B, 7E, and 7F), chBG219P (Figures 7E and 7F), or chBG346P (Figures 7E and 7F) bind to normal activated human T cells expressing MUC1. However, antibodies HMFG1 (Figures 7C and 7D) and 16A (Figures 7G and 7H), which target the MUC1 N-terminus, bind to the majority of activated T cells.Figures 8A-8D show that chimeric (chBG138P) and humanized (huBG138P-Hz2 and huBG138P-Hz4) versions of antibody BG138P retain similar binding properties to BG138P and do not bind to normal activated human T cells expressing MUC1 (Figures 8A and 8B), whereas antibody HMFG1, which targets the MUC1 N-terminus (Figures 8C and 8D), binds to normal activated T cells.

[0149] The results indicate that, compared with antibodies targeting the MUC1 N-terminus, which bind to both cancer cells and normal T cells, antibodies targeting the MUC1 membrane proximal region specifically target cancer cells and not normal T cells, and therefore may result in an optimized safety profile when used as an antitumor therapy in humans.

[0150] Example 7. chBG138P-induced MUC1 internalization in cancer cell lines chBG138P and human IgG were labeled using pHrodo iFL STP Ester (amine-reactive dye, Invitrogen, reference number P36013) according to the manufacturer's instructions. Cancer cell lines T-47D, HCC827, and H1975 were incubated with pHrodo-labeled chBG138P (final concentration 10 μg / ml) or human IgG (human immunoglobulin for intravenous injection, Hualan) as a negative control for 60 minutes at 4°C in the dark. Cells were washed twice with DPBS at 4°C. Half of the cells were kept at 4°C and the other half were transferred to 37°C. Cells were incubated at 4°C or 37°C for 5 hours. 1 μl / well of 7-AAD (7-amino-actinomycin D) was added to all groups. Cells were incubated on ice in the dark for 30 minutes. Cells were washed and fixed with 1% paraformaldehyde (PFA) in DPBS before FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress software. The percentage of pHrodo-positive cells was calculated according to the manufacturer's instructions (Invitrogen, Reference Number P36013).

[0151] As shown in Figures 9A-9C, approximately 10-20% of cells in all three cancer cell lines, T-47D (9A), HCC827 (9B), and H1975 (9C), were pHrodo-positive after 5 hours of incubation at 37°C. These results indicate that the antibody chBG138P, which targets the membrane-proximal region of MUC1, induced internalization by MUC1-expressing human cancer cell lines, whereas the negative control hIgG induced little internalization. This internalization property may be beneficial for the design of monoclonal or bispecific antibody therapies to avoid membrane target loss and drug resistance. Furthermore, the potential for internalization holds promise for the development of antibody-drug conjugates containing the MUC1 antibodies disclosed herein to treat MUC1-expressing cancers.

[0152] Example 8. Humanization of mouse anti-human MUC1 antibodies BG138P and BG219P Humanization of BG138P A CDR-grafting strategy was employed for the humanization of the murine anti-human MUC1 BG138P antibody. The murine BG138P antibody heavy and light chain variable domain sequences (abbreviated as VH and VL, respectively) were aligned with the germline sequences of human antibodies using the IgBlast tool, and the most homologous human germline framework was selected as the acceptor framework for CDR grafting. To maintain the canonical structure of the CDRs, the murine version of the BG138P antibody structure was modeled using Schrodinger software and used to select potential backmutation sites to maintain binding affinity. Each backmutation site was screened by constructing multiple variants without that mutation to assess the necessity of retaining that site in the humanized version. The identified important backmutation sites were then combined to construct a second round of humanized variants for affinity characterization.

[0153] Specifically, for the BG138P VH sequence, the germline sequence of IGHV1-3*01 and J region H4 (JH4) was selected as the acceptor framework. For the BG138P VL sequence, Vκ1-27*01 and J region κ2 were selected as the acceptor framework. Antibody variants, including a CDR-grafted version (designated with the suffix "Hz0"), a version with all backmutation sites (designated with the suffix "Bz0"), and multiple variants with reduced backmutation sites (designated with the suffixes "Bz1" to "Bz16"), were constructed in a proprietary IgG1 / Cκ eukaryotic expression vector, produced using the Expi293™ Expression System (Thermofisher Scientific), and purified using MabSelect PrismA™ Protein A chromatography resin (Cytiva). The antibody variant list and corresponding VH / VL sequences from this round of screening are summarized in Table 7 ("X" indicates the CDR position for grafting). [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0154] All generated antibody variants were characterized by SPR affinity against human and cynomolgus monkey MUC1 SEA domain protein (SEQ ID NO: 2) using a Biacore 2000 (Cytiva) system. Variants with a greater than 2-fold loss in affinity compared to chimeric BG138P (calculated by Kd at a single concentration binding kinetics) were selected as Tier 1 backmutation sites, and variants with a 1.5- to 2-fold loss in affinity were selected as Tier 2 backmutation sites. The SPR binding affinity data are summarized in Tables 8 and 9. [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2]

[0155] Motif analysis revealed that the amino acids "DG" (Kabat numbers 56-57) at the boundary between CDRL2 and FR3 are an aspartate isomerization motif. To eliminate the risk of this post-translational modification (PTM), several mutation variants were constructed. Four mutations (DG to EG, AG, TG, and SG; these variants are referred to as BG138P-PTM-m1 to m4 in Tables 10 and 11) were designed and introduced into the chimeric BG138P to remove this isomerization motif. SPR binding characterization against human and cynomolgus monkey MUC1 SEA proteins was performed as described above. The results are shown in Tables 10 and 11. All variants exhibited binding equivalent to that of the chimeric BG138P antibody. Mutation m4 (DG to SG) was ultimately selected for use in the humanized BG138P antibody. [Table 10] [Table 11]

[0156] The Tier 1 and Tier 2 backmutation sites of VH and VL were combined for the construction of a second round of humanized variants (all of which contained a "DG" to "SG" PTM removal mutation). The final combined humanized variants (called huBG138P-Hz1-Hz4 or abbreviated as Hz1-Hz4) are summarized in Table 12, and the sequences are listed in Table 14. [Table 12]

[0157] SPR assays were then performed to evaluate the binding affinity of huBG138P-Hz1-Hz4 to human and cynomolgus monkey MUC1 SEA. SPR assays showed that all variants maintained binding equivalent to chimeric BG138P. The data are summarized in Table 13. [Table 13]

[0158] FACS binding (iQue3™ FACS, Sartorius) assays were then performed using HEK293-hMUC1 and HEK293-cynoMUC1 to determine the cell-based binding activity of Hz1–Hz4. The FACS binding data for chimeric BG138P and its humanized antibody huBG138P-Hz1–Hz4 (hIgG1 was used as a control) were processed using GraphPad Prism software and are shown in Figures 10A and 10B. The results show that BG138P-Hz2 and BG138P-Hz4 maintain cell-based binding activity to both human and cynomolgus MUC1 similar to that of chimeric BG138P, whereas BG138P-Hz1 and BG138P-Hz3 maintain cell-based binding activity to human MUC1 similar to that of chimeric BG138P, but exhibit lower cell-based binding activity to cynomolgus MUC1 than chimeric BG138P.

[0159] The HCDR, LCDR, VH and VL amino acid sequences of huBG138P-Hz2 are shown in Table 14. [Table 14]

[0160] Humanization of BG219P For humanization of BG219P, human germline IgG genes were searched for sequences that shared a high degree of homology with the protein sequence of the BG219P variable region by performing sequence comparisons against the IMGT human immunoglobulin gene database. Human IGHV and IGKV genes, which are frequently present in the human antibody repertoire and share high homology with murine BG219P, were selected as templates for humanization.

[0161] Humanization was performed by CDR grafting followed by the incorporation of significant back mutations. The humanized antibody was engineered in a human IgG1 wild-type format using an expression vector developed in-house. In the first round of humanization, mutations from murine to human amino acid residues in the framework regions were guided by 3D structural analysis, and structurally important murine framework residues were retained in the first round of humanization design to maintain the canonical structure of the CDRs. Among all 19 variants generated, BG219P-Bz0, a CDR-grafted version of the antibody variant containing all back mutation sites, possesses a theoretical binding activity similar to that of the parent murine antibody BG219P.

[0162] Specifically, BG219P-Bz0 was generated as described herein. Human germline variable genes IGKV1-39*01 and IGKJ2*01 and human germline variable genes IGHV3-23*01 and IGHJ6*01 were selected as the acceptor frameworks for the BG219P VL and VH sequences. The LCDR of mouse BG219P was grafted onto the frameworks of human germline variable genes IGKV1-39*01 and IGKJ2*01, retaining the D17E, A43S, I48V, T69P, and F71Y mouse framework residues. The resulting amino acid and DNA sequences of BG219P-Bz0 VL are shown in Table 16. The HCDRs of murine BG219P were grafted into the frameworks of the human germline variable genes IGHV3-23*01 and IGHJ6*01, retaining the S30N, S49A, A93T, and K94R murine framework residues. The resulting amino acid and DNA sequences of BG219P-Bz0 VH are shown in Table 16.

[0163] To further improve the biophysical properties for therapeutic use in humans, several additional amino acid changes in the CDR regions of both VH and VL were made starting from the humanized BG219P antibody huBG219P-Bz0. Considerations included removing post-translational modifications while maintaining binding activity and improving thermal stability (Tm).

[0164] More than 30 humanized BG219P (also called huBG219P) variants were constructed using an in-house IgG1 / Cκ eukaryotic expression vector containing the human wild-type IgG1 and kappa chain constant regions, respectively, with easily adapted subcloning sites. Variants were generated by transient transfection of plasmids into ExpiCHO-s cells (Thermofisher Scientific). The conditioned medium was collected, and the variants were purified using MabSelect™ SuRe columns (Cytiva), followed by buffer exchange with UF / DF. All purified antibodies were stored in small aliquots in a freezer at -80°C.

[0165] For affinity determination, the antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding profiles of the anti-MUC1 antibodies determined by SPR are summarized in Table 15. huBG219P-E39 and huBG219P-E43 have similar binding affinities with dissociation constants of 35.2 pM and 30.3 pM, respectively, which are comparable to that of chimeric BG219P (39.8 pM). The sequences of huBG219P-E39 and huBG219P-E43 are shown in Table 16. [Table 15] [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4]

[0166] To assess the binding activity of anti-MUC1 antibodies to native MUC1 on live cells, ZR-75 cells were used in a FACS-based binding assay. Live ZR-75 cells were seeded in 96-well plates and incubated with a dilution series of chimeric or humanized BG219P. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC2 values ​​for dose-dependent binding to human native MUC1 were calculated. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 11 and Table 17, the humanized BG219P antibodies huBG219P-Bz0, E39, and E43 retained comparable binding affinity to native MUC1 compared to chimeric BG219P. [Table 17]

[0167] Example 9. Generation of scFvs of mouse anti-human MUC1 mAbs BG138P, BG219P, and BG346P To generate scFv constructs, the heavy and light chain variable regions (VH, VL) were linked via an artificial amino acid linker. For clone BG138P, the VH and VL from BG138P-Hz2 were linked by a (GGGGS)4 linker (SEQ ID NO: 83) with a histidine tag (i.e., HHHHHH (SEQ ID NO: 85)) fused at the C-terminus to generate the final construct VH-(GGGGS)4-VL-HHHHHH, designated scFv1-78P. For clone BG219P, the VH and VL of murine BG219P were used to generate VH-(GGGGS)4-VL-HHHHHH; further humanization work was based on this format, the details of which are disclosed below. For clone BG346P, the VH and VL of murine BG346P were first linked by a (GGGGS)3 linker (SEQ ID NO: 84) to generate VH-(GGGGS)3-VL-HHHHHH, and further engineering work based on this construct is disclosed below.

[0168] Humanization and engineering of BG138P scFv To test whether there was room for further improvement of the physical properties of scFv1-78P, 40 constructs with additional single mutations in either VL or VH were tested for their yield, purity, and thermal stability. For the experiments, scFv1-78P was fused onto a C-terminal Fc tag to produce scFv-Fc (scFv 野生型 A series of scFv1-78P mutants were also fused onto a C-terminal F tag to produce scFv-Fc (scFv 変異体 The resulting scFv (referred to as scFv-Fc) was overexpressed in Expi293™ cells (Thermo Fisher) and purified using a MabSelect SuRe™ column (Cytiva). 変異体 Yield and purity of scFv-Fc 野生型 -Fc. 21 scFv 変異体 -Fc, scFv野生型 The two scFvs showed better yields than the -Fc. 変異体 -Fc showed good yield and purity, but both of them were similar to scFv 野生型 -Fc did not show better thermal stability than -Fc.

[0169] Humanization and engineering of BG219P scFv The VH and VL of mouse BG219P were linked to generate an scFv construct as described above. For humanization, the frameworks of the mouse VH and VL were compared to a human germline database. The closest human germlines, IGHV3-21*01+IGHJ4 and IGKV1-33*01+IGKJ2, were selected for humanization. The six CDRs in the mouse scFv were directly grafted into the selected human germline without additional mutations in the framework, resulting in the construct scFv2-14P. Characterization of the affinity and physical properties is disclosed below.

[0170] To remove potential post-translational deamidation sites in the CDR-H2 region of scFv2-14P without affecting the affinity of the scFv, the labile Asp was mutated to Glu to mimic the structure of the CDR-H2 loop of scFv2-14P. The resulting mutant scFv2-57P showed comparable yields from Expi293™ cells and similar affinity for soluble recombinant MUC1 protein and MUC1+ cell lines compared to scFv2-14P. The data are shown in Table 18. [Table 18]

[0171] Humanization and engineering of BG346P scFv The VH and VL sequences from mouse BG346P were linked via the artificial linker (GGGGS)3 (SEQ ID NO: 84) to generate mouse scFv BG346P. Humanization of the mouse BG346P scFv was performed using a framework swap strategy. The CDR residues of BG346P were grafted into a series of frameworks listed in Table 19, with additional mutations to the frameworks listed. [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4]

[0172] The humanized scFv constructs listed above were overexpressed in Expi293™ cells (Thermo Fisher). To select stable scFv constructs, the supernatant of Expi293™ cells overexpressing scFv proteins was kept at 60°C for 2 hours. The heated scFv proteins were tested for their ability to bind to the recombinant MUC1 SEA domain. After the heat stress experiment, the stable scFv scFv3-65P was selected for further manipulation. A longer artificial linker, (GGGGS)4 (SEQ ID NO: 83), was introduced into scFv3-65P to replace the original (GGGGS)3 (SEQ ID NO: 84) linker, allowing for better scFv expression. The resulting scFv was designated scFv3-96P. In the scFv3-96P background, amino acids with potential post-translational modification risks were replaced with amino acids with similar side chains to generate constructs scFv3-98P, scFv3-99P, and scFv3-00P through scFv3-07P. Furthermore, to minimize immunogenicity, the mouse back mutations introduced in scFv3-65P were mutated back to human amino acids relative to scFv3-96P to generate constructs scFv3-08P through scFv3-19P. The relative affinities of scFv3-98P, scFv3-99P, and scFv3-00P through scFv3-19P were compared to scFv3-96P, and any beneficial mutations were then selected. The data are presented in Table 20. [Table 20]

[0173] After testing each individual mutation for its effect on affinity, mutations that were not detrimental to scFv affinity were selected and combined. For amino acids with PTM risk, the goal was to remove as many of them as possible without affecting affinity. For mouse mutations, the goal was to revert as many of them to human amino acids as possible to minimize immunogenicity. Finally, the construct with the least PTM risk and the fewest number of mouse mutations that maintained affinity was selected as the final scFv. This was BG346P-scFv3-20P (or abbreviated as scFv3-20P), as shown in Table 21. [Table 21]

[0174] The sequences of the four scFvs are listed in Table 22. [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 23]

[0175] Determination of affinity of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P The binding affinity and kinetic constants of the purified scFv fragments were determined by surface plasmon resonance (Biacore 8K) at 25°C. MUC1-SEA-mIgG2a was coupled to a CM5 Biocore sensor derivatized with a monoclonal rabbit anti-mouse Fc antibody (catalog number 29215281) by amine coupling. Then, the scFv fragments scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P were flowed at a rate of 30 μl / min. Association of the scFv with the MUC1 protein was monitored for 2 min, and dissociation of the scFv fragments in 1× HBS buffer (Cytiva, catalog: BR100669) was monitored for 10 min. Ka and Kd were determined by fitting the real-time sensorgrams to a 1:1 binding model using Biacore™ Insight Evaluation software. D ) is K D The binding kinetic parameters of the scFv proteins to the MUC1 protein are shown in Table 24. [Table 24]

[0176] Example 10. Binding activity of scFv fragments to native MUC1 To assess the binding ability of anti-MUC1 scFv to MUC1 on the surface of live cells, HEK293 cells were engineered to overexpress full-length MUC1 protein as described above. For the assay, 1 × 10 6 HEK293-MUC1 cells were seeded in a 96-well plate. To generate scFv dose-response curves, serially diluted scFv proteins (4.2 pm - 250 nm) were added to the cells, and bound scFv was detected using the His-tag antibody ifluor488 (Cat. No. A01800). Flow Cytometry Instruments-iQue® 3 (Sartorius) was used to measure the MFI (median fluorescence intensity) of each cell population and determine the EC of each scFv.50 and Emax were determined using a four-parameter logistic model. The results are shown in Table 25.

[0177] The results show that all scFvs maintained affinity for the full-length MUC1 protein on the cell surface. scFv1-78P, scFv2-14P, and scFv2-57P showed better affinity than scFv3-20P. [Table 25]

[0178] Example 11. Epitope mapping of humanized scFv fragments To assess whether the three scFv fragments, scFv1-78P, scFv2 (scFv2-14P and scFv2-57P share the same epitope bin and are therefore collectively referred to as "scFv2" in this example), and scFv3-20P, could compete with each other for binding to their respective epitopes on MUC1, a binding competition assay was performed using a tandem method. Briefly, MUC1-SEA-mIgG2a was immobilized on a CM5 Biocore sensor derivatized with a monoclonal rabbit anti-mouse Fc antibody (catalog no. 29215281) by amine coupling. The first scFv fragment was then flowed at a rate of 10 μl / min. Capture of the first scFv by MUC1-SEA-mIgG2a was monitored for 1.5 minutes. The second scFv fragment was then flowed at a rate of 10 μl / min, and capture was monitored for 2 minutes. Competition between scFv1-78P and scFv2 (scFv2-14P, scFv2-57P) is shown in Figure 12, while scFv3-20P belongs to a different binning.

[0179] The results show that the epitopes of scFv1-78P, scFv2-14P, and scFv2-57P at least partially overlap, but the epitope of scFv3-20P is distinct from the other three.

[0180] Example 12. Physical properties of humanized scFv fragments The hydrophobicity, thermal stability, and aggregation tendency of the humanized scFv fragments were assessed as follows.

[0181] Hydrophobicity evaluation To determine the hydrophobicity of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P using HIC, 50 μg of sample at 1 mg / ml was diluted with mobile phase A solution (1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0) to achieve a final ammonium sulfate concentration of approximately 1 M before analysis. A Thermo Fisher mabpac HIC-10 column was used with a linear gradient of mobile phase A and mobile phase B solution (50 mM sodium phosphate, pH 7.0) over 29 minutes at a flow rate of 0.5 mg / min. Peak retention times were monitored at A280 absorbance. The results are summarized in Table 26. scFv1-78P (room temperature, 14.2 min) and scFv3-20P (room temperature, 9.7 min) showed higher hydrophilicity than scFv2-14P (room temperature, 20.5 min) and scFv2-57P (room temperature, 20.6 min). All four scFv domains showed acceptable hydrophobicity. [Table 26]

[0182] Thermal stability evaluation The thermal stability (described by the thermal unfolding transition midpoint, Tm (°C) (melting temperature)) of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P was measured by extrinsic fluorescence. Tm was determined using the QuantStudio™ 6 Flex System from Applied Biosystems. 20 μl of sample at 1 mg / ml was mixed with 20 μl of 40X SYPRO Orange. The plate was scanned from 25°C to 95°C at a rate of 0.9°C / min. Tm was obtained using the first derivative of the raw data from the QuantStudio™ 6 Flex System Analysis software. The results are summarized in Table 27. scFv1-78P (70.0°C) and scFv3-20P (69.8°C) showed Tm approximately 10°C higher than scFv2-14P (60.0°C) and scFv2-57P (60.2°C). All four scFv domains showed acceptable thermal stability. [Table 27]

[0183] Agglomeration tendency evaluation To determine the aggregation tendency of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P, static light scattering intensity was measured using the Uncle™ system (Unchained Labs). During the measurement, approximately 8.8 μl of protein sample was loaded into a cuvette at 1 mg / ml. The sample was held at 25°C for 120 seconds and then heated to 95°C at a rate of 0.3°C / min. Scattering data were collected at a 90° angle with a laser wavelength of 266 nm. Tagg (aggregation temperature) was analyzed and calculated using Uncle Analysis Software. The results are summarized in Table 28. scFv3-20P (63.8°C) showed a much higher Tagg than scFv1-78P (56.3°C), scFv2-14P (54.5°C), and scFv2-57P (53.1°C). All four scFv domains showed acceptable Tagg. [Table 28]

Claims

1. An anti-MUC1 antibody or antigen-binding fragment thereof that specifically binds to human MUC1, (i) A heavy chain variable region comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; (ii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 56, and (f) an LCDR3 of SEQ ID NO: 9; (iii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (iv) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 78, (b) an HCDR2 of SEQ ID NO: 79, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (v) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; (vi) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29, or (vii) The anti-MUC1 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 74, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO:

29.

2. (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 20, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 21; (iii) A heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 30, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 31; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 59; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (vi) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 58, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 59; (vii). a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 58, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (viii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 62; (ix) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 66; (x) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 68; (xi) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 71, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 72; (xii) A heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 75, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 76; or (xiii) The anti-MUC1 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 80, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:

81.

3. (i) A heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; (ii) A heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (iii) A heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; (iv). A heavy chain variable region (VH) comprising SEQ ID NO: 57, and a light chain variable region (VL) comprising SEQ ID NO: 59; (v) A heavy chain variable region (VH) comprising SEQ ID NO: 57, and a light chain variable region (VL) comprising SEQ ID NO: 60; (vi). A heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 59; (vii). A heavy chain variable region (VH) comprising SEQ ID NO: 58, and a light chain variable region (VL) comprising SEQ ID NO: 60; (viii). A heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; (ix). A heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; (x) A heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; (xi). A heavy chain variable region (VH) comprising SEQ ID NO: 71, and a light chain variable region (VL) comprising SEQ ID NO: 72; (xii) A heavy chain variable region (VH) comprising SEQ ID NO: 75 and a light chain variable region (VL) comprising SEQ ID NO: 76, or (xiii) The anti-MUC1 antibody or antigen-binding fragment thereof according to claim 2, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 80, and a light chain variable region (VL) comprising SEQ ID NO:

81.

4. The anti-MUC1 antibody or antigen-binding fragment thereof of claim 3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 10, 11, 20, 21, 30, 31, 57, 58, 59, 60, 61, 62, 66, 68, 71, 72, 75, 76, 80, or 81 have been inserted, deleted, or substituted.

5. The anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human-engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

6. 6. The antibody or antigen-binding fragment thereof of claim 5, comprising an scFv comprising a VH comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, and a VL comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:

81.

7. The antibody or antigen-binding fragment thereof of claim 6, comprising an scFv comprising a VH comprising SEQ ID NO: 57, SEQ ID NO: 71, SEQ ID NO: 75, or SEQ ID NO: 80, and a VL comprising SEQ ID NO: 60, SEQ ID NO: 72, SEQ ID NO: 76, or SEQ ID NO:

81.

8. (i) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 57, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 60; (ii) a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 71, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 72; (iii) A VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 75, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 76; or (iv) The antibody or antigen-binding fragment thereof of claim 7, comprising an scFv comprising a VH comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 80, and a VL comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:

81.

9. (i). A VH comprising SEQ ID NO: 57, and a VL comprising SEQ ID NO: 60; (ii) A VH comprising SEQ ID NO: 71 and a VL comprising SEQ ID NO: 72; (iii) A VH comprising SEQ ID NO: 75 and a VL comprising SEQ ID NO: 76, or (iv) The antibody or antigen-binding fragment thereof of claim 8, comprising an scFv comprising a VH comprising SEQ ID NO: 80 and a VL comprising SEQ ID NO:

81.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within SEQ ID NO: 57, 60, 71, 72, 75, 76, 80, or 81 have been inserted, deleted, or substituted.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the VH and VL of the scFv are linked via an amino acid linker.

12. The antibody or antigen-binding fragment thereof of claim 11, wherein the amino acid linker comprises SEQ ID NO: 83 or 84.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the scFv comprises SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 77, or SEQ ID NO:

82.

14. The anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

15. The anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which is hypoglycosylated, non-glycosylated, or hypofucosylated.

16. The anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising increased bisecting GlcNAc structures.

17. The anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the Fc domain is IgG1.

18. A pharmaceutical composition comprising the anti-MUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. An isolated nucleic acid encoding the anti-MUC1 antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

20. A vector comprising the nucleic acid of claim 19.

21. 21. A host cell comprising the nucleic acid of claim 19 or the vector of claim 20.

22. 22. A process for producing an anti-MUC1 antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 21 and recovering the antibody or antigen-binding fragment from the culture.

Images