MUC1 antibodies and methods of use

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human MUC1, as well as pharmaceutical compositions comprising the antibodies.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT application No. PCT / CN2023 / 080961, filed March 12, 2023, entitled “MUC1 Antibodies and Methods of Use”; PCT / CN2023 / 079507, filed March 3, 2023, entitled “MUC1 and CD16A Antibodies and Methods of Use”; and PCT / CN2023 / 107724, filed July 17, 2023, entitled “MUC1 and CD16A Antibodies and Methods of Use”, all of which are hereby incorporated by reference.

[0003] sequence list

[0004] This application is submitted together with an electronic sequence list. The sequence list is provided as a file titled "01368-0023-00PCT_SL.xml" created on February 27, 2024, with a size of 121,339 bytes. The information in the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0005] This article discloses antibodies or antigen-binding fragments thereof that bind to human MUC1 and their methods of use. Background Technology

[0006] Mucin 1 (MUC1; also known as CA15-3, EMA, MCD, PEM, PUM, KL-6, MAM6, MCKD, PEMT, CD227, H23AG, MCKD1, ADMCKD, 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 lungs, breasts, stomach, pancreas, and uterus. MUC1 plays a crucial role in forming a protective mucus barrier on these epithelial surfaces.

[0007] MUC1 is translated as a single polypeptide whose extracellular portion undergoes self-cleavage into two subunits at the SEA (sea urchin spermatin, enterokinase, and aggregatein) domain. The distal extracellular membrane subunit, containing 20-125 20-amino acid repeats (variable number tandem repeats, VNTRs), forms a heterodimeric complex with the proximal membrane subunit via strong non-covalent interactions. The proximal membrane subunit consists of a 58-amino acid (aa) extracellular domain, a 28-aa transmembrane domain, and a 72-aa cytoplasmic tail, which intersects with multiple oncogenic signaling pathways.

[0008] MUC1 is abnormally overexpressed in a variety of common human cancers, including lung cancer, breast cancer, colon cancer, gastric cancer, esophageal cancer, and ovarian cancer. In addition to upregulation, changes in MUC1 hypoglycosylation and cellular localization are also associated with cancer. Furthermore, cleaved distal membrane subunits tend to detach from the surface of cancer cells, accompanied by increased plasma levels.

[0009] Given its overexpression in various human cancers, MUC1 is an attractive tumor-associated antigen. However, previous attempts to target the distal membrane (MUC1 N-terminal) subunits (such as AS1402 (huHMFG-1) and BrevaRex (AR-20.5)) have been unsuccessful. This is partly because the circulating pool of shed MUC1 prevents antibodies from targeting the surface of tumor cells expressing MUC1. Conversely, the non-shedding proximal membrane (MUC1 C-terminal) subunits (which function as oncoproteins) represent an attractive target for developing antibody-based therapeutics.

[0010] This disclosure provides an anti-MUC1 antibody that targets the proximal region of the MUC1 membrane (particularly the MUC1-SEA domain, which is adjacent to the transmembrane region of the MUC1-C terminus) with minimal interference from shed MUC1. Summary of the Invention

[0011] This disclosure relates to anti-MUC1 antibodies and their antigen-binding fragments.

[0012] In its implementation, this disclosure relates to an anti-MUC1 antibody and its antigen-binding fragment, said anti-MUC1 antibody and its antigen-binding fragment specifically binding to cancer cells and not to normal cells.

[0013] In an embodiment, this disclosure relates to an anti-MUC1 antibody or an antigen-binding fragment thereof, said anti-MUC1 antibody or antigen-binding fragment thereof comprising an antibody or a binding fragment thereof that specifically binds to the SEA domain (SEQ ID NO:1) of human MUC1 at amino acids 1036 to 1155 (SEQ ID NO:2).

[0014] In its implementation, this disclosure relates to an anti-MUC1 antibody or antigen-binding fragment that specifically binds to human MUC1, said anti-MUC1 antibody or antigen-binding fragment comprising:

[0015] (i). The heavy chain variable region comprising (a) HCDR1 (heavy chain complementarity determination region 1) of SEQ ID NO:4, (b) HCDR2 of SEQ ID NO:5 and (c) HCDR3 of SEQ ID NO:6, and the light chain variable region comprising (d) LCDR1 (light chain complementarity determination region 1) of SEQ ID NO:7, (e) LCDR2 of SEQ ID NO:8 and (f) LCDR3 of SEQ ID NO:9;

[0016] (ii). The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:4, (b) HCDR2 of SEQ ID NO:5 and (c) HCDR3 of SEQ ID NO:6, and the light chain variable region comprising (d) LCDR1 of SEQ ID NO:7, (e) LCDR2 of SEQ ID NO:56 and (f) LCDR3 of SEQ ID NO:9;

[0017] (iii). The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:14, (b) HCDR2 of SEQ ID NO:15 and (c) HCDR3 of SEQ ID NO:16, and the light chain variable region comprising (d) LCDR1 of SEQ ID NO:17, (e) LCDR2 of SEQ ID NO:18 and (f) LCDR3 of SEQ ID NO:19;

[0018] (iv). The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:78, (b) HCDR2 of SEQ ID NO:79 and (c) HCDR3 of SEQ ID NO:16, and the light chain variable region comprising (d) LCDR1 of SEQ ID NO:17, (e) LCDR2 of SEQ ID NO:18 and (f) LCDR3 of SEQ ID NO:19;

[0019] (v). The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:25 and (c) HCDR3 of SEQ ID NO:26, and the light chain variable region comprising (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:28 and (f) LCDR3 of SEQ ID NO:29;

[0020] (vi) The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:25, and (c) HCDR3 of SEQ ID NO:26, and the light chain variable region comprising: (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:65, and (f) LCDR3 of SEQ ID NO:29; or

[0021] (vii). The heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:74 and (c) HCDR3 of SEQ ID NO:26, and the light chain variable region comprising (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:65 and (f) LCDR3 of SEQ ID NO:29.

[0022] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment comprises:

[0023] (i) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:10 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:11;

[0024] (ii) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:20 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:21;

[0025] (iii) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:30 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:31;

[0026] (iv) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:57 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:59;

[0027] (v). A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:57 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:60;

[0028] (vi) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:58 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:59;

[0029] (vii) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:58 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:60;

[0030] (viii) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:61 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:62;

[0031] (ix). A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:61 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:66;

[0032] (x) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:61 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:68;

[0033] (xi). A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:71 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:72;

[0034] (xii). A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:75, and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:76; or

[0035] (xiii) A heavy chain variable region (VH) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:80 and a light chain variable region (VL) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:81.

[0036] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment comprises:

[0037] (i) The heavy chain variable region (VH) containing SEQ ID NO:10 and the light chain variable region (VL) containing SEQ ID NO:11;

[0038] (ii). The heavy chain variable region (VH) containing SEQ ID NO:20 and the light chain variable region (VL) containing SEQ ID NO:21;

[0039] (iii) A heavy chain variable region (VH) comprising SEQ ID NO:30 and a light chain variable region (VL) comprising SEQ ID NO:31;

[0040] (iv). The heavy chain variable region (VH) containing SEQ ID NO:57 and the light chain variable region (VL) containing SEQ ID NO:59;

[0041] (v). The heavy chain variable region (VH) containing SEQ ID NO:57 and the light chain variable region (VL) containing SEQ ID NO:60;

[0042] (vi) The heavy chain variable region (VH) containing SEQ ID NO:58 and the light chain variable region (VL) containing SEQ ID NO:59;

[0043] (vii). Containing the heavy chain variable region (VH) of SEQ ID NO:58 and the light chain variable region (VL) of SEQ ID NO:60;

[0044] (viii). The heavy chain variable region (VH) containing SEQ ID NO:61 and the light chain variable region (VL) containing SEQ ID NO:62;

[0045] (ix). The heavy chain variable region (VH) containing SEQ ID NO:61 and the light chain variable region (VL) containing SEQ ID NO:66;

[0046] (x). Containing the heavy chain variable region (VH) of SEQ ID NO:61 and the light chain variable region (VL) of SEQ ID NO:68;

[0047] (xi). Containing the heavy chain variable region (VH) of SEQ ID NO:71 and the light chain variable region (VL) of SEQ ID NO:72;

[0048] (xii). Containing the heavy chain variable region (VH) of SEQ ID NO:75 and the light chain variable region (VL) of SEQ ID NO:76; or

[0049] (xiii) The heavy chain variable region (VH) containing SEQ ID NO:80 and the light chain variable region (VL) containing SEQ ID NO:81.

[0050] In the implementation scheme, in the anti-MUC1 antibody or its antigen-binding fragment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids in 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.

[0051] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, single-chain antibody (scFv), Fab fragment, Fab' fragment, or F(ab')2 fragment.

[0052] In an embodiment, the antibody or its antigen-binding fragment comprises an scFv, said scFv comprising a heavy chain variable region (VH) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, and a VL containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76, or SEQ ID NO:81.

[0053] In an embodiment, the antibody or its antigen-binding fragment comprises scFv, said scFv comprising VH having an amino acid sequence having SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75 or SEQ ID NO:80 and VL having an amino acid sequence having SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76 or SEQ ID NO:81.

[0054] In the implementation scheme, the antibody or its antigen-binding fragment comprises an scFv, said scFv comprising:

[0055] (i) A VH containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:57, and a VL containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:60;

[0056] (ii) A VH comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:71, and a VL comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:72;

[0057] (iii) A VH comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 75, and a VL comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 76; or

[0058] (iv) VH containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:80 and VL containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:81.

[0059] In the implementation scheme, the antibody or its antigen-binding fragment comprises an scFv, said scFv comprising:

[0060] (i) VH having the amino acid sequence of SEQ ID NO:57 and VL having the amino acid sequence of SEQ ID NO:60;

[0061] (ii). VH having the amino acid sequence of SEQ ID NO:71 and VL having the amino acid sequence of SEQ ID NO:72;

[0062] (iii) VH having the amino acid sequence of SEQ ID NO:75 and VL having the amino acid sequence of SEQ ID NO:76; or

[0063] (iv). VH having the amino acid sequence of SEQ ID NO:80 and VL having the amino acid sequence of SEQ ID NO:81.

[0064] In the implementation scheme, in the anti-MUC1 antibody or its antigen-binding fragment, one, two, three, four, five, six, seven, eight, nine or ten amino acids in SEQ ID NO:57, 60, 71, 72, 75, 76, 80 or 81 have been inserted, deleted or substituted.

[0065] In this embodiment, the VH and VL of the scFv of the anti-MUC1 antibody or its antigen-binding fragment are linked via an amino acid linker. The amino acid linker may have the amino acid sequence of SEQ ID NO: 83 or 84.

[0066] In the implementation scheme, the antibody or its antigen-binding fragment comprises an scFv having an amino acid sequence having SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:77 or SEQ ID NO:82.

[0067] In the embodiments, the antibody or its antigen-binding fragment comprises an scFv having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:70, 73, 77, or 82.

[0068] In the implementation scheme, in the anti-MUC1 antibody or its antigen-binding fragment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids in SEQ ID NO:70, 73, 77, or 82 have been inserted, deleted, or substituted.

[0069] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment exhibits antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In the implementation scheme, ADCC is against target cells expressing MUC1.

[0070] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment has reduced glycosylation, no glycosylation, or low fucosylation.

[0071] In the implementation scheme, the anti-MUC1 antibody or its antigen-binding fragment comprises an increased bipartite GlcNac structure.

[0072] In one embodiment, the anti-MUC1 antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 isotype. In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain constant region of a subclass of IgG1, IgG2, IgG3, or IgG4 and / or a light chain constant region of type κ or λ. In one embodiment, the antibody or its antigen-binding fragment comprises an Fc domain of wild-type human IgG1 (also known as human IgG1wt or huIgG1) or IgG2. In another embodiment, the antibody or its antigen-binding fragment comprises an IgG1 Fc domain.

[0073] In its implementation, this disclosure relates to a pharmaceutical composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0074] In its implementation, this disclosure relates to an isolated nucleic acid that encodes the anti-MUC1 antibody or its antigen-binding fragment disclosed herein.

[0075] In the implementation scheme, this disclosure relates to a vector containing the nucleic acid disclosed herein.

[0076] In the implementation scheme, this disclosure relates to a host cell that contains the nucleic acids or vectors disclosed herein.

[0077] In an embodiment, this disclosure relates to a method for generating an anti-MUC1 antibody or an antigen-binding fragment thereof, the method comprising culturing the host cells disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

[0078] In one embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises one or more complementarity-determining regions (CDRs) containing amino acid sequences 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.

[0079] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region (HCDR) comprising one or more complementarity-determining regions, said HCDR 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 (LCDR) comprising one or more complementarity-determining regions, said LCDR 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.

[0080] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region (HCDR) comprising three complementarity-determining regions, said HCDR1 comprising the amino acid sequence of SEQ ID NO:4; SEQ ID NO:14, SEQ ID NO:24 or SEQ ID NO:78; HCDR2 comprising 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 HCDR3 comprising 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 (LCDR) comprising three complementarity-determining regions, said LCDR1 comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:17 or SEQ ID NO:27; LCDR2 comprising 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 HCDR3 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:26; and HCDR4 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:26; and HCDR5 comprising the amino acid sequence of SEQ ID NO:7, SEQ ID NO:17 or SEQ ID NO:27; and HCDR6 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:26; and HCDR3 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:26; and HCDR4 comprising the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:26; and HCDR3 comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:26; and HC LCDR3 is the amino acid sequence of NO:19 or SEQ ID NO:29.

[0081] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises:

[0082] (a) A heavy chain variable region (HCDR) comprising three complementary determinant regions, wherein the HCDR is

[0083] HCDR1 containing the amino acid sequence of SEQ ID NO:4

[0084] HCDR2 containing the amino acid sequence of SEQ ID NO:5, and

[0085] HCDR3 containing the amino acid sequence of SEQ ID NO:6; or

[0086] HCDR1 containing the amino acid sequence of SEQ ID NO:14

[0087] HCDR2 containing the amino acid sequence of SEQ ID NO:15, and

[0088] HCDR3 containing the amino acid sequence of SEQ ID NO:16; or

[0089] HCDR1 containing the amino acid sequence of SEQ ID NO:24

[0090] HCDR2 containing the amino acid sequence of SEQ ID NO:25, and

[0091] HCDR3 containing the amino acid sequence of SEQ ID NO:26; or

[0092] HCDR1 containing the amino acid sequence of SEQ ID NO:24

[0093] HCDR2 containing the amino acid sequence of SEQ ID NO:74, and

[0094] HCDR3 containing the amino acid sequence of SEQ ID NO:26; or

[0095] HCDR1 containing the amino acid sequence of SEQ ID NO:78

[0096] HCDR2 containing the amino acid sequence of SEQ ID NO:79, and

[0097] HCDR3 containing the amino acid sequence of SEQ ID NO:16; and / or

[0098] (b) A light chain variable region (LCDR) comprising three complementary determinant regions, wherein the LCDR is

[0099] LCDR1 contains the amino acid sequence of SEQ ID NO:7.

[0100] LCDR2 containing the amino acid sequence of SEQ ID NO:8, and

[0101] LCDR3 containing the amino acid sequence of SEQ ID NO:9; or

[0102] LCDR1 contains the amino acid sequence of SEQ ID NO:17.

[0103] LCDR2 containing the amino acid sequence of SEQ ID NO:18, and

[0104] LCDR3 containing the amino acid sequence of SEQ ID NO:19; or

[0105] LCDR1 contains the amino acid sequence of SEQ ID NO:27.

[0106] LCDR2 containing the amino acid sequence of SEQ ID NO:28, and

[0107] LCDR3 containing the amino acid sequence of SEQ ID NO:29; or

[0108] LCDR1 contains the amino acid sequence of SEQ ID NO:7.

[0109] LCDR2 containing the amino acid sequence of SEQ ID NO:56, and

[0110] LCDR3 containing the amino acid sequence of SEQ ID NO:9; or

[0111] LCDR1 contains the amino acid sequence of SEQ ID NO:27.

[0112] LCDR2 containing the amino acid sequence of SEQ ID NO:65, and

[0113] LCDR3 containing the amino acid sequence of SEQ ID NO:29.

[0114] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:4, (b) HCDR2 of SEQ ID NO:5 and (c) HCDR3 of SEQ ID NO:6, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:7, (e) LCDR2 of SEQ ID NO:8 and (f) LCDR3 of SEQ ID NO:9.

[0115] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:4, (b) HCDR2 of SEQ ID NO:5, and (c) HCDR3 of SEQ ID NO:6, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:7, (e) LCDR2 of SEQ ID NO:56, and (f) LCDR3 of SEQ ID NO:9.

[0116] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:14, (b) HCDR2 of SEQ ID NO:15, and (c) HCDR3 of SEQ ID NO:16, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:17, (e) LCDR2 of SEQ ID NO:18, and (f) LCDR3 of SEQ ID NO:19.

[0117] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:78, (b) HCDR2 of SEQ ID NO:79, and (c) HCDR3 of SEQ ID NO:16, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:17, (e) LCDR2 of SEQ ID NO:18, and (f) LCDR3 of SEQ ID NO:19.

[0118] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:25, and (c) HCDR3 of SEQ ID NO:26, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:28, and (f) LCDR3 of SEQ ID NO:29.

[0119] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:25, and (c) HCDR3 of SEQ ID NO:26, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:65, and (f) LCDR3 of SEQ ID NO:29.

[0120] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:24, (b) HCDR2 of SEQ ID NO:74, and (c) HCDR3 of SEQ ID NO:26, and a light chain variable region comprising: (d) LCDR1 of SEQ ID NO:27, (e) LCDR2 of SEQ ID NO:65, and (f) LCDR3 of SEQ ID NO:29.

[0121] In one embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: (a) an amino acid sequence having 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, or SEQ ID NO:80; and / or (b) a heavy chain variable region comprising SEQ ID NO:11, 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 (b) an amino acid sequence that 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 (c) an antigen-binding fragment comprising 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, or SEQ ID NO:80. The light chain variable region of the amino acid sequence of SEQ ID NO:72, SEQ ID NO:76 or SEQ ID NO:81, or the amino acid sequence that 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.

[0122] In another embodiment, the anti-MUC1 antibody or its antigen-binding fragment comprises: (a) an amino acid sequence comprising 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 comprising one, two, or three amino acid substitutions in the amino acid sequence comprising 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 (b) a heavy chain variable region comprising 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, or SEQ ID NO:80, and / or (b) an amino acid sequence comprising 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, or SEQ ID NO:80, and / or (c) an amino acid sequence comprising 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, or (c) an amino acid sequence comprising SEQ ID NO:10, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:59, SEQ ID NO:6 The light chain variable region may contain one, two, three, four, or five amino acid substituted amino acid sequences in the amino acid sequences of SEQ ID NO:76 or SEQ ID NO:81, or in the amino acid sequences 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. In another embodiment, the amino acid substitutions are conserved amino acid substitutions.

[0123] In one implementation, the anti-MUC1 antibody is used at a concentration of 1 x 10⁻⁶. -6 M to 1x 10 -10 M or up to 1x 10 -11 Binding affinity of M (K) D () binds to MUC1. In another embodiment, the anti-MUC1 antibody binds at approximately 1 x 10⁻⁶. -6 M, approximately 1 x 10 -7 M, approximately 1 x 10 -8 M, approximately 1 x 10 -9 M, approximately 1 x 10 -10 M or approximately 1x10 -11 M of K D Combined with MUC1.

[0124] In another embodiment, the anti-human MUC1 antibody or its antigen-binding fragment exhibits cross-species binding activity against cynomolgus monkey MUC1.

[0125] In some embodiments, this disclosure relates to isolated nucleic acids comprising nucleotide sequences encoding an amino acid sequence encoding 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 comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, or SEQ ID NO:63, and encodes the VH region of the antibody or antigen-binding fragment of this 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 comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% identical 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 the antibody or antigen-binding fragment of this disclosure.

[0126] In some embodiments, this disclosure provides an anti-human MUC1 antibody or an antigen-binding fragment thereof that exhibits specific binding and high affinity to human MUC1. Attached Figure Description

[0127] Figure 1 This is a schematic diagram of MUC1-SEA-mIgG2a (top image) and MUC1-SEA-huIgG1 (bottom image), where "N" is the N-terminus and "C" is the C-terminus.

[0128] Figures 2A-2F The binding affinity of purified MUC1 antibody to human and cynomolgus monkey MUC1-overexpressing cells was demonstrated by FACS assay, with human IgG1 as a negative control. Figure 2A and 2B The binding affinity of the chimeric anti-MUC1 monoclonal antibody BG138P to human and cynomolgus monkey cells overexpressing MUC1 was demonstrated. Figure 2C and 2D The binding affinity of chimeric BG346P to human and cynomolgus monkey cells overexpressing MUC1 was demonstrated. Figure 2Eand 2F The binding affinity of chimeric BG219P to human and cynomolgus monkey cells overexpressing MUC1 was demonstrated.

[0129] Figures 3A-3C Epitope binning assays performed via competitive SPR assays are shown, in which purified human MUC1-mFc antigen flows across the chip surface and is captured by anti-mouse IgG antibodies.

[0130] Figures 4A-4F The effect of soluble MUC1 on the binding of MUC1 antibody to MUC1-expressing cells was demonstrated. Figure 4A -C shows 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 -F shows 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.

[0131] Figures 5A-5C The anti-MUC1 monoclonal antibody chBG138P, which targets the proximal region of the MUC1 membrane, was shown to bind to MUC1-positive cancer cell lines. Figures 5A-5C This demonstrates that chBG138P binds to the MUC1-expressing tumor cell line HCC827 in a dose-dependent manner. Figure 5A H1975 Figure 5B ) and T-47D ( Figure 5C (Human IgG1 was used as a negative control).

[0132] Figure 6 This diagram illustrates the FACS gating strategy for T cell binding assays. The dashed boxes indicate the proportion of antibody-bound T cells.

[0133] Figures 7A-7H This demonstrates the chimeric anti-MUC1 monoclonal antibody chBG138P targeting the proximal region of the MUC1 membrane. Figure 7A , 7B 7E and 7F), chBG219P ( Figure 7E and 7F ) or chBG346P ( Figure 7E and 7F ) does not bind to activated T cells, while the antibody HMFG1, which targets the distal portion of the MUC1 membrane, does not. Figure 7C and 7D ) and 16A ( Figure 7G and7H It can bind to normal T cells.

[0134] Figures 8A-8D The diagram illustrates chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4. Figure 8A and 8B ) does not bind to normal T cells, while the antibody HMFG1, which targets the distal portion of the MUC1 membrane, does not. Figure 8C and 8D It binds to normally activated T cells.

[0135] Figures 9A-9C This is a bar graph showing the internalization of MUC1 in chBG138P-induced cancer cell lines.

[0136] Figure 10A and 10B The chimeric BG138P and its humanized antibodies huBG138P-Hz1 to huBG138P-Hz4 were shown to bind to FACS in human and cynomolgus monkey cells overexpressing MUC1.

[0137] Figure 11 The humanized antibodies huBG219P-Bz0, huBG219P-E39, and huBG219P-E43 showed comparable binding to the MUC1 overexpressing cell line ZR-75-1 as the chimeric antibody chBG219P.

[0138] Figure 12 The results of the binding competition assays among scFv1-78P, scFv2 (scFv2-14P, scFv2-57P) and scFv3-20P are shown.

[0139] definition

[0140] Unless otherwise expressly defined below or elsewhere in this document, all other technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art.

[0141] As used herein (including in the appended claims), unless the context clearly specifies otherwise, the singular forms of words such as “a”, “an” and “the” include their corresponding plural referents.

[0142] Unless the context clearly specifies otherwise, the term “or” is used to mean the term “and / or” and is used interchangeably with the term “and / or”.

[0143] Unless otherwise specified or apparent from the context, as used herein, the term “about” refers to a value or composition within an acceptable range of error for a particular value or composition as determined by one of ordinary skill in the art, in part depending on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to practice in the art. “About” may mean a range up to 10% (i.e., ±10%). Thus, “about” can be understood as greater than or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of a specified value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or processes, the term may mean a value up to an order of magnitude or up to 5 times. When a particular value or composition is provided in this disclosure, unless otherwise stated, the word “about” shall be assumed to be within the acceptable range of error for said particular value or composition.

[0144] 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 in accession number P15941.

[0145] As used herein, when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, the terms "administration" and "administering" mean contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, subject, cell, tissue, organ, or biological fluid. Cellular treatment encompasses contact between the reagent and cells, as well as contact between the reagent and a fluid, wherein the fluid is in contact with the cells.

[0146] The terms “subject” or “patient” in this document include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., including patients who are at risk of having the conditions described herein).

[0147] In one aspect, “treating” any disease or condition means improving the disease or condition (i.e., slowing or stopping or reducing the development of at least one of the disease or its clinical symptoms). In another aspect, “treat,” “treating,” or “treatment” means reducing or improving at least one bodily parameter, including those that the patient may not be able to discern. In yet another aspect, “treat,” “treating,” or “treatment” means regulating a disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of bodily parameters), or both.

[0148] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen. Within an antigen, the variable region of the antibody interacts with the antigen at many sites through non-covalent forces. Generally, the more interactions, the stronger the affinity.

[0149] As used herein, the term "antibody" ("Ab") refers to a polypeptide of the immunoglobulin family that binds nonvalently, reversibly, and in a specific manner to a corresponding antigen. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated 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 hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four framework regions (FRs) arranged from 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 binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0150] The locations of the CDR and frame regions can be determined using various well-known definitions in the art, such as those of Kabat, Chothia, AbM, and IMGT (see, for example, 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)).

[0151] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, anti-idiotype (anti-Id) antibodies, and humanized antibodies. Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0152] The term "chimeric antibody" refers to a molecule made up of domains from different species, that is, the fusion of the variable domain of an antibody from a host species (e.g., mouse, rabbit, camel, etc.) with the constant domain of an antibody from a different species (e.g., human).

[0153] In some embodiments, the anti-MUC1 antibody includes at least one antigen-binding site. In some embodiments, the anti-MUC1 antibody includes an antigen-binding fragment from the MUC1 antibody described herein. In some embodiments, the anti-MUC1 antibody is isolated or recombinant. In some embodiments, the anti-MUC1 antibody also encompasses a multispecific antibody targeting MUC1 as at least one arm and targeting other antigens as the other arm.

[0154] The terms “monoclonal antibody” or “mAb” or “Mab” herein refer to a group of substantially homogeneous antibodies, meaning that the antibody molecules in this group are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody formulations typically comprise a variety of different antibodies with different amino acid sequences in their variable domains, particularly their CDRs, which are typically specific to different epitopes. The modifier “monoclonal” indicates that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; US 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 Immunologia 1993. The antibodies disclosed herein can be any class of immunoglobulins, including IgG, IgM, IgD, IgE, IgA, and any of their subclasses, such as IgG1, IgG2, IgG3, and IgG4. Hybridomas that produce monoclonal antibodies can be cultured in vitro or in vivo. High-titer monoclonal antibodies can be obtained through in vivo production, in which cells derived from an individual hybridoma 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 same type IgM or IgG can be purified from this ascites or from culture supernatant using column chromatography methods well known to those skilled in the art.

[0155] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50–70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain defines a constant region primarily responsible for effector function. Human light chains are typically classified as κ and λ light chains. Furthermore, human heavy chains are generally classified as α, δ, ε, γ, or μ, and antibody isotypes are defined as IgA, IgD, IgE, IgG, and IgM, respectively.

[0156] In both the light and heavy chains, the variable and constant regions are linked by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids.

[0157] Each light chain / heavy chain (VL / VH) pair has a variable region that forms an antibody binding site. Therefore, typically, a complete antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are usually identical in the primary sequence.

[0158] Typically, both heavy and light chain variable domains contain three hypervariable regions, also known as "complementarity-determining regions (CDRs)," located between relatively conservative frame regions (FRs). CDRs are usually arranged through frame regions to enable binding to specific epitopes. Typically, 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 the CDR and frame regions can be determined using various well-known definitions in the art, such as those of Kabat, Chothia, AbM, and IMGT (see, for example, 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) number (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme)). The definition of antigen combination sites is also described in the following: 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, under Kabat, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions from Kabat and Chothia, the CDR is composed 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. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (according to Kabat numbering). Under IMGT, the CDR region of the antibody can be determined using the IMGT / DomainGapAlign procedure.

[0159] The term “hypervariant region” refers to the amino acid residues of the antibody responsible for antigen binding. The hypervariant region contains amino acid residues from the “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. (Defining the CDR region of an antibody by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (Defining the CDR region of an antibody by structure). The terms “frame” or “FR” residues refer to those variable domain residues other than the hypervariant residues defined herein as CDR residues.

[0160] Unless otherwise indicated, “antigen-binding fragment” means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound to the full-length antibody, such as 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; bisomatic antibodies; 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)).

[0161] As used herein, "specifically binding to" an antigen (e.g., a protein) means that the antibody exhibits preferential binding to the target compared to other proteins, but this specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of the antigen in a heterogeneous population of proteins and other biological agents, such as in biological samples, blood, serum, plasma, or tissue samples. Therefore, under certain specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind to other antigens present in the sample in significant amounts. In one aspect, under specified immunoassay conditions, an antibody or its antigen-binding fragment specifically binds to a particular antigen at least ten times as much as background levels and does not specifically bind to other antigens present in the sample in significant amounts.

[0162] As used herein, an "antigen-binding domain" comprises at least six CDRs (or three CDRs for a single-domain antibody) 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 specifically binding to a first epitope and a second antigen-binding domain specifically binding to a second epitope. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with their antigen-binding domains targeting each specific epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) containing multiple antigen-binding domains, such as 2, 3, 4, or more antigen-binding domains specifically binding to a first epitope and 2, 3, 4, or more antigen-binding domains specifically binding to a second epitope.

[0163] The term "human antibody" in this document refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain mouse carbohydrate chains. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only the sequence of mouse or rat immunoglobulin proteins, respectively.

[0164] The term "humanized" or "humanized antibody" refers to an antibody form containing sequences derived from non-human (e.g., mouse, rabbit, camel, etc.) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies will contain at least one and usually two variable domains, where all or almost all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins, and all or almost all FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. When it is necessary to distinguish humanized antibodies from parental (e.g., rodent) antibodies, the prefix "hum," "hu," "Hu," or "h" is added to the antibody clone name. Humanized forms of rodent antibodies will typically contain the same CDR sequence as the parental rodent antibody, although certain amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0165] The term "corresponding human phylogenetic 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 immunoglobulin variable region sequences. A corresponding human phylogenetic sequence can also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A corresponding human phylogenetic sequence can be a frame region only, a complementarity-determining region only, a frame and complementarity-determining region, a variable region or sequence or subsequence, or other combinations thereof. Sequence identity can be determined using the methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human phylogenetic nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. Additionally, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant versions of human germline sequences, or antibodies containing common frame sequences derived from human frame sequence analysis, for example, as described in Knappik et al., J.Mol.Biol.296:57-86, 2000.

[0166] The terms "equilibrium dissociation constant," "KD," or "M" refer to the dissociation rate constant (kd, time constant). -1 Divide by the association rate constant (ka, time) -1 M -lThe equilibrium dissociation constant can be measured using any method known in the art. The antibodies disclosed herein will typically have a dissociation constant of less than about 10. -7 Or 10 -8 M, for example, less than about 10 -9 M or 10 -10 M, in some respects, is less than approximately 10. -11 M, 10 -12 M or 10 -13 The equilibrium dissociation constant of M.

[0167] As used herein, the terms “cancer” or “tumor” have the broadest meaning as understood in the art and refer to a physiological disorder in mammals typically characterized by disordered cell growth. In the context of this disclosure, cancer is not limited to a particular type or location.

[0168] In the context of this disclosure, when referring to an amino acid sequence, the term "conservative substitution" means the replacement of an original amino acid with a new amino acid that substantially does not alter the chemical, physical, and / or functional properties of the antibody or fragment, such as its binding affinity to MUC1. Common amino acid conservative substitutions are well known in the art.

[0169] As used herein, the term "knob-into-hole" technique refers to the technique of guiding the pairing of amino acids between two peptides in vitro or in vivo by introducing a spatial protrusion (knob) into one peptide and a cavity (socket) into the other peptide at the interface of two interacting peptides. For example, this technique has been used at the Fc:Fc binding interface of antibodies, C... L :C H A mortise and tenon joint is introduced at the I-interface or VH / VL-interface (see, for example, US 2011 / 0287009, US2007 / 0178552, WO 96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science, 6:781-788). In some embodiments, the mortise and tenon joint ensures the correct pairing of two different heavy chains during the manufacture of the multispecific antibody. For example, a multispecific antibody having mortise and tenon amino acids in the Fc region may further include a single variable domain linked to each Fc region or may further include different heavy chain variable domains paired with similar or different light chain variable domains. The mortise and tenon joint technique can also be used in the VH or VL regions to additionally ensure correct pairing.

[0170] An example of an algorithm suitable for determining sequence identity percentages 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 analysis is publicly available from 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. These short words match or satisfy a positive threshold score T when compared to words of the same length in a database sequence. T is called the neighboring word score threshold. These initial neighboring word hits serve as the starting value for the search to find longer HSPs containing them. Word hits extend in both directions along each sequence until the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hits cease extending in each direction when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of two strands. For amino acid sequences, the BLAST program uses a word length of 3 by default, and an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) for alignment (B) 50, expected value (E) 10, M = 5, N = -4 and comparison of the two strands.

[0171] The BLAST algorithm also performs statistical analysis on 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 minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in the comparison of 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, then the nucleic acid is considered similar to the reference sequence.

[0172] Alternatively, 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)) can be used to determine the percentage identity between two amino acid sequences using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970) (which has been incorporated into the GAP program of the GCG software package) can be used, employing 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 to determine the percentage identity between two amino acid sequences.

[0173] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides, or polymers thereof, in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methyl phosphonates, chiral methyl phosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0174] In the context of nucleic acids, the term "operably linked" refers to a functional relationship between two or more segments of polynucleotides (e.g., DNA). Typically, it refers to the functional relationship between a transcriptional regulatory sequence and the transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in a suitable host cell or other expression system, then that promoter or enhancer sequence is operably linked to the coding sequence. Generally, the promoter transcriptional regulatory sequence operably linked to the transcribed sequence is physically contiguous with the transcribed sequence; that is, they act in cis. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous with or located in close proximity to the coding sequence that they enhance the transcription of.

[0175] In some aspects, this disclosure provides compositions, such as pharmaceutically acceptable compositions, comprising an anti-MUC1 antibody as described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceuticalally acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0176] The compositions disclosed herein may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, liposomes, and suppositories. A suitable form depends on the intended administration method and therapeutic application. A suitable administration method is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0177] As used herein, the term "therapeutic effective amount" refers to the amount of antibody sufficient to affect such treatment against a disease, condition, or symptom when administered to a subject to treat a disease, or at least one clinical symptom of a disease or condition. "Therapeutic effective amount" can vary depending on the antibody, the disease, condition, and / or the symptoms of the disease or condition, the severity of the symptoms, the age of the subject being treated, and / or the weight of the subject being treated. In any given case, an appropriate amount will be obvious to those skilled in the art or can be determined through routine experiments. In the case of combination therapies, "therapeutic effective amount" refers to the total amount of the combination components.

[0178] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic ailment or condition. Such administration encompasses the co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses the co-administration of each active ingredient in multiple or separate containers or formulations (e.g., capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Additionally, "combination therapy" encompasses the sequential use of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the combination of drugs in treating the ailment or condition described herein.

[0179] As used herein, the phrase "in combination with" means administering the anti-MUC1 antibody to the subject concurrently with, before, or immediately after administration of an additional therapeutic agent. In some embodiments, the anti-MUC1 antibody is administered as a co-formulation with the additional therapeutic agent. Detailed Implementation

[0180] This disclosure provides anti-human MUC1 antibodies and antigen-binding fragments thereof. This disclosure also provides antibodies possessing desired binding affinity, desired internalization, and other desired properties. Anti-human MUC1 antibodies can be used to construct multispecific antibodies with additional functionalities 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, anti-human MUC1 antibodies and their constructs, or pharmaceutical compositions comprising them, can be used to treat cancers and related conditions expressing MUC1.

[0181] Anti-MUC1 antibody

[0182] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to human MUC1. In embodiments, the antibody or antigen-binding fragment thereof specifically binds to the SEA domain of the human MUC1 protein, which is the proximal membrane portion. In embodiments, the antibody or antigen-binding fragment thereof has no or significantly reduced interference from soluble MUC1 compared to antibodies targeting the N-terminus of MUC1. The antibodies or antigen-binding fragments of this disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated by the methods described below.

[0183] In one embodiment, the antibody or antigen-binding fragment disclosed herein specifically binds to MUC1 and comprises a VH domain having an amino acid sequence of SEQ ID NO: 10, 20, 30, 57, 58, 61, 71, 75, or 80. In another embodiment, the antibody or antigen-binding fragment specifically binds to MUC1 and comprises an HCDR having an amino acid sequence of any one of the HCDRs listed in Tables 2, 7, 14, 16, 22, and 23 below. In one aspect, the antibody or antigen-binding fragment specifically binds to MUC1, and the antibody comprises one, two, three, or more HCDRs (or consists of HCDRs) having an amino acid sequence of any one of the HCDRs listed in Tables 2, 7, 14, 16, 22, and 23 below.

[0184] In one embodiment, the antibody or antigen-binding fragment disclosed herein specifically binds to MUC1 and comprises a VL domain having an amino acid sequence of SEQ ID No: 11, 21, 31, 59, 60, 62, 66, 68, 72, 76, or 81. In another embodiment, the antibody or antigen-binding fragment specifically binds to MUC1 and comprises an LCDR having an amino acid sequence of any one of the LCDRs listed in Tables 2, 7, 14, 16, 22, and 23. In yet another embodiment, the antibody or antigen-binding fragment specifically binds to MUC1 and comprises one, two, three, or more LCDRs (or constitute thereof) having an amino acid sequence of any one of the LCDRs listed in Tables 2, 7, 14, 16, 22, and 23.

[0185] In the implementation, the antibody or antigen-binding fragment disclosed herein contains modified amino acids that have at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR region with the CDR regions disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, it includes amino acid sequence variations, wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR region have been changed when compared with the CDR regions depicted in the sequences in Tables 2, 7, 14, 16, 22, and 23.

[0186] In the implementation scheme, the antibody or antigen-binding fragments disclosed herein include antibody or antigen-binding fragments in which amino acids or nucleic acids encoding amino acids have been modified, but which have at least 60%, 70%, 80%, 90%, 95%, or 99% identity with the sequences disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, it includes changes in the amino acid sequence in which no more than 1, 2, 3, 4, or 5 amino acids in the variable region have been modified when compared with the variable region depicted in the sequences disclosed in Tables 2, 7, 14, 16, 22, and 23, while retaining substantially the same therapeutic activity.

[0187] This disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chain, and full-length light chain of antibodies that specifically bind to MUC1. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0188] In the embodiments, the antibodies or antigen-binding fragments disclosed herein exhibit cross-reactivity with both human and cynomolgus MUC1 (cynomolgus MUC1, SEQ ID NO:3). In the embodiments, the antibodies or antigen-binding fragments target the proximal region of the MUC1 membrane, where interference from detached MUC1 cells is minimal. In the embodiments, the antibodies or antigen-binding fragments can be used to treat cancers expressing MUC1.

[0189] This 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. Therefore, additional antibodies and antigen-binding fragments can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibiting binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of the antibodies and antigen-binding fragments of this disclosure to MUC1 demonstrates that the test antibody can compete with said antibodies or their antigen-binding fragments for binding to MUC1. Without being bound by any theory, such antibodies can bind to the same or related (e.g., structurally similar or spatially proximate) epitopes on MUC1 with competing antibodies or their antigen-binding fragments. In one aspect, antibodies that bind to the same epitopes on MUC1 as the antibodies or their antigen-binding fragments of this disclosure are human or humanized monoclonal antibodies. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0190] In one implementation, the anti-MUC1 antibody or its antigen-binding fragment as disclosed herein can be used to construct a multispecific antibody with additional functionality such as binding to a second human tumor-associated antigen (TAA), immune checkpoint inhibition, or immune stimulation.

[0191] In some implementations, the antibody or its antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or an F(ab')2 fragment.

[0192] In one embodiment, the antibody or its antigen-binding fragment is in scFv format, comprising VH-VL in the N-terminal to C-terminal direction or VL-VH in the N-terminal to C-terminal direction. In some embodiments, VH and VL are linked via amino acid linkers such as those described herein. In some embodiments, VH or VL is any one of the VH or VL described in Tables 2, 7, 14, 16, 22, and 23. Other scFvs of this disclosure contain amino acids that have been varied but have at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR region as disclosed in Tables 2, 7, 14, 16, 22, and 23. In some aspects, it includes amino acid sequence variations wherein no more than 1, 2, 3, 4, or 5 amino acids in the CDR region have been varied when compared to the CDR regions depicted in the sequences in Tables 2, 7, 14, 16, 22, and 23.

[0193] Amino acid linkers

[0194] In embodiments, the anti-MUC1 antibody or its antigen-binding fragment disclosed herein is used to construct multispecific antibodies, which may be, for example, bispecific tetravalent antibodies. The domains and / or regions of the polypeptide chain of the bispecific tetravalent antibody may be separated by linker regions of various lengths. In some embodiments, the antigen-binding domains are separated from each other by linker regions (CL, CH1, hinge, CH2, CH3, or the entire Fc region). For example, the polypeptide chain may contain the sequences VL1-CL-(linker)VH2-CH1, VH-linker-VL. Such linker regions may contain randomly sorted amino acids or a restricted set of amino acids. Such linker regions may be flexible or rigid (see US2009 / 0155275).

[0195] Multispecific antibodies have been developed via genetic fusion of two single-chain Fv (scFv) or Fab fragments (with or without flexible linkers) (Mallender et al., J. Biol. Chem. 1994 269:199-206; Mack et al., Proc. Natl. Acad. Sci. USA. 1995 92:7021-5; Zapata et al., Protein Eng. 1995 8:1057-62); via dimerization devices such as leucine zippers (Kostelny et al., J. Immunol. 1992 148:1547-53; de Kruifetal J. Biol. Chem. 1996 271:7630-4) and Ig. C / CH1 domain (Muller et al., FEBS Lett. 422:259-64); constructed via bisomal antibodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., Bio / Technology (NY) 1996 14:192-6); Fab-scFv fusion (Schoonjans et al., J. Immunol. 2000 165:7050-7); and microantibody formats (Pack et al., Biochemistry 1992 31:1579-84; Pack et al., Bio / Technology 1993 11:1271-7).

[0196] Dimerization-specific amino acids

[0197] In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid change. The dimerization-specific amino acid change can lead to a "mortar and pestle" interaction and can increase the likelihood of correct assembly of the desired multivalent antibody. The dimerization-specific amino acid may be within the CH1 domain or the CL domain, or a combination thereof. Suitable dimerization-specific amino acids for pairing the CH1 domain with other CH1 domains (CH1-CH1) and the CL domain with other CL domains (CL-CL) can be found at least in the disclosures of WO2014082179, WO2015181805, and WO2017059551. The dimerization-specific amino acid may also be within the Fc domain and may be combined with dimerization-specific amino acids within the CH1 or CL domains. In one embodiment, this disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0198] Further changes to the framework of the Fc zone

[0199] In various respects, the effector function of an antibody can be altered by replacing at least one amino acid residue with a different amino acid residue. For example, one or more amino acids can be replaced with different amino acid residues to give the antibody an altered affinity for an effector ligand, while retaining the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in U.S. Patents 5,624,821 and 5,648,260 to Winter et al.

[0200] On the other hand, one or more amino acid residues may be replaced by one or more different amino acid residues, resulting in altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC) of the antibody. This method is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0201] On another front, one or more amino acid residues are varied to alter the antibody's ability to fix complement. This method is described, for example, in Bodmer et al., publication WO 94 / 29351. In a specific aspect, for the IgG1 subclass and the κ isotype, one or more amino acids of the antibody or its antigen-binding fragment of this disclosure are replaced by one or more allotropic amino acid residues. The allotropic amino acid residues also include, but are not limited to, constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and constant regions of the light chains of the κ isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).

[0202] On the other hand, the Fc region can be modified by altering one or more amino acids to increase the ability of antibody-mediated antibody-dependent cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor. This method is described, for example, in Presta's publication WO00 / 42072. Furthermore, binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with modified binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0203] On the other hand, the glycosylation of the antibody is modified. For example, glycosylated-free antibodies (i.e., antibodies lacking or having reduced glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for an "antigen". Such carbohydrate modification 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, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation at said sites. Such glycosylation can increase the antibody's affinity for the antigen. This method is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

[0204] Alternatively or concurrently, antibodies with altered glycosylation patterns can be prepared, such as hypofucosylated antibodies with reduced amounts of fucose residues or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. This carbohydrate modification can be achieved, for example, by expressing antibodies in host cells with altered glycosylation pathways. Cells with altered glycosylation pathways have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in this cell line exhibit hypofucosylation. Presta's publication WO 03 / 035835 describes a variant CHO cell line, Lecl3 cells, which has the ability to attach fucose to Asn(297)-linked carbohydrates with reduced fucosylation, resulting in hypofucosylation of antibodies expressed in said host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Umana et al.'s WO99 / 54342 describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), resulting in antibodies expressed in engineered cell lines exhibiting increased bipartite GlcNac structures, which leads to increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0205] On the other hand, if reduced ADCC is desired, many previous reports have shown that the human antibody subclass IgG4 has only moderate ADCC and almost no 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 increased temperatures (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al., 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operatively linking an antibody to an IgG4 Fc engineered with a combination of alterations that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is the dynamic separation of its two heavy chains in solution to form haptens, which leads to the in vivo production of bispecific antibodies via a process known as “Fab arm exchange” (Vander Neut Kolfschoten M et al., 2007 Science, 317:1554-157). A serine mutation at position 228 (EU numbering system) to proline appears to inhibit the separation of the IgG4 heavy chains (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19). Reports indicate that some amino acid residues in the hinge region and γFc region influence the interaction between the antibody and the Fcγ receptor (Chappel SM et al., 1991 Proc. Natl. Acad. Sci. USA, 88: 9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9: 115-119; Armour, KL et al., 1999 Eur J Immunol, 29: 2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6: 443-446; Arnold JN, 2007 Annu Rev immunol, 25: 21-50). In addition, some rare IgG4 isoforms in the human population can also cause different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19).To generate antibodies with low ADCC and CDC but good stability, the hinge and Fc regions of human IgG4 may be modified, introducing numerous alterations. These modified IgG4 Fc molecules can be found in SEQ ID NO:83-88 of U.S. Patent No. 8,735,553 to Li et al.

[0206] Antibody production

[0207] Antibodies and their antigen-binding fragments can be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, through hybridoma or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0208] This disclosure further provides polynucleotides encoding antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or segments containing complementarity-determining regions as described herein.

[0209] The polynucleotides disclosed herein can encode variable region sequences of anti-MUC1 antibodies. They can also encode both variable and constant regions of the antibody. Some sequences encode polypeptides containing variable regions of both the heavy and light chains of the exemplified anti-MUC1 antibody.

[0210] This disclosure also provides expression vectors and host cells for generating anti-MUC1 antibodies. The choice of expression vector depends on the intended host cells in which the vector will be expressed. The expression vector may contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-MUC1 antibody chain or an antigen-binding fragment. In some aspects, except under induction-controlled conditions, inducible promoters are employed to prevent the expression of the inserted sequence. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be amplified under non-inducible conditions without deviating from a population whose expression product is better tolerated by the host cells. In addition to the promoter, other regulatory elements may be included for efficient expression of the anti-MUC1 antibody or its antigen-binding fragment. These elements may include an ATG start codon and an adjacent ribosome binding site or other sequences. Furthermore, expression efficiency can be enhanced by including enhancers suitable for the cell system used (see, for example, 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.

[0211] The host cells used to carry and express anti-MUC1 antibody vectors can be prokaryotic or eukaryotic. *Escherichia coli* (E. coli) is a prokaryotic host suitable for cloning and expressing the polynucleotides disclosed herein. Other suitable microbial hosts include bacilli, such as *Bacillus subtilis*, and other Enterobacteriaceae, such as *Salmonella*, *Serratia*, and various *Pseudomonas* species. Expression vectors can also be prepared from these prokaryotic hosts, typically containing expression control sequences (e.g., origin of replication) compatible with the host cell. Additionally, any number of well-known promoters can be present, such as lactose promoter systems, tryptophan (trp) promoter systems, β-lactamase promoter systems, or promoter systems derived from bacteriophage λ. Promoters are typically optionally controlled by operon sequences and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-MUC1 antibodies. Combinations of insect cells with baculovirus vectors can also be used.

[0212] In other respects, mammalian host cells are used to express and produce the anti-MUC1 antibody disclosed herein. Examples include hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These cells include any normally dead or normally or abnormally immortalized 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, HEK 293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures to express peptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, 1987. Expression vectors used for mammalian host cells may include expression control sequences, such as origin of replication, promoters, and enhancers (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell-type specific, stage-specific, and / or tunable or modulotropic. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as the human immediate early CMV promoter), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0213] Methods of detection and diagnosis

[0214] The antibody or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for detecting MUC1. In one aspect, the antibody or antigen-binding fragment can be used to detect the presence of MUC1 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In some aspects, the biological sample includes cells or tissues. In other aspects, such tissues include normal and / or cancerous tissues that express MUC1 at higher levels relative to other tissues.

[0215] In one aspect, this disclosure provides a method for detecting the presence of MUC1 in a biological sample. In some aspects, 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 may include, but is not limited to, urine, tissue, saliva, or blood.

[0216] This also includes methods for diagnosing conditions associated with MUC1 expression. In some aspects, the methods include contacting test cells with an anti-MUC1 antibody; determining the expression level (quantitative or qualitative) of MUC1 expressed by the test cells by detecting the binding of the anti-MUC1 antibody to the MUC1 peptide; and comparing the expression level of the test cells with the MUC1 expression level in control cells (e.g., normal cells of the same tissue origin as the test cells or non-MUC1-expressing cells), wherein a higher MUC1 expression level in the test cells compared to the control cells indicates the presence of conditions associated with MUC1 expression.

[0217] Pharmaceutical compositions and formulations

[0218] Compositions comprising an anti-MUC1 antibody or its antigen-binding fragment or polynucleotide are also provided, including pharmaceutical formulations, wherein the polynucleotide contains a sequence encoding an anti-MUC1 antibody or antigen-binding fragment. These compositions may further comprise a suitable carrier, such as a pharmaceutically acceptable excipient, including buffers well known in the art.

[0219] Pharmaceutical formulations of anti-MUC1 antibodies or antigen-binding fragments as described herein are prepared by mixing such antibody or antigen-binding fragment, having the desired degree of purity, with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10-residue polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; 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 nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers further include interstitial drug dispersants, such as soluble, neutrally active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc. Certain exemplary sHASEGPs (including rHuPH20) and methods of use are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans such as chondroitinase.

[0220] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter of which comprises histidine-acetate buffer.

[0221] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a molded article, such as a membrane or microcapsule.

[0222] Formulations intended for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

[0223] equivalent

[0224] It should be understood that although the anti-human 4Ig-B7H3 antibody and its antigen-binding fragment have been described in conjunction with their detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0225] It should be understood that one, some, any, or all of the features of the various embodiments disclosed herein may be combined to form further embodiments of this disclosure. These and other aspects of this disclosure will be apparent to those skilled in the art.

[0226] Example

[0227] Example 1. Generation of anti-MUC1 monoclonal antibodies targeting the proximal region of the MUC1 membrane

[0228] MUC1 recombinant protein for immunoassay and binding assays

[0229] The 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 them. The coding region of the SEA domain (sea urchin spermatin, enterokinase, and aggregate protein) (SEQ ID NO:2) of full-length human MUC1, consisting of amino acids (AA) 1036-1155, was amplified by PCR and cloned into a pcDNA3.4-based expression vector (Invitrogen, Carlsbad, CA, USA). The C-terminus of this vector was fused to either the Fc domain of mouse IgG2a or the Fc domain of the human IgG1 heavy chain, resulting in two recombinant fusion protein expression plasmids: MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1. A schematic diagram of the MUC1 fusion protein is shown in [image / image / description]. Figure 1For the generation of recombinant fusion proteins, 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 (catalog: 17549852, Cytiva Life Sciences), followed by purification using a HiLoad 16 / 600 Superdex 200pg size size exclusion column (catalog: 28989335, Cytiva Life Sciences). Both MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 proteins were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at -80°C.

[0230] Table 1. Sequence of MUC1

[0231]

[0232] To generate cell lines that stably express human or cynomolgus monkey MUC1 for antibody production, screening, and validation.

[0233] Cell lines that stably express human MUC1 were generated and validated, including PT67 / human MUC1 cell line (internal generated cell line), HEK293 / human MUC1 cell line (HEK293 was obtained from ATCC CRL-1573) and HCT116 / human MUC1 cell line (ATCC CCL-247).

[0234] Cell lines that stably express cynomolgus monkey MUC1 (SEQ ID NO:3) were generated and validated, including HEK293 / cynomolgus monkey MUC1, L929 / cynomolgus monkey MUC1 cell line (L929 was obtained from ATCC CCL-1), HCT116 / cynomolgus monkey MUC1 cell line and Daudi / cynomolgus monkey MUC1 cell line (Daudi was obtained from ATCC CCL-213).

[0235] To generate cell lines stably expressing human or cynomolgus monkey MUC1, an exotrophic vector was constructed using the retroviral construct PFBneo (STRATAGENE, catalog number 217561-51). Following the manufacturer's instructions, the retroviral construct was transfected into PLAT-E cells (Cyagen, catalog number IPMPC-01001) using Lipofectamine 2000 (Invitrogen, reference number 52758). Viral supernatants were collected at 24, 48, and 72 hours post-transfection and filtered (0.45 μm) before use. The resulting exotrophic virus was used to transduce the bitropic packaged cell line PT67 in the presence of polybrene (final concentration: 8 μg / ml). After three rounds of transduction, PT67 cells were selected for 7 days in G418 (final concentration: 1 mg / ml). To collect the bitropic virus generated from PT67 cells, the medium was replaced with fresh DMEM complete medium without G418 when the cells became 100% confluent. Virus was collected daily for 3 days. Cell lines were infected with a virus containing human or cynomolgus monkey MUC1. After 3 rounds of transduction, infected cells were selected for 7 days in G418 (final concentration: 1 mg / ml).

[0236] immunity

[0237] To generate antibodies against MUC1, 30 cohorts of inbred BALB / C, MRL strain mice were immunized with different MUC1 antigens. Each cohort underwent an immunization strategy comprising a unique combination of MUC1 antigens (including proteins and cell lines from Example 1), dosage, route of injection, adjuvant, and timing of immunization. A total of 5 animals from 6 cohorts were immunized. Animals received immunizations at different time points between 0 and 90 days. To monitor the immune response, serum titrated by ELISA and FACS was typically performed 30–90 days after 2–6 immunizations. Antibodies binding to the MUC1 antigen were screened in the serum. The MUC1-specific antibody response in each animal was measured, and animals with sufficient anti-MUC1 Ig titers were selected for a final booster on day 4.

[0238] Hybridoma fusion and screening

[0239] Lymphatic organs, including the spleen and lymph nodes, were isolated from mice immunized as described above. Hybridomas were generated by fusion with SP2 / 0-derived immortalized mouse myeloma cells via PEG-based fusion. The resulting cells were seeded in 96-well cell culture plates using standard 1640 medium supplemented with HAT for hybridoma selection. After 10–13 days of culture and replacement of the growth medium, hybridoma culture supernatants were collected from each well and screened to identify wells containing secreted MUC1-specific antibodies. All supernatants were initially screened against the recombinant protein huMUC1-SEA-huIgG1 (from Example 1). Antibody binding to the recombinant protein huMUC1-SEA-huIgG1 was measured by ELISA. MUC1 antibody screening was performed on the supernatants from the culture wells of three hybridoma fusions. In summary, 2 μg / mL huMUC1-SEA-huIgG1 was coated onto a 96-well ELISA plate, and 50 μl of hybridoma culture supernatant was added for incubation for 30–60 min. After washing, the plate was incubated with a secondary anti-mouse IgG Fc Ab conjugated to HRP. After incubation and washing, the plate was developed with HRP substrate, and absorbance was measured.

[0240] Hybridomas from positive wells were transferred to 24-well plates with fresh culture medium and grown for 2–3 days. They were then screened again by flow cytometry to confirm the binding of the antibody to human MUC1 and cynomolgus monkey MUC1 overexpressing cell lines.

[0241] Antibodies (Abs) binding to human MUC1 and cynomolgus monkey MUC1-overexpressing cell lines were measured using flow cytometry (FACS). Briefly, 100 μl of hybridoma culture supernatant was co-incubated with either human MUC1-overexpressing or cynomolgus monkey MUC1-overexpressing cells for 30–60 min, washed, and then incubated with a secondary anti-mouse IgG Fc Ab conjugated to APCs. After incubation and washing, fluorescence was measured by flow cytometry.

[0242] Subcloning and Sequence Analysis

[0243] Selected anti-MUC1 Ab secretory hybridoma subclones were subcloned once or twice to ensure monoclonalness. In short, positive hybridoma clones were subcloned via limiting dilution. After 7–10 days, the culture supernatant was screened by ELISA and flow cytometry as previously described to confirm binding to human and cynomolgus monkey MUC1 Ab. Stable hybridoma subclones were cultured in vitro for cell cryopreservation and for cloning and sequencing of the antibody VH and VL genes.

[0244] Subcloned anti-MUC1 Ab secretory hybridomas were lysed using lysis buffer. The lysates containing mRNA were then transferred to 96-well deep-well plates for mRNA isolation, cDNA synthesis, and DNA sequencing using standard sequencing technology (Sanger sequencing). Typically, total RNA from the cell lysates was prepared according to the manufacturer's instructions, and cDNA was generated by reverse transcription of the mRNA using SuperMix (Invitrogen) first-strand synthesis with SuperScript III. The sequence of the BG138P antibody is listed in Table 2.

[0245] Single B screening

[0246] Immunized mice were sacrificed and spleens were harvested. Enriched plasma cells were loaded onto a 14K microarray. On-array screening was performed using hMUC1 beads, cynomolgus monkey MUC1 beads, and HEK293-cynomolgus monkey MUC1 cells. Hit cells were selected and exported to 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. Sequences of BG219P and BG346P are listed in Table 2.

[0247] Table 2. Mouse antibody sequences against human MUC1

[0248]

[0249]

[0250]

[0251]

[0252] Large-scale expression and purification of chimeric BG219P, BG138P, and BG346P

[0253] Chimeric antibodies chBG219P, chBG138P, and chBG346P were produced by transiently transfecting internally generated plasmids containing heavy and light chains into ExpiCHO-s cells. Conditioned medium was harvested and processed using a MabSelect SuRe column (Cytiva), followed by POROS. TM Antibodies were purified using a 50 HS column (Thermofisher Scientific) and a G-25 desalting column (Cytiva). All purified antibodies were stored in aliquots at -80°C.

[0254] Example 2. Binding kinetics and affinity determination of anti-MUC1 antibody by SPR

[0255] Using BIAcore TM The binding kinetics of chimeric anti-MUC1 antibodies were characterized using a SPR assay on the T-200 (GE Life Sciences). In short, 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 flowed through the chip surface and was captured by the anti-human IgG antibody. Then, serially diluted human or cynomolgus monkey MUC1-SEA protein was flowed through the chip surface, and changes in surface plasmon resonance signal were analyzed to calculate the association rate (kJ / kE) using a one-to-one Langmuir binding model (BIA evaluation software, GE Life Sciences). on ) and dissociation rate (k off Equilibrium dissociation constant (K) D ) is calculated as k off / k on The binding affinity profiles of chimeric anti-MUC1 antibodies chBG138P, chBG346P, and chBG219P are shown in Table 3 below. ChBG138P, chBG346P, and chBG219P all exhibited high affinity for human and cynomolgus monkey MUC1-SEA.

[0256] Table 3. Antigen binding affinity of chBG138P, chBG346P and chBG219P

[0257]

[0258] Example 3. Determining the binding affinity of anti-MUC1 antibody to MUC1 expressed in a stable cell line.

[0259] The binding affinity of chimeric anti-MUC1 antibodies to human and cynomolgus monkey MUC1-overexpressing cell lines (HEK293 / human MUC1 and HEK293 / cynomolgus monkey MUC1) was determined by FACS. Briefly, human or cynomolgus monkey MUC1-overexpressing cells were incubated with serially diluted purified antibodies, washed, and then incubated with anti-human IgG secondary antibodies conjugated to APCs. After incubation and washing, fluorescence was measured by flow cytometry. A summary of the binding affinity of the chimeric anti-MUC1 antibodies is shown in Table 4 below. Figures 2A-2F (with hIgG1 as a negative control). The results showed that all three chimeric anti-MUC1 antibodies had good binding affinity for human and cynomolgus monkey MUC1 expressed in stable cell lines.

[0260] Table 4. Cell binding affinity of chBG138P, chBG346P, and chBG219P

[0261]

[0262] Example 4. Epitope binning of anti-MUC1 antibody

[0263] Epitope binning of chimeric anti-MUC1 antibodies was determined by competitive SPR assay. In short, anti-mouse IgG Fc antibodies were immobilized on an activated CM5 biosensor chip. Purified huMUC1-SEA-mIgG2a (human MUC1 linked with mouse IgG2aFc) antigen flowed across the chip surface and was captured by anti-mouse IgG antibodies. Reference MUC1-SEA Ab5F3 (Cancer Immunol Immunother. 2020 July; 69(7):1337-1352) was first injected under saturated antigen-binding conditions, followed by injection of chBG138P (…). Figure 3A ), chBG219P Figure 3B ) or chBG346P ( Figure 3C The sensor map of the sub-bin is shown in... Figures 3A-3C As shown in Table 5 below, the three chimeric MUC1 antibodies were divided into two epitope bins within the MUC1-SEA domain. ChBG138P and chBG219P bind to the same epitope on the MUC1-SEA, as does 5F3, which has a different MUC1 epitope than chBG346P. More specifically, chBG138P and chBG219P bind to epitope A, while chBG346P binds to epitope B.

[0264] Table 5. Position boxes for chBG138P, chBG346P, and chBG219P

[0265] Ab number box chBG138P A chBG346P B chBG219P A

[0266] Example 5. Chimeric anti-MUC1 antibodies chBG138P, chBG219P, and chBG346P showed reduced interference from soluble MUC1.

[0267] 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 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 IC50 value of soluble MUC1 blocking the binding of MUC1 antibody to MUC1-expressing cells was determined. 50 The values ​​are shown in Table 6, and the blocking curve is shown in... Figures 4A-4F In the study, HMFG1 was a positive control, and mIgG and hIgG1 were negative controls. The profile indicated that the binding of HMFG to MUC1-expressing cells was easily interfered with at high, medium, and low antibody concentrations (i.e., 30, 3, and 0.3 μg / ml), but at low antibody concentrations (i.e., 0.3 μg / ml), the binding of chBG138P, chBG219P, and chBG346P was only slightly interfered with. Figure 4A -F). In summary, the overview in Figure 4 indicates that the binding of the MUC1 antibody to cells expressing MUC1 showed significantly reduced interference from soluble MUC1 compared to HMFG1 (Abcam) targeting the N-terminus of MUC1.

[0268] Table 6. IC50 of soluble MUC1 blocking activity 50

[0269]

[0270]

[0271] (NA: Not available. Due to extremely low interference, IC) 50 The data cannot be well fitted and retrieved, such as Figure 4A , 4B (As shown by the binding curves in 4E and 4F.)

[0272] Example 6. Anti-MUC1 monoclonal antibodies targeting the proximal region of the MUC1 membrane bind to cancer cell lines but not to normal T cells, while antibodies HMFG1 or 16A targeting the N-terminus of MUC1 can bind to normal T cells.

[0273] To evaluate whether anti-MUC1 monoclonal antibodies targeting the proximal region of the MUC1 membrane could differentially bind to MUC1-expressing tumor cells and normal cells (such as activated T cells) expressing MUC1, a FACS binding assay was performed. For tumor cell line binding assays, cells were collected and stained with either anti-human MUC1 antibody chBG138P or a control (human IgG1) for 1 hour. The cells were then washed twice and subsequently treated with a secondary antibody (Alexa). Cells were stained with 647 anti-human IgG (Fc) for 30 minutes. Cells were washed and fixed in 1% paraformaldehyde (PFA) DPBS solution, followed by FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress software. Figures 5A-5C As shown, the chimeric antibody chBG138P, targeting the proximal region of the human MUC1 membrane, binds to the MUC1-expressing tumor cell line HCC827 in a dose-dependent manner. Figure 5A H1975 Figure 5B ) and T-47D ( Figure 5C (Human IgG1 as a negative control) This indicates that antibodies targeting the proximal region of the MUC1 membrane can be used to target cancer cells and thus be applied to the treatment of cancers that express or overexpress MUC1.

[0274] To evaluate whether monoclonal antibodies targeting the proximal region of the MUC1 membrane could avoid binding to normal cells expressing MUC1, an activated T-cell binding assay was performed. 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. The stimulated PBMCs were then subjected to FACS staining. The cell suspension was then subjected to LIVE / DEAD... TMCells were pre-incubated with a fixative dead cell staining kit (Invitrogen, reference number L34964) and Fc receptor blocking solution (100 μg / mL human IgG in FACS buffer), followed by staining with anti-human antibodies. Cells were washed twice and incubated for 1 hour with 10 μg / mL anti-MUC1 monoclonal antibody targeting the proximal region of the MUC1 membrane, or HMFG1 antibody targeting the N-terminus of MUC1 (as a positive control, Abcam, catalog number ab215670) or 16A (as a positive control, Biolegend, catalog number 355608). Cells were then washed and stained for 30 minutes with PE-CY7 anti-human αβTCR (eBioscience, catalog number 25-9986-42) and AF647 anti-human IgG Fc (Biolegend, reference number 409320). Cells were washed and fixed with 1% PFA in DPBS solution, followed by FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress software. Figure 6 and 7A As shown in -7H, the chimeric antibody chBG138P ( Figure 7A , 7B 7E and 7F), chBG219P ( Figure 7E and 7F ) or chBG346P ( Figure 7E and 7F Neither of these antibodies binds to normally activated human T cells expressing MUC1. However, the antibody HMFG1, which targets the N-terminus of MUC1, does not bind. Figure 7C and 7D ) and 16A ( Figure 7G and 7H It binds to a significant portion of activated T cells. Figures 8A-8D The chimeric (chBG138P) and humanized (huBG138P-Hz2 and huBG138P-Hz4) versions of the antibody BG138P were shown to retain similar binding properties to BG138P, and did not bind to normally activated human T cells expressing MUC1. Figure 8A and 8B ), while the antibody HMFG1, which targets the N-terminus of MUC1 ( Figure 8C and 8D It binds to normally activated T cells.

[0275] The results indicate that, compared to antibodies targeting the N-terminus of MUC1 that bind to both cancer cells and normal T cells, antibodies targeting the proximal region of the MUC1 membrane specifically target cancer cells while preserving normal T cells, and therefore provide an optimized safety profile when used as an anti-tumor therapy in humans.

[0276] Example 7. ChBG138P induces internalization of MUC1 in cancer cell lines.

[0277] Following the manufacturer's instructions, chBG138P and human IgG were labeled using pHrodo iFL STP ester (amine reactive dye, Invitrogen, reference number P36013). Cancer cell lines T-47D, HCC827, and H1975 were incubated at 4°C for 60 min in the dark with pHrodo-labeled chBG138P (final concentration 10 μg / ml) or human IgG (human immunoglobulin from Hualan for intravenous injection) as a negative control. 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 h. 1 μl / well of 7-AAD (7-amino-actinomycin D) was added to all groups. Cells were incubated in the dark on ice for 30 min. Cells were washed and fixed with 1% paraformaldehyde (PFA) in DPBS solution, and then subjected to 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).

[0278] like Figures 9A-9C As shown, for all three cancer cell lines T-47D (9A), HCC827 (9B), and H1975 (9C), approximately 10-20% of cells were pHrodo positive after incubation at 37°C for 5 hours. These results indicate that the antibody chBG138P, targeting the proximal region of the MUC1 membrane, induces internalization in human cancer-derived cell lines expressing MUC1, while the negative control hIgG induces almost no internalization. This internalization property may be advantageous for designing monoclonal antibody or bispecific antibody therapies to avoid loss of the membrane target and drug resistance. Furthermore, the potential for internalization retains the possibility of developing antibody-drug conjugates, including the MUC1 antibody disclosed herein, for the treatment of cancers expressing MUC1.

[0279] Example 8. Humanization of mouse anti-human MUC1 antibodies BG138P and BG219P

[0280] Humanization of BG138P

[0281] For the humanization of the mouse anti-human MUC1 BG138P antibody, a CDR (Continuous Receptor Domain) transplantation strategy was employed. The variable domain sequences (VH and VL) of the mouse BG138P antibody heavy and light chains were aligned with the human antibody germline sequence using Ig blast, and the human germline framework with the highest homology was selected as the recipient framework for CDR transplantation. To maintain the canonical CDR structure, the mouse version of the BG138P antibody structure was modeled using Schrodinger software and used to select potential reversion mutation sites to maintain binding affinity. Each reversion mutation site was screened by constructing multiple variants without the mutations to evaluate the necessity of maintaining the site in the humanized version. Then, the identified key reversion mutation sites were combined to construct a second round of humanized variants for affinity characterization.

[0282] Specifically, for the BG138P VH sequence, the IGHV1-3*01 and J region H4 (JH4) germline sequences were selected as the receptor framework. And for the BG138P VL sequence, Vκ1-27*01 and J region κ2 were selected as the receptor framework. Antibody variants, including a CDR-only transplantation version (named with the suffix "Hz0"), a version with all reversion mutation sites (named with the suffix "Bz0"), and multiple variants with reduced reversion mutation sites (named with the suffixes "Bz1"-"Bz16"), were constructed using an internal IgG1 / Cκ eukaryotic expression vector and expressed using Expi293. TM The expression system (Thermofisher Scientific) was generated and used with MabSelectPrismA. TM Protein A was purified using chromatographic resin (Cytiva). The list of antibody variants screened in this round and their corresponding VH / VL sequences are summarized in Table 7 (where "X" indicates the CDR position for transplantation).

[0283] Table 7. Humanized design for screening BG138P reversion mutation sites

[0284]

[0285]

[0286]

[0287] All generated antibody variants were characterized for SPR affinity of the human and cynomolgus monkey MUC1SEA domain protein (SEQ ID NO:2) using a Biacore 2000 (Cytiva) system. Variants exhibiting a greater than 2-fold loss of affinity compared to chimeric BG138P (calculated by Kd from single-concentration binding kinetics) were selected as Layer 1 reversion mutation sites, and variants exhibiting a 1.5–2-fold loss of affinity were selected as Layer 2 reversion mutation sites. SPR binding affinity data are summarized in Tables 8 and 9.

[0288] Table 8. SPR binding affinity of BG138P humanized variant to human MUC1 SEA protein

[0289]

[0290]

[0291] Table 9. SPR binding affinity between the humanized variant of BG138P and the cynomolgus monkey MUC1 SEA protein

[0292]

[0293] Motif analysis revealed that the amino acid “DG” (Kabat numbers 56-57) at the CDRL2 and FR3 boundary is an aspartic acid isomerization motif, and several mutant variants were constructed to remove the risk of this post-translational modification (PTM). Four mutations (DG to EG, AG, TG, and SG, which are named BG138P-PTM-m1 to m4 in Tables 10 and 11) were designed and introduced into chimeric BG138P to remove this isomerization motif. SPR binding characterization of human and cynomolgus monkey MUC1 SEA proteins was performed as described above, and the results are shown in Tables 10 and 11. All variants showed binding comparable to the chimeric BG138P antibody. Finally, mutant m4 (DG to SG) was selected for the humanized BG138P antibody.

[0294] Table 10. SPR binding affinity of BG138P PTM-removed variant to human MUC1 SEA protein

[0295]

[0296] Table 11. SPR binding affinity of BG138P PTM-removed variant to cynomolgus monkey MUC1 SEA protein

[0297] Injection Variable Capture 1 Solution Analyte 1 solution 1:1 combination with ka kd KD(M) BG138P - Molding Crab-eating macaque MUC1 SEA 1.07E+06 7.12E-03 6.63E-09 BG138P-PTM-m1 Crab-eating macaques MUC1 SEA 1.19E+06 7.51E-03 6.32E-09 BG138P-PTM-m2 Crab-eating macaques MUC1 SEA 1.02E+06 6.99E-03 6.85E-09 BG138P-PTM-m3 Crab-eating macaques MUC1 SEA 9.47E+05 6.62E-03 6.99E-09 BG138P-PTM-m4 Crab-eating macaques MUC1 SEA 9.64E+05 6.50E-03 6.75E-09

[0298] The layer 1 and layer 2 reversion mutation sites of VH and VL were combined to construct a second round of humanized variants, all of which included PTM removal mutations from “DG” to “SG”. The final combined humanized variants (referred to as huBG138P-Hz1 to Hz4 or abbreviated as Hz1 to Hz4) are summarized in Table 12, and the sequences of the variants are listed in Table 14.

[0299] Table 12. Hz1-Hz4 with combinations of reversion mutation sites in layers 1 and 2

[0300]

[0301]

[0302] SPR assays were then performed to evaluate the binding affinity of huBG138P-Hz1 to Hz4 to human and cynomolgus monkey MUC1SEA. SPR assays showed that all variants maintained binding affinity comparable to chimeric BG138P, and the data are summarized in Table 13.

[0303] Table 13. SPR binding affinity of BG138P humanized variants Hz1 to Hz4 with human and cynomolgus monkey MUC1 SEA protein

[0304]

[0305] Then, FACS binding was performed using HEK293-hMUC1 and HEK293-cynomolgus monkey MUC1 (iQue3). TM FACS (Sartorius) assays were performed to determine the cell-based binding activity of Hz1 through Hz4. FACS binding data for chimeric BG138P and its humanized antibody huBG138P-Hz1 through Hz4 (with hIgG1 as a control) were processed using GraphPad Prism software and displayed. Figure 10A and 10B The results indicated that the cell-based binding activities of BG138P-Hz2 and BG138P-Hz4 to human and cynomolgus monkey MUC1 remained similar to those of chimeric BG138P. Furthermore, the cell-based binding activities of BG138P-Hz1 and BG138P-Hz3 to human MUC1 remained similar to those of chimeric BG138P, but their cell-based binding activities to cynomolgus monkey MUC1 were lower than those of chimeric BG138P.

[0306] The HCDR, LCDR, VH, and VL amino acid sequences of huBG138P-Hz2 are provided in Table 14.

[0307] Table 14. Sequence listing of humanized variants of BG138P

[0308]

[0309] Humanization of BG219P

[0310] For the humanization of BG219P, sequences sharing high homology with the variable region of BG219P were searched in the human immunoglobulin gene database in IMGT by sequence comparison. Human IGHV and IGKV genes, which are frequently present in human antibody libraries and highly homologous to mouse BG219P, were selected as templates for humanization.

[0311] Humanization was achieved through CDR transplantation followed by the incorporation of key reversion mutations. The humanized antibody was engineered into a human IgG1 wild-type format using an internally developed expression vector. In the initial rounds of humanization, 3D structural analysis guided mutations of mouse-to-human amino acid residues in the framework region, and structurally important mouse framework residues for maintaining the canonical structure of the CDR were preserved in the first round of humanization design. BG219P-Bz0 (the antibody variant with all reversion mutation sites among all 19 generated CDR-transplanted versions) is a variant with a theoretical binding capacity close to that of the parent mouse antibody BG219P.

[0312] 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 recipient frameworks for the BG219P VL and VH sequences. The LCDR of mouse BG219P was transplanted into 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 amino acid and DNA sequences of the resulting BG219P-Bz0 VL are shown in Table 16. The HCDR of mouse BG219P was transplanted into the frameworks of human germline variable genes IGHV3-23*01 and IGHJ6*01, retaining the S30N, S49A, A93T, and K94R mouse framework residues. The amino acid and DNA sequences of the obtained BG219P-Bz0 VH are shown in Table 16.

[0313] Starting with the humanized BG219P antibody huBG219P-Bz0, several additional amino acid changes were made in the CDR regions of VH and VL to further improve its biophysical properties for human therapeutic use. Considerations included removal of post-translational modifications and improvement of thermal stability (Tm) while maintaining binding activity.

[0314] More than thirty humanized BG219P (also known as huBG219P) variants were constructed using an internal IgG1 / Cκ eukaryotic expression vector. These vectors contain constant regions of the human wild-type IgG1 and Cκ chains, respectively, and have readily adaptable subcloning sites. The variants were generated by transiently transfecting plasmids into ExpiCHO-s cells (Thermofisher Scientific). Conditioned media were harvested and processed using MabSelect. TM The variants were purified using a SuRe column (Cytiva), followed by buffer replacement via UF / DF. All purified antibodies were stored in aliquots at -80°C.

[0315] For affinity assays, antibodies were captured on the anti-human Fc surface and used for affinity determination based on surface plasmon resonance (SPR) technology. The results of the SPR assays of the binding profiles of anti-MUC1 antibodies are summarized in Table 15. huBG219P-E39 and huBG219P-E43 exhibited similar binding affinities, with dissociation constants of 35.2 pM and 30.3 pM, respectively, comparable to the dissociation constant of chimeric BG219P (39.8 pM). The sequences of huBG219P-E39 and huBG219P-E43 are provided in Table 16.

[0316] Table 15. Comparison of binding affinity between huBG219P and MUC1 SEA-Fc by SPR

[0317] Antibody <![CDATA[k on (M-1s-1)]]> <![CDATA[k off (s-1)]]> <![CDATA[K D (nM)]]> chBG219P 1.44E+06 5.74E-05 3.98E-011 huBG219P-E39 1.40E+06 4.92E-05 3.52E-011 huBG219P-E43 1.61E+06 4.86E-05 3.03E-011

[0318] Table 16. Sequence List

[0319]

[0320]

[0321]

[0322] To evaluate the binding activity of anti-MUC1 antibodies to native MUC1 on live cells, a FACS-based binding assay was performed using ZR-75 cells. Live ZR-75 cells were seeded in 96-well plates and incubated with a series of dilutions of chimeric or humanized BG219P. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. The dose-dependent binding activity of EC1s to native human MUC1 was determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. 50 Value. For example... Figure 11As shown in Table 17, the humanized BG219P antibodies huBG219P-Bz0, E39, and E43 retained considerable binding affinity to native MUC1 compared to chimeric BG219P.

[0323] Table 17. Comparison of binding affinity between chBG219P and huBG219P and native MUC1 by FACS

[0324] Ab number <![CDATA[ZR-75 binds to EC 50 (nM)]]> chBG219P 12.3 huBG219P-bz0 20.9 huBG219P-E39 14.0 huBG219P-E43 15.7

[0325] Example 9. Generation of scFv of mouse anti-human MUC1 mAb BG138P, BG219P and BG346P

[0326] To generate the scFv construct, the heavy and light chain variable regions (VH, VL) are linked via artificial amino acid linkers. For clone BG138P, VH and VL from BG138P-Hz2 are linked via a (GGGGS)4 linker (SEQ ID NO:83), and a histidine tag (i.e., HHHHHH (SEQ ID NO:85)) is fused to the C-terminus to form the final construct VH-(GGGGS)4-VL-HHHHHH, named scFv1-78P. For clone BG219P, VH and VL from mouse BG219P are used to generate VH-(GGGGS)4-VL-HHHHHH, and additional humanization work is performed based on this format, details of which are disclosed below. For clone BG346P, the VH and VL of mouse BG346P are initially connected via (GGGGS)3 connector (SEQ ID NO:84) to generate VH-(GGGGS)3-VL-HHHHHH, and additional engineering work based on this construct is disclosed below.

[0327] BG138P scFv Humanization and Engineering

[0328] To test whether there was room for further improvement in the physical properties of scFv1-78P, yield, purity, and thermal stability tests were performed on forty constructs with additional single mutations in VL or VH. For the experiment, scFv1-78P was fused to the C-terminal Fc tag, producing scFv-Fc (referred to as scFv). 野生型 -Fc), and a series of scFv1-78P mutants are also fused to the C-terminal Fc tag, producing scFv-Fc (called scFv 突变体 -Fc). Protein in Expi293 TM Overexpressed in Thermo Fisher cells and used MabSelect SuRe TM Purification was performed using a Cytiva column. The resulting scFv was then purified.突变体 The yield and purity of -Fc are related to scFv 野生型 -Fc comparison. Twenty-one scFv 突变体 -Fc and scFv 野生型 -Fc showed better yield compared to scFv. 突变体 -Fc showed good yield and purity; however, compared with scFv 野生型 Compared to -Fc, none of them showed good thermal stability.

[0329] BG219P scFv Humanization and Engineering

[0330] The VH and VL frames of mouse BG219P were linked to generate the scFv construct as described above. For humanization, the mouse VH and VL frames were compared with human genus databases. The closest human genus lines IGHV3-21*01+IGHJ4 and IGKV1-33*01+IGKJ2 were selected for humanization. The six CDRs in the mouse scFv were directly transplanted into the selected human genus lines without additional mutations in the frame, resulting in the construct scFv2-14P. Affinity and physical property characterization are disclosed below.

[0331] To remove a potential post-translational deamidation site in the CDR-H2 region of scFv2-14P without affecting the affinity of scFv, the vulnerable Asp was mutated to Glu to mimic the structure of the CDR-H2 loop in scFv2-14P. Compared to scFv2-14P, the resulting mutant scFv2-57P showed better performance in Expi 293. TM The protein showed comparable yields in cells and similar affinity for soluble recombinant MUC1 protein and MUC1+ cell lines. Data are shown in Table 18.

[0332] Table 18. Post-translational site removal of scFv2-14P

[0333]

[0334] (NA: Not applicable. No further mutations will be added.)

[0335] BG346P scFv Humanization and Engineering

[0336] VH and VL from mouse BG346P were linked via an artificial linker (GGGGS)3 (SEQ ID NO:84) to generate mouse scFv BG346P. Humanization of mouse BG346P scFv was performed using a frame exchange strategy. CDR residues of BG346P were transplanted into a series of frames listed in Table 19, and additional mutations were performed on the frames as listed.

[0337] Table 19. Framework used in the humanization of BG346P scFv

[0338]

[0339]

[0340]

[0341]

[0342] (NA: Not applicable. No further mutations will be added.)

[0343] The humanized scFv constructs listed above are available in Expi293. TM Overexpression in Thermo Fisher cells. To select a stable scFv construct, Expi293 cells overexpressing the scFv protein were used. tm Cell supernatants were incubated at 60°C for two hours. The ability of heated scFv protein to bind to the recombinant MUC1 SEA domain was tested. After heat stress experiments, the stable scFv scFv3-65P was selected for further engineering. A longer artificial linker (GGGGS)4 (SEQ ID NO:83) was introduced into scFv3-65P, replacing the original (GGGGS)3 (SEQ ID NO:84) linker, thereby allowing for better scFv expression. The resulting scFv was named 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 to scFv3-07P. In addition, to minimize immunogenicity, the mouse reversion mutation introduced in scFv3-65P was mutated back to human amino acids in the background of scFv3-96P, generating constructs scFv3-08P to scFv3-19P. The relative affinities of scFv3-98P, scFv3-99P, and scFv3-00P to scFv3-19P were compared with scFv3-96P, and any beneficial mutations were selected. The data are presented in Table 20.

[0344] Table 20. Additional mutations tested in humanized BG346P

[0345]

[0346]

[0347] After testing the effect of each individual mutation on affinity, mutations that were harmless to scFv affinity were selected and combined. For amino acids with PTM risk, the goal was to remove them as much as possible without affecting affinity. For mouse mutations, the goal was to transfer as many human amino acids as possible back to minimize immunogenicity. Finally, the construct with the lowest PTM risk and the fewest number of mouse mutations maintaining affinity was selected as the final scFv, which is BG346P-scFv3-20P (or abbreviated as scFv3-20P), as shown in Table 21.

[0348] Table 21. Combinatorial mutations tested in humanized BG346P

[0349]

[0350] The sequences of the four scFvs are listed in Table 22.

[0351] Table 22. Sequences of selected scFv fragments

[0352]

[0353]

[0354]

[0355] Table 23. Sequences of amino acid linkers in scFv

[0356] SEQ ID NO Notes sequence SEQ ID NO:83 AA connector GGGGSGGGGSGGGGSGGGGS SEQ ID NO:84 AA connector GGGGSGGGGSGGGGS

[0357] Affinity determination of scFv1-78P, scFv2-14P, scFv2-57P and scFv3-20P

[0358] 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, which was derivatized via amine conjugation with a monoclonal rabbit anti-mouse Fc antibody (catalog number 29215281). The scFv fragments scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P were then flowed through the sensor at a rate of 30 μL / min. Association of scFv with MUC1 protein was monitored for 2 min, and dissociation of the scFv fragments in 1x HBS buffer (Cytiva, catalog number BR100669) was monitored for 10 min.

[0359] Ka and Kd are obtained by using Biacore. TMThe Insight evaluation software fits real-time sensor images to a 1:1 scale and combines this with a model to determine the model's performance. This is combined with the dissociation equilibrium constant (K... D The kinetic rate is calculated as: K D (M) = kd / ka. The binding kinetics of scFv protein and MUC1 protein are shown in Table 24.

[0360] Table 24. Biacore binding affinity of scFv at 25℃

[0361] Antibody Ka(Ms-1) Kd(S-1) KD (moles) scFv1-78P 1.85e+6 2.60e-3 1.40e-9 scFv2-14P 8.15e+5 4.25e-4 5.22e-10 scFv2-57P 3.97e+5 4.35e-4 1.10e-9 scFv3-20P 3.01e+5 2.02e-2 6.71e-8

[0362] Example 10. Binding activity of scFv fragment to native MUC1

[0363] To evaluate the binding ability of anti-MUC1 scFv to MUC1 on the surface of living cells, HEK293 cells were engineered to overexpress the full-length MUC1 protein, as described above. For the assay, 1x10 6 HEK293-MUC1 cells were seeded into 96-well plates. To generate scFv dose-response curves, serially diluted scFv protein (4.2 pm to 250 nm) was added to the cells, and scFv binding was detected using the His-tagged antibody ifluor 488 (catalog number A01800). Flow cytometry was used. 3. The median fluorescence intensity (MFI) of each cell population was measured using Sartorius, and the EC of each scFv was determined using a four-parameter logistic model. 50 And Emax. The results are shown in Table 25.

[0364] The results indicated 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 compared to scFv3-20P.

[0365] Table 25. FACS and MFI of dose-dependent binding of scFv fragment to native MUC1

[0366] Antibody <![CDATA[EC 50 (nM)]]> MFI scFv1-78P 3.873 309523 scFv2-14P 5.083 401851 scFv2-57P 5.247 402602 scFv3-20P 16.15 226489

[0367] Example 11. Epitope localization of humanized scFv fragments

[0368] To evaluate whether the three scFv fragments, scFv1-78P, scFv2 (scFv2-14P and scFv2-57P are collectively referred to as "scFv2" in this embodiment because they share the same epitope bin), and scFv3-20P, could compete with each other for binding to the corresponding epitopes on MUC1, a tandem binding competition assay was performed. Briefly, MUC1-SEA-mIgG2a was immobilized on a CM5 Biocore sensor derivatized via amine conjugation with a monoclonal rabbit anti-mouse Fc antibody (catalog number 29215281). The first scFv fragment was then flowed through at a rate of 10 μl / min. Capture of the first scFv by MUC1-SEA-mIgG2a was monitored for 1.5 min. Then, the second scFv fragment was flowed through at a rate of 10 μl / min, and capture was monitored for 2 min. The competition between scFv1-78P and scFv2 (scFv2-14P, scFv2-57P) is shown in Figure 12 In the middle, scFv3-20P belongs to another compartment.

[0369] The results indicate that the epitopes of scFv1-78P, scFv2-14P, and scFv2-57P at least partially overlap, while the epitope of scFv3-20P is different from the other three epitopes.

[0370] Example 12. Physical properties of humanized scFv fragments

[0371] The hydrophobicity, thermal stability, and aggregation tendency of the humanized scFv fragments are evaluated as follows.

[0372] Hydrophobicity assessment

[0373] 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 (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 Mabpac HIC-10 column from Thermo Fisher was used, with mobile phase A and mobile phase B (50 mm sodium phosphate, pH 7.0) flowing linearly at a rate of 0.5 mg / min over 29 min. Peak retention time was monitored at A280 absorbance. The results are summarized in Table 26. scFv1-78P (RT, 14.2 min) and scFv3-20P (RT, 9.7 min) showed higher hydrophilicity than scFv2-14P (RT, 20.5 min) and scFv2-57P (RT, 20.6 min). All four scFv domains showed acceptable hydrophobicity.

[0374] Table 26. Retention Time of HIC Columns

[0375] Sample Name Retention time (min) scFv1-78P 14.2 scFv2-14P 20.5 scFv2-57P 20.6 scFv3-20P 9.7

[0376] Thermal stability assessment

[0377] The thermal stability of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P was measured by exogenous fluorescence and described by the thermal expansion transition midpoint Tm (°C) (melting temperature). QuantStudio from Applied Biosystems was used. TM Tm was determined using a 6Flex system. 20 μl of a 1 mg / mL sample was mixed with 20 μl of 40X SYPRO Orange. The plate was scanned from 25 °C to 95 °C at a rate of 0.9 °C / min. The measurement was performed using a system from QuantStudio. TM 6. The first derivatives of the raw data from the Flex system analysis software were used to assign Tm. The results are summarized in Table 27. scFv1-78P (70.0℃) and scFv3-20P (69.8℃) showed Tm approximately 10℃ higher than scFv2-14P (60.0℃) and scFv2-57P (60.2℃). All four scFv domains showed acceptable thermal stability.

[0378] Table 27. Tm values ​​from thermal displacement measurements

[0379] Sample Name Tm (°C) scFv1-78P 70.0 scFv2-14P 60.0 scFv2-57P 60.2 scFv3-20P 69.8

[0380] Aggregation tendency assessment

[0381] To determine the aggregation tendency of scFv1-78P, scFv2-14P, scFv2-57P, and scFv3-20P, Uncle... TM The static light scattering intensity was measured using the Unchained Labs system. During the measurement, approximately 8.8 μl of protein sample (1 mg / ml) was loaded into a cuvette; the sample was held at 25 °C for 120 s and then heated to 95 °C at a rate of 0.3 °C / min. Scattering data were collected at a 90° angle with a laser wavelength of 266 nm. The Tagg (aggregation temperature) was analyzed and calculated using Unclear analysis software. The results are summarized in Table 28. scFv3-20P (63.8 °C) showed a 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.

[0382] Table 28. Tagg values ​​from SLS

[0383] Sample Name Tagg (°C) scFv1-78P 56.3 scFv2-14P 54.5 scFv2-57P 53.1 scFv3-20P 63.8

Claims

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

29.

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

81.

3. The anti-MUC1 antibody or its antigen-binding fragment as described in claim 2, wherein the anti-MUC1 antibody or its antigen-binding fragment comprises: (i) The heavy chain variable region (VH) containing SEQ ID NO:10 and the light chain variable region (VL) containing SEQ ID NO:11; (ii). The heavy chain variable region (VH) containing SEQ ID NO:20 and the light chain variable region (VL) containing 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). The heavy chain variable region (VH) containing SEQ ID NO:57 and the light chain variable region (VL) containing SEQ ID NO:59; (v). The heavy chain variable region (VH) containing SEQ ID NO:57 and the light chain variable region (VL) containing SEQ ID NO:60; (vi) The heavy chain variable region (VH) containing SEQ ID NO:58 and the light chain variable region (VL) containing SEQ ID NO:59; (vii). Containing the heavy chain variable region (VH) of SEQ ID NO:58 and the light chain variable region (VL) of SEQ ID NO:60; (viii). The heavy chain variable region (VH) containing SEQ ID NO:61 and the light chain variable region (VL) containing SEQ ID NO:62; (ix). The heavy chain variable region (VH) containing SEQ ID NO:61 and the light chain variable region (VL) containing SEQ ID NO:66; (x). Containing the heavy chain variable region (VH) of SEQ ID NO:61 and the light chain variable region (VL) of SEQ ID NO:68; (xi). Containing the heavy chain variable region (VH) of SEQ ID NO:71 and the light chain variable region (VL) of SEQ ID NO:72; (xii). Containing the heavy chain variable region (VH) of SEQ ID NO:75 and the light chain variable region (VL) of SEQ ID NO:76; or (xiii) The heavy chain variable region (VH) containing SEQ ID NO:80 and the light chain variable region (VL) containing SEQ ID NO:

81.

4. The anti-MUC1 antibody or its antigen-binding fragment as described in claim 3, wherein one, two, three, four, five, six, seven, eight, nine, or ten amino acids in 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 its antigen-binding fragment as described in any one of claims 1 to 4, wherein the anti-MUC1 antibody or its antigen-binding fragment 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. The antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the antibody or antigen-binding fragment thereof comprises scFv, the scFv comprising a VH containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75, or SEQ ID NO:80, and a VL containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as 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 as claimed in claim 6, wherein the antibody or antigen-binding fragment thereof comprises scFv, wherein the scFv comprises a VH containing SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:75 or SEQ ID NO:80 and a VL containing SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76 or SEQ ID NO:

81.

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

81.

9. The antibody or antigen-binding fragment thereof as claimed in claim 8, wherein the antibody or antigen-binding fragment thereof comprises scFv, and the scFv comprises: (i) VH containing SEQ ID NO:57 and VL containing SEQ ID NO:60; (ii). VH containing SEQ ID NO:71 and VL containing SEQ ID NO:72; (iii) VH containing SEQ ID NO:75 and VL containing SEQ ID NO:76; or (iv). VH containing SEQ ID NO:80 and VL containing SEQ ID NO:

81.

10. The antibody or antigen-binding fragment thereof as claimed in claim 9, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids in 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 as claimed in 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 as claimed in claim 11, wherein the amino acid linker comprises SEQ ID NO: 83 or 84.

13. The antibody or antigen-binding fragment thereof as claimed in 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 as claimed in any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

15. The anti-MUC1 antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or low fucosylation.

16. The anti-MUC1 antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof comprises an augmented bipartite GlcNac structure.

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

18. A pharmaceutical composition comprising an anti-MUC1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

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

20. A vector comprising the nucleic acid as described in claim 19.

21. A host cell comprising the nucleic acid as described in claim 19 or the vector as described in claim 20.

22. A method for generating an anti-MUC1 antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell as described in claim 21 and recovering the antibody or antigen-binding fragment from the culture.

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