MUC1 and CD16A Antibodies and Methods of Use

Abstract

The present disclosure provides multispecific antibodies and antigen-binding fragments thereof that bind to human MUC1 and CD16A, pharmaceutical compositions comprising the antibodies, and uses of the antibodies or compositions to treat diseases such as cancer. In embodiments, the present disclosure relates to multispecific antibodies or antigen-binding fragments thereof that comprise a first antigen-binding domain that specifically binds to human MUC1 and a second antigen-binding domain that specifically binds to human CD16A. In embodiments, the present disclosure relates to multispecific antibodies or antigen-binding fragments, wherein the first antigen-binding domain has higher selectivity over human CD16B.

Description

[Technical Field]

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

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as file entitled "01368-0054-00PCT_SL," created February 27, 2024, and is 179,126 bytes in size. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] Disclosed herein are multispecific antibodies or antigen-binding fragments thereof that bind to human MUC1 and human CD16A, and compositions comprising said antibodies. [Background technology]

[0004] Mucin 1 (MUC1; also known as CA15-3, EMA, MCD, PEM, PUM, KL-6, MAM6, MCKD, PEMT, CD227, H23AG, MCKD1, ADMCKD, and ADTKD2) is a highly glycosylated heterodimeric membrane-bound protein belonging to the mucin family. It is normally expressed on the apical surface of glandular or luminal epithelial cells in various tissues, where it lubricates and moisturizes the epithelial cell surface and protects against pathogens. However, in most human epithelial cancers, including pancreatic, breast, ovarian, lung, and colon cancers, MUC1 is hypoglycosylated and aberrantly overexpressed. Furthermore, because cancer cells lose apical-basal polarity, MUC1 is expressed over the entire surface of tumor cells. Given these properties, MUC1 is considered a promising therapeutic target for human cancers.

[0005] The MUC1 heterodimer consists of a longer N-terminal extracellular domain (ECD) (MUC1-N) and a shorter subunit (MUC1-C) containing a 69-amino acid C-terminal cytoplasmic domain, a 28-amino acid hydrophobic transmembrane domain, and a 58-amino acid short ECD. The two subunits are noncovalently linked by hydrogen interactions. MUC1-N is often shed from the cell surface and released into the circulation. Elevated levels of shed MUC1 are observed in the serum of patients with various cancers. Previous MUC1-N-targeting therapeutics, such as AS1402 (huHMFG-1) and BrevaRex (AR-20.5), have failed in clinical trials, likely due to the shedding of anti-MUC1 antibodies, which prevented them from binding to MUC1 on the cancer cell surface. To overcome this issue, antibodies that bind to MUC1-C may be a more promising strategy for efficiently targeting MUC1-expressing cancer cells.

[0006] CD16A (also known as FcγRIIIa) is a low-affinity receptor for IgG1 and IgG3, and is expressed on natural killer (NK) cells, macrophages, and some circulating monocytes. CD16A itself can induce activation signals and kill antibody-opsonized target cells via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] The ADCC mechanism contributes to the therapeutic efficacy of several widely used tumor-targeting monoclonal antibodies (mAbs), such as trastuzumab and rituximab. This role of CD16A is also supported by evidence of clinical responses to therapeutic antibodies influenced by CD16A polymorphisms. Patients with the homozygous high-affinity variant (CD16A-158V / V) exhibit better clinical responses in multiple indications than patients with the heterozygous (CD16A-158V / F) or homozygous low-affinity variant (CD16A-158F / F).

[0008] To date, there are various strategies to enhance NK cell responses to tumor-targeting therapeutics. The most common strategy is genetic manipulation and glycoengineering of antibody Fc regions to enhance interaction with CD16A. Another strategy is to address antibody formats. Bispecific or trispecific killer engagers have been generated to efficiently target innate immune cells, such as NK cells, to tumor cells. These formats better bridge NK cells and tumor cells, allowing high-affinity CD16A engagement, resulting in superior killing activity compared to antibodies.

[0009] The present disclosure provides targeting MUC1-C and CD16A via multispecific antibodies that recruit innate immune cells to MUC1-expressing cells and are useful for treating MUC1-expressing cancers. Summary of the Invention

[0010] The present disclosure relates to multispecific anti-MUC1xCD16A antibodies and antigen-binding fragments thereof.

[0011] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to human MUC1 and a second antigen-binding domain that specifically binds to human CD16A.

[0012] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein the first antigen-binding domain has higher selectivity than human CD16B.

[0013] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment, wherein the first antigen-binding domain that specifically binds to human MUC1 is: (i) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; (iii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; or (iv) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19.

[0014] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment, wherein the first antigen-binding domain is: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 30, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 31; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 62; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 66; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; or (vi) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 20, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 21.

[0015] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are inserted, deleted, or substituted within one or more of SEQ ID NOs: 30, 31, 61, 62, 66, 68, 10, 11, 20, and 21.

[0016] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment, wherein the first antigen-binding domain is: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 61 and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 10 and a light chain variable region (VL) comprising SEQ ID NO: 11; or (vi) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising SEQ ID NO: 20 and a light chain variable region (VL) comprising SEQ ID NO: 21.

[0017] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment, wherein the second antigen-binding domain that specifically binds to human CD16A is: (i) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; or (ii) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111.

[0018] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment, wherein the second antigen-binding domain is: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 115; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 117; or (iv) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 119.

[0019] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are inserted, deleted, or substituted within one or more of SEQ ID NOs: 112, 115, 117, and 119.

[0020] A multispecific antibody or antigen-binding fragment, wherein the second antigen-binding domain is: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 112; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 115; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 117; or (iv) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising SEQ ID NO: 119.

[0021] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment comprising: (i) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (ii) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (iii) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (iv) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; and a second antigen-binding domain that specifically binds to human CD16A comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (v) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; (vi) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; (vii) a first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; and a second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; or (viii) A multispecific antibody or antigen-binding fragment comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; and a second antigen-binding domain that specifically binds to human CD16A comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111.

[0022] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment comprising: (i) The first antigen-binding domain that specifically binds to human MUC1 is: a) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; b) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; c) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; d) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii) and a second antigen-binding domain that specifically binds to human CD16A, comprising: a) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; or b) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111.

[0023] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment comprising: (i) The first antigen-binding domain that specifically binds to human MUC1 is: a) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; b) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; c) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; d) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (ii) and a second antigen-binding domain that specifically binds to human CD16A, comprising: a) a heavy chain variable region (VH) comprising SEQ ID NO: 112; b) a heavy chain variable region (VH) comprising SEQ ID NO: 115; c) a heavy chain variable region (VH) comprising SEQ ID NO: 117; or d) a heavy chain variable region (VH) comprising SEQ ID NO: 119.

[0024] In embodiments, the present disclosure provides a multispecific antibody or antigen-binding fragment comprising: (i) The first antigen-binding domain that specifically binds to human MUC1 is: a) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; b) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; c) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; d) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii) and a second antigen-binding domain that specifically binds to human CD16A, comprising: a) a heavy chain variable region (VH) comprising SEQ ID NO: 112; b) a heavy chain variable region (VH) comprising SEQ ID NO: 115; c) a heavy chain variable region (VH) comprising SEQ ID NO: 117; or d) a heavy chain variable region (VH) comprising SEQ ID NO: 119.

[0025] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment that is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0026] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein the multispecific antibody is a bispecific antibody.

[0027] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein the multispecific antibody is BG1222P (SEQ ID NO: 143, SEQ ID NO: 145, and SEQ ID NO: 147).

[0028] In embodiments, the present disclosure relates to multispecific antibodies or antigen-binding fragments, wherein the antibodies or antigen-binding fragments have antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0029] In embodiments, the present disclosure relates to multispecific antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof are reduced or aglycosylated, or hypofucosylated.

[0030] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an increase in bisecting GlcNAc structures.

[0031] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein the Fc domain is an IgG1 with reduced effector function.

[0032] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment, wherein the Fc domain is IgG4.

[0033] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment thereof comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of a CDR, VH, VL, or complete chain disclosed herein.

[0034] In embodiments, the present disclosure relates to a multispecific antibody or antigen-binding fragment thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been inserted, deleted, or substituted within the amino acid sequence of one or more of the CDRs, VH, VL, or full chains disclosed herein.

[0035] In embodiments, the present disclosure relates to a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may include histidine / histidine HCl, trehalose dihydrate, and polysorbate 20.

[0036] In embodiments, the present disclosure relates to a method of treating cancer comprising administering to a patient in need thereof an effective amount of a multispecific antibody or antigen-binding fragment disclosed herein. In embodiments, the present disclosure relates to an isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment disclosed herein.

[0037] In embodiments, the present disclosure relates to a vector comprising the nucleic acid disclosed herein.

[0038] In embodiments, the present disclosure relates to a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0039] In embodiments, the present disclosure relates to a process for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing a host cell disclosed herein and recovering the antibody or antigen-binding fragment from the culture.

[0040] In embodiments, the multispecific antibodies of the present disclosure are of the IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the antibodies of the present disclosure comprise the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2.

[0041] In one embodiment, the multispecific antibody of the present disclosure is administered in a concentration of 1 x 10 -6 M~1×10 -10 Binding affinity (K D In another embodiment, the antibodies of the disclosure bind to MUC1 at about 1 x 10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, or approximately 1 x 10 -10 Binding affinity (K D ) binds to MUC1.

[0042] In embodiments, the anti-human MUC1 multispecific antibodies of the disclosure exhibit cross-species binding activity for cynomolgus monkey MUC1.

[0043] In embodiments, the antibodies of the disclosure have potent Fc-mediated effector functions, hi some embodiments, the antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against MUC1-expressing target cells. [Brief explanation of the drawings]

[0044] [Figure 1] Schematic diagram of MUC1-SEA-mIgG2a (top) and MUC1-SEA-huIgG1 (bottom), where "N" indicates the N-terminus and "C" indicates the C-terminus. [Figure 2A] Figure 1 shows the binding affinity of purified MUC1 antibodies to human MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric anti-MUC1 monoclonal antibody BG138P to human cells overexpressing MUC1. [Figure 2B] Figure 1 shows the binding affinity of purified MUC1 antibodies to human MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric BG346P to human cells overexpressing MUC1. [Figure 2C] Figure 1 shows the binding affinity of purified MUC1 antibodies to cynomolgus monkey MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric anti-MUC1 monoclonal antibody BG138P to cynomolgus monkey cells overexpressing MUC1. [Figure 2D] Figure 1 shows the binding affinity of purified MUC1 antibodies to cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric BG346P to cynomolgus MUC1-overexpressing cells. [Figure 2E] Figure 1 shows the binding affinity of purified MUC1 antibodies to human MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric BG219P to human cells overexpressing MUC1. [Figure 2F] Figure 1 shows the binding affinity of purified MUC1 antibodies to cynomolgus MUC1-overexpressing cells by FACS assay using human IgG1 as a negative control. Figure 2 shows the binding affinity of chimeric BG219P to cynomolgus MUC1-overexpressing cells. [Figure 3A] We demonstrate the determination of epitope binning by a competitive SPR assay, in which purified human MUC1-mFc antigen was flowed over the chip surface and captured with an anti-mouse IgG antibody. [Figure 3B] We demonstrate the determination of epitope binning by a competitive SPR assay, in which purified human MUC1-mFc antigen was flowed over the chip surface and captured with an anti-mouse IgG antibody. [Figure 3C] We demonstrate the determination of epitope binning by a competitive SPR assay, in which purified human MUC1-mFc antigen was flowed over the chip surface and captured with an anti-mouse IgG antibody. [Figure 4A] Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG219P at a concentration (30 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 4B] Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG219P at a concentration (3 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 4C] Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG219P at a concentration (0.3 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 4D]Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG346P at a concentration (30 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 4E] Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG346P at a concentration (3 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 4F] Figure 1 shows the effect of soluble MUC1 on MUC1 antibody binding to MUC1-expressing cells. The binding profiles of chBG138P and chBG346P at a concentration (0.3 μg / ml) in the presence of soluble MUC1 are shown. HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 5A] This shows that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. This shows that chBG138P binds to the MUC1-expressing tumor cell line HCC827 in a dose-dependent manner (human IgG1 was used as a negative control). [Figure 5B] Figure 1 shows that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. Figure 2 shows that chBG138P binds to the MUC1-expressing tumor cell line H1975 in a dose-dependent manner (human IgG1 was used as a negative control). [Figure 5C] Figure 1 shows that the anti-MUC1 monoclonal antibody chBG138P, which targets the MUC1 membrane proximal region, binds to MUC1-positive cancer cell lines. Figure 2 shows that chBG138P binds to the MUC1-expressing tumor cell line T-47D in a dose-dependent manner (human IgG1 was used as a negative control). [Figure 6] Figure 1 shows a schematic of the FACS gating strategy for the T cell binding assay. The dashed box indicates the percentage of antibody-binding T cells. [Figure 7A]We show that a chimeric anti-MUC1 monoclonal antibody, chBG138P, which targets the MUC1 membrane proximal region, does not bind to activated T cells. [Figure 7B] We show that a chimeric anti-MUC1 monoclonal antibody, chBG138P, which targets the MUC1 membrane proximal region, does not bind to activated T cells. [Figure 7C] This shows that the MUC1 membrane distal portion-targeting antibody HMFG1 can bind to normal T cells. [Figure 7D] This shows that the MUC1 membrane distal portion-targeting antibody HMFG1 can bind to normal T cells. [Figure 7E] We show that chimeric anti-MUC1 monoclonal antibodies, chBG138P, chBG219P, or chBG346P, which target the MUC1 membrane proximal region, do not bind to activated T cells. [Figure 7F] We show that chimeric anti-MUC1 monoclonal antibodies, chBG138P, chBG219P, or chBG346P, which target the MUC1 membrane proximal region, do not bind to activated T cells. [Figure 7G] This shows that antibody 16A, which targets the membrane distal portion of MUC1, is able to bind to normal T cells. [Figure 7H] This shows that antibody 16A, which targets the membrane distal portion of MUC1, is able to bind to normal T cells. [Figure 8A] This shows that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 do not bind to normal T cells. [Figure 8B] This shows that chBP138P and the humanized MUC1 antibodies BG138P-hz2 and BG138P-hz4 do not bind to normal T cells. [Figure 8C] We demonstrate that the MUC1 membrane-distal targeting antibody HMFG1 binds to normal activated T cells. [Figure 8D] We demonstrate that the MUC1 membrane-distal targeting antibody HMFG1 binds to normal activated T cells. [Figure 9] 1 shows that the humanized antibodies huBG219P-Bz0, huBG219P-E39, and huBG219P-E43 bind to the MUC1-overexpressing cell line ZR-75-1 to a similar extent compared to the chimeric antibody chBG219P. [Figure 10] ELISA analysis of a representative top clone, BG523P, compared to LS21 is shown. [Figure 11A] FACS analysis of a representative top clone, BG523P, compared to LS21 is shown. [Figure 11B] FACS analysis of a representative top clone, BG523P, compared to LS21 is shown. [Figure 11C] FACS analysis of a representative top clone, BG523P, compared to LS21 is shown. [Figure 12A] 1 shows FACS binding of BG523P and BG524P to NK92mi / CD16A 158F, a CD16A 158F overexpressing cell line. [Figure 12B] FACS binding of BG523P, BG525P, and BG526P to NK92mi / CD16A F158 cells is shown. [Figure 13] 1 shows the FACS binding signals of 300 nM BG523P, BG525P, and BG526P against CD16B-overexpressing cell lines NK92mi / CD16B NA1 and NK92mi / CD16B NA2. [Figure 14A] Figure 1 shows FACS-based human IgG competition for the binding of NK92mi / CD16A 158F to BG523P in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. Figure 2 shows the effect of IgG competition on the binding of BG523P to NK92mi / CD16A 158F. [Figure 14B] Figure 1 shows FACS-based human IgG competition for the binding of NK92mi / CD16A 158F to the humanized VHH of BG523P (BG525P) in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. Figure 2 shows the effect of IgG competition on the binding of BG525P to NK92mi / CD16A 158F. [Figure 14C] Figure 1 shows FACS-based human IgG competition for the binding of NK92mi / CD16A 158F to the humanized VHH of BG523P (BG526P) in the presence or absence of 10 mg / mL recombinant CB6 human IgG1. Figure 2 shows the effect of IgG competition on the binding of BG526P to NK92mi / CD16A 158F. [Figure 15] FIG. 1 is a schematic diagram of the MUC1×CD16A multispecific antibody BG1222P format. [Figure 16] 1 shows the binding affinity of the MUC1×CD16A multispecific antibody BG1222P to the MUC1-expressing tumor cell line T47D. [Figure 17] (A) Comparison of the binding of MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF to human CD16A F158-overexpressing NK92mi cells in the absence of competition with human IgG. (B) Comparison of the binding of MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF to human CD16A F158-overexpressing NK92mi cells in the presence of competition with human IgG. (C) Comparison of the binding of MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF to human CD16A V158-overexpressing NK92mi cells in the absence of competition with human IgG. D shows a comparison of the binding of MUC1xCD16A multispecific antibodies BG1222P and huBG219P-E39-AF to human CD16A V158-overexpressing NK92mi cells in the presence of competition with human IgG. [Figure 18]Figure 1 shows a comparison of antibody-dependent cellular cytotoxicity (ADC) activity mediated by NK92mi / CD16A F158 between the MUC1 x CD16A multispecific antibodies BG1222P and huBG219P-E39-AF. Activity was characterized in the following cases: A) in T47D cells without competition with human IgG; B) in HCC827 cells without competition with human IgG; C) in H358 cells without competition with human IgG; D) in MDA-MB-453 cells without competition with human IgG; E) in T47D cells with competition with human IgG; F) in HCC827 cells with competition with human IgG; G) in H358 cells with competition with human IgG; and H) in MDA-MB-453 cells with competition with human IgG. [Figure 19] Figure 1 shows a comparison of antibody-dependent cellular cytotoxicity (ADC) activity mediated by NK92mi / CD16A V158 between the MUC1 x CD16A multispecific antibodies BG1222P and huBG219P-E39-AF. Activity was characterized in the following cases: A) without competition with human IgG in T47D cells; B) without competition with human IgG in HCC827 cells; C) without competition with human IgG in H358 cells; D) without competition with human IgG in MDA-MB-453 cells; E) with competition with human IgG in T47D cells; F) with competition with human IgG in HCC827 cells; G) with competition with human IgG in H358 cells; and H) with competition with human IgG in MDA-MB-453 cells. [Figure 20] 1 shows a comparison of the cytolytic activity of the MUC1×CD16A multispecific antibody BG1222P and various anti-MUC1 defucosylated antibodies against the cancer cell line T47D in human whole blood. [Figure 21](A) Comparison of the cytolytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against T47D cells in human whole blood. (B) Comparison of the cytolytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against HCC827 cells in human whole blood. (C) Comparison of the cytolytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against H358 cells in human whole blood. (D) Comparison of the cytolytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against MDA-MB-453 cells in human whole blood. [Figure 22] (A) Comparison of the phagocytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against T47D cells (mediated by human M2 macrophages). (B) Comparison of the phagocytic activity of the MUC1×CD16A multispecific antibodies BG1222P and huBG219P-E39-AF against MDA-MB-453 cells (mediated by human M2 macrophages). [Figure 23] A. MUC1×CD16A multispecific antibody BG1222P and daratumumab induced NK fratricide in NK cells. B. MUC1×CD16A multispecific antibody BG1222P and daratumumab induced NK fratricide in NK cells. [Figure 24] 1 shows the pharmacokinetic profile of the MUC1×CD16A multispecific antibody BG1222P in cynomolgus monkeys after iv injection of 5 mg / kg and 25 mg / kg of BG1222P. [Figure 25A]This figure shows the effect of soluble MUC1 on the binding of the MUC1 x CD16A multispecific antibody to MUC1-expressing cells. Briefly, human MUC1-expressing cells were incubated with 30 μg / ml BG1222P in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co. LTD). After washing, the cells were incubated with an anti-human IgG secondary Ab, and fluorescence was measured by flow cytometry. The binding profile of BG1222P at various concentrations (30 μg / ml) in the presence of soluble MUC1 is shown. HuVH-HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 25B] This figure shows the effect of soluble MUC1 on the binding of the MUC1 x CD16A multispecific antibody to MUC1-expressing cells. Briefly, human MUC1-expressing cells were incubated with 3 μg / ml BG1222P in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co. LTD). After washing, the cells were incubated with an anti-human IgG secondary Ab, and fluorescence was measured by flow cytometry. The binding profile of BG1222P at a concentration (3 μg / ml) in the presence of soluble MUC1 is shown. HuVH-HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. [Figure 25C] Figure 25 shows the effect of soluble MUC1 on the binding of the MUC1 x CD16A multispecific antibody to MUC1-expressing cells. Briefly, human MUC1-expressing cells were incubated with 0.3 μg / ml BG1222P in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co. LTD). After washing, the cells were incubated with an anti-human IgG secondary Ab, and fluorescence was measured by flow cytometry. Figures 25A-C show the binding profile of BG1222P at a concentration of 0.3 μg / ml in the presence of soluble MUC1. HuVH-HMFG1 was used as a positive control, and mIgG and hIgG1 were used as negative controls. DETAILED DESCRIPTION OF THE INVENTION

[0045] definition Unless specifically defined below or elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art.

[0046] As used in this specification, including the appended claims, singular terms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0047] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0048] Unless otherwise specified or clear from the context, as used herein, the term "about" refers to a value or composition that falls within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to practice in the art. "About" can also mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% more or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or up to 5 times greater. When a specific value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" should be assumed to be within the tolerance range of that specific value or composition.

[0049] The term "MUC1" or "mucin 1," also known as CA15-3, EMA, MCD, PEM, PUM, KL-6, MAM6, MCKD, PEMT, CD227, H23AG, MCKD1, ADMCKD, or ADTKD2, is a member of the mucin family. The amino acid sequence of human MUC1 is listed as SEQ ID NO: 1 and can also be found under accession number P15941.

[0050] The term "CD16A" refers to a type I membrane protein with two Ig-like domains with low affinity for IgG, also known as FCGRIIIA and FCGR3A. The amino acid sequence of human CD16A (P08637) can be found in the Uniprot database at Uniprot P08637.

[0051] As used herein, the terms "administration" and "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with a cell as well as contact of a reagent with a biological fluid, where the biological fluid is in contact with the cell.

[0052] The term "subject" or "patient" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient having or at risk of having a disorder described herein).

[0053] In one aspect, "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0054] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable region of the antibody interacts with the antigen through non-covalent forces at multiple sites. Generally, the more interactions, the stronger the affinity.

[0055] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0056] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0057] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0058] In some embodiments, the anti-MUC1 antibody comprises at least one antigen-binding site. In some embodiments, the anti-MUC1 antibody comprises an antigen-binding fragment derived from a MUC1 antibody described herein. In some embodiments, the anti-MUC1 antibody is isolated or recombinant.

[0059] In some embodiments, the anti-CD16A antibody comprises at least one antigen-binding site, at least the variable region. In some embodiments, the anti-CD16A antibody comprises an antigen-binding fragment derived from a CD16A antibody described herein. In some embodiments, the anti-CD16A antibody is isolated or recombinant.

[0060] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, etc., and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0061] Generally, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light chain" (approximately 25 kDa) and one "heavy chain" (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the antibody isotype is defined as IgA, IgD, IgE, IgG, and IgM, respectively.

[0062] Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including an additional "D" region of about 10 amino acids.

[0063] The variable regions of each light / heavy chain (VL / VH) pair form the antibody binding site. Thus, an intact antibody generally has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites generally have the same primary sequence.

[0064] Typically, both heavy and light chain variable domains contain three hypervariable regions, also called "complementarity-determining regions (CDRs)," which are located between relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, both light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (FR3), CDR-3 (CDR3), and FR-4 (FR4). The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (see "IMGT" numbering scheme)).The definition of antigen-binding sites is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). For example, in Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.In IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) (numbering according to Kabat). In IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0065] The term "hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen binding. A hypervariable region comprises amino acid residues from a "CDR" (e.g., LCDR1, LCDR2, and LCDR3 in the light-chain variable domain and HCDR1, HCDR2, and HCDR3 in the heavy-chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining antibody CDR regions by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining antibody CDR regions by structure). The term "framework" or "FR" residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0066] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules, e.g., 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)).

[0067] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target relative to other proteins, although this specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of an antigen in a heterogeneous population of proteins and other biologics, for example, in a biological sample, blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least twice as much as background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least 10 times as much as background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0068] As used herein, an "antigen-binding domain" comprises at least six CDRs (or, in the context of a single-domain antibody, three CDRs) and specifically binds to an epitope. The "antigen-binding domain" of a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain that specifically binds to a first epitope and a second antigen-binding domain that also comprises at least three CDRs and specifically binds to a second epitope. Multispecific antibodies can be bispecific, trispecific, tetraspecific, etc., with an antigen-binding domain directed to each specific epitope. Multispecific antibodies can be multivalent (e.g., a bispecific tetravalent antibody) comprising multiple antigen-binding domains, for example, two, three, four, or more antigen-binding domains that specifically bind to a first epitope and two, three, four, or more antigen-binding domains that specifically bind to a second epitope.

[0069] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0070] The terms "humanized" or "humanized antibody" refer to forms of antibodies comprising sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). Where necessary to distinguish humanized antibodies from rodent parent antibodies, the name of the antibody clone is prefixed with "hum," "hu," "Hu," or "h." Humanized forms of rodent antibodies generally contain the same CDR sequences of the parent rodent antibody, but can contain certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0071] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. Corresponding human germline sequence 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. The corresponding human germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable regions, or other combinations of sequences or subsequences. Sequence identity can be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid or amino acid sequence of the reference variable region. Furthermore, if the antibody contains a constant region, the constant region also is derived from such a human sequence, e.g., a human germline sequence, or a mutated version of a human germline sequence, or an antibody containing a consensus framework sequence derived from human framework sequence analysis, e.g., as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000.

[0072] "Equilibrium dissociation constant" or "K D The term "M" refers to the dissociation rate constant (kd, time -1 ) is the association rate constant (ka, time -1 , Ml). The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure generally have an equilibrium dissociation constant of about 10 -7 Less than or equal to 10 -8 Less than m, e.g., about 10 -9 Less than M or 10 -10M or less, and in some embodiments, about 10 -11 Under M, 10 -12 Less than M or 10 -13 It is less than M.

[0073] As used herein, the terms "cancer" or "tumor" have the broadest meaning understood in the art and refer to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. In the context of this disclosure, cancer is not limited to any particular type or location.

[0074] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" refers to the substitution of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, e.g., its binding affinity to MUC1 or CD16A. Common conservative changes of amino acids are well known in the art.

[0075] As used herein, the term "knob-into-hole" technology refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide (at the interface where they interact). For example, knob-into-hole technology can be used to engineer the Fc:Fc binding interface of an antibody, C L :C HIn some embodiments, knob-into-hole amino acids have been introduced into the VH / VL interface or the VH / VL interface (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knob-into-hole amino acids ensure the correct pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. Knob-into-hole technology can also be used with VH or VL regions to ensure correct pairing.

[0076] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value, if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, M=5, N=−4, and a comparison of both strands.

[0077] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0078] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0079] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0080] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences operably linked to a transcriptional sequence are physically contiguous to the transcriptional sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0081] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-MUC1xCD16A multispecific antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all solvents, dispersion media, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0082] The compositions disclosed herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. One preferred method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0083] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case will be apparent to one of ordinary skill in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined components.

[0084] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration in multiple or separate containers or formulations (e.g., capsules, powders, and liquids) for each active ingredient. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, "combination therapy" encompasses the use of each type of therapeutic agent in a sequential manner, either at about the same time or at different times. In either case, the treatment regimen provides the beneficial effect of the drug combination in treating the conditions or disorders described herein.

[0085] As used herein, the term "in combination with" means that the anti-MUC1xCD16A multispecific antibody is administered to a subject simultaneously with, immediately before, or immediately after the administration of an additional therapeutic agent. In certain embodiments, the anti-MUC1xCD16A multispecific antibody is administered as a combination with an additional therapeutic agent.

[0086] The present disclosure provides antibodies, antigen-binding fragments, and anti-MUC1xCD16A multispecific antibodies. Additionally, the present disclosure provides antibodies that have desirable pharmacokinetic properties and other desirable attributes, and thus can be used to reduce the likelihood of or treat cancer. The present disclosure further provides pharmaceutical compositions comprising the antibodies, as well as methods of making and using such pharmaceutical compositions for the prevention and treatment of cancer and related diseases.

[0087] Anti-MUC1 antibody The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to MUC1. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated as described below.

[0088] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having an amino acid sequence listed in Table 2 and / or Table 8. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies or antigen-binding fragments comprise an HCDR having the amino acid sequence of any one of the HCDRs listed in Table 2 and Table 8. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies comprise (or alternatively consist of) one, two, three, or more HCDRs having the amino acid sequence of any of the HCDRs listed in Table 2 and Table 8.

[0089] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies or antigen-binding fragments comprise a VL domain having an amino acid sequence listed in Table 2 and / or Table 8. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies or antigen-binding fragments comprise an LCDR having the amino acid sequence of any one of the LCDRs listed in Table 2 and Table 8. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to MUC1, wherein the antibodies or antigen-binding fragments comprise (or alternatively consist of) one, two, three, or more LCDRs having the amino acid sequence of any of the LCDRs listed in Table 2 and Table 8.

[0090] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acid alterations in the CDR regions that are at least 60%, 70%, 80%, 90%, 95%, or 99% identical to the CDR regions disclosed in Tables 2 and 8. In some embodiments, this includes amino acid alterations in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids are altered in the CDR regions when compared to the CDR regions shown in the sequences in Tables 2 and 8.

[0091] Other antibodies of the disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been altered but are at least 60%, 70%, 80%, 90%, 95%, or 99% identical to the sequences set forth in Tables 2 and 8. In some embodiments, this includes amino acid sequence alterations in which no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 amino acids have been altered in the variable regions when compared to the variable regions set forth in the sequences set forth in Tables 2 and 8, while retaining substantially the same therapeutic activity.

[0092] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds MUC1. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0093] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human MUC1. In certain aspects, the antibodies and antigen-binding fragments can bind to the same epitope of MUC1.

[0094] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-MUC1 antibodies described in Tables 2 and 8. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibit binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to MUC1 demonstrates that the test antibody can compete with that antibody or antigen-binding fragment thereof for binding to MUC1. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on MUC1 as the competing antibody or antigen-binding fragment thereof. In certain embodiments, an antibody that binds to the same epitope on MUC1 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0095] Anti-CD16A antibody The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CD16A. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated as described below.

[0096] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having an amino acid sequence listed in Table 23. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies or antigen-binding fragments comprise an HCDR having the amino acid sequence of any one of the HCDRs listed in Table 23. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to CD16A, wherein the antibodies comprise (or alternatively consist of) one, two, three, or more HCDRs having the amino acid sequence of any of the HCDRs listed in Table 23.

[0097] Other antibodies or antigen-binding fragments thereof of the present disclosure contain amino acids that have alterations but that have at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR regions to the CDR regions disclosed in Table 23. In some embodiments, it contains amino acid alterations in which no more than 1, 2, 3, 4, or 5 amino acids are altered in the CDR regions when compared to the CDR regions set forth in the sequences of Table 23.

[0098] Other antibodies of the disclosure include those with amino acid or nucleic acid encoding amino acid alterations but with at least 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequences set forth in Table 23. In some embodiments, the antibodies contain amino acid sequence alterations resulting in no more than 1, 2, 3, 4, or 5 amino acids being altered in the variable regions when compared to the variable regions set forth in the sequences of Table 23 while retaining substantially the same therapeutic activity.

[0099] The present disclosure also provides nucleic acid sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to CD16A. Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0100] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human CD16A. In certain embodiments, the antibodies and antigen-binding fragments can bind to the same epitope of CD16A.

[0101] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-CD16A antibodies listed in Table 23. Accordingly, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in binding assays (e.g., competitively inhibit binding in a statistically significant manner). The ability of a test antibody to inhibit the binding of an antibody and antigen-binding fragment thereof of the present disclosure to CD16A demonstrates that the test antibody can compete with that antibody or antigen-binding fragment thereof for binding to CD16A. Without being bound by any one theory, such antibodies may bind to the same or a related (e.g., structurally similar or spatially proximal) epitope on CD16A as the competing antibody or antigen-binding fragment thereof. In certain embodiments, antibodies that bind to the same epitope on CD16A as the antibodies or antigen-binding fragments thereof of the present disclosure are human or humanized monoclonal antibodies. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0102] Anti-MUC1×CD16A multispecific antibody In embodiments, the anti-MUC1 and anti-CD16A antibodies disclosed herein can be combined into an anti-MUC1 x CD16A multispecific antibody. The antibody is a multispecific antibody, e.g., comprises multiple antigen-binding domains, where at least one antigen-binding domain sequence specifically binds to MUC1 as a first epitope and a second antigen-binding domain sequence specifically binds to CD16A as a second epitope. In embodiments, the multispecific antibody comprises a third, fourth, or fifth antigen-binding domain. In embodiments, the multispecific antibody is a bispecific, trispecific, or tetraspecific antibody. In each embodiment, the multispecific antibody comprises at least one anti-MUC1 antigen-binding domain and at least one anti-CD16A antigen-binding domain.

[0103] In one embodiment, a multispecific antibody is a bispecific antibody. As used herein, a bispecific antibody specifically binds to only two antigens. A bispecific antibody comprises a first antigen-binding domain that specifically binds to MUC1 and a second antigen-binding domain that specifically binds to CD16A. This includes bispecific antibodies comprising a heavy chain variable domain and a light chain variable domain that specifically bind to MUC1 as a first epitope and a heavy chain variable domain that specifically binds to CD16A as a second epitope. In another embodiment, a bispecific antibody comprises an antigen-binding fragment that specifically binds to MUC1 and an antigen-binding fragment that specifically binds to CD16A. When a bispecific antibody comprises an antigen-binding fragment, the antigen-binding fragment can be a Fab, F(ab')2, Fv, or single-chain Fv (ScFv).

[0104] Previous work (Coloma and Morrison Nature Biotech. 15:159-163 (1997)) described tetravalent bispecific antibodies engineered by fusing DNA encoding a single-chain anti-dansyl antibody Fv (scFv) to the C-terminus (CH3-scFv) or hinge (hinge-scFv) of an anti-dansyl antibody IgG3. The present disclosure provides multivalent antibodies (e.g., tetravalent antibodies) having at least two antigen-binding domains, which can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibodies herein contain three to eight, preferably four, antigen-binding domains that specifically bind to at least two antigens.

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

[0106] Multispecific antibodies have been synthesized through 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 domains (Muller et al., FEBS Lett. 422:259-64), with or without the use of flexible linkers; diabodies (Holliger et al., (1993) Proc. Nat. Acad. Sci. USA. 1998 90:6444-8; Zhu et al., Bio / Technology (NY) 1996 14:192-6); Fab-scFv fusions (Schoonjans et al., J. Immunol. 2000 165:7050-7); and miniantibody formats (Pack et al. They have been constructed by genetically fusing two single-chain Fv (scFv) or Fab fragments (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) by various methods (Pack et al., Biochemistry 1992 31:1579-84; Pack et al., Bio / Technology 1993 11:1271-7).

[0107] The bispecific tetravalent antibodies disclosed herein comprise a linker region of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues between one or more of their antigen-binding domains, CL domain, CH1 domain, hinge region, CH2 domain, CH3 domain, or Fc region. In some embodiments, the linker region is composed of the amino acids glycine and serine.The linkers are GS, GGS, GSG, SGG, GGG, GGGS (SEQ ID NO: 149), SGGG (SEQ ID NO: 150), GGGGS (SEQ ID NO: 151), GGGGSGS (SEQ ID NO: 152), GGGGSGS (SEQ ID NO: 153), GGGGSGGS (SEQ ID NO: 154), GGGGSGGGGS (SEQ ID NO: 155), GGGSGGGGSGGGGS (SEQ ID NO: 156), AKTTPKLEEGEFSEAR (SEQ ID NO: 157), AKTTPKLEEGEFS EARV (SEQ ID NO: 158), AKTTPKLGG (SEQ ID NO: 159), SAKTTPKLGG (SEQ ID NO: 160), AKTTPKLEEGEFSEARV (SEQ ID NO: 161), SAKTTP (SEQ ID NO: 162), SAKTTPKLGG (SEQ ID NO: 163), RADAAP (SEQ ID NO: 164), RADAAPTVS (SEQ ID NO: 165), RADAAAAAGGPGS (SEQ ID NO: 166), RADAAAA(G4S)4 (SEQ ID NO: 167), SAKTTP (SEQ ID NO: 168). SEQ ID NO: 168), SAKTTPKLGG (SEQ ID NO: 169), SAKTTPKLEEGEFSEARV (SEQ ID NO: 170), ADAAP (SEQ ID NO: 171), ADAAPTVSIFPP (SEQ ID NO: 172), TVAAP (SEQ ID NO: 173), TVAAPSVFIFPP (SEQ ID NO: 174), QPKAAP (SEQ ID NO: 175), QPKAAPSVTLFPP (SEQ ID NO: 176), AKTTPP (SEQ ID NO: 177), AKTTPPSVTPLAP (SEQ ID NO: 178) ), AKTTAP (SEQ ID NO: 179), AKTTAPSVYPLAP (SEQ ID NO: 180), ASTKGP (SEQ ID NO: 181), ASTKGPSVFPLAP (SEQ ID NO: 182), GENKVEYAPALMALS (SEQ ID NO: 183), GPAKELTPLKEAKVS (SEQ ID NO: 184), or GHEAAAVMQVQYPAS (SEQ ID NO: 185), or any combination thereof (see WO2007 / 024715).

[0108] Dimerization-specific amino acids In one embodiment, the multivalent antibody comprises at least one dimerization-specific amino acid modification. The dimerization-specific amino acid modification may result in a "knob-into-hole" interaction, increasing the likelihood of correct assembly of the desired multivalent antibody. The dimerization-specific amino acid may be in the CH1 domain or the CL domain, or a combination thereof. Suitable dimerization-specific amino acids are used to pair a CH1 domain with another CH1 domain (CH1-CH1) and a CL domain with another CL domain (CL-CL), and can be found in at least WO2014082179, WO2015181805, and WO2017059551. The dimerization-specific amino acid may also be in the Fc domain or may be combined with a dimerization-specific amino acid in the CH1 or CL domain. In one embodiment, the present disclosure provides a bispecific antibody comprising at least one dimerization-specific amino acid pair.

[0109] Further modifications to the Fc region framework In some embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with a different amino acid residue, resulting in an antibody with altered affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The affinity-altered effector ligand can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0110] In another embodiment, one or more amino acid residues can be substituted with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al.

[0111] In yet another embodiment, one or more amino acid residues are altered to modify the antibody's ability to fix complement. This approach is described, for example, in publication WO 94 / 29351 by Bodmer et al. In certain embodiments, one or more amino acids of an antibody or antigen-binding fragment thereof of the present disclosure are replaced with one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues include, but are not limited to, the heavy chain constant regions of the IgG1, IgG2, and IgG3 subclasses and the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0112] In another embodiment, the Fc region is modified by modifying one or more amino acids to enhance the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This approach is described, for example, in publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0113] In yet another embodiment, the glycosylation of the multispecific antibody is modified. For example, an aglycosylated antibody (i.e., an antibody lacking or reduced glycosylation) can be generated. Altering glycosylation can increase, for example, the affinity of the antibody for an "antigen." Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0114] Additionally or alternatively, antibodies can be made that have an altered type of glycosylation (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues or increased bisecting GlcNAc structures). Such altered glycosylation patterns have been shown to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation pathway. Cells with altered glycosylation pathways have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. See, for example, Hang et al. EP 1,176,195 by Umana et al. describes a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, such that the expressed antibodies exhibit hypofucosylation. WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). WO 99 / 54342 by Umana et al. describes cell lines that have been engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0115] In another aspect, if reduced ADCC is desired, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little CDC effector function (Moore GL, et al., 2010 MAbs, 2:181-189). However, native IgG4 has been found to be less stable under stress conditions, such as in acidic buffers or at elevated temperatures (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al., 1998 Biochemistry, 37:9266-9273; Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably binding an antibody to an IgG4 Fc engineered with a combination of modifications that reduce FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biological drugs, one of the more undesirable intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form antibody halves, which generates bispecific antibodies in vivo through a process called "Fab arm exchange" (Van der Neut Kolfschoten M., et al., 2007 Science, 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) appeared to inhibit IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al., 2002 Immunol, 105:9-19).Some amino acid residues in the hinge and gamma Fc region have been reported to affect antibody interaction with Fcγ receptors (Chappel SM, et al., 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al., 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev Immunol, 25:21-50). Furthermore, some IgG4 isoforms that occur rarely in the human population may also result in different physicochemical properties (Brusco, A. et al., 1998 Eur J Immunogenet, 25:349-55; Aalberse et al., 2002 Immunol, 105:9-19). To generate multispecific antibodies with low ADCC and CDC but good stability, it is possible to modify the hinge and Fc regions of human IgG4 and introduce several alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 by Li et al.

[0116] antibody production Antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including, but not limited to, recombinant expression of antibody tetramers, chemical synthesis, and enzymatic digestion, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0117] The present disclosure further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementarity determining regions described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, and SEQ ID NO: 63. In some aspects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 33, and SEQ ID NO: 64.

[0118] The polynucleotides of the present disclosure can encode variable region sequences of anti-MUC1xCD16A antibodies. They can also encode both the variable and constant regions of the antibodies. Some of the sequences encode polypeptides containing both the heavy and light chain variable regions of the exemplified anti-MUC1xCD16A antibodies.

[0119] The present disclosure also provides expression vectors and host cells for producing anti-MUC1xCD16A antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. The expression vector may contain a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the anti-MUC1xCD16A antibody chain or antigen-binding fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions without biasing the population toward coding sequences whose expression products are better tolerated by the host cell. In addition to a promoter, other regulatory elements can be included for efficient expression of the anti-MUC1xCD16A antibody or antigen-binding fragment thereof. These elements may include an ATG initiation codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be increased by incorporating enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994, and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0120] Host cells for harboring and expressing anti-MUC1xCD16A antibody vectors can be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other suitable microbial hosts include bacilli such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors typically containing expression control sequences compatible with the host cell (e.g., an origin of replication) can also be constructed for these prokaryotic hosts. Additionally, various well-known promoters can be present, such as the lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system, or promoter systems derived from phage lambda. Promoters typically control expression, optionally via operator sequences, and contain ribosome binding site sequences for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-MUC1xCD16A antibodies. Insect cells can also be used in conjunction with baculovirus vectors.

[0121] In other embodiments, mammalian host cells are used to express and produce the anti-MUC1xCD16A antibodies of the present disclosure. Examples include hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines harboring exogenous expression vectors. These include any normal mortal, or normal or abnormal immortal, animal or human cells. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells can include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or tunable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP poly III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0122] Bispecific antibody production The bispecific antibodies disclosed herein can be produced using a knob-into-hole (KiH) design, which introduces mutations into the core CH3 domain interface. The resulting heterodimers have reduced CH3 melting temperatures (69°C or lower). In contrast, the ZW heterodimer Fc design has a thermal stability of 81.5°C, comparable to the wild-type CH3 domain. Specific methods for producing the antibodies disclosed herein are described in the Examples.

[0123] Methods of detection and diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for a variety of applications, including, but not limited to, methods for detecting MUC1. In one embodiment, the antibodies or antigen-binding fragments are useful for detecting the presence of MUC1 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues. In other embodiments, such tissues include normal and / or cancerous tissues that express MUC1 at higher levels than other tissues.

[0124] In one aspect, the present disclosure provides a method for detecting the presence of MUC1 in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-MUC1xCD16A antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample can include, but is not limited to, urine, tissue, sputum, or blood.

[0125] Also included are methods for diagnosing disorders associated with MUC1 expression. In certain embodiments, the methods include contacting a test cell with an anti-MUC1xCD16A antibody; determining (quantitatively or qualitatively) the expression level of MUC1 expressed by the test cell by detecting binding of the anti-MUC1xCD16A antibody to a MUC1 polypeptide; and comparing the expression level by the test cell with the expression level of MUC1 in a control cell (e.g., a normal cell or a non-MUC1-expressing cell of the same tissue origin as the test cell), wherein a higher level of MUC1 expression in the test cell compared to the control cell indicates the presence of a disorder associated with MUC1 expression.

[0126] Pharmaceutical Compositions and Formulations Also provided are compositions, including pharmaceutical formulations, comprising an anti-MUC1xCD16A antibody or antigen-binding fragment thereof, or a polynucleotide comprising a sequence encoding an anti-MUC1xCD16A antibody or antigen-binding fragment. These compositions can further comprise a suitable carrier, e.g., a pharmaceutically acceptable excipient such as a buffer, which are well known in the art.

[0127] Pharmaceutical formulations of the anti-MUC1xCD16A antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and may include buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol), low molecular weight (less than about 10 residues) polypeptides; proteins; e.g., hydroxybenzoates ... Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, interstitial drug dispersants such as serum albumin, gelatin, or immunoglobulin; 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), for example, human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0128] In embodiments, the formulation comprises L-histidine / L-histidine hydrochloride monohydrate, trehalose, and polysorbate 20. In embodiments, the anti-MUC1xCD16A antibody formulation comprises 10 mg / mL anti-MUC1xCD16A antibody, 20 mM histidine / histidine HCl, 240 mM trehalose dihydrate, and 0.02% polysorbate 20, and is an isotonic solution with a pH of approximately 5.5, after constitution with sterile water for injection.

[0129] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0130] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0131] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0132] equivalent While the anti-human 4Ig-B7H3 antibody and antigen-binding fragments thereof have been described with reference to detailed descriptions thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0133] It should be understood that one, some, any, or all of the features of the various embodiments disclosed herein may be combined to form additional embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. [Example]

[0134] Example 1. Generation of anti-MUC1 monoclonal antibodies targeting the MUC1 membrane proximal region MUC1 recombinant protein for immunization and binding assays A cDNA encoding full-length human MUC1 (SEQ ID NO: 1) was synthesized by Genewiz (Suzuki, China) based on its Uniprot sequence (UniprotKB: P15941) and purchased from Genewiz. The coding region for the SEA domain (sea urchin sperm protein, enterokinase, agrin) (SEQ ID NO: 2), consisting of amino acids (AA) 1036–1155 of full-length human MUC1, was PCR-amplified and cloned into a pcDNA3.4-based expression vector (Invitrogen, Carlsbad, CA, USA) so that the C-terminus was fused to either the Fc domain of mouse IgG2a or the Fc domain of human IgG1 heavy chain, resulting in two recombinant fusion protein expression plasmids, MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1, respectively. A schematic diagram of the MUC1 fusion proteins is shown in Figure 1. To produce the recombinant fusion proteins, the MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 plasmids were transiently transfected into Expi293 cells (Thermo Fisher Scientific, 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 removed by centrifugation. MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 were purified using a Protein A column (Cat. No. 17549852, Cytiva Life Sciences) followed by purification using a HiLoad16 / 600 Superdex 200pg size-exclusion column (Cat. No. 28989335, Cytiva Life Sciences). The MUC1-SEA-mIgG2a and MUC1-SEA-huIgG1 proteins were dialyzed against phosphate-buffered saline (PBS) and stored in small aliquots in a -80°C freezer. [Table 1-1] [Table 1-2]

[0135] Cell lines stably expressing human or cynomolgus MUC1 were generated for antibody generation, screening, and validation. Cell lines stably expressing human MUC1 were generated and validated, including the PT67 / human MUC1 cell line (an internally generated cell line), the HEK293 / human MUC1 cell line (HEK293 obtained from ATCC CRL-1573), and the HCT116 / human MUC1 cell line (ATCC CCL-247).

[0136] Cell lines stably expressing cynomolgus MUC1 (SEQ ID NO: 3) were generated and validated, including the HEK293 / cynomolgus MUC1, L929 / cynomolgus MUC1 cell line (L929 obtained from ATCC, CCL-1), HCT116 / cynomolgus MUC1 cell line, and Daudi / cynomolgus MUC1 cell line (Daudi obtained from ATCC, CCL-213).

[0137] To generate cell lines stably expressing human or cynomolgus MUC1, an ectotrophic vector was constructed using the retroviral construct PFBneo (STRATAGENE, catalog no. 217561-51). Transfection of the retroviral construct into PLAT-E cells (Cyagen, catalog no. IPMPC-01001) was performed using Lipofectamine 2000 (Invitrogen, reference no. 52758) according to the manufacturer's instructions. Viral supernatants were collected 24, 48, and 72 hours after transfection and filtered (0.45 μm) before use. The ectotrophic virus produced above was used to transduce the dual-tropic packaging cell line PT67 in the presence of polybrene (final concentration: 8 μg / ml). After three rounds of transduction, PT67 cells were selected with G418 (final concentration: 1 mg / ml) for 7 days. To harvest the dual tropism virus generated from PT67 cells, the medium was replaced with fresh complete DMEM medium without G418 when the cells reached 100% confluence. Virus was collected once daily for three days. The cell lines were infected with viruses containing human or cynomolgus MUC1. After three rounds of transduction, cells were selected with G418 (final concentration: 1 mg / ml) for seven days.

[0138] immunization To generate antibodies against MUC1, cohorts of 30 inbred mice (BALB / C, MRL strains) were immunized with different MUC1 antigens. Each cohort received a unique immunization strategy, including a unique combination of MUC1 antigen (including the protein and cell line described in Example 1), dose, injection route, adjuvant, and immunization time. A total of five animals were immunized in six cohorts. Animals were immunized over various time periods ranging from 0 to 90 days. To monitor the immune response, titrated sera were screened by ELISA and FACS, usually after two to six immunizations over a 30-90 day period. Sera were screened for antibody binding to the MUC1 antigen. MUC1-specific antibody responses were measured in each animal, and animals with sufficient anti-MUC1 Ig titers were selected for a final boost four days later.

[0139] Hybridoma fusion and screening Lymphoid organs, including spleens and lymph nodes, were isolated from immunized mice as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells derived from SP2 / 0 using PEG-based fusion. For hybridoma selection, the resulting cells were plated in 96-well cell culture plates using standard 1640 medium supplemented with HAT. After 10–13 days of culture and growth medium changes, hybridoma culture supernatants were collected from individual wells and screened to identify wells that secreted MUC1-specific antibodies. Initially, all supernatants were screened against the recombinant protein huMUC1-SEA-huIgG1 (from Example 1). Antibody binding to the recombinant protein huMUC1-SEA-huIgG1 was measured by ELISA. Supernatants from culture wells of three hybridoma fusions were screened for MUC1 antibodies. Briefly, 2 μg / mL huMUC1-SEA-huIgG1 was coated onto a 96-well ELISA plate, co-incubated with 50 μl of hybridoma culture supernatant for 30–60 min, washed, and incubated with an HRP-conjugated anti-mouse IgG Fc secondary Ab. After incubation and washing, the plate was developed with HRP substrate, and absorbance was measured.

[0140] Hybridomas from positive wells were transferred to 24-well plates in fresh culture medium, grown for 2–3 days, and then rescreened by flow cytometry to identify antibodies that bound to cell lines overexpressing human MUC1 and cynomolgus MUC1.

[0141] Antibody (Ab) binding to cell lines overexpressing human MUC1 and cynomolgus MUC1 was measured by FACS. Briefly, 100 μl of hybridoma culture supernatant and human MUC1-overexpressing or cynomolgus MUC1-overexpressing cells were co-incubated for 30–60 min, washed, and incubated with an APC-conjugated anti-mouse IgG Fc secondary Ab. After incubation and washing, fluorescence was measured by flow cytometry.

[0142] Subcloning and sequence analysis Selected anti-MUC1 Ab screening hybridomas were subcloned once or twice to ensure monoclonality. Briefly, positive hybridoma clones were subcloned by limiting dilution. After 7–10 days, culture supernatants were screened by ELISA and flow cytometry as described above to confirm human and cynomolgus monkey MUC1 Ab binding. Stable hybridoma subclones were cultured in vitro for cell cryopreservation, antibody VH and VL gene cloning, and sequencing.

[0143] After subcloning, the anti-MUC1 Ab-secreting hybridomas were lysed in cell lysis buffer. The mRNA-containing lysates were transferred to a 96-well deep-well plate for mRNA isolation, cDNA synthesis, and DNA sequencing using standard sequencing techniques (Sanger sequencing). Generally, total RNA was prepared from cell lysates, and cDNA was generated by reverse transcription of mRNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) according to the manufacturer's instructions. The sequence of the BG138P antibody is listed in Table 2.

[0144] Single B Screening Immunized mice were sacrificed and spleens were harvested. Enriched plasma cells were loaded onto a 14K chip. hMUC1 beads, cynomolgus MUC1 beads, and HEK293-cynomolgus MUC1 cells were used for on-chip screening. Hits were selected and transferred to lysis buffer. Single-cell RNA was purified and Ig sequences were recovered using a BLI cDNA synthesis kit (BERKELEY LIGHTS) according to the manufacturer's instructions. The sequences of BG219P and BG346P are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0145] Large-scale expression and purification of chimeras BG219P, BG138P, and BG346P Chimeric antibodies chBG219P, chBG138P, and chBG346P were produced by transient transfection of ExpiCHO-s cells with in-house generated heavy and light chain-containing plasmids. Conditioned medium was collected, and antibodies were purified using a MabSelect SuRe column (Cytiva), followed by a POROS™ 50 HS column (Thermofisher Scientific) and a G-25 desalting column (Cytiva). All purified antibodies were stored in small aliquots at -80°C in a freezer.

[0146] Example 2. Measurement of binding kinetics and affinity of anti-MUC1 antibodies by SPR The chimeric anti-MUC1 antibodies were characterized for binding kinetics by SPR assay using a BIAcore™ T-200 (GE Life Sciences). Briefly, mouse anti-human IgG Fc antibodies were immobilized on an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences). Purified chimeric anti-MUC1 antibodies were flowed over the chip surface and captured with anti-human IgG antibodies. Serial dilutions of human or cynomolgus MUC1-SEA proteins were then flowed over the chip surface, and changes in surface plasmon resonance signals were analyzed to determine the association rate (k ) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). on ) and dissociation rate (k off The equilibrium dissociation constant (K D ) as the ratio k off / k on The binding affinity profiles of the chimeric anti-MUC1 antibodies chBG138P, chBG346P, and chBG219P are shown in Table 3 below. ChBG138P, chBG346P, and chBG219P exhibited high affinity for both human MUC1-SEA and cynomolgus monkey MUC1-SEA. [Table 3]

[0147] Example 3. Measurement of binding affinity of anti-MUC1 antibodies to MUC1 expressed in stably expressing cell lines The binding affinity of the chimeric anti-MUC1 antibodies to human and cynomolgus MUC1-overexpressing cell lines (HEK293 / human MUC1 and HEK293 / cynomolgus MUC1) was measured by FACS. Briefly, human or cynomolgus MUC1-overexpressing cells were incubated with serially diluted purified antibodies, washed, and then incubated with an APC-conjugated anti-human IgG secondary antibody. After incubation and washing, fluorescence was measured by flow cytometry. The binding affinity profiles of the chimeric anti-MUC1 antibodies are shown in Table 4 below and Figures 2A–2F (hIgG1 was used as a negative control). These results demonstrated that all three chimeric anti-MUC1 antibodies had good binding affinity to both human MUC1 and cynomolgus MUC1 expressed in stable cell lines. [Table 4]

[0148] Example 4. Epitope binning of anti-MUC1 antibodies Epitope binning of chimeric anti-MUC1 antibodies was determined by competitive SPR assay. Briefly, an anti-mouse IgG Fc antibody was immobilized on an activated CM5 biosensor chip. Purified huMUC1-SEA-mIgG2a (human MUC1 linked to mouse IgG2a Fc) antigen was flowed over the chip surface and captured by an anti-mouse IgG antibody. The reference MUC1-SEA Ab 5F3 (Cancer Immunol Immunother. 2020 Jul;69(7):1337-1352) was injected first under saturating antigen binding conditions, followed by injections of chBG138P (Figure 3A), chBG219P (Figure 3B), or chBG346P (Figure 3C). The sensorgrams for epitope binning are shown in Figures 3A-3C. As shown in Table 5 below, the three chimeric MUC1 antibodies were grouped into two epitope bins within the MUC1-SEA domain. ChBG138P and chBG219P bind to the same epitope on MUC1-SEA as 5F3, a different MUC1 epitope from chBG346P. More specifically, chBG138P and chBG219P bind to epitope bin A, while chBG346P binds to bin B. [Table 5]

[0149] Example 5. Chimeric anti-MUC1 antibodies chBG138P, chBG219P, and chBG346P show reduced interference with soluble MUC1 The presence of soluble MUC1 in the specific binding of MUC1 antibody to MUC1-expressing cells was determined by competitive FACS assay. Briefly, human MUC1-expressing cells were incubated with 30, 3, and 0.3 μg / ml of MUC1 antibody in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co. LTD). After washing, the cells were incubated with an anti-human IgG secondary antibody, and fluorescence was measured by flow cytometry. The IC of soluble MUC1 blocking the binding of MUC1 antibody to MUC1-expressing cells was 0.01. 50The values ​​are shown in Table 6, and the blocking curves are shown in Figures 4A–4F, where HMFG1 is the positive control and mIgG and hIgG1 are negative controls. The profiles indicate that HMFG binding to MUC1-expressing cells can be easily interfered with at high, medium, and low antibody concentrations (i.e., 30, 3, and 0.3 μg / ml), whereas the binding of chBG138P, chBG219P, and chBG346P is only slightly interfered with at low antibody concentrations (i.e., 0.3 μg / ml) (Figures 4A–F). Collectively, the profiles in Figure 4 indicate that the binding of MUC1 antibodies to MUC1-expressing cells showed significantly reduced interference by soluble MUC1 compared with HMFG1 (Abcam), which targets the MUC1 N-terminus. [Table 6]

[0150] Example 6. Anti-MUC1 monoclonal antibodies targeting the MUC1 membrane proximal region bind to cancer cell lines but not to normal T cells, whereas the MUC1 N-terminally targeted antibodies HMFG1 or 16A can bind to normal T cells. To evaluate whether anti-MUC1 monoclonal antibodies targeting the MUC1 membrane proximal region can differentially bind to MUC1-expressing tumor cells versus MUC1-expressing normal cells, such as activated T cells, a FACS binding assay was performed. For tumor cell line binding experiments, cells were stained with the anti-human MUC1 antibody chBG138P or a control (human IgG1) for 1 hour and then harvested. Next, cells were washed twice and subsequently stained with a secondary antibody (Alexa Fluor® 647 anti-human IgG Fc) for 30 minutes. After washing and fixing with 1% paraformaldehyde (PFA) in DPBS, FACS analysis was performed. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.), and the data were analyzed using NovoExpress software. As shown in Figures 5A-5C, the chimeric antibody chBG138P targeting the human MUC1 membrane proximal region bound to the MUC1-expressing tumor cell lines HCC827 (Figure 5A), H1975 (Figure 5B), and T-47D (Figure 5C) in a dose-dependent manner (human IgG1 was used as a negative control), indicating that antibodies targeting the MUC1 membrane proximal region can be used to target cancer cells and therefore can be applied to the treatment of MUC1-expressing cancers.

[0151] To evaluate whether monoclonal antibodies targeting the membrane-proximal region of MUC1 can avoid binding to normal cells expressing MUC1, we performed an activated T cell binding assay. Briefly, human peripheral blood mononuclear cells (PBMCs) from six healthy donors purchased from Allcells or Stemcell were stimulated with 1 μg / ml PHA-L for 3 days. FACS staining was then performed using the stimulated PBMCs. The cell suspension was preincubated with the LIVE / DEAD™ Fixable Dead Cell Stain Kit (Invitrogen, reference number L34964) and Fc receptor blocking solution (100 μg / ml human IgG in FACS buffer) before staining with anti-human antibodies. Cells were washed twice and incubated with 10 μg / mL anti-MUC1 monoclonal antibody targeting the MUC1 membrane-proximal region, or the MUC1 N-terminal targeting HMFG1 antibody (as a positive control, Abcam, catalog no. ab215670) or 16A antibody (as a positive control, Biolegend, catalog no. 355608) for 1 hour. Next, cells were washed and stained with PE-CY7 anti-human αβ TCR (eBioscience, catalog no. 25-9986-42) or AF647 anti-human IgG Fc (Biolegend, reference no. 409320) for 30 minutes. Cells were washed and fixed with 1% PFA in DPBS before FACS analysis. All flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) and analyzed using NovoExpress software. As shown in Figures 6 and 7A-7H, the chimeric antibodies chBG138P (Figures 7A, 7B, 7E, and 7F), chBG219P (Figures 7E and 7F), and chBG346P (Figures 7E and 7F) do not bind to normal, activated human T cells expressing MUC1. However, the MUC1-N-terminally targeted antibodies HMFG1 (Figures 7C and 7D) and 16A (Figures 7G and 7H) bind to the majority of activated T cells.Figures 8A-8D show that the chimeric (chBG138P) and humanized versions (huBG138P-Hz2 and huBG138P-Hz4) of antibody BG138P retain similar binding properties to BG138P but do not bind to MUC1-expressing normal activated human T cells (Figures 8A and 8B), whereas the MUC1-N-terminally targeted antibody HMFG1 (Figures 8C and 8D) binds to normal activated T cells.

[0152] The results show that, compared with MUC1-N-terminal-targeting antibodies that bind to both cancer cells and normal T cells, antibodies targeting the MUC1 membrane proximal region specifically target cancer cells without affecting normal T cells, and may therefore confer an optimized safety profile when used as an antitumor therapy in humans.

[0153] Example 7. Humanization of mouse anti-human MUC1 antibody BG219P For humanization of BG219P, we performed sequence comparison with the IMGT human immunoglobulin gene database and searched for human germline IgG genes for sequences that shared a high degree of homology with the protein sequence of the BG219P variable region. The human IGVH and IGKV genes, which are frequently present in the human antibody repertoire and show high homology to murine BG219P, were selected as templates for humanization.

[0154] Humanization was performed by CDR grafting followed by the incorporation of key backmutations. The humanized antibody was engineered as a wild-type human IgG1 antibody using an expression vector developed in-house. In the first round of humanization, mutations from murine to human amino acid residues in the framework regions were guided by 3D structural analysis. To maintain the canonical structure of the CDRs, structurally important murine framework residues were retained in the first round of humanization design. Among all 19 variants generated, the CDR-grafted antibody variant BG219P-Bz0, which contains all backmutation sites, has a theoretical binding affinity similar to that of the parent murine antibody BG219P.

[0155] 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 acceptor frameworks for the VL and VH sequences of BG219P. The LCDR of mouse BG219P was grafted onto the frameworks of human germline variable genes IGKV1-39*01 and IGKJ2*01, retaining the D17E, A43S, I48V, T69P, and F71Y mouse framework residues. The resulting amino acid and DNA sequences of BG219P-Bz0 VL are shown in Table 8. The HCDRs of murine BG219P were grafted onto the frameworks of the human germline variable genes IGHV3-23*01 and IGHJ6*01, retaining the S30N, S49A, A93T, and K94R murine framework residues. The resulting amino acid and DNA sequences of BG219P-Bz0 VH are shown in Table 8.

[0156] Starting with the humanized BG219P antibody huBG219P-Bz0, several additional amino acid changes were made to the CDR regions of both VH and VL to further improve its biophysical properties for therapeutic use in humans. Considerations included removing post-translational modifications while maintaining binding activity and improving thermal stability (Tm).

[0157] More than 30 humanized BG219P (also referred to as huBG219P) variants were constructed using an in-house IgG1 / Cκ eukaryotic expression vector containing human wild-type IgG1 and kappa chain constant regions, each with easily adaptable subcloning sites. The variants were produced by transient transfection of the plasmids into ExpiCHO-s cells (Thermofisher Scientific). The conditioned medium was collected, and the variants were purified using MabSelect™ SuRe columns (Cytiva), followed by buffer exchange with UF / DF. All purified antibodies were stored in small aliquots in a -80°C freezer.

[0158] For affinity determination, the antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The results of the SPR binding profiles of the anti-MUC1 antibodies are summarized in Table 7. huBG219P-E39 and huBG219P-E43 have similar binding affinities with dissociation constants of 35.2 pM and 30.3 pM, respectively, which are comparable to that of chimeric BG219P (39.8 pM). The sequences of huBG219P-E39 and huBG219P-E43 are shown in Table 8. [Table 7] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0159] To assess 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 dilution series of chimeric antibodies or humanized BG219P. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC20 values ​​for dose-dependent binding to human native MUC1 were calculated. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 9 and Table 9, the humanized BG219P antibodies huBG219P-Bz0, E39, and E43 retained comparable binding affinity to native MUC1 compared to chimeric BG219P. [Table 9]

[0160] Example 8. Generation of anti-CD16A VHHs Human CD16A recombinant protein and cell lines for immunization and assays A 6-histidine-tagged recombinant extracellular domain (ECD) fragment of human CD16A protein (V158) (SEQ ID NO: 101)—designated human CD16A-His6(V158)—was purchased from a commercial source (Sino Biologics) and used as an antigen to immunize alpacas. Recombinant hexa-histidine-tagged ECD fragments of human CD16A(F158) (SEQ ID NO: 102), human CD16B(NA1) (SEQ ID NO: 103), human CD16B(NA2) (SEQ ID NO: 104), human CD16B(SH) (SEQ ID NO: 105), and cynomolgus CD16 (SEQ ID NO: 106)—referred to as human CD16A-His6(F158), human CD16B-His6(NA1), human CD16B-His6(NA2), human CD16B-His6(SH), and cynomolgus CD16-His6, respectively—were purchased from a commercial source (SinoBiologics) and used in various in vitro assays.

[0161] To facilitate screening and detection, a DNA fragment of human CD16A (V158) ECD (AA1-208 of SEQ ID NO: 101) was fused to a C-terminal human IgG1 mf Fc tag (SEQ ID NO: 107), mouse IgG2a Fc tag, or alpaca IgG2b Fc tag and transiently expressed in Expi293 cells (Thermofisher Scientific). Culture supernatants were collected, clarified, and affinity-purified using a Protein A column (Cytiva). The final product was buffer-exchanged into DPBS by ultrafiltration / diafiltration (UF / DF) and stored at -80°C.

[0162] To evaluate the binding activity of antibodies against CD16A expressed on live cells, NK92mi (ATCC, CRL-2407) cells were engineered to overexpress human CD16A (NK92mi / CD16A F158 and NK92mi / CD16A V158) by cotransfection with expression plasmids containing CD16A (F158 or V158) and FcRγ cDNA. NK92mi / CD16B(NA1)- and NK92mi / CD16B(NA2)-expressing cell lines were similarly prepared from CD16B(NA1)- or CD16B(NA2)-expressing plasmids.

[0163] Immunization and Screening An external research institute immunized one alpaca with recombinant human CD16A-His6 (V158) protein as an antigen, and constructed an immune VHH phage library from isolated alpaca peripheral blood mononuclear cells (PBMCs) after the third immunization (Pardon Els et al. (2014) Nature Protocols). Phage display selection was performed using standard protocols (Silacci et al. (2005) Proteomics, 5, 2340-50; Zhao et al. (2014) PLoS One, 9, e111339). Briefly, human CD16A (V158)-specific binders were enriched in panning rounds 1 and 2 using 10 μg / ml of human CD16A-V158-Alpaca IgG2b immobilized on immunotubes. The immunotubes were blocked for 1 hour with 5% milk powder (w / v) in PBS supplemented with 1% Tween® 20 (MPBST). After washing with PBST (PBS buffer supplemented with 0.05% Tween® 20), 1 × 10 13 (Round 1) or 2 x 10 12Phages (round 2) were first depleted with human CD16B-His6(NA2) in MPBST for 1 hour and then incubated with antigen for 1 hour. After washing with PBST, bound phages were eluted with 100 mM triethylamine (Sigma-Aldrich). The eluted phages were used to infect mid-logarithmic phase E. coli TG1 bacteria and plated on 2xYT (yeast extract tryptone) agar plates supplemented with 2% glucose and 100 μg / mL ampicillin. After three rounds of selection, individual clones were selected, and phage-containing supernatants were prepared using standard protocols. Anti-human CD16A antibodies were screened using phage ELISA.

[0164] For phage ELISA, Maxisorp immunoplates were coated with recombinant human CD16A-His6(V158) protein as the antigen and blocked with 5% milk powder (w / v) in PBS buffer. Phage supernatants were blocked with MPBST for 30 min and added to the wells of the ELISA plate for 1 h. After washing with PBST, bound phages were detected using HRP-conjugated anti-M13 antibody (GE Healthcare) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA).

[0165] Positive clones from the phage ELISA were sequenced and recovered. Six anti-CD16A VHH variants were constructed by fusing their open reading frames to a eukaryotic expression vector, which added a C-terminal human IgG1 mf Fc (SEQ ID NO: 107) tag. The plasmids were transfected into ExpiCHO-s cells (Thermofisher Scientific) using the MAX Titer protocol. The Fc-tagged VHH variants (VHH-Fc) were purified by MabSelect SuRe (Cytiva) followed by SPHP column (Cytiva). The final product was buffer-exchanged into DPBS by UF / DF and stored at -80°C for subsequent use, including binding analysis.

[0166] For antigen ELISA, Maxisorp immunoplates were coated with antigen (human CD16A(V158), human CD16A(F158), human CD16B(NA1), human CD16B(NA2), human CD16B(SH), or cynomolgus monkey CD16) and blocked with 3% BSA (w / v) in PBS buffer (blocking buffer). Monoclonal VHH-Fc antibodies were blocked with blocking buffer for 30 min and added to the ELISA plate wells for 1 h. After washing with PBST, bound antibodies were detected using an HRP-conjugated anti-human IgG antibody (Sigma, A0170) and 3,3',5,5'-tetramethylbenzidine substrate (catalog: 00-4201-56, eBioscience, USA).

[0167] Flow cytometry revealed that NK92mi / CD16A V158 cells, NK92mi / CD16B(NA1) cells, and NK92mi / CD16B(NA2) cells (10 5 Cells (cells / well) were incubated with various concentrations of IgG-like antibodies, followed by binding with Alexa Fluro-647-labeled anti-human IgGFc antibodies (catalog: 409320, BioLegend, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte™ 8HT, Merck-Millipore, USA).

[0168] Following the procedures disclosed above, 44 positive clones were sequenced and recovered, and one representative positive anti-CD16A variant, BG523P (VHH AA SEQ ID NO: 112, VHH DNA SEQ ID NO: 113), was obtained from six VHH-Fc fusion clones. The binding affinity of BG523P to CD16A was also confirmed by antigen ELISA. The results of ELISA and FACS analysis of BG523P relative to the positive control LS21 are shown in Tables 10-12 and Figures 10 and 11A-11C. More specifically, the FACS binding data of BG523P versus the human CD16A-specific binder LS21 (SEQ ID NO: 108, patent EP1888645B1) as a positive control to the NK92mi / CD16A cell line are shown in Table 10, demonstrating that BG523P has binding specificity to NK92mi / CD16A cells. Figure 10 shows that BG523P exhibited higher binding to human CD16A, CD16B (NA1), and cynomolgus monkey CD16 at 1 μg / ml compared to LS21. Figure 11A shows that BG523P specifically binds to NK92mi / CD16A cells. Figures 11B and 11C show that BG523P exhibits weak binding to NK92mi / CD16B at high concentrations. BG523P exhibits slight binding activity to CD16B (NA1) in ELISA and FACS assays (Figure 10, Table 11), but its binding affinity was significantly reduced (calculated EC 50 (The binding values ​​were approximately 40-fold lower than those for CD16A in the FACS assay.) This result suggests that BG523P can selectively bind to CD16A over CD16B. Figure 10 also shows that BG523P hardly binds to the CD16B SH allotype. Considering that the predominant variants of human CD16B are the NA1 and NA2 allotypes and that the frequency of the SH allotype is rare, reported to be less than 0.05 in Caucasians, the binding properties of CD16B to the SH allotype were not further characterized. [Table 10] [Table 11] [Table 12]

[0169] Example 9. Humanization of anti-human CD16A VHH BG523P For humanization of BG523P, human germline IgG genes were searched for sequences sharing high homology with the cDNA sequence of the variable region of BG523P by blasting the human immunoglobulin gene databases on the IMGT (http: / / www.imgt.org / IMGT_vquest / share / textes / index.html) and NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) websites. Human IGVH genes, which are frequently present in the human antibody repertoire (Glanville et al., 2009 PNAS 106:20216-20221) and share high homology with BG523P, were selected as templates for humanization.

[0170] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and the humanized VHH variants were engineered as VHH-Fc variants using an in-house expression vector for subsequent binding and biophysical stability analyses. In the initial round of humanization, mutations from camelid-derived amino acid residues to human-derived amino acid residues in the framework regions were introduced based on simulated 3D structures. To maintain the canonical structure of the CDRs, structurally important camelid-derived framework residues were retained in the first version of humanized VHH, BG523P. Among the many variants, BG524P is the preferred humanized VHH, retaining the most camelid-derived residues. Specifically, HCDR1 (SEQ ID NO: 109) and HCDR3 (SEQ ID NO: 111) of BG523P were grafted onto the framework of the human germline mutant gene IGVH3-7, retaining five camelid-derived framework residues (F37, R45, V78, P84, and A94 according to Kabat numbering), while introducing one mutation into HCDR2 to remove a potential isomerization site. The sequence of BG524P is shown in Table 23 as SEQ ID NOs: 109, 114, 111, and 115-116.

[0171] The humanized BG523P variant contains the Fc region of a human IgG1 variant (SEQ ID NO: 107) fused to the N-terminus of the Fc as VHH-Fc using an in-house developed expression vector containing easily adaptable subcloning sites. Expression and preparation of the humanized BG523P VHH-Fc antibody was achieved by transfection of the construct into ExpiCHO-s cells and purification using a Protein A column. The purified VHH-Fc antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0172] For affinity determination, VHH-Fc antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding profile results of anti-CD16A VHHs determined by SPR are summarized in Table 13. BG524P showed slightly improved binding affinity to CD16A 158V and CD16A 158F compared to BG523P, with dissociation constants of 0.08 nM and 0.08 nM, respectively. Meanwhile, BG524P maintained selectivity for CD16A over CD16B as characterized by SPR. [Table 13]

[0173] The CD16A 158F overexpressing cell line NK92mi / CD16A F158 is used to assess the ability of anti-CD16A VHH-Fc antibodies to bind to native CD16A on live cells. Live NK92mi / CD16A 158F cells were seeded into 96-well plates and incubated with a dilution series of anti-CD16A VHH-Fc. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC2 values ​​for dose-dependent binding to human native CD16A were obtained. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figure 12A and Table 14, BG524P showed improved binding affinity to native CD16A 158F, but a reduced Emax. [Table 14]

[0174] To determine whether humanized BG523P maintained the optimal biophysical stability of BG523P, the melting temperature (Tm) and aggregation temperature (Tagg) of BG524P were determined and compared to those of BG523P. BG524P had inferior Tm and Tagg compared to those of BG523P (Table 15).

[0175] Melting temperatures (Tm) were measured using a high-throughput MicroCal™ VP-Capillary DSC (Malvern Instruments, Northampton, MA). Thermograms of each protein (350 μL at 0.5 mg / mL) were acquired from 20°C to 100°C using a scan rate of 60°C / hr. The buffer-alone thermogram was subtracted from each protein sample. The results show the transition temperature (Tm) midpoint and calorimetric enthalpy (ΔH) values ​​for the samples.

[0176] The aggregation temperature, Tagg (°C), represents the colloidal stability of the sample and was obtained by monitoring the onset of aggregation with an SLS266 using UNCLE™ (Unchained Lab, Pleasanton, CA). The sample was loaded into a Uni and the temperature was increased from 15°C to 95°C. Back-reflecting optics cannot detect near-ultraviolet light scattering by protein aggregates, and therefore only unscattered light reaches the detector. Therefore, the reduction in back-reflected light is a direct measure of aggregation in the sample. [Table 15]

[0177] BG524P was further engineered by introducing mutations in the CDRs and back mutations in the framework regions to improve biophysical properties, remove PTM sites, and restore binding Emax to native CD16A for therapeutic use in humans.

[0178] Taken together, successfully engineered versions of humanized monoclonal antibodies BG525P (SEQ ID NOs: 109-111 and 117-118) and BG526P (SEQ ID NOs: 109, 114, 111, and 119-120) were derived from the above mutational process and, as characterized in detail (Tables 16-18 and Figure 12B), both retain the binding affinity to CD16A, selectivity over CD16B, and optimal biophysical stability of the parent clones. [Table 16] [Table 17] [Table 18]

[0179] Example 10. Binding of anti-CD16A VHH to native CD16B To evaluate the ability of anti-CD16A VHHs to bind to native CD16B on live cells, NK92mi cells were engineered to overexpress human CD16B NA1 or NA2. Live NK92mi / CD16B cells were seeded into 96-well plates and incubated with 300 nM anti-CD16A VHH-Fc. Goat anti-human IgG was used as a secondary antibody to detect binding of anti-CD16A VHH-Fc to the cell surface. As shown in Figure 13 and Table 19, the binding signals of the humanized VHH-Fc to CD16B were comparable to or lower than those of the parent clones and significantly lower than their corresponding binding signals to CD16A (Table 17, Figures 12A-12B). [Table 19]

[0180] Example 11. Human IgG competition for VHH binding to native CD16A To assess the effect of human IgG competition on the ability of anti-CD16A VHH-Fc to bind native CD16A on live cells, a FACS-based assay was performed in the presence or absence of human IgG. Live NK92mi / CD16A cells were seeded into 96-well plates and incubated at 37°C with a dilution series of biotinylated anti-CD16A VHH-Fc alone or with 10 mg / ml of human IgG1 antibody CB6 (anti-SARS-Covid19 antibody) (SEQ ID NOs: 121-122). Streptavidin-AF647 was used as a secondary antibody to detect binding of biotinylated anti-CD16A VHH-Fc to the cell surface. EC of dose-dependent binding to human native CD16A. 50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Figures 14A-14C and Table 20, binding of BG525P and BG526P to CD16A 158F was similarly affected by the presence of human IgG compared to binding of the parental BG523P. [Table 20]

[0181] Example 12. Binding affinity of humanized CD16A to cynomolgus monkey CD16 by SPR For affinity determination, VHH-Fc was captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The binding profile results of anti-CD16A VHH-Fc determined by SPR are summarized in Table 21. Humanized anti-CD16A VHH-Fcs retained cross-reactivity to cynomolgus monkey CD16. [Table 21] [Table 22-1] [Table 22-2] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] [Table 25-1] [Table 25-2]

[0182] Example 13. Generation and production of MUC1-targeting antibodies Generation and production of MUC1 x CD16A multispecific antibody BG1222P To construct the MUC1 x CD16A multispecific antibody BG1222P, the anti-CD16A VHH BG526P and the anti-MUC1 antibody huBG219P-E39 were assembled in a "2 + 1" IgG-like bispecific format, with knob-into-hole (KiH) mutations to promote Fc dimerization (Figure 15). Specifically, tandem BG526P VHHs, flanked by a 2G4S linker (GGGGSGGGGS, SEQ ID NO: 72), were fused to the N-terminus of the hinge region of a human IgG1 constant domain (chain 1, SEQ ID NOs: 143-144) carrying the T366W (EU numbering) mutation for the "knob" mutation, the C220S (EU numbering) mutation for the removal of a free cysteine, and the M252Y / S254T / T256E mutations for half-life extension. For the MUC1-binding arm, the humanized MUC1 antibody huBG219P-E39 VH region was fused to the constant region of human IgG1 carrying T366S / L368A / Y407V (EU numbering) for "hole" mutations and M252Y / S254T / T256E for half-life extension (chain 2, SEQ ID NOs: 145-146). The light chain of BG1222P was generated from the VL region of the humanized MUC1 antibody huBG219P-E39 fused to the constant region of the human kappa chain (chain 3, SEQ ID NOs: 147-148). These constructs were prepared using an in-house developed expression vector containing easily adaptable subcloning sites or pcDNA3.4.

[0183] All three plasmids were co-transfected into ExpiCHO-s cells (Thermofisher Scientific). The plasmid ratio was optimized to improve the purity of the starting material and facilitate downstream purification procedures. The bispecific antibody was first captured with MabSelect SuRe Lx (Cytiva) and further purified using two ion exchange columns, Capto S ImpAct and Capto Q ImpRes (Cytiva), to remove most impurities and aggregates. The final product was buffer-exchanged into DPBS or histidine buffer using a G25 desalting column (Cytiva) and stored at -80°C.

[0184] Generation of multiple anti-human MUC1 reference Abs Multiple anti-human MUC1 reference antibody sequences were extracted from published literature and patents, as summarized in Table 25. The reference antibodies were constructed using an in-house IgG1 / Cκ eukaryotic expression vector.

[0185] Defucosylated versions of antibodies (referred to with the suffix -AF), including the reference antibodies mentioned above and huBG219P-E39-AF, were produced using the ExpiCHO transient expression system (Thermofisher Scientific) for use in the assays. To inhibit fucosylation, 2F-peracetyl-fucose (catalog no. 344827, EMD Millipore) was added to the growth medium at a final concentration of 100 μM prior to inoculation. Conditioned medium was collected, and defucosylated antibodies were purified using a MabSelect SuRe column (Cytiva) followed by an SPHP column (Cytiva). All purified antibodies were buffer-exchanged into DPBS via UF / DF, aliquoted into small amounts for later assays, and stored at -80°C.

[0186] Example 14. Determination of binding kinetics and affinity of MUC1xCD16A multispecific antibodies For affinity determination, a surface plasmon resonance (SPR) technology-based assay was developed to characterize the binding affinity of the bispecific antibody. Briefly, neutravidin (netrAvidin) was immobilized on a CM5 chip surface, and then biotin-labeled anti-human Fc VHH was flowed over the surface and captured by the immobilized neutravidin. The MUC1×CD16A multispecific antibody was captured by the anti-human Fc VHH / neutravidin complex on the chip surface, and serial dilutions of CD16A or hMUC1-SEA-mFc (human MUC1-SEA domain linked to mouse IgG2a Fc) were flowed over the surface. Binding responses were calculated by subtracting the RU from a reference flow cell without bispecific antibody. The binding profile determined by SPR for the MUC1×CD16A multispecific antibody is summarized in Table 26. The multispecific antibody BG1222P exhibited high binding affinity to its targets, human CD16A (158V and 158F) and hMUC1-SEA. [Table 26]

[0187] Example 15. Determination of binding affinity of MUC1xCD16A multispecific antibodies Binding affinity of MUC1×CD16A multispecific antibodies to MUC1-expressing cancer cell lines The binding affinity of purified BG1222P to MUC1-expressing cancer cell lines and NK92mi was determined by FACS. Briefly, the MUC1-expressing tumor cell line, T47D, was incubated with serially diluted purified BG1222P, washed, and then incubated with an APC-conjugated anti-human IgG secondary antibody. After incubation and washing, fluorescence was measured by flow cytometry. The binding affinity of BG1222P to T47D is shown in Table 27 below and Figure 16. The results demonstrate that BG1222P has high binding affinity to MUC1-expressing cancer cell lines. [Table 27]

[0188] Binding affinity of MUC1×CD16A multispecific antibodies to NK92mi / CD16A F158 and NK92mi / CD16A V158 cells To evaluate the binding activity of BG1222P and huBG219P-E39-AF antibodies to CD16A expressed on live cells, NK92mi (ATCC) cells were engineered to overexpress human CD16A (NK92mi / CD16A F158 and NK92mi / CD16A V158) by cotransfection with expression plasmids containing CD16A (F158 or V158 allele) and FcRγ cDNA. Serial dilutions of purified, biotinylated bispecific antibodies were incubated with NK92mi / CD16A F158 or NK92mi / CD16A V158 cells in the presence or absence of 10 mg / ml human IgG at 37°C for 45 min. After washing twice with FACS buffer, diluted Alexa Fluor 647 streptavidin (Invitrogen #S32357) was added and incubated with the cells for 60 minutes at 4°C in the dark. After washing twice with FACS buffer, the cells were resuspended in FACS buffer and acquired on a BD FACS Celesta. Titration curves were generated using a sigmoidal linear dose-response nonlinear fit from GraphPad, and the EC values ​​of representative antibodies were calculated. 50 The results are shown in Table 28 and Figures 17A-17D. BG1222P exhibits strong binding affinity to both F158 and V158 human CD16A-overexpressing cell lines. Human IgG competition impairs the binding Emax of huBG219P-E39-AF, but not that of BG1222P. The results indicate that the CD16A binding affinity of BG1222P can be better maintained than that of huBG219P-E39-AF in the presence of high levels of IgG in the circulation or tumor. The CD16A binding activity of BG1222P is less impaired by IgG competition than that of huBG219P-E39-AF. [Table 28]

[0189] Example 16. Antibody-dependent cellular cytotoxicity activity of MUC1 x CD16A multispecific antibodies MUC1 + A nanoluc release assay was performed to determine the antibody-dependent cytotoxicity (ADC) activity of the BG1222P and huBG219P-E39-AF antibodies against NK92mi / CD16A F158 and NK92mi / CD16A V158 cell lines. Several cancer cell lines with different MUC1 expression levels—T-47D (MUC1 high), HCC827 (MUC1 medium), H358 (MUC1 low), and MDA-MB-453 (MUC1 negative)—were engineered to express nanoluc intracellularly by retroviral transduction and used as target cells. Upon lysis of target cells by effector cells, nanoluc was released into the culture medium. Cytotoxicity was assessed by measuring nanoluc in the supernatant using a Nano-Glo Luciferase Assay Kit (Promega, Madison, Wis.). Briefly, effector and target cells were added to a V-bottom 96-well plate with serial dilutions of bispecific antibodies in the presence or absence of 10 mg / ml human IgG1 at an E:T ratio of 2:1 and co-cultured at 37°C for 20–24 h. Specific lysis was determined using the following formula: Percentage of specific lysis = [Luminescence (sample) – Luminescence (spontaneous)] / [Luminescence (maximum) – Luminescence (spontaneous)] × 100%. Luminescence (spontaneous) represents the luminescence counts from the supernatant of effector cells and antibody-free target cells. Luminescence (maximum) represents the luminescence counts released after total cell lysis induced by the addition of Triton®-X-100. As shown in Tables 29 and 30 and Figures 18A-19H, BG1222P induced lysis of MUC1-expressing cell lines, but not the MUC1-negative cell line MDA-MB-453, in a dose-dependent manner in the presence or absence of human IgG. The results demonstrate that the ADCC activity of BG1222P is superior to that of huBG219P-E39-AF. [Table 29] [Table 30]

[0190] Example 17. Cytolytic activity of MUC1 x CD16A multispecific antibodies against MUC1-expressing cells in a human whole blood assay The cytolytic activity of BG1222P in human whole blood was also evaluated using a nanoluc release assay. Briefly, 100 μL / well of human whole blood from healthy donors was mixed with target cells (T-47D / nanoluc, HCC827 / nanoluc, H358 / nanoluc, and MDA-MB-453 / nanoluc) (2000 cells / well) and a dilution series of bispecific antibodies described in Example 16 in a U-bottom 96-well plate. The total volume was 200 μL / well. After incubation at 37°C for 18–20 hours, nanoluc released into the supernatant was measured using the Nano-Glo Luciferase Assay Kit. Specific lysis was determined using the formula described in Example 16.

[0191] To compare the cytolytic activity of bispecific antibodies with that of anti-MUC1 monoclonal IgG1 antibodies, the following antibodies with engineered Fc (defucosylation, -AF) were produced with reference to the published sequences: gatipotuzumab-AF, clivatuzumab-AF, HuVH-HMFG1-AF, MUC1 5F3-hFc-AF, and MUC1 3D1-hFc-AF. Removal of the core fucose of the Fc glycan (defucosylation) has been shown to significantly increase FcγRIIIa binding affinity and, consequently, the cytotoxic activity of the antibody. These antibodies were compared with the bispecific antibodies in the human whole blood assay described above using T-47D / nanoluc as target cells. As shown in Table 31 and Figure 20, BG1222P inhibited EC 50 and Emax, it exhibits much stronger cytolytic activity than the aforementioned anti-MUC1 defucosylated antibody.

[0192] In addition to T47D cells, the cytolytic activity of BG1222P against target cells with different levels of MUC1 expression was evaluated in a human whole blood assay. As shown in Table 32 and Figures 21A-21D, BG1222P specifically induced lysis of MUC1-expressing cell lines, but not the MUC1-negative cell line, MDA-MB-453, in a dose-dependent manner in human whole blood. The results demonstrate that the cytolytic activity of BG1222P is superior to that of huBG219P-E39-AF. [Table 31] [Table 32]

[0193] Example 18. Phagocytic activity of MUC1 x CD16A multispecific antibodies The phagocytic activity of BG1222P was evaluated using human PBMC-derived M2 macrophages as effector cells. M2 macrophage generation was performed according to the protocol described by Leidi et al. (Journal of Immunology, (2009) 182(7), 4415-4422). Briefly, human PBMCs (Sailybio) were cultured in complete RPMI 1640 medium supplemented with 30 ng / ml human M-CSF (Peprotech) in 6-well plates (Corning) for 4 days. Nonadherent and loosely adherent cells were gently washed away to retain adherent cells, and half of the medium was replaced and cultured for an additional 2–3 days. For M2 polarization, 10 ng / ml IL-10 (Peprotech) was added during the final 48 hours of culture.

[0194] Target cells (T-47D and MDA-MB-453) were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) according to the manufacturer's instructions. Target cells and M2 macrophages were plated at a 2:1 ratio in a U-bottom 96-well plate with bispecific antibodies in the presence or absence of 10 mg / ml human IgG. After 2 hours of incubation at 37°C, cells were stained with anti-CD11b-BV421 and subjected to flow cytometry. The percentage of macrophages that underwent antibody-dependent cellular phagocytosis of target cells was determined by FACS of double-positive (CFSE+ and CD11b+) cells after gating on CD11b+ M2 macrophages. As shown in Table 33 and Figures 22A-22B, BG1222P exhibited superior phagocytic activity to huBG219P-E39-AF in a dose-dependent manner in the MUC1-expressing cell line T47D, both with and without the addition of human IgG. BG1222P did not exhibit activity in the MUC1-negative cell line MDA-MB-453. [Table 33]

[0195] Example 19. NK fratricide of MUC1 x CD16A multispecific antibodies A flow cytometry-based assay was set up to determine the NK fratricidal activity of BG1222P. Primary NK cells were isolated from PBMCs of healthy donors using an NK cell isolation kit from Miltenyi Biotec (Germany) according to the manufacturer's instructions. Isolated NK cells were cultured with serial dilutions of BG1222P in V-bottom 96-well plates. Daratumumab, known to exhibit NK fratricidal activity at the cellular level and in patients, was used as a positive control. After 5 hours of incubation at 37°C, cells were stained with anti-CD3-BV421, annexin V-FITC, 7-AAD, and anti-CD56-AF647. The percentages of apoptotic and dead NK cells were determined by FACS for annexin V-positive and double-positive (annexin V+ and 7-AAD+) cells after gating on CD3-CD56+ NK cells. As shown in Figures 23A-23B, BG1222P did not exhibit fratricidal tendencies in NK cells from either of the two donors. In contrast, daratumumab exhibited dose-dependent killing activity against NK cells from both donors.

[0196] Example 20. Pharmacokinetic profile of MUC1xCD16A multispecific antibody in cynomolgus monkeys Blood samples were collected from cynomolgus monkeys at 0, 0.5, 1, 4, 8 hours, and 1, 3, 7, 10, 14, 21, and 28 days after intravenous infusion of 5 mg / kg or 25 mg / kg BG1222P. Serum was then separated by centrifugation (4°C, 3000 × g, 15 min). BG1222P concentrations were measured using an in-house developed ELISA ligand-binding assay. Briefly, HuMUC1-SEA-mFc was used as the capture reagent, and biotin-labeled CD16A-V158 with a His tag was used as the detection reagent for BG1222P. The resulting pharmacokinetic profiles and parameters are shown in Figure 24 and Table 34, respectively. In the 5 mg / kg dose group, BG1222P levels were below the lower limit of quantitation (0.78 μg / mL) at 21 days post-dose. Anti-drug antibodies (ADAs) were detected in serum from day 10 in the 5 mg / kg dose group, indicating a potential impact on the pharmacokinetic curve. In the 25 mg / kg dose group, BG1222P exhibited much lower clearance, and the impact of ADAs was minimal. Even under these circumstances, a relatively long terminal elimination half-life of BG1222P was observed after administration of 5 to 25 mg / kg, ranging from 4.25 to 10.2 days. The clearance range of 4.3 to 9.1 days suggests that BG1222P was slowly eliminated from the body. The volume of distribution (V z ) approximated the physiological serum volume in cynomolgus monkeys, indicating that BG1222P was primarily located in the serum volume. [Table 34] [Table 35]

[0197] Example 21. MUC1xCD16A multispecific antibodies show reduced interference by soluble MUC1. The effect of soluble MUC1 on the specific binding of the MUC1 × CD16A multispecific antibody to MUC1-expressing cells was determined by competitive FACS assay. Briefly, human MUC1-expressing cells were incubated with 30, 3, or 0.3 μg / ml BG1222P in the presence of serially diluted soluble MUC1 (Shanghai Linc-Bio Science Co. LTD). After washing, the cells were incubated with an anti-human IgG secondary antibody, and fluorescence was measured by flow cytometry. The IC of soluble MUC1 blocking BG1222P binding to MUC1-expressing cells was 0.01. 50 The values ​​are shown in Table 36, and the blocking curves are shown in Figures 25A-25C. The profiles indicate that HuVH-HMFG binding to MUC1-expressing cells can be easily interfered with at high, medium, and low antibody concentrations (i.e., 30, 3, and 0.3 μg / ml), whereas BG1222P binding is only slightly interfered with at low antibody concentrations (0.3 μg / ml) (Figures 25A-25C). Collectively, the profiles in Figure 25 indicate that BG1222P binding to MUC1-expressing cells showed significantly reduced interference by soluble MUC1 compared with HuVH-HMFG1, which targets the membrane-distal portion of MUC1. [Table 36]

Claims

1. A multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding domain that specifically binds to human MUC1 and a second antigen-binding domain that specifically binds to human CD16A.

2. 2. The multispecific antibody or antigen-binding fragment of claim 1, wherein the first antigen-binding domain has higher selectivity than human CD16B.

3. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the first antigen-binding domain that specifically binds to human MUC1 is: (i) a heavy chain variable region comprising (a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 24, (b) a HCDR2 of SEQ ID NO: 25, and (c) a HCDR3 of SEQ ID NO: 26, and (d) a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 27, (e) a LCDR2 of SEQ ID NO: 28, and (f) a LCDR3 of SEQ ID NO: 29; (ii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; (iii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; or (iv) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO:

19.

4. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the first antigen-binding domain is: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 30, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 31; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 62; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 66; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 61, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; or (vi) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 20, and a light chain variable region (VL) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

21.

5. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are inserted, deleted or substituted within one or more of SEQ ID NOs: 30, 31, 61, 62, 66, 68, 10, 11, 20 and 21.

6. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the first antigen-binding domain is: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; (ii) A heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; (iii) A heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; (v) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; or (vi) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO:

21.

7. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the second antigen-binding domain that specifically binds to human CD16A is: (i) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; or (ii) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO:

111.

8. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the second antigen-binding domain is: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 112; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 115; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 117; or (iv) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:

119.

9. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are inserted, deleted or substituted within one or more of SEQ ID NOs: 112, 115, 117 and 119.

10. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the second antigen-binding domain is: (i). A heavy chain variable region (VH) comprising SEQ ID NO: 112; (ii). A heavy chain variable region (VH) comprising SEQ ID NO: 115; (iii) A heavy chain variable region (VH) comprising SEQ ID NO: 117; or (iv) The multispecific antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) comprising SEQ ID NO:

119.

11. 10. A multispecific antibody or antigen-binding fragment according to any one of the preceding claims, comprising: (i) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (ii) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (iii) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (iv) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; (v) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; (vi) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 65, and (f) an LCDR3 of SEQ ID NO: 29; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; (vii) The first antigen-binding domain that specifically binds to human MUC1 comprises a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO: 111; or (viii) A heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; and the second antigen-binding domain that specifically binds to human CD16A comprises (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO:

111.

12. 10. A multispecific antibody or antigen-binding fragment according to any one of the preceding claims, comprising: (i). The first antigen-binding domain that specifically binds to human MUC1 is selected from the following: a) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; b) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; c) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; d) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii). And, the second antigen-binding domain that specifically binds to human CD16A is: a) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 110, and (c) an HCDR3 of SEQ ID NO: 111; or b) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 109, (b) an HCDR2 of SEQ ID NO: 114, and (c) an HCDR3 of SEQ ID NO:

111.

13. 10. A multispecific antibody or antigen-binding fragment according to any one of the preceding claims, comprising: (i). The first antigen-binding domain that specifically binds to human MUC1 is selected from the following: a) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 24, (b) an HCDR2 of SEQ ID NO: 25, and (c) an HCDR3 of SEQ ID NO: 26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 27, (e) an LCDR2 of SEQ ID NO: 28, and (f) an LCDR3 of SEQ ID NO: 29; b) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO:24, (b) an HCDR2 of SEQ ID NO:25, and (c) an HCDR3 of SEQ ID NO:26, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO:27, (e) an LCDR2 of SEQ ID NO:65, and (f) an LCDR3 of SEQ ID NO:29; c) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 4, (b) an HCDR2 of SEQ ID NO: 5, and (c) an HCDR3 of SEQ ID NO: 6, and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 7, (e) an LCDR2 of SEQ ID NO: 8, and (f) an LCDR3 of SEQ ID NO: 9; or d) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 14, (b) an HCDR2 of SEQ ID NO: 15, and (c) an HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising (d) an LCDR1 of SEQ ID NO: 17, (e) an LCDR2 of SEQ ID NO: 18, and (f) an LCDR3 of SEQ ID NO: 19; (ii). And, the second antigen-binding domain that specifically binds to human CD16A is: a) a heavy chain variable region (VH) comprising SEQ ID NO: 112; b) a heavy chain variable region (VH) comprising SEQ ID NO: 115; c) a heavy chain variable region (VH) comprising SEQ ID NO: 117; or d) a heavy chain variable region (VH) comprising SEQ ID NO:

119.

14. 10. A multispecific antibody or antigen-binding fragment according to any one of the preceding claims, comprising: (i). The first antigen-binding domain that specifically binds to human MUC1 is selected from the following: a) a heavy chain variable region (VH) comprising SEQ ID NO: 30, and a light chain variable region (VL) comprising SEQ ID NO: 31; b) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 62; c) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 66; d) a heavy chain variable region (VH) comprising SEQ ID NO: 61, and a light chain variable region (VL) comprising SEQ ID NO: 68; e) a heavy chain variable region (VH) comprising SEQ ID NO: 10, and a light chain variable region (VL) comprising SEQ ID NO: 11; or f) a heavy chain variable region (VH) comprising SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21; (ii). And, the second antigen-binding domain that specifically binds to human CD16A is: a) a heavy chain variable region (VH) comprising SEQ ID NO: 112; b) a heavy chain variable region (VH) comprising SEQ ID NO: 115; c) a heavy chain variable region (VH) comprising SEQ ID NO: 117; or d) a heavy chain variable region (VH) comprising SEQ ID NO:

119.

15. 10. A multispecific antibody or antigen-binding fragment according to any one of the preceding claims, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

16. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the multispecific antibody is a bispecific antibody.

17. 10. The multispecific antibody or antigen-binding fragment according to any one of the preceding claims, wherein the multispecific antibody is BG1222P (SEQ ID NO: 143, SEQ ID NO: 145, and SEQ ID NO: 147).

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

19. 10. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is reduced or aglycosylated or hypofucosylated.

20. 10. The multispecific antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises an increase in bisecting GlcNAc structures.

21. 10. The multispecific antibody or antigen-binding fragment of any one of the preceding claims, wherein the Fc domain is IgG1.

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

23. 10. A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier.

24. 24. The pharmaceutical composition of claim 23, comprising histidine / histidine HCl, trehalose dihydrate, and polysorbate 20.

25. An isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 22.

26. A vector comprising the nucleic acid of claim 25.

27. 27. A host cell comprising the nucleic acid of claim 25 or the vector of claim 26.

28. 28. A process for producing a multispecific antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 27 and recovering the antibody or antigen-binding fragment from the culture.

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