Bispecific antibodies against MUC17 and CD3 and uses thereof

Abstract

The present invention relates to bispecific antibodies capable of specifically binding to MUC17 and CD3, which are capable of modulating the function of immune cells and can be used as medicaments for the treatment of gastrointestinal malignancies. In addition, polynucleotides encoding the bispecific antibodies, vectors, host cells and pharmaceutical compositions comprising the bispecific antibodies are also provided.

Description

Bispecific antibodies targeting MUC17 and CD3 and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of Chinese Application No. 202310092717.2, filed on February 3, 2023, and Chinese Application No. 202310308001.1, filed on March 23, 2023, the entire contents of which are incorporated herein for all purposes. Technical Field

[0003] The present application relates to the field of antibodies, and in particular, to bispecific antibodies targeting MUC17 and CD3. Background Art

[0004] Bispecific antibodies (BsAbs) are a class of antibody molecules that can simultaneously bind to two different antigens or two distinct epitopes on the same antigen. These two unique antigen-binding sites facilitate dual target binding, leveraging the synergistic effects of two monoclonal antibodies. Bispecific antibodies can act as a bridge between target cells and functional molecules, generating targeted effector functions. They may offer advantages over the combined use of two monoclonal antibodies in preclinical research and clinical treatment, and hold broad application prospects in areas such as tumor immunotherapy and autoimmune diseases.

[0005] Killing tumor cells through bispecific antibody-mediated cytotoxicity is a hot topic in current immunotherapy research. This is achieved by leveraging the ability of bispecific antibodies to simultaneously bind to effector cells and tumor-associated antigens, directly triggering immune cells to specifically kill tumor cells. Among them, CD3 is a surface-specific molecule present on all T lymphocytes that can recruit effector T cells with killing properties. MUC17 is a tumor-specific antigen that is widely expressed on a variety of tumor cells, while in normal tissues it is only expressed in intestinal tissue. Therefore, by targeting CD3 and MUC17, specific killing of MUC17-positive tumors can be achieved.

[0006] Summary of the Invention

[0007] The present application provides an anti-MUC17 / anti-CD3 bispecific, a nucleic acid molecule encoding the same, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.

[0008] In a first aspect, the present application provides an anti-MUC17 / anti-CD3 bispecific antibody comprising:

[0009] (a) a first antigen-binding portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL form an anti-CD3 antigen-binding domain; wherein the anti-CD3 antigen-binding domain comprises HCDR1, HCDR2, and HCDR3 of the VH set forth in SEQ ID NO. 9 and LCDR1, LCDR2, and LCDR3 of the VL set forth in SEQ ID NO. 10;

[0010] (b) a second antigen-binding portion, comprising a VHH that specifically binds to MUC17, wherein the VHH comprises: CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO.11, or CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO.12, or CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO.13, or CDR1, CDR2, and CDR3 of the sequence shown in SEQ ID NO.14.

[0011] In some embodiments, based on the Kabat numbering system, the HCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.15; the HCDR2 comprises the sequence shown in SEQ ID NO.16; and the HCDR3 comprises the sequence shown in SEQ ID NO.17.

[0012] In some embodiments, based on the Chothia numbering system, the HCDR1 of the first antigen-binding portion comprises the sequence shown in SEQ ID NO.33; the HCDR2 comprises the sequence shown in SEQ ID NO.34; and the HCDR3 comprises the sequence shown in SEQ ID NO.35.

[0013] In some embodiments, based on the IMGT numbering system, the HCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.51; the HCDR2 comprises the sequence shown in SEQ ID NO.52; and the HCDR3 comprises the sequence shown in SEQ ID NO.53.

[0014] In some embodiments, based on the Kabat numbering system, the LCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.18; LCDR2 comprises the sequence shown in SEQ ID NO.19; and LCDR3 comprises the sequence shown in SEQ ID NO.20.

[0015] In some embodiments, based on the Chothia numbering system, LCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.36; LCDR2 comprises the sequence shown in SEQ ID NO.37; and LCDR3 comprises the sequence shown in SEQ ID NO.38.

[0016] In some embodiments, based on the IMGT numbering system, LCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.54; LCDR2 comprises the sequence shown in SEQ ID NO.55; and LCDR3 comprises the sequence shown in SEQ ID NO.56.

[0017] In some embodiments, the HCDR1 of the first antigen binding portion comprises the sequence shown in any one of SEQ ID NO.15, 33 or 51; the HCDR2 comprises the sequence shown in any one of SEQ ID NO.16, 34 or 52; and the HCDR3 comprises the sequence shown in any one of SEQ ID NO.17, 35 or 53.

[0018] In some embodiments, LCDR1 of the first antigen binding portion comprises the sequence shown in any one of SEQ ID NO.18, 36 or 54; LCDR2 comprises the sequence shown in any one of SEQ ID NO.19, 37 or 55; and LCDR3 comprises the sequence shown in any one of SEQ ID NO.20, 38 or 56.

[0019] In some embodiments, based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.21; the CDR2 comprises the sequence shown in SEQ ID NO.22; and the CDR3 comprises the sequence shown in SEQ ID NO.23.

[0020] In some embodiments, based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO. 24; the CDR2 comprises the sequence shown in SEQ ID NO. 25; and the CDR3 comprises the sequence shown in SEQ ID NO. 26.

[0021] In some embodiments, based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO. 27; the CDR2 comprises the sequence shown in SEQ ID NO. 28; and the CDR3 comprises the sequence shown in SEQ ID NO. 29.

[0022] In some embodiments, based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.30; the CDR2 comprises the sequence shown in SEQ ID NO.31; and the CDR3 comprises the sequence shown in SEQ ID NO.32.

[0023] In some embodiments, based on the Chothia numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.39; the CDR2 comprises the sequence shown in SEQ ID NO.40; and the CDR3 comprises the sequence shown in SEQ ID NO.41.

[0024] In some embodiments, based on the Chothia numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.42; the CDR2 comprises the sequence shown in SEQ ID NO.43; and the CDR3 comprises the sequence shown in SEQ ID NO.44.

[0025] In some embodiments, based on the Chothia numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.45; the CDR2 comprises the sequence shown in SEQ ID NO.46; and the CDR3 comprises the sequence shown in SEQ ID NO.47.

[0026] In some embodiments, based on the Chothia numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.48; the CDR2 comprises the sequence shown in SEQ ID NO.49; and the CDR3 comprises the sequence shown in SEQ ID NO.50.

[0027] In some embodiments, based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.57; the CDR2 comprises the sequence shown in SEQ ID NO.58; and the CDR3 comprises the sequence shown in SEQ ID NO.59.

[0028] In some embodiments, based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.60; the CDR2 comprises the sequence shown in SEQ ID NO.61; and the CDR3 comprises the sequence shown in SEQ ID NO.62.

[0029] In some embodiments, based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO. 63; the CDR2 comprises the sequence shown in SEQ ID NO. 64; and the CDR3 comprises the sequence shown in SEQ ID NO. 65.

[0030] In some embodiments, based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO. 66; the CDR2 comprises the sequence shown in SEQ ID NO. 67; and the CDR3 comprises the sequence shown in SEQ ID NO. 68.

[0031] In some embodiments, the CDR1 of the second antigen binding portion comprises a sequence as shown in any one of SEQ ID NO. 21, 24, 27, 30, 39, 42, 45, 48, 57, 60, 63 or 66; the CDR2 comprises a sequence as shown in any one of SEQ ID NO. 22, 25, 28, 31, 40, 43, 46, 49, 58, 61, 64 or 67; and the CDR3 comprises a sequence as shown in any one of SEQ ID NO. 23, 26, 29, 32, 41, 44, 47, 50, 59, 62, 65 or 68.

[0032] In some embodiments, the first antigen binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0033] (1) the sequences represented by SEQ ID NOs. 15, 16, 17, 18, 19 and 20, respectively, based on the Kabat numbering system; or

[0034] (2) sequences represented by SEQ ID NOs. 33, 34, 35, 36, 37, and 38, respectively, based on the Chothia numbering system; or

[0035] (3) the sequences represented by SEQ ID NOs. 51, 52, 53, 54, 55, and 56, respectively, based on the IMGT numbering system; or

[0036] (4) A sequence having at least 90% identity with the sequence shown in (1) to (3) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

[0037] In some embodiments, the second antigen binding portion comprises CDR1, CDR2, and CDR3 of the following sequence:

[0038] (1) the sequences represented by SEQ ID NOs. 21, 22 and 23, respectively, based on the Kabat numbering system; or

[0039] (2) the sequences represented by SEQ ID NOs. 24, 25 and 26, respectively, based on the Kabat numbering system; or

[0040] (3) the sequences represented by SEQ ID NOs. 27, 28 and 29, respectively, based on the Kabat numbering system; or

[0041] (4) the sequences represented by SEQ ID NOs. 30, 31 and 32, respectively, based on the Kabat numbering system; or

[0042] (5) the sequences represented by SEQ ID NOs. 39, 40, and 41, respectively, based on the Chothia numbering system; or

[0043] (6) the sequences represented by SEQ ID NOs. 42, 43, and 44, respectively, based on the Chothia numbering system; or

[0044] (7) the sequences represented by SEQ ID NOs. 45, 46 and 47, respectively, based on the Chothia numbering system; or

[0045] (8) the sequences represented by SEQ ID NOs. 48, 49 and 50, respectively, based on the Chothia numbering system; or

[0046] (9) the sequences represented by SEQ ID NOs. 57, 58, and 59, respectively, based on the IMGT numbering system; or

[0047] (10) the sequences represented by SEQ ID NOs. 60, 61 and 62, respectively, based on the IMGT numbering system; or

[0048] (11) the sequences represented by SEQ ID NOs. 63, 64, and 65, respectively, based on the IMGT numbering system; or

[0049] (12) the sequences represented by SEQ ID NOs. 66, 67, and 68, respectively, based on the IMGT numbering system; or

[0050] (13) A sequence having at least 90% identity with the sequence shown in (1) to (12) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

[0051] In some embodiments, the VH of the first antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.9; and the VL of the first antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.10.

[0052] In some embodiments, the second antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs. 11-14.

[0053] In some embodiments, the bispecific antibody comprises a heavy chain comprising an anti-CD3 VH, a light chain comprising an anti-CD3 VL, and a heavy chain comprising an anti-MUC17 VHH.

[0054] In some embodiments, the heavy chain comprising the anti-CD3 VH comprises a sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO.1, the light chain comprising the anti-CD3 VL comprises a sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO.2, and the heavy chain comprising the anti-MUC17 VHH comprises a sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO.3, 4, 5 or 6.

[0055] In some embodiments, the bispecific antibody is a humanized antibody.

[0056] In some embodiments, the bispecific antibody specifically binds to human and monkey MUC17 proteins; preferably, the KD of the bispecific antibody binding to human and monkey MUC17 is better than 1.00E-9M.

[0057] In a second aspect, the present application provides an isolated nucleic acid molecule encoding the bispecific antibody of the first aspect.

[0058] In a third aspect, the present application provides a vector comprising the nucleic acid molecule described in the second aspect.

[0059] In a fourth aspect, the present application provides a host cell comprising the vector described in the third aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (e.g., Escherichia coli), fungi (e.g., yeast), insect cells or mammalian cells (e.g., CHO cell line or 293T cell line).

[0060] In a fifth aspect, the present application provides a method for preparing the bispecific antibody of the first aspect, comprising culturing the host cell of the fourth aspect, and isolating the bispecific antibody expressed by the cell.

[0061] In the sixth aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, or the product prepared by the method described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0062] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent; preferably, the additional therapeutic agent is an anti-tumor agent; more preferably, the anti-tumor agent is a PD-1 axis binding antagonist, a small molecule anti-tumor agent or a cell therapy agent.

[0063] In the seventh aspect, the present application provides the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, the product prepared by the method described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect for the preparation of a medicament for treating cancer, tumor or infectious disease; wherein the cancer or tumor is selected from solid tumors and blood tumors.

[0064] In some embodiments, the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor, or the cancer or tumor expresses MUC17 protein on its cell surface.

[0065] In some embodiments, the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer, and central nervous system cancer.

[0066] In some embodiments, the cancer is gastric cancer or pancreatic cancer.

[0067] In some embodiments, the drug is used in combination with another therapeutic agent or with surgery; wherein the additional therapeutic agent or the surgery is selected from radiation therapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, surgical intervention, immunostimulatory antibodies, immunomodulatory drugs, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.

[0068] In an eighth aspect, the present application provides a method for treating cancer, tumor, or infectious disease, comprising administering to a patient in need thereof an effective amount of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the product prepared by the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect; wherein the cancer or tumor is selected from solid tumors and blood tumors.

[0069] In some embodiments, the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor, or the cancer or tumor expresses MUC17 protein on its cell surface.

[0070] In some embodiments, the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer, and central nervous system cancer.

[0071] In some embodiments, the cancer is gastric cancer or pancreatic cancer.

[0072] In the ninth aspect, the present application provides the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, the host cell described in the fourth aspect, the product prepared by the method described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, for use in the pre-treatment of cancer, tumor or infectious disease; wherein the cancer or tumor is selected from solid tumors and blood tumors.

[0073] In some embodiments, the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor, or the cancer or tumor expresses MUC17 protein on its cell surface.

[0074] In some embodiments, the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer, and central nervous system cancer.

[0075] In some embodiments, the cancer is gastric cancer or pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Unless otherwise defined herein, scientific and technical terms related to the present application shall have the meanings understood by those of ordinary skill in the art.

[0077] Figure 1 is a structural diagram of the bispecific antibody.

[0078] FIG2A is an ELISA test showing the binding activity of Bis57, Bis58, Bis59 and Bis92 to human MUC17 protein.

[0079] FIG2B shows the binding activity of Bis57, Bis58, Bis59 and Bis92 to monkey MUC17 protein (mFc-tagged cynomolgus monkey MUC17 protein extracellular domain fusion protein) detected by ELISA.

[0080] FIG3A shows the ELISA assay for the binding activity of Bis57, Bis58, Bis59 and Bis92 to human CD3e-His.

[0081] FIG3B shows the binding activity of Bis57, Bis58, Bis59 and Bis92 to monkey CD3e-His detected by ELISA.

[0082] Figures 4A to 4C show the binding reactions of Bis57, Bis58, Bis59 and Bis92 to human endogenous tumor cell lines NUGC4, SNU16 and ASPC1 detected by FACS.

[0083] FIG5A is a FACS analysis of the binding reaction of Bis57, Bis58, Bis59 and Bis92 to human MUC17 overexpressing cells FlpinCHO-huMUC17-D1.

[0084] FIG5B is a FACS analysis of the binding reaction of Bis57, Bis58, Bis59 and Bis92 to monkey MUC17 overexpressing cells FlpinCHO-cynoMUC17-D1.

[0085] FIG6 is a graph showing the binding reaction of Bis57, Bis58, Bis59 and Bis92 to human Jurkat cells detected by FACS.

[0086] 7A and 7B show the binding activities of Bis57, Bis58, Bis59 and Bis92 to human and monkey PBMCs detected by FACS.

[0087] Figure 8A shows the activation of Bis57, Bis58, Bis59, and Bis92 after co-incubation of NUGC4 and Jurkat-luc using a luciferase reporter gene assay.

[0088] Figure 8B shows the activation of Bis57, Bis58, Bis59, and Bis92 after co-incubation of MDA-231 and Jurkat-luc using a luciferase reporter gene assay.

[0089] FIG9 shows the activation effect of bispecific antibodies Bis57 and Bis59 on T cells.

[0090] FIG10 shows the expression levels of MUC17 on the surfaces of tumor cell lines ASPC-1, NUGC4 and SNU-16 detected by FACS.

[0091] 11A to 11C are evaluations of the killing activity of Bis57 and Bis59 against tumor cell lines SNU16, ASPC1, and NUGC4.

[0092] FIG11D to FIG11E are evaluations of the killing activity of Bis58 and Bis92 against tumor cell lines NUGC4 and ASPC1.

[0093] FIG12A shows that the bispecific antibodies Bis57 and Bis59 stimulate PBMC to secrete IFNγ in the presence of SNU16 cells.

[0094] FIG12B shows that the bispecific antibodies Bis58 and Bis92 stimulate PBMC to secrete IFNγ in the presence of ASPC1 cells.

[0095] FIG12C shows that the bispecific antibodies Bis57 and Bis59 stimulate PBMC to secrete TNFα in the presence of ASPC1 cells.

[0096] FIG12D shows that the bispecific antibodies Bis58 and Bis92 stimulate PBMC to secrete TNFα in the presence of NUGC4 cells.

[0097] FIG12E shows that the bispecific antibodies Bis57 and Bis59 stimulate PBMC to secrete IL6 in the presence of NUGC4 cells.

[0098] FIG12F shows that the bispecific antibodies Bis58 and Bis92 stimulate PBMC to secrete IL6 in the presence of ASPC1 cells.

[0099] FIG13 shows the pharmacokinetics in wild-type C57 mice.

[0100] FIG14A is a PBMC reconstitution model used to evaluate the tumor inhibition ability of bispecific antibodies.

[0101] FIG14B shows the changes in mouse body weight during the drug administration period. DETAILED DESCRIPTION

[0102] The present application will be further described below with reference to specific examples, and the advantages and features of the present application will become clearer as the description proceeds. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or those recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0103] The embodiments of the present application are merely exemplary and do not constitute any limitation on the scope of the present application. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0104] Definitions and Explanations of Terms

[0105] Unless otherwise defined herein, scientific and technical terms related to the present application shall have the meanings understood by those of ordinary skill in the art.

[0106] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0107] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0108] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the term."

[0109] The term "MUC17" herein refers to a member of the mucin family, which includes over 20 members. Mucins are large, heavily glycosylated, membrane-bound proteins that are expressed almost exclusively in the intestine. Their general function is to protect epithelial cells from environmental influences and to regulate cell proliferation and survival. MUC17 is highly expressed in pancreatic adenocarcinoma tissue. MUC17 is also expressed in pancreatic cancer, appendiceal cancer, and some colon cancers. Its expression is undetectable in normal pancreas, pancreatitis, or cell lines derived from other cancers.

[0110] The term "CD3" (cluster of differentiation 3) herein refers to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). In mammals, the CD3 molecule is a six-chain multiprotein complex, including a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain, wherein the CD3ζ chain is the intracellular tail of the CD3 molecule, and all of the CD3γ, CD3δ, and CD3ε chains contain an extracellular domain (ECD) expressed on the surface of T cells. Exemplary sequences of human CD3 include human CD3ε protein (NCBI Ref Seq No. NP_000724 or NCBI: AAH49847.1), human CD3δ protein (NCBI Ref Seq No. NP_000723), and human CD3γ protein (NCBI Ref Seq No. NP_000064). Exemplary sequences of non-human CD3 include Macaca fascicularis (monkey) CD3 epsilon protein (NCBI Ref Seq No. NP_001270544), Macaca fascicularis (monkey) CD3 delta protein (NCBI Ref Seq No. NP_001274617), Macaca fascicularis (monkey) CD3 gamma protein (NCBI Ref Seq No. NP_001270839); mouse CD3 epsilon protein (NCBI Ref Seq No. NP_031674), mouse CD3 delta protein (NCBI Ref Seq No. NP_038515), mouse CD3 gamma protein (NCBI Ref Seq No. AAA37400); Rattus norvegicus (rat) CD3 epsilon protein (NCBI Ref Seq No. NP_001101610), Rattus norvegicus (rat) CD3 delta protein (NCBI Ref Seq No. No.NP_037301), Rattus norvegicus (rat) CD3γ protein (NCBI Ref Seq No.NP_001071114).

[0111] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an affinity of 1×10 -7 M or lower, about 1×10 -8M or lower, about 1×10 -9 M or lower, about 1×10 -10 M or less, 1×10 -11 M or lower or 1×10 -12 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis. For example, see Example 5 herein for methods for obtaining KD values.

[0112] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0113] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0114] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0115] The term "antibody" herein includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0116] As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0117] As used herein, the term "nanoantibody" refers to a naturally occurring heavy chain antibody lacking a light chain that exists in camels. Cloning its variable region can produce a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0118] The terms "nanobody" and "single domain antibody" (sdAb) are used interchangeably and have the same meaning. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0119] For further description of “heavy chain antibodies” and “nanobodies”, see: Hamers-Casterman et al., Nature. 1993; 363; 446-8; the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO 03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and the other prior art mentioned in these applications.

[0120] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0121] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0122] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0123] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0124] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0125] The term "Fd" herein refers to an antibody composed of a VH and CH1 domain. The term "Fv" herein refers to an antibody fragment composed of a single-arm VL and VH domain. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0126] The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0127] The term "diabody" herein refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0128] The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or a tracer; the term "conjugated antibody" herein refers to an antibody that is conjugated to a therapeutic agent or a tracer.

[0129] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0130] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0131] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0132] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0133] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0134] The term "IMGT numbering system" herein generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0135] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0136] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0137] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0138] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at the Cys226 position or from Pro230 to its carboxyl terminal. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can, for example, be present in the production or purification process of an antibody, or removed by recombinant engineering of a nucleic acid encoding an antibody heavy chain. Therefore, the Fc region may include or may not include Lys447.

[0139] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0140] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0141] 1) Alanine (A), serine (S), threonine (T);

[0142] 2) Aspartic acid (D), glutamic acid (E);

[0143] 3) Asparagine (N), glutamine (Q);

[0144] 4) Arginine (R), Lysine (K), Histidine (H);

[0145] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0146] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0147] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0148] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0149] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0150] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0151] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid (SEQ ID NO: 1) molecule of the present application. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecule of the present application. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0152] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes the nucleotide bases of the modification of the residue of the sugar or phosphate backbone bonding or chemical modification with derivative.Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for in vitro and / or in vivo, such as in a host or patient, directly expressing the antibody of the present application.Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified.For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into the subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823B1).Herein, "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0153] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0154] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0155] The term "pharmaceutical composition" herein refers to a formulation that is in a form that allows for the effective biological activity of the active ingredient contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. The "pharmaceutical composition" may further comprise an additional therapeutic agent, such as an anti-tumor agent; more preferably, the anti-tumor agent may be a PD-1 axis binding antagonist, a small molecule anti-tumor agent, or a cell therapy agent, wherein the cell therapy agent may be CAR-T, CAR-NK, etc.

[0156] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals being treated for a disease or condition.

[0157] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject of treatment, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0158] The term "immunostimulatory antibodies" herein refers to antibodies that promote anti-tumor immunity by directly modulating immune function, i.e., by blocking otherwise inhibitory targets or enhancing immunostimulatory proteins. These include: 1) antagonistic antibodies that target inhibitory immune checkpoints and agonistic antibodies that enhance immunostimulatory proteins.

[0159] The term "immunomodulatory drug" herein may include, for example, thymosin alpha 1. Principle: Thymosin alpha 1 (Tα1) is a naturally occurring thymosin peptide that acts as an endogenous regulator of the innate and adaptive immune systems. It is used worldwide to treat conditions associated with immune dysfunction, including viral infections such as hepatitis B and C, certain cancers, and for vaccine enhancement. In particular, recent advances in immunomodulatory research have indicated a beneficial effect of Tα1 treatment in septic patients (Wu et al., Critical Care, 2013, 17: R8).

[0160] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0161] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0162] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

[0163] Example

[0164] Example 1 Design and Construction of Bispecific Antibodies

[0165] A bispecific antibody molecule targeting MUC17×CD3 was constructed using an anti-MUC17 humanized nanobody sequence and an anti-CD3 humanized antibody sequence, wherein the MUC17 variable region sequence is derived from patent PCT / CN2022 / 126410, and the CD3 antibody variable region sequence is derived from patent (PCT / CN2022 / 118334). The bispecific antibody comprises three chains, namely a heavy chain containing the anti-CD3 humanized antibody VH, a light chain containing the anti-CD3 humanized antibody VL, and a heavy chain of the anti-MUC17 humanized nanobody, and its structure is shown in Figure 1. To reduce homologous mispairing, the molecule adopts an asymmetric structure, and KIH mutations are introduced in the Fc region of each of the two heavy chains. At the same time, to reduce the antibody's ADCC and CDC functions, avoid damage to T cells and potential toxic side effects, mutations L234A, L235A, and G237A are introduced in the heavy chain constant region. The constructed bispecific antibodies were named Bis 57, Bis 58, Bis 59, and Bis 92, and the amino acid sequences of each chain are shown in Table 1. The sequence of the control antibody AMG199 was derived from the published patent WO2019133961, and the specific sequence is shown in Table 1.

[0166] Table 1 Bispecific antibody amino acid sequence list

[0167] Table 2 Variable region sequences of bispecific antibodies

[0168] Table 3 CDR analysis results of bispecific antibodies

[0169] Example 2 Design and expression of MUC17 antigen

[0170] The amino acid sequence encoding a truncated extracellular region of human MUC17 protein (UniProt: Q685J3) was cloned into a His-tagged pTT5 vector (Ubigene, VT2202). Plasmids were prepared using a plasmid extraction kit and transiently expressed in Expi 293F cells (Gibco, A14527) to obtain the antigen and detection protein of this application. The preparation method for the truncated extracellular region of cynomolgus macaque MUC17 protein is similar to that for human recombinant proteins. The cynomolgus macaque MUC17 sequence is from UniProt No. A0A2K5WH09. The specific sequence information of the recombinant protein is shown below:

[0171] Human MUC17 ECD4131-his (His-tagged human MUC17 protein extracellular domain fusion protein) (SEQ ID NO.69):

[0172] Cyno MUC17 ECD3577-mFc (mFc-tagged cynomolgus monkey MUC17 protein extracellular domain fusion protein) (SEQ ID NO.70):

[0173] Example 3 Bispecific Antibody Expression and Purification

[0174] 3.1 Transfection of double antibody plasmid

[0175] The light and heavy chain nucleotide sequences encoding AMG199, Bis57, Bis58, Bis59, and Bis92 were cloned into the pTT5 vector. The plasmids and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (Thermofisher, Catalog No. A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day of transfection, the cell supernatant was collected.

[0176] 3.2 Expression and purification of bispecific antibodies

[0177] 3.2.1 Purification of AMG199 control bispecific antibody

[0178] After collecting the culture supernatant, the protein was purified using AKTA Pure Protein A affinity and molecular sieve purification. The resulting antibodies were quantitatively and qualitatively analyzed by SDS-PAGE, SEC-HPLC, and CE-SDS. The specific purification method is as follows: Initial purification was performed using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The Protein A column was first equilibrated with 3–5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and the clarified culture supernatant was then loaded at a flow rate of 8 mL / min. After loading, the column was washed with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3–5 column volumes. Proteins bound to the Protein A column were eluted with an eluent (20 mM citrate buffer, pH 3.5), and protein elution was monitored by the A280 UV absorbance peak. The eluted protein was collected, neutralized to pH 5-6 by adding 1M pH 8.0 Tris-HCl, and dialyzed into molecular sieve buffer (10 mM Hac, 150 mM NaCl, pH 5.5). Next, the protein was purified using molecular sieves (purchased from Boglund) and the target sample was collected. After concentration, the sample was dialyzed into 559 buffer (10 mM Hac, 9% sucrose, pH 5.5), sterile filtered using a 0.22 μm filter, and stored aseptically to obtain the purified AMG199 antibody.

[0179] 3.2.2 Bispecific Antibody Purification Methods

[0180] After collecting the culture supernatant, the protein was purified using Protein A affinity and KappaSelect affinity purification using AKTA Pure. The resulting antibody was quantitatively and qualitatively analyzed by SEC-HPLC and CE-SDS. The specific purification method is as follows.

[0181] 1. Initial purification was performed using a Protein A column (Mabselect SuRe™, purchased from Cytiva). The Protein A column was equilibrated with 3-5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading, the column was eluted with a high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) for 3-5 column volumes. Protein bound to the Protein A column was eluted with an eluent (20 mM citrate buffer, pH 3.5), and protein elution was monitored by the A280 UV absorbance peak. The eluted protein was collected and neutralized to pH 5-6 by adding 1 M Tris-HCl, pH 8.0.

[0182] 2. Purify using HiTrap KappaSelect (purchased from Cytiva). First, equilibrate with 3 to 5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and then load the pre-purified protein solution at a flow rate of 5 mL / min. After loading, elute with PBS for 3 to 5 column volumes, followed by 10 column volumes of 10 mM phosphate buffer (pH 7.4). Elute the protein bound to the KappaSelect column with eluent (50 mM glycine, pH 3.4), and monitor the protein elution by the A280 UV absorption peak. Collect the eluted protein and neutralize it to pH 5-6 by adding 1 M pH 8.0 Tris-HCl. After concentration, dialyze and exchange the buffer into 559 buffer (10 mM Hac, 9% sucrose, pH 5.5). Sterile filter using a 0.22 μm filter and store aseptically to obtain the purified bispecific antibody.

[0183] 3.3 Purity testing of bispecific antibodies

[0184] 3.3.1 SEC-HPLC analysis

[0185] The SEC-HPLC method was used to analyze the test samples, characterize the molecular size uniformity of the bispecific antibodies, and determine the purity of the bispecific antibodies. The HPLC used in this method was Agilent 1260, the chromatographic column was TSKgel G3000SWXL from Tosoh Bioscience, the mobile phase was 200mM phosphate buffer, pH 7.0 / isopropanol (v / v 9:1) (batch number: 20220616101), the detection temperature was 25°C, the flow rate was 0.5mL / min, the detection wavelength was 280nm, the target protein was diluted 10 times with DI water, the sample load was 50μg, and the analysis time was 40 minutes. For the SEC-HPLC data, the chromatogram was analyzed by manual integration, and the protein purity was calculated according to the area normalization method. The main peak was considered to be a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragment. The purity information of the obtained bispecific antibodies is shown in Table 4 below.

[0186] Table 4 Purity of bispecific antibodies detected by SEC-HPLC

[0187] 3.3.2 CE-SDS analysis

[0188] Non-reducing CE-SDS analysis was used to analyze the test samples, determine the purity of the bispecific antibody, and characterize the size homogeneity of the test samples. The capillary electrophoresis instrument used for this method was an AB Sciex PA 800Plus, with a PDA detector, a detection wavelength of 220 nm, a capillary detection effective length of 20 cm, a protein separation voltage of 15.0 kV, and a 40-min NR-CE SDS detection time. The sample size was 100 μg. For the NR-CE-SDS method, the protein sample was dissolved in SDS Sample Buffer (20 mM PB, 5 mM citric acid, 1% SDS, pH 6.5) to 94 μL. 5 μL of 100 mmol / L NEM and 1 μL of a 10 kDa marker were added, and the sample was incubated at 70°C for 10 min. Chromatograms were analyzed using manual integration of the NR-CE SDS data, and protein purity was calculated using area normalization. The purity information of the obtained bispecific antibodies is shown in Table 5.

[0189] Table 5 Purity of bispecific antibodies detected by NR-CE-SDS

[0190] Example 4 Binding Activity Determination of Bispecific Antibodies

[0191] 4.1 ELISA detection of binding of bispecific antibodies to human and monkey MUC17 proteins

[0192] Human MUC17-his protein was diluted with PBS to a final concentration of 2 μg / mL, then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking buffer [PBS + 2% (w / w) BSA] for 2 hours at room temperature. The blocking buffer was discarded, and 50 μl of a bispecific antibody starting at 27 nM and a 6-fold serial dilution of positive and negative control antibodies were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-conjugated secondary antibody (purchased from Merck, Cat. No. AP113P) was added and incubated at 37°C for 1 hour, followed by washing five times with PBST. TMB substrate (50 μl) was added to each well, incubated at room temperature for 10 minutes, and then stop buffer (1.0 M HCl) was added to each well. The OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). 450nmFigure 2A shows the binding activity of the bispecific antibodies to the human MUC17 protein. The results showed that the negative controls anti-FITC×CD3 and anti-FITC-hIgG1 (derived from the literature J Biol Chem. 1990 Jan 5; 265(1):133-8) did not bind to MUC17, while the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all effectively bound to the human MUC17-his protein.

[0193] The monkey MUC17-mFc protein was subjected to ELISA detection and data analysis according to the method of Example 4.1. The analysis results are shown in Figure 2B, and the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all have good binding activity to the monkey MUC17 protein.

[0194] 4.2 ELISA detection of binding of bispecific antibodies to human and monkey CD3e proteins

[0195] Human CD3e-His protein (Sino Biological, CAT#10977-H08H) was diluted in PBS to a final concentration of 1 μg / mL. 50 μl was then added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking buffer (PBS + 2% (w / w) BSA) for 2 hours at room temperature. The blocking buffer was discarded, and 50 μl of a bispecific antibody starting at 270 nM and a six-fold serial dilution of positive and negative control antibodies were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-conjugated secondary antibody (Merck, Cat. No. AP113P) was added and incubated at 37°C for 1 hour. The plate was then washed five times with PBST. 50 μl of TMB substrate was added to each well and incubated at room temperature for 10 minutes. Then, 50 μl of stop buffer (1.0 M HCl) was added to each well. OD was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) 450nm The binding activity of the bispecific antibodies to human CD3e protein is shown in Figure 3A. The results showed that the negative control anti-FITC-hIgG1 did not bind to CD3e, and the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all effectively bound to the human CD3e-his protein.

[0196] Monkey CD3-his protein (ACRO, CAT#CDE-C5226) was subjected to ELISA and data analysis according to the method of Example 4.2. The analysis results are shown in Figure 3B, and the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all have good binding activity to monkey CD3e protein.

[0197] 4.3 Flow cytometry (FACS) detection of the binding of bispecific antibodies to human MUC17

[0198] Endogenous tumor cells NUGC4 were expanded and cultured in T-175 culture flasks until the logarithmic growth phase. The culture medium was removed and the cells were washed twice with PBS buffer. The cells were trypsinized and then digested with complete culture medium. The cells were pipetted to a single cell suspension. After cell counting, the cells were centrifuged and the cell pellet was resuspended in FACS buffer (PBS + 2% fetal bovine serum) to 2×10 6 For every milliliter of cells, 100 μl was added to each well of a 96-well FACS reaction plate, centrifuged, the supernatant was discarded, 50 μl of the antibody sample to be tested (100 nM as the starting concentration, 3-fold serial dilution) was added to each well, mixed with the cells, and incubated at 4°C for 1 hour. Washed three times by centrifugation with PBS buffer, 50 μl of Alexa Fluor® was added to each well. Incubate with 647AffiniPure Goat Anti-Human IgG, an Fcγ fragment-specific secondary antibody (purchased from Jackson, Cat. No. 109-605-098) at 4°C for 1 hour. Wash cells three times with PBS buffer by centrifugation, resuspend in 100 μl of PBS, and analyze using FACS (FACS Canto™, purchased from BD Biosciences). Data were analyzed using FlowJo software to obtain the mean fluorescence intensity (MFI). GraphPad Prism 8 software was then used for data fitting and EC50 calculation. As shown in Figure 4A, the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all specifically bound to NUGC4 cells.

[0199] The same method was used to detect the binding of bispecific antibodies to the gastric cancer cell line SNU16 and the pancreatic cancer cell line ASPC1, which both moderately express MUC17 ( Figure 10 , showing the expression levels of MUC17 in NUGC4 cells, SNU16 cells, and ASPC1 cells). The results showed that the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all had excellent binding activity to SNU16 ( Figure 4B ) and ASPC-1 ( Figure 4C ), and their binding ability was stronger than that of AMG199.

[0200] 4.4 Flow cytometry (FACS) detection of the binding of bispecific antibodies to MUC17-overexpressing cells

[0201] The nucleotide sequence encoding the human MUC17 fragment was cloned into the pcDNA5 vector (purchased from Universal), and plasmids were prepared to construct an overexpression cell line. Monoclonal cell lines with high fluorescence intensity were selected for subsequent testing. The constructed overexpression cell line was named FlpinCHO-huMUC17-D1.

[0202] FlpinCHO-huMUC17-D1 was subjected to FACS analysis and data analysis according to the method of Example 4.3. The antibody samples were diluted threefold using a starting concentration of 1350 nM. As shown in Figure 5A , the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 effectively bound to FlpinCHO-huMUC17-D1 cells. Recombinant cells overexpressing monkey MUC17 (FlpinCHO-cynoMUC17-D1) were subjected to FACS analysis and data analysis according to the same method. As shown in Figure 5B , the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 effectively bound to FlpinCHO-cynoMUC17-D1 cells.

[0203] 4.5 Flow cytometry (FACS) detection of the binding of bispecific antibodies to Jurkat cells

[0204] To test the binding ability of bispecific antibodies to cell surface CD3, we used FACS to perform binding assays on Jurkat cells. Jurkat cells were cultured and harvested. Antibody samples were diluted three-fold starting at a concentration of 1350 nM and analyzed by FACS using the same protocol as in Example 4.3. The results, shown in Figure 6, show that bispecific antibodies Bis57, Bis58, Bis59, and Bis92 effectively bound to Jurkat cells, albeit with weaker binding than AMG199. This weak binding to CD3 may help mitigate drug side effects.

[0205] 4.6 Flow cytometry (FACS) detection of the binding of bispecific antibodies to human and monkey PBMCs

[0206] After overnight culture, human PBMC (SailyBio, CAT#XFB-HP010B) and monkey PBMC (Hekai Bio, CAT#5208) were collected, Fc Block (BD, 564220) was added, and FACS detection and data analysis were performed according to the method of Example 4.3. The results are shown in Figures 7A to 7B. The bispecific antibodies Bis57, Bis58, Bis59, and Bis92 can effectively bind to human and monkey PBMC cells, but the binding ability is weaker than that of AMG199. The weaker CD3 binding ability helps to reduce the cytokine storm of the drug.

[0207] 4.7 Luciferase reporter gene assay to detect the effect of bispecific antibodies on T cell activity

[0208] NUGC4 (MUC17 positive cells) and Jurkat-luc cells (Jurkat cells overexpressing luciferase gene) were cultured and collected, and resuspended to 5×10 4 / 25μL. The antibody sample to be tested was diluted 4-fold with a starting concentration of 25nM. At the same time, 25μL of NUGC4 and 25μL of Jurkat-luc cells were mixed with 50μL of antibody diluent and added to a 96-well white-bottom microplate (corning, CAT#3610). After incubation at 37°C for 5 hours, 50μL of Nano-Glo Luciferase Reagent (Promega, CAT#N1130) was added. After incubation at room temperature at 450rpm for 10 minutes, the results were read using an Envision microplate reader (Perkin Elmer, Envision2105). As shown in Figure 8A, the bispecific antibodies Bis57, Bis58, Bsi59, and Bis92 were all able to effectively activate Jurkat cells. The same method was used to detect the activation of MDA-231 (MUC17-negative cells) after co-incubation with Jurkat-luc. The results are shown in Figure 8B. None of the antibodies were able to activate Jurkat cells. This suggests that the activation of T cells by MUC17 / CD3 bispecific antibody is specifically mediated by MUC17.

[0209] 4.8 In vitro testing of bispecific antibodies for T cell activation in tumor cell killing assays

[0210] SNU16 tumor cells were seeded in a 96-well plate at a ratio of 1:10 to PBMCs (purchased from Allcells, Cat. No. PB004F-C). 5,000 tumor cells were plated in 50 μL / well and 50,000 PBMCs were plated in 50 μL / well, for a total volume of 100 μL / well. The test antibody was diluted to the desired concentration in RPMI-1640 medium and added to the plate at a volume of 10 μL / well. The plate was incubated at 37°C, 5% CO₂ in a humidified incubator for 48 hours. After 48 hours, the plate was removed from the plate, allowed to return to room temperature, and then centrifuged at 350 g for 7 minutes, discarding the supernatant. The plate was rinsed once with 100 μL of PBS and centrifuged at 350 g for 5 minutes. The supernatant was discarded and this step repeated once. The detection antibody was diluted with PBS containing 1% BSA at a ratio of 1:60, and 60 μL of the above detection antibody dilution solution was added to each well, and the cells were stained at 4°C for 30 minutes. The antibody FITC anti-human CD3 (purchased from BD, product number 300440) was used to label T cells, the antibody APC anti-human CD4 (purchased from BD, product number 300537) was used to label CD4-positive cells, and the antibody Brilliant Violet 421 was used to label CD4-positive cells. TM Anti-human CD69 (purchased from BD, Catalog No. 310930) was used to label CD69-positive cells, and PE anti-human CD25 (purchased from BD, Catalog No. 302606) was used to label CD25-positive cells. After staining, the cells were centrifuged at 350g for 5 minutes, the supernatant discarded, and the cells were rinsed twice with PBS. Flow cytometry analysis was then performed directly. As shown in Figure 9, in the T cell activation assay, Bis 57 and Bis 59 upregulated CD69 and CD25 on both CD4-positive and CD8-positive T cells less than the control antibody AMG199.

[0211] Example 5 Affinity detection of bispecific antibodies

[0212] The binding strength of antibodies to antigens was determined using a Protein A capture assay using a BIAcore 8K instrument. Protein A was first immobilized onto a CM4 chip (GE, BR-1005-34) using the amino coupling method. Following the instructions for the Amine Coupling Kit (GE, BR100633), the chip was activated for approximately 600 seconds using a mixture of NHS and EDC using HBS-EP+, pH 7.4, as the mobile phase. Protein A was then diluted to 50 μg / mL with 10 mM sodium acetate, pH 4.5, and injected for 600 seconds. Finally, any remaining active sites were blocked with ethanolamine. Then, the affinity between the antibody and the antigen was determined by a multi-cycle kinetic method. In each cycle, the antibody to be tested was first captured using a Protein A chip, and then a single concentration of antigen protein was injected, and the binding and dissociation processes of the antibody and antigen protein were recorded. Finally, the chip was regenerated with Glycine pH 1.5. The mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate was 30μL / min, the regeneration time was 30s, and the detection temperature was 25°C. Finally, according to the 1:1 binding model, the data were analyzed and the antibody-antigen binding kinetic parameters were fitted, including the association rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax. The binding rates (Ka), dissociation rates (Kd), and binding affinities (KD) of the bispecific antibodies Bis 57, Bis 58, Bis 59, Bis 92, and AMG199 to the MUC17 protein are shown in Table 6.

[0213] Table 6: Affinity of bispecific antibodies to MUC17 protein detected by SPR (Biacore)

[0214] Example 6: Bispecific Antibody-Mediated Tumor Cell Killing in Vitro

[0215] Bispecific antibody-mediated PBMC killing of tumor cells was performed by quantitatively measuring cell proliferation. Cell Titer glo was used to measure ATP content in cells. ATP is an indicator of viable cell metabolism and is directly proportional to the number of cells in culture. Four different tumor cell lines were used, including three tumor cell lines with moderate MUC17 expression (SNU16, NUGC4, and ASPC-1; MUC17 expression levels are shown in Figure 10) and a MUC17-negative control cell line, MDA-MB-231.

[0216] 5,000 tumor cells / well and 50,000 PBMCs (Allcells, PB004F-C) / well were diluted with RPMI Medium 1640 containing 10% FBS and added to a 96-well plate. Control and test antibodies were diluted to varying concentrations in RPMI Medium 1640 and added to the 96-well plate. The final antibody concentration in the reaction system started at 5 nM and was serially diluted 4-fold. After incubation of the CD3 bispecific antibody with PBMCs and tumor cells at 37°C in a 5% CO2 incubator for 2 days, the ATP in live cells was quantified using the CTG kit (Promega, G7573), reflecting antibody-mediated tumor cell killing by PBMCs. Tumor cell killing rate = (100 * (culture medium well - experimental well) / (culture medium well - PBMC well))%. As shown in Figures 11A to 11E , the bispecific antibodies Bis57, Bis58, Bis59, and Bis92 all showed significant cytotoxicity against MUC17-positive tumor cells. Specific cytotoxicity data are shown in Table 7 below.

[0217] Table 7 PBMC-mediated in vitro tumor cell killing EC50

[0218] Note: NT stands for Not Rated

[0219] Example 7: Bispecific Antibody-Mediated Cytokine Secretion

[0220] T cells are activated under the mediation of bispecific antibodies and release cytokines while killing target cells. The cell supernatant in Example 6 was collected, centrifuged at 3000rpm for 10 minutes, and then frozen in a -80 degree refrigerator for use. The secretion levels of cytokines IFNγ (Cisbio, 62HIFNGPEH) and TNFα (Cisbio, 62HTNFAPEH) in the cell supernatant were determined by HTRF. The secretion level of IL-6 (BD, 555220) was detected by ELISA. The steps are detailed in the instructions in the kit. The results are shown in Figures 12A to 12F. The bispecific antibodies Bis57, Bis58, Bis59 and Bis92 can effectively induce PBMC to secrete IFNγ, TNFα and IL6 when PBMC and MUC17-positive tumor cells coexist, and the cytokine secretion is significantly lower than that of AMG199. The level of cytokine release reflects the activity of the multispecific antibody, which is generally positively correlated with the activation level of T cells, as well as the in vitro killing activity and in vivo tumor suppression activity. At the same time, the stronger the T cell activation activity of the multispecific antibody, the higher the risk of cytokine storm (CRS) after entering the human body.

[0221] Example 8: Pharmacokinetics of bispecific antibodies in mice

[0222] To compare the pharmacokinetic differences between different antibodies, SPF female wild-type C57 mice, 6-8 weeks old, weighing approximately 18-20 g, were used in this example. Three mice were used in each group and a single tail vein injection of a 1 mg / kg dose of a control antibody (AMG199) and the bispecific antibodies (Bis57 and Bis59) prepared in Example 3 was administered.

[0223] Mice were maintained on a standard diet and were not deprived of food or water. Drugs were diluted with normal saline. Orbital blood was collected before dosing and at 0.25, 2, 8, 24, 72, 120, 168, 240, 336, 504, and 672 hours after administration. Blood samples were placed in microtubes and allowed to rest for approximately 30 minutes. The samples were then centrifuged at 12,000 rpm for 5 minutes at 4°C. Serum was separated into low-absorption centrifuge tubes, labeled with the compound code and time point, and stored frozen at -80°C until analysis.

[0224] The human MUC17 protein was coated, and the concentration of antibodies in the serum of each control and test group was determined by indirect enzyme-linked immunosorbent assay. Pharmacokinetic parameters were calculated based on the blood drug concentration of each animal at different time points. Specific results are shown in Table 8 and Figure 13. The results show that the molecules of the present application have good PK performance. The half-lives of Bis 57 and Bis59 were 248 and 309 hours, respectively, which were significantly higher than the 130 hours of the control group AMG199. The terminal exposure of Bis 57 and Bis59 was 1575 and 1825 h*μg / mL, slightly higher than the 1118 h*μg / mL of AMG199.

[0225] Table 8 Pharmacokinetic parameters of wild-type C57 mice

[0226] Example 9: PBMC-reconstructed mouse efficacy model

[0227] The anti-tumor efficacy of the bispecific antibody in vivo was evaluated using a human PBMC-reconstructed NPG mouse model (NPG: female 5-6 weeks, Beijing Weitongda Biotechnology Co., Ltd.). NUGC4 cells were cultured to the logarithmic growth phase, collected by centrifugation, and plated at 10×10 6 / mouse were subcutaneously inoculated, and human PBMC cells were resuscitated on the same day and cultured at 5×10 6 / mice were intravenously inoculated. The mice were then raised normally. When the tumor volume of the tumor-bearing mice reached 100mm 3Around 14:00, mice were randomly divided into groups of 8 per group, and the reconstruction rate was tested simultaneously. The day of grouping was defined as day 0 of the experiment. Subsequently, the test antibody and control antibody were intravenously administered twice a week at equimolar doses for a total of 6 times. Tumors were observed and measured twice a week, and tumor volume was calculated according to the following formula: Tumor volume (TV) = 1 / 2 (height * width) 2 The width was defined as the smaller of the two measurements, and the height was defined as the larger of the two measurements. Throughout the dosing period, the weight of the mice was recorded twice weekly, and the weight change was calculated. As shown in Figures 14A and 14B , after one week of dosing, both the bispecific antibodies Bis57 and Bis59 were able to effectively inhibit tumor growth, with no significant change in mouse weight during the dosing period.

[0228] Table 9 Effects of the test substances on tumor volume in mice transplanted with human PBMCs using NUGC4 cells

Claims

1. An anti-MUC17 / anti-CD3 bispecific antibody comprising: (a) a first antigen binding portion, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL form an anti-CD3 antigen binding domain; wherein, The anti-CD3 antigen binding domain comprises HCDR1, HCDR2 and HCDR3 in VH as shown in SEQ ID NO.9 and LCDR1, LCDR2 and LCDR3 in VL as shown in SEQ ID NO.10; and (b) a second antigen binding portion, which comprises a VHH that specifically binds to MUC17, wherein the VHH comprises: CDR1, CDR2 and CDR3 in the sequence shown in SEQ ID NO.11, or CDR1, CDR2 and CDR3 in the sequence shown in SEQ ID NO.12, or CDR1, CDR2 and CDR3 in the sequence shown in SEQ ID NO.13, or CDR1, CDR2 and CDR3 in the sequence shown in SEQ ID NO.

14.

2. The bispecific antibody according to claim 1, wherein: (1) Based on the Kabat numbering system, the HCDR1 of the first antigen-binding portion comprises the sequence shown in SEQ ID NO.15; the HCDR2 comprises the sequence shown in SEQ ID NO.16; and the HCDR3 comprises the sequence shown in SEQ ID NO.17; or Based on the Chothia numbering system, the HCDR1 of the first antigen-binding portion comprises the sequence shown in SEQ ID NO.33; the HCDR2 comprises the sequence shown in SEQ ID NO.34; and the HCDR3 comprises the sequence shown in SEQ ID NO.35; or Based on the IMGT numbering system, the HCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.51; HCDR2 comprises the sequence shown in SEQ ID NO.52; HCDR3 comprises the sequence shown in SEQ ID NO.53; and / or (2) Based on the Kabat numbering system, LCDR1 of the first antigen-binding portion comprises the sequence shown in SEQ ID NO.18; LCDR2 comprises the sequence shown in SEQ ID NO.19; and LCDR3 comprises the sequence shown in SEQ ID NO.20; or Based on the Chothia numbering system, LCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.36; LCDR2 comprises the sequence shown in SEQ ID NO.37; LCDR3 comprises the sequence shown in SEQ ID NO.38; or Based on the IMGT numbering system, LCDR1 of the first antigen binding portion comprises the sequence shown in SEQ ID NO.54; LCDR2 comprises the sequence shown in SEQ ID NO.55; and LCDR3 comprises the sequence shown in SEQ ID NO.

56.

3. The bispecific antibody according to claim 1, wherein: Based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.21; CDR2 comprises the sequence shown in SEQ ID NO.22; and CDR3 comprises the sequence shown in SEQ ID NO.23; or Based on the Kabat numbering system, the CDR1 of the second antigen binding portion comprises the CDR1 shown in SEQ ID NO. CDR2 comprises the sequence shown in SEQ ID NO.25; CDR3 comprises the sequence shown in SEQ ID NO.26; or Based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.27; the CDR2 comprises the sequence shown in SEQ ID NO.28; and the CDR3 comprises the sequence shown in SEQ ID NO.29; or Based on the Kabat numbering system, the CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.30; the CDR2 comprises the sequence shown in SEQ ID NO.31; and the CDR3 comprises the sequence shown in SEQ ID NO.32; or Based on the Chothia numbering system, CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.39; CDR2 comprises the sequence shown in SEQ ID NO.40; and CDR3 comprises the sequence shown in SEQ ID NO.41; or Based on the Chothia numbering system, CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.42; CDR2 comprises the sequence shown in SEQ ID NO.43; CDR3 comprises the sequence shown in SEQ ID NO.44; or Based on the Chothia numbering system, CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.45; CDR2 comprises the sequence shown in SEQ ID NO.46; and CDR3 comprises the sequence shown in SEQ ID NO.47; or Based on the Chothia numbering system, CDR1 of the second antigen-binding portion comprises the sequence shown in SEQ ID NO.48; CDR2 comprises the sequence shown in SEQ ID NO.49; and CDR3 comprises the sequence shown in SEQ ID NO.50; or Based on the IMGT numbering system, CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.57; CDR2 comprises the sequence shown in SEQ ID NO.58; CDR3 comprises the sequence shown in SEQ ID NO.59; or Based on the IMGT numbering system, CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.60; CDR2 comprises the sequence shown in SEQ ID NO.61; and CDR3 comprises the sequence shown in SEQ ID NO.62; or Based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.63; CDR2 comprises the sequence shown in SEQ ID NO.64; and CDR3 comprises the sequence shown in SEQ ID NO.65; or Based on the IMGT numbering system, the CDR1 of the second antigen binding portion comprises the sequence shown in SEQ ID NO.66; CDR2 comprises the sequence shown in SEQ ID NO.67; and CDR3 comprises the sequence shown in SEQ ID NO.

68.

4. The bispecific antibody according to claim 2, wherein: The first antigen binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequence: (1) based on the Kabat numbering system, the sequences shown in SEQ ID NOs. 15, 16, 17, 18, 19 and 20, respectively; or (2) Based on the Chothia numbering system, they are shown in SEQ ID NOs. 33, 34, 35, 36, 37 and 38, respectively. or (3) sequences represented by SEQ ID NOs. 51, 52, 53, 54, 55 and 56, respectively, based on the IMGT numbering system; or (4) A sequence having at least 90% identity with the sequence shown in (1) to (3) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

5. The bispecific antibody according to claim 3, wherein The second antigen binding portion comprises CDR1, CDR2 and CDR3 of the following sequence: (1) based on the Kabat numbering system, the sequences shown in SEQ ID NOs. 21, 22 and 23, respectively; or (2) based on the Kabat numbering system, the sequences shown in SEQ ID NOs. 24, 25 and 26, respectively; or (3) based on the Kabat numbering system, the sequences shown in SEQ ID NOs. 27, 28 and 29, respectively; or (4) the sequences shown in SEQ ID NOs. 30, 31 and 32, respectively, based on the Kabat numbering system; or (5) based on the Chothia numbering system, the sequences shown in SEQ ID NOs. 39, 40 and 41, respectively; or (6) based on the Chothia numbering system, the sequences shown in SEQ ID NOs. 42, 43 and 44, respectively; or (7) based on the Chothia numbering system, the sequences shown in SEQ ID NOs. 45, 46 and 47, respectively; or (8) based on the Chothia numbering system, the sequences shown in SEQ ID NOs. 48, 49 and 50, respectively; or (9) the sequences represented by SEQ ID NOs. 57, 58 and 59, respectively, based on the IMGT numbering system; or (10) based on the IMGT numbering system, the sequences shown in SEQ ID NOs. 60, 61 and 62, respectively; or (11) based on the IMGT numbering system, the sequences shown in SEQ ID NOs. 63, 64 and 65, respectively; or (12) based on the IMGT numbering system, the sequences shown in SEQ ID NOs. 66, 67 and 68, respectively; or (13) A sequence having at least 90% identity with the sequence shown in (1) to (12) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

6. The bispecific antibody according to any one of claims 1 to 5, wherein The VH of the first antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.9; the VL of the first antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.

10.

7. The bispecific antibody according to any one of claims 1 to 6, wherein The second antigen binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs. 11-14.

8. The bispecific antibody according to any one of claims 1 to 7, wherein The bispecific antibody comprises a heavy chain comprising an anti-CD3 VH, a light chain comprising an anti-CD3 VL, and a heavy chain comprising an anti-MUC17 VHH.

9. The bispecific antibody according to claim 8, wherein The heavy chain of the anti-CD3 VH includes a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.1, the light chain of the anti-CD3 VL includes a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.2, and the heavy chain of the anti-MUC17 VHH includes a sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO.3, 4, 5 or 6. 10 . The bispecific antibody according to claim 1 , which is a humanized antibody.

11. The bispecific antibody according to any one of claims 1 to 10, which specifically binds to human or monkey MUC17 protein; preferably, the KD of the bispecific antibody binding to human or monkey MUC17 is better than 1.00E-9M.

12. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule of claim 12.

14. A host cell comprising the vector of claim 13; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterial, fungal, insect cell or mammalian cell. 15 . A method for preparing the bispecific antibody of any one of claims 1 to 11 , comprising culturing the host cell of claim 14 , and isolating the bispecific antibody expressed by the host cell.

16. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 11, or the nucleic acid molecule according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, or the product prepared by the method according to claim 15, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, further comprising an additional therapeutic agent; preferably, the additional therapeutic agent is an anti-tumor agent; more preferably, the anti-tumor agent is a PD-1 axis binding antagonist, a small molecule anti-tumor agent or a cell therapy agent.

18. Use of the bispecific antibody according to any one of claims 1 to 11, or the nucleic acid molecule according to claim 12, or the vector according to claim 13, or the host cell according to claim 14, or the product obtained by the method according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating cancer, tumor or infectious disease; wherein the cancer or tumor is selected from solid tumors and blood tumors.

19. The use of claim 18, wherein the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor.

20. The method of claim 18, wherein the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer, and central nervous system cancer.

21. The use according to claim 18, wherein The drug is used in combination with another therapeutic agent or with surgery; wherein the other therapeutic agent or the surgery is selected from radiotherapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, surgery, immunostimulatory antibodies, immunomodulatory drugs, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.

22. A method for treating cancer, a tumor or an infectious disease, comprising administering to a patient in need thereof an effective amount of the bispecific antibody of any one of claims 1 to 11, or the nucleic acid molecule of claim 12, or the vector of claim 13, or the host cell of claim 14, or the product prepared by the method of claim 15, or the pharmaceutical composition of claim 16; wherein the cancer or tumor is selected from solid tumors and blood tumors.

23. The method of claim 22, wherein the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor.

24. The method of claim 22, wherein the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer, and central nervous system cancer.

25. The bispecific antibody of any one of claims 1 to 11, or the nucleic acid molecule of claim 12, or the vector of claim 13, or the host cell of claim 14, or the product obtained by the method of claim 15, or the pharmaceutical composition of claim 16, for pre-treatment of cancer, tumor or infectious disease; wherein the cancer or tumor is selected from solid tumors and blood tumors.

26. The bispecific antibody, nucleic acid molecule, vector, host cell, product or pharmaceutical composition of claim 25, wherein the cancer or tumor is a MUC17-positive cancer or a MUC17-positive tumor.

27. The bispecific antibody, nucleic acid molecule, vector, host cell, product or pharmaceutical composition of claim 25, wherein the cancer or tumor is selected from the group consisting of gastric cancer, pancreatic cancer, small intestine cancer, large intestine cancer, rectal cancer, colon cancer, colorectal cancer, esophageal cancer, breast cancer, non-small cell lung cancer, adenocarcinoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, thyroid cancer, bladder cancer, cervical cancer, blood cancer, skin cancer, epithelial cancer, brain cancer and central nervous system cancer.

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