Anti-HER2 / TROP2 antibodies and uses thereof

Abstract

The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates.

Description

Technical Field

[0001] The present disclosure relates to antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (eg, bispecific antibodies), and antibody drug conjugates.

[0002] Sequence Listing

[0003] This application contains a sequence listing which has been filed herewith and is hereby incorporated by reference in its entirety. The .xml copy created on November 28, 2023 is named 52501-0006WO1 and is 41,620 bytes in size. Background Art

[0004] Human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER2 is overexpressed in various cancer types such as breast cancer and gastric cancer, and has been reported to be a negative prognostic factor in breast cancer.

[0005] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. TROP2 is a protein closely associated with tumors. It primarily promotes tumor cell growth, proliferation, and metastasis by regulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, pancreatic cancer, oral squamous cell carcinoma, and ovarian cancer. This protein can promote tumor cell proliferation, invasion, metastasis, spread, and other processes.

[0006] Given the important roles of HER2 and TROP2 in cancer, there is a need to develop therapeutic agents targeting HER2 and / or TROP2. Summary of the Invention

[0007] The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates involving these antibodies or antigen-binding fragments thereof.

[0008] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to HER2 (human epidermal growth factor receptor 2), the antibody or antigen-binding fragment thereof comprising:

[0009] a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence,

[0010] The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 22, 23 and 24, respectively.

[0011] In some embodiments, the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 22, 23, and 24, respectively.

[0012] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to HER2, comprising a heavy chain antibody variable domain (VHH) comprising an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is set forth in SEQ ID NO: 1.

[0013] In some embodiments, the antibody or antigen-binding fragment specifically binds to HER2.

[0014] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0015] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises human IgG Fc.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0018] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described herein.

[0019] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to TROP2 (tumor-associated calcium signal transducer 2), the antibody or antigen-binding fragment thereof comprising:

[0020] a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence;

[0021] wherein the selected VHH CDR1, 2 and 3 amino acid sequences are one of the following:

[0022] (1) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 26, and 27, respectively;

[0023] (2) the selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, and 30, respectively; and

[0024] (3) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively.

[0025] In some embodiments, the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 25, 26, and 27, respectively.

[0026] In some embodiments, the VHH comprises CDR1, 2, 3 whose amino acid sequences are shown in SEQ ID NOs: 28, 29, and 30, respectively.

[0027] In some embodiments, the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 31, 32, and 33, respectively.

[0028] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NO: 2-12.

[0029] In some embodiments, the VHH comprises the sequence of SEQ ID NO:2.

[0030] In some embodiments, the VHH comprises the sequence of SEQ ID NO:3.

[0031] In some embodiments, the VHH comprises the sequence of SEQ ID NO:4.

[0032] In some embodiments, the VHH comprises the sequence of SEQ ID NO:10.

[0033] In some embodiments, the VHH comprises the sequence of SEQ ID NO:12.

[0034] In some embodiments, the antibody or antigen-binding fragment specifically binds to TROP2.

[0035] In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

[0036] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof described herein.

[0037] In some embodiments, the antibody or antigen-binding fragment comprises human IgG Fc.

[0038] In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0039] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof described herein.

[0040] In one aspect, the present disclosure relates to a multispecific antibody or antigen-binding fragment thereof, comprising: a first VHH (VHH1) that specifically binds to HER2; and a second VHH (VHH2) that specifically binds to TROP2.

[0041] In some embodiments, the multispecific antibody or antigen-binding fragment thereof further comprises: a third VHH (VHH3), wherein the VHH3 specifically binds to HER2; and a fourth VHH (VHH4), wherein the VHH4 specifically binds to TROP2.

[0042] In some embodiments, the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are Figure 33 Listed in.

[0043] In some embodiments, the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is set forth in SEQ ID NO:1.

[0044] In some embodiments, the VHH2 and / or the VHH4 comprise complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are Figure 34 Listed in.

[0045] In some embodiments, the VHH3 and the VHH4 comprise an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 2-12.

[0046] In some embodiments, the multispecific antibody or antigen-binding fragment thereof comprises human IgG Fc.

[0047] In some embodiments, the VHH1 and the VHH3 are linked to the N-terminus or the C-terminus of the human IgG Fc.

[0048] In some embodiments, said VHH2 and said VHH4 are linked to the N-terminus or C-terminus of said human IgG Fc.

[0049] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0050] (a) a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain antibody variable domain (VHH1), a first hinge region, a first CH2, a first CH3, and a second VHH (VHH2); and

[0051] (b) a second polypeptide comprising, from N-terminus to C-terminus, a third VHH (VHH3), a second hinge region, a second CH2, a second CH3 and a fourth VHH (VHH4),

[0052] wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

[0053] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 18; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 18.

[0054] In some embodiments, the VHH2 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence.

[0055] In some embodiments, the VHH4 is connected to the C-termini of the second CH2 and the second CH3 via a second linker peptide sequence.

[0056] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

[0057] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0058] (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and

[0059] (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3,

[0060] wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

[0061] In some embodiments, the VHH1 is connected to the N-terminus of the VHH2 via a first linker peptide sequence.

[0062] In some embodiments, the VHH3 is linked to the N-terminus of the VHH4 via a second linker peptide sequence.

[0063] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 17; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 17.

[0064] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

[0065] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0066] (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH2, VHH1, a first hinge region, a first CH2, and a first CH3; and

[0067] (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH4, VHH3, a second hinge region, a second CH2, and a second CH3,

[0068] wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

[0069] In some embodiments, the VHH2 is linked to the N-terminus of the VHH1 via a first linker peptide sequence.

[0070] In some embodiments, the VHH4 is linked to the N-terminus of the VHH3 via a second linker peptide sequence.

[0071] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 15 or 21; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 15 or 21.

[0072] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

[0073] In one aspect, the present disclosure relates to a polypeptide complex comprising

[0074] (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH2, a first hinge region, a first CH2, a first CH3, and VHH1; and

[0075] (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH4, a second hinge region, a second CH2, a second CH3 and VHH3,

[0076] wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

[0077] In some embodiments, the first polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 16 or 20; and / or wherein the second polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 16 or 20.

[0078] In some embodiments, the VHH1 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence.

[0079] In some embodiments, the VHH3 is connected to the C-terminus of the second CH2 and the second CH3 via a second linker peptide sequence.

[0080] In some embodiments, the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

[0081] In some embodiments, the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are Figure 33 Listed in.

[0082] In some embodiments, the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is set forth in SEQ ID NO:1.

[0083] In some embodiments, the VHH2 and / or the VHH4 comprise complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR 1, 2, and 3 amino acid sequences are Figure 34 Listed in.

[0084] In some embodiments, said VHH2 and / or said VHH4 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein said selected VHH sequence is selected from the group consisting of SEQ ID NOs: 2-12.

[0085] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein.

[0086] In some embodiments, the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

[0087] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0088] In one aspect, the disclosure relates to a cell comprising a vector described herein.

[0089] In some embodiments, the cells are CHO cells.

[0090] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0091] In one aspect, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising

[0092] (a) culturing the cells described herein under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment; and

[0093] (b) collecting the antibody or the antigen-binding fragment produced by the cell.

[0094] In one aspect, the present disclosure relates to a T cell engager (TCE) comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein.

[0095] In one aspect, the present disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein.

[0096] In one aspect, the present disclosure relates to a CAR-T, CAR-NK or CAR-NKT cell comprising a CAR described herein.

[0097] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein covalently bound to a therapeutic agent.

[0098] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0099] In some embodiments, the drug to antibody ratio (DAR) is 4.

[0100] In one aspect, the present disclosure relates to a method of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, a polypeptide complex described herein, a TCE described herein, a CAR described herein, a CAR-T or CAR-NK or CAR-NKT cell described herein, or an antibody drug conjugate described herein.

[0101] In some embodiments, the subject has a HER2-expressing cancer.

[0102] In some embodiments, the subject has a cancer that expresses TROP2.

[0103] In some embodiments, the cancer is gastric cancer, cervical cancer, esophageal cancer, thyroid cancer, bile duct cancer, colon cancer, rectal cancer, lung cancer, breast cancer, kidney cancer, hepatocellular carcinoma, renal cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or an advanced solid tumor.

[0104] In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0105] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, a polypeptide complex described herein, a TCE described herein, a CAR described herein, a CAR-T or CAR-NK or CAR-NKT cell described herein, or an antibody drug conjugate described herein.

[0106] In one aspect, the present disclosure relates to a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, a polypeptide complex described herein, a TCE described herein, a CAR described herein, a CAR-T or CAR-NK or CAR-NKT cell described herein, or an antibody-drug conjugate described herein.

[0107] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, a polypeptide complex described herein, a TCE described herein, a CAR described herein, a CAR-T or CAR-NK or CAR-NKT cell described herein, or an antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.

[0108] In one aspect, the present disclosure relates to an engineered antibody or antigen-binding fragment thereof comprising a serine residue at position 220 of the heavy chain according to EU numbering.

[0109] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an engineered hinge region covalently bound to a therapeutic agent, wherein the engineered hinge region comprises a C220S mutation, wherein the therapeutic agent is bound to the cysteine ​​residue at position 226 or 229 according to EU numbering.

[0110] In one aspect, the present disclosure relates to an antibody drug conjugate (ADC) comprising an engineered hinge region covalently bound to a therapeutic agent, wherein the engineered hinge region comprises a serine at position 220, wherein the therapeutic agent is bound to a cysteine ​​residue at position 226 or 229 according to EU numbering.

[0111] In some embodiments, the drug to antibody ratio (DAR) is 1-4.

[0112] In some embodiments, the ADC further comprises a VHH, wherein the VHH is linked to the engineered hinge region.

[0113] In some embodiments, the ADC comprises:

[0114] (1) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH2, a second hinge region, a second CH2, and a second CH3;

[0115] (2) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3; or

[0116] (3) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, a second hinge region, a second CH2, a second CH3, and VHH4.

[0117] In one aspect, the present disclosure relates to an antibody drug conjugate (ADC) comprising an engineered hinge region covalently bound to a therapeutic agent, wherein the therapeutic agent is bound to a cysteine ​​residue at position 220, 226, or 229 according to EU numbering.

[0118] In some embodiments, the drug to antibody ratio (DAR) is 4-6.

[0119] In some embodiments, the ADC comprises:

[0120] (1) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH2, a second hinge region, a second CH2, and a second CH3;

[0121] (2) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3; or

[0122] (3) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, a second hinge region, a second CH2, a second CH3, and VHH4.

[0123] In one aspect, the present disclosure relates to a polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH2, a second hinge region, a second CH2, and a second CH3.

[0124] In one aspect, the present disclosure relates to a polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, a second hinge region, a second CH2, a second CH3, and VHH4.

[0125] In one aspect, the present disclosure relates to a polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3.

[0126] In some embodiments, the VHH1 and the VHH3 specifically bind to a first tumor antigen, and the VHH2 and the VHH4 specifically bind to a second tumor antigen.

[0127] In one aspect, the present disclosure relates to an antibody drug conjugate (ADC) comprising a polypeptide complex described herein covalently bound to a payload.

[0128] In some embodiments, the payload is selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radionuclide, a biologically active protein, a synthetic polymer, an enzyme, a nucleic acid (eg, DNA or RNA), and fragments thereof.

[0129] In some embodiments, each of the first hinge region and the second hinge region consists of 2 cysteines.

[0130] In some embodiments, each of the first hinge region and the second hinge region consists of 3 cysteines.

[0131] In some embodiments, each of the first hinge region and the second hinge region consists of 2 cysteines, wherein the drug to antibody ratio (DAR) is 1.0-4.0.

[0132] In some embodiments, each of the first hinge region and the second hinge region consists of 3 cysteines, wherein the drug to antibody ratio (DAR) is 4.0-6.0.

[0133] In one aspect, the disclosure relates to a method of diagnosing a disease or condition, wherein the method comprises incubating a sample with a composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody or antigen-binding fragment thereof described herein, or a polypeptide complex described herein.

[0134] As used herein, the term "antibody" refers to any antigen binding molecule that contains at least one (e.g., one, two, three, four, five or six) complementary determining region (CDR) (e.g., any one CDR from the three CDRs of an immunoglobulin light chain or any one CDR from the three CDRs of an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies and humanized antibodies. In certain embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, bifunctional antibodies, linear antibodies and multispecific antibodies formed by antibody fragments.

[0135] As used herein, the term "human antibody" refers to an antibody encoded by endogenous nucleic acids derived from humans (e.g., rearranged human immunoglobulin heavy chain or light chain loci). In certain embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In certain embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In certain embodiments, human antibodies are produced in bacteria or yeast cells. In certain embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0136] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different species (e.g., antibodies from two different mammalian species such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0137] As used herein, the term "humanized antibody" refers to a non-human antibody containing the minimum sequence derived from non-human (e.g., mouse) immunoglobulin and containing a sequence derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (receptor antibody), wherein the hypervariable (e.g., CDR) region residues of the receptor antibody are replaced by hypervariable (e.g., CDR) region residues from non-human antibodies (e.g., donor antibodies), and the non-human antibodies are, for example, mice, rats, or rabbit antibodies with desired specificity, affinity, and ability. In certain embodiments, the Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In certain embodiments, humanized antibodies can contain residues not found in receptor antibodies or donor antibodies. These modifications can be carried out to further optimize antibody performance. In certain embodiments, humanized antibodies contain at least one and typically all variable domains substantially in two variable domains, wherein all or substantially all hypervariable loops (CDR) correspond to those regions of non-human (e.g., mouse) immunoglobulin, and all or substantially all framework regions are those regions of human immunoglobulin. Humanized antibodies can also contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0138] As used herein, the term "antigen-binding protein construct" is: (i) a single polypeptide comprising at least one antigen-binding domain; or (ii) a complex of two or more polypeptides (e.g., the same polypeptide or different polypeptides) that together form at least one or more antigen-binding domains. Non-limiting examples and aspects of antigen-binding protein constructs are described herein. Additional examples and aspects of antigen-binding protein constructs are known in the art. In some embodiments, the antigen-binding protein construct has 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 antigen-binding domains.

[0139] As used herein, the term "antigen binding domain" refers to one or more protein domains (e.g., formed by amino acids from a single polypeptide or formed by amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some instances, the antigen binding domain may bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain may be an antibody or a fragment thereof. An example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. In some embodiments, the antigen binding domain is a VHH. Non-limiting examples of antigen binding domains are described herein. Additional examples of antigen binding domains are known in the art. In some instances, the antigen binding domain can bind to a single antigen.

[0140] As used herein, the term "bispecific antibody" refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

[0141] As used herein, the term "multispecific antibody" refers to an antibody that is capable of binding to two or more different epitopes. The epitopes can be on the same antigen or on different antigens.

[0142] As used herein, "VHH" refers to the variable domain of a heavy chain antibody or a single domain antibody. In some embodiments, the VHH is a humanized VHH.

[0143] As used herein, when referring to an antibody, the phrases "specific binding (specifically binding)" and "specific binding (specifically binds)" mean that the antibody interacts with its target molecule (e.g., HER2), preferably with other molecules, because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, reagents typically identify molecules including specific structures rather than all molecules, and bind to the molecules. The antibody that specifically binds to the target molecule can be referred to as a target-specific antibody. For example, the antibody that specifically binds to the HER2 molecule can be referred to as a HER2-specific antibody or an anti-HER2 antibody.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0145] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1A 、 1B and 1C show the top VHH-Fc clones and CHO cells stably transfected with human HER2 (CHO-h-HER2 stable cells, Figure 1A ), SKBR3 cells (breast cancer cells, Figure 1B ) and N87 cells (gastric cancer cells, Figure 1C Whole cell binding (WCB) of HER2 cells was determined by flow cytometry using an AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the median fluorescence intensity (MFI) of AlexaFluor 488. Herceptin and Perjeta are reference anti-HER2 antibodies from Roche. 4A8, 11F6, and 11D5 are anti-human HER2 VHH-Fc clones.

[0147] Figure 2A ELISA binding of the top three VHH-Fc clones to human HER2-domain IV is shown. 96-well plates were coated overnight with 2.5 μg / ml of human HER2-domain IV-his protein, and binding of anti-HER2 VHH-Fc clones 4A8, 11F6, and 11D5, as well as reference anti-HER2 antibodies Herceptin and Perjeta (from Roche), was detected by anti-human IgG Fc-HRP. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450 nm.

[0148] Figure 2BELISA binding of VHH-Fc clones 11F6 and 11D5 to human HER2 domains II+III+IV is shown. 96-well plates were coated overnight with 3 μg / ml of human HER2 domains II+III+IV-his protein, and binding of anti-HER2 VHH-Fc clones 11F6 and 11D5 was detected using anti-human IgG Fc-HRP. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the OD at 450 nm.

[0149] Figure 2C ELISA binding of VHH-Fc clones 11F6 and 11D5 to human HER2 domain II is shown. 96-well plates were coated overnight with 3 μg / ml of human HER2 domain II-his protein, and binding of anti-HER2 VHH-Fc clones 11F6 and 11D5 was detected using anti-human IgG Fc-HRP. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the OD at 450 nm.

[0150] Figure 3A and 3B Figure 2 shows anti-HER2 antibody-mediated killing of cancer cells with high HER2 expression in the presence of human PBMCs. Anti-HER2 VHH-Fc clones 4A8, 11F6, and 11D5, as well as the reference antibody Perjeta, were added at varying concentrations to cells cultured 1 day prior to 15 x 10 3 AU-565 and SKBR3 cells were seeded in 96-well plates at 0.3 x 10 6 1:20 ratio of tumor cells to PBMCs was added, and cell viability was measured 1 day later by adding CCK-8 and reading at OD 450 nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of OD at 450 nm.

[0151] Figure 4 The effect of anti-HER2 antibodies on the proliferation of AU-565 cells is shown. Anti-HER2 VHH-Fc clones 4A8, 11F6 and 11D5 and the reference antibody Herceptin were added at different concentrations to cells cultured 1 day prior to 15 x 10 3 AU-565 cells were seeded per well in a 96-well plate in RPMI 1640 medium supplemented with 2% FBS. One day later, cell viability was measured by adding CCK-8 and reading the OD at 450 nm. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the OD at 450 nm.

[0152] Figure 5A and 5BThe expression of TROP2 VHH Fc clones in CHO cells stably transfected with human TROP2 (CHO-h-TROP2 stable cells, Figure 5A ) and 293T cells stably transfected with mouse TROP2 ( Figure 5B Whole-cell binding of IgG2 (p361) to IgG4 (p361) was determined by flow cytometry using an AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. The x-axis represents the antibody concentration in nanomolar units. The y-axis represents the median fluorescence intensity of AlexaFluor 488. Immu-132 is a reference anti-TROP2 antibody from Gilead. 8B5, 1E9, 1H11, 4D3, 5G11, 4C6, 8F10, 3H9, 6B7, and 1H6 are anti-human TROP2 VHH-Fc clones.

[0153] Figure 6 Shown is whole-cell binding of the leading TROP2 VHH-Fc clone to endogenously expressed TROP2 in SKBR3 cells, as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the median fluorescence intensity of AlexaFluor 488. Immu-132 is a reference anti-TROP2 antibody from Gilead Sciences.

[0154] Figure 7 Leading anti-TROP2 VHH-Fc antibodies compete with Immu-132 for whole-cell binding. CHO-h-TROP2 stable cells were pre-incubated with anti-TROP2 VHH-Fc antibodies at 60 nM for 30 minutes. After washing, Immu-132 was added at different concentrations and incubated for 30 minutes. Binding was detected by adding AlexaFluor488-conjugated goat anti-human IgG F(ab)2 fragment-specific secondary antibodies. The X-axis is the value of the Immu-132 concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor488. 4C6, 3H9, and 1H11 are anti-TROP2 VHH-Fc clones. The negative control (Ctrl) is an anti-human PD-L1 VHH-Fc antibody.

[0155] Figure 8Figure 2 shows the binding of humanized variants of the 3H9 VHH-Fc clone to whole-cell CHO-h-TROP2 stable cells, as determined by flow cytometry using an AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the median fluorescence intensity of AlexaFluor 488. 3H9 is an alpaca VHH-Fc clone, and 3H9-hv1, hv2, hv3, and hv4 are four different humanized variants.

[0156] Figure 9A 、 9B Figures 9C and 9C show whole-cell binding of humanized variants of the 4C6 VHH-Fc clone to CHO-h-TROP2 stable cells, as determined by flow cytometry using an AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the median fluorescence intensity of AlexaFluor 488. 4C6 is an alpaca VHH-Fc clone, and 4C6-hv1, hv2, hv3, hv4, hv5, hv6, hv7, hv8, hv9, and hv10 are 10 different humanized variants. Immu-132 is a reference anti-TROP2 antibody from Gilead Sciences.

[0157] Figure 10A and 10B Figure 2 shows the binding of humanized variants of the 1H11 VHH-Fc clone to whole-cell CHO-h-TROP2 stable cells, as determined by flow cytometry using an AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. The X-axis represents the antibody concentration in nanomolar units. The Y-axis represents the median fluorescence intensity of AlexaFluor 488. 1H11 is an alpaca VHH-Fc clone, and 1H11-hv2, hv3, hv4, hv5, hv6, hv7, hv8, hv9, and hv10 are nine different humanized variants. Immu-132 is a reference anti-TROP2 antibody from Gilead Sciences.

[0158] Figure 11A and 11B Humanized variants of the 1H11 VHH-Fc clone are shown in NCI-H1975 cells ( Figure 11A ) and Colo205 cells ( Figure 11BWhole-cell binding of endogenously expressed TROP2 in WT cells (Figure 5A), as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis plots the antibody concentration in nanomolar units. The Y-axis plots the median fluorescence intensity of AlexaFluor 488. 1H11 is an alpaca VHH-Fc clone, and 1H11-hv6, hv7, hv8, hv9, and hv10 are five different humanized variants. Immu-132 is a reference anti-TROP2 antibody from Gilead Sciences.

[0159] Figures 12A-12F Schematic diagrams of different formats of monospecific VHH-Fc constructs (12A and 12B) and BsAb constructs (12C-12F) are shown.

[0160] Figures 13A-13B Shown are the whole-cell binding of different forms of monospecific anti-HER2 (11D5) or anti-TROP2 (1H11-hv10) and bispecific anti-HER2 / TROP2 VHH-Fc clones to CHO-h-HER2 stable cells (13A) and CHO-h-TROP2 stable cells (13B), as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488. The construct names are shown in the figure.

[0161] Figures 14A-14C Schematic diagrams of two different formats of the monospecific VHH-Fc construct 1H11-hv8 (14A) and BsAb constructs (14B and 14C) are shown.

[0162] Figures 15A-15E Shown are the binding affinities of BsAb 1H11-hv8_Fc_11D5 or monospecific Abs to h-HER2 ECD or h-TROP2 ECD, as determined by BLI binding assay. In brief, the sensor is loaded with 300 nM of antibody and then immersed in a solution of a recombinant protein (his-tagged) containing 200 nM h-HER2 ECD. After 130 seconds of association, the sensor is moved and immersed in another solution of h-TROP2 (his-tagged) containing 200 nM. The Y axis is the displacement in nm, and the X axis is the time in seconds.

[0163] Figure 16AFigure 3. Binding of different monospecific anti-HER2 (11D5) or anti-TROP2 (1H11-hv8) and bispecific anti-HER2 / TROP2 VHH-Fc clones to whole-cell SKBR3 cells, as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. Antibody names are indicated in the figure. Immu-132 is an anti-TROP2 reference antibody from Gilead Sciences. Herceptin is an anti-HER2 reference antibody from Roche. The x-axis is the value of the antibody concentration in nanomolar units. The y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0164] Figure 16B Figure 2 shows the killing of SKBR3 cells by anti-HER2 or anti-TROP2 monospecific or bispecific antibodies in the presence of a secondary antibody (Fab) conjugated to vc-MMAE against human IgG Fc. Viable cells were measured by adding CCK-8 reagent and reading at OD 450 nm. The antibody names are shown in the figure. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450 nm.

[0165] Figure 17A Figure 2 shows the binding of different monospecific anti-HER2 (11D5) or anti-TROP2 (1H11-hv8) and bispecific anti-HER2 / TROP2 VHH Fc clones to whole-cell NCI-H441 cells, as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The antibody names are indicated in the figure. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0166] Figure 17B Shown is the killing of anti-HER2 or anti-TROP2 monospecific or bispecific antibodies mediated by NCI-H441 cells in the presence of a secondary antibody (Fab) conjugated with vc-MMAE against human IgG Fc. Viable cells were measured by adding cell counting kit-8 (CCK-8) reagent and reading at OD 450nm. The antibody names are shown in the figure. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450nm.

[0167] Figure 18AFigure 2 shows the binding of different monospecific anti-HER2 (11D5) or anti-TROP2 (1H11-hv8) and bispecific anti-HER2 / TROP2 VHH Fc clones to whole-cell HCC202 cells, as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The antibody names are indicated in the figure. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0168] Figure 18B Shown in the presence of anti-human IgG Fc vc-MMAE conjugated secondary antibody (Fab), to the anti-HER2 or anti-TROP2 monospecific or bispecific antibody-mediated killing of HCC202 cells. Viable cells are measured by adding cell counting-8 (CCK-8) reagent and reading at OD 450nm. Antibody name is shown in the figure. X-axis is the value of antibody concentration in nanomolar units. Y-axis is the value of OD at 450nm.

[0169] Figure 19A 、 20A , 21A, 22A and 23A show the HIC-HPLC results of 11D5_MMAE (19A), 1H11_MMAE (20A), BsAb_MMAE (1H11-hv8_Fc_11D5_MMAE, 21A), Herceptin_MMAE (22A) and Immu-132_MMAE (23A).

[0170] Figure 19B 、 20B , 21B, 22B and 23B show the SEC-HPLC results of 11D5_MMAE (19B), 1H11_MMAE (20B), BsAb_MMAE (1H11-hv8_Fc_11D5_MMAE, 21B), Herceptin_MMAE (22B) and Immu-132_MMAE (23B).

[0171] Figure 24A The killing effect of Immu-132-MMAE on NCI-H441 cells at different DARs (DAR3.5, 4.0 and 4.4) is shown. Viable cells were measured by adding cell counting kit-8 (CCK-8) reagent and reading at OD 450nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450nm.

[0172] Figure 24BThe killing effect of Herceptin-MMAE on AU-565 cells at different drug-to-antibody ratios (DAR) (DAR 3.6, 4.0, and 4.3) is shown. Viable cells were measured by adding cell counting kit-8 (CCK-8) reagent and reading at OD 450nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450nm.

[0173] Figures 25A-25B Figure 25A shows the binding of Herceptin and Immu-132 to whole cells of AU-565 cells (25A) and N87 cells (25B), as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0174] Figures 26A-26C Figure 2 shows the killing effect of vc-MMAE-conjugated anti-HER2 (11D5_MMAE and Herceptin_MMAE) or anti-TROP2 (1H11_MMAE and Immu-132_MMAE) monospecific antibodies or anti-HER2 / TROP2 BsAb (1H11-hv8_Fc_11D5_MMAE) on SKBR3 cells (26A), AU-565 cells (26B) and N87 cells (26C). Viable cells were measured by adding cell counting kit-8 (CCK-8) reagent and reading at OD 450nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450nm.

[0175] Figures 27A-27B Figure 27A shows the binding of Herceptin and Immu-132 to whole-cell SKOV-3 cells (27A) and OE-19 cells (27B), as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0176] Figures 28A-28B Figure 2 shows the killing effect of vc-MMAE-conjugated anti-HER2 (11D5_MMAE and Herceptin_MMAE) or anti-TROP2 (1H11_MMAE and Immu-132_MMAE) monospecific antibodies or anti-HER2 / TROP2 BsAb (BsAb_MMAE) on SKOV-3 cells (28A) and OE-19 cells (28B). Viable cells were measured by adding Cell Counter-8 (CCK-8) reagent and reading at OD 450nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of OD at 450nm.

[0177] Figures 29A-29D Figure 2 shows the binding of Herceptin and Immu-132 to whole cells of NCI-H1975 cells (29A), Colo205 cells (29B), T-47D cells (29C), and A431 cells (29D), as determined by flow cytometry using AlexaFluor 488-conjugated anti-human IgG Fc as the secondary antibody. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the median fluorescence intensity of AlexaFluor 488.

[0178] Figures 30A-30E Figure 30: Anti-HER2 (11D5_MMAE and Herceptin_MMAE) or anti-TROP2 (1H11_MMAE and Immu-132_MMAE) monospecific antibodies or anti-HER2 / TROP2 BsAb (BsAb_MMAE) conjugated to vc-MMAE showed killing of NCI-H441 cells (30A), NCI-H1975 cells (30B), Colo205 cells (30C), T-47D cells (30D) and A431 cells (30E). Viable cells were measured by adding cell counting-8 (CCK-8) reagent and reading at OD 450nm. The X-axis is the value of the antibody concentration in nanomolar units. The Y-axis is the value of the OD at 450nm.

[0179] Figure 31 Figure 2 shows tumor volume data from an in vivo efficacy study of A431 tumors in Nu / Nu nude mice. Treatment groups are indicated in the figure legend, with N = 7 mice per group. *P < 0.01, **P < 0.001 compared to negative control by 2-way ANOVA with Tukey's multiple comparison analysis. The X-axis represents the number of days treated. The Y-axis represents tumor volume.

[0180] Figure 32 Figure 2 shows tumor volume data from an in vivo efficacy study of OE-19 tumors in immunodeficient B-NDG mice. Treatment groups are indicated in the figure legend, with N = 7 mice per group. *P < 0.05, ***P < 0.0001 compared to negative control by 2-way ANOVA with Tukey's multiple comparison analysis. The X-axis represents the number of days treated. The Y-axis represents tumor volume.

[0181] Figure 33 (SEQ ID NOs: 39, 23, and 24) show the CDR sequences of exemplary anti-HER2 antibodies described in this disclosure.

[0182] Figure 34 (SEQ ID NOs: 25-33) show the CDR sequences of exemplary anti-TROP2 antibodies described in this disclosure.

[0183] Figure 35 (SEQ ID NO: 1-12) list the amino acid sequences of VHHs as described in the present disclosure.

[0184] Figures 36A-36F Schematic diagrams of BsAb-ADCs with two or three cysteines in each hinge region in N-, C-terminal format (36A and 36B) or tandem format (36C and 36D) or monospecific VHH_Fc-ADC (36E and 36F) are shown. DETAILED DESCRIPTION

[0185] A bispecific antibody or antigen-binding fragment thereof is an artificial protein that can simultaneously bind to two different epitopes (e.g., on two different antigens). In some embodiments, a bispecific antibody or antigen-binding fragment thereof can have two arms (arms A and B).

[0186] The present disclosure relates to anti-HER2 antibodies or antigen-binding fragments thereof, anti-TROP2 antibodies or antigen-binding fragments thereof, antigen-binding protein constructs (e.g., bispecific antibodies or antigen-binding fragments thereof) that specifically bind to two different antigens (e.g., HER2 and TROP2), and antibody drug conjugates.

[0187] Heavy chain single variable domain (VHH) antibodies

[0188] Monoclonal antibodies and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, Camelidae (e.g., llama) can produce conventional antibodies consisting of two heavy chains and two light chains that are combined in a Y shape (e.g., IgG1) with a disulfide bond. However, Camelidae also produces two unique IgG subclasses: IgG2 and IgG3, also referred to as heavy chain IgG. These antibodies consist of only two heavy chains that lack the CH1 region but still carry the antigen-binding domain known as VHH (or nanobody) at its N-terminus. Conventional Ig requires the association of the variable regions from both heavy and light chains to allow for the high diversity of antigen-antibody interactions. Although the separated heavy and light chains still show this ability, when compared with paired heavy and light chains, they exhibit very low affinity. The unique feature of heavy chain IgG is that its monomeric antigen-binding domain is capable of binding to antigens with specificity, affinity, and specifically diversity, which is comparable to conventional antibodies that do not need to be paired with another district. This characteristic is primarily due to major variations in the amino acid sequences of the variable regions of the two heavy chains, which induce profound conformational changes compared to conventional Igs. The major substitutions in the variable regions prevent the light chains from binding to the heavy chains, but also prevent unbound heavy chains from being retrieved by immunoglobulin binding proteins.

[0189] The single variable domain (namely VHH, sdAb or nanobody) of these antibodies is the minimum antigen-binding domain produced by the adaptive immune system. It has been found that the third complementary determining region (CDR3) of the variable region of these antibodies is twice as long as that of conventional antibodies. This increases the interaction surface with the antigen and the diversity of antigen-antibody interactions, which compensates for the disappearance of light chain. In the case of long complementary determining region 3 (CDR3), VHH can extend into the protein cleft that conventional antibodies cannot reach, including the site of functional interest, such as the active site of an enzyme or the receptor binding ravine on a viral surface. In addition, other cysteine ​​residues make the structure more stable, therefore increasing the intensity of interaction.

[0190] Compared to conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHH provides many other advantages, including higher stability, solubility, expression yield and refolding ability and better in vivo tissue penetration. In addition, compared to the VH domains of conventional antibodies, VHH does not show an inherent tendency to bind to the light chain. This is conducive to inducing heavy chain antibodies in the presence of functional light chain loci. Further, since VHH does not bind to the VL domain, it is much easier to reformat VHH into a bispecific antibody construct than to reformat it into a construct containing a conventional VH-VL pair or a single domain based on the VH domain.

[0191] Anti-HER2 antibodies and antigen-binding fragments

[0192] Human epidermal growth factor receptor 2 (HER2) (also known as ERBB2) is a transmembrane receptor belonging to the epidermal growth factor receptor subfamily of receptor protein tyrosine kinases. HER2 is overexpressed in various cancer types such as breast cancer and gastric cancer, and has been reported to be a negative prognostic factor in breast cancer.

[0193] HER2 is a member of the human epidermal growth factor receptor (HER / EGFR / ERBB) family. However, unlike other members of the ERBB family, HER2 does not directly bind to the ligand. When HER2 concentration is high, such as in cancer, HER2 activation is produced by heterodimerization with another ERBB member or by homodimerization. The amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years, protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patients.

[0194] Detailed reviews of HER2 and its role in cancer can be found in the following: Krishnamurti, Uma, and Jan F. Silverman. “HER2 in breast cancer: a review and update.” Advances in anatomic pathology 21.2 (2014): 100-107; Gutierrez, Carolina, and Rachel Schiff. “HER2: biology, detection, and clinical implications.” Archives of pathology & laboratory medicine 135.1 (2011): 55-62; Oh, Do-Youn, and Yung-Jue Bang. “HER2-targeted therapies—arole beyond breast cancer.” Nature Reviews Clinical Oncology Clinical Oncology)》17.1(2020):33-48, each of which is incorporated herein by reference in its entirety.

[0195] The present disclosure provides, for example, anti-HER2 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof. The present disclosure also provides VHHs of these antibodies. These VHHs can be used in various multispecific antibody constructs as described herein. The present disclosure also provides antigen-binding protein constructs containing antigen-binding regions derived from these anti-HER2 antibodies.

[0196] The CDR sequences of 11D5 and 11D5-derived antibodies (eg, humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 22, 23, and 24, respectively. The amino acid sequence of the VHH domain of the 11D5 antibody is shown in SEQ ID NO: 1.

[0197] Also provided are the amino acid sequences of various modified or humanized VHHs. Because there are different ways to modify or humanize llama antibodies (e.g., different amino acid substitutions can be made for the modified sequence), an antibody can have more than one version of a humanized sequence. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 1.

[0198] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two, or three VHH domain CDRs selected from the group consisting of SEQ ID NOs: 22-24.

[0199] In some embodiments, the antibody may have a heavy chain antibody variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR3 amino acid sequence. The selected VHH CDR1, 2, 3 amino acid sequences are Figure 33 Shown in.

[0200] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) comprising one, two, or three of the following: a VHH CDR1 having zero, one, or two amino acid insertions, deletions, or substitutions; a VHH CDR2 having zero, one, or two amino acid insertions, deletions, or substitutions; and a VHH CDR3 having zero, one, or two amino acid insertions, deletions, or substitutions, wherein VHH CDR1, VHH CDR2, and VHH CDR3 are selected from Figure 33 CDRs in .

[0201] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the following: the CDRs of SEQ ID NO: 22 with zero, one, or two amino acid insertions, deletions, or substitutions; the CDRs of SEQ ID NO: 23 with zero, one, or two amino acid insertions, deletions, or substitutions; and the CDRs of SEQ ID NO: 24 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0202] Insertions, deletions and substitutions may be within a CDR sequence, or at one or both ends of a CDR sequence.In some embodiments, CDRs are determined based on the IMGT definition.

[0203] In some embodiments, the antibody or antigen-binding fragment thereof contains a heavy chain antibody variable domain (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 1.

[0204] In order to determine the identity percentage of two amino acid sequences or two nucleotide sequences, sequence is compared for the purpose of best comparison (for example, can introduce room for best comparison in one or both of the first and second amino acid or nucleotide sequences, and for the purpose of comparison, non-homologous sequences can be ignored).Then the amino acid residue or the nucleotide at corresponding amino acid position or nucleotide position are compared.When the position in the first sequence is occupied by the amino acid residue or nucleotide identical with the corresponding position in the second sequence, these molecules are identical at said position.The identity percentage between two sequences is the function of the quantity of the same position shared by said sequences and the length of each room, needs to introduce said function to carry out the best comparison of two sequences.For the purpose of illustration, the determination of the identity percentage between the comparison of sequence and two sequences can for example be completed using Blossum62 scoring matrix, wherein gap penalty is 12, gap extension penalty is 4 and frameshift gap penalty is 5.

[0205] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain antibody variable domains (VHH). VHH comprises Figure 33 The CDRs shown, or with Figure 35 sequence shown.

[0206] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH) CDR1 of SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH) CDR2 of SEQ ID NO: 23. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH) CDR3 of SEQ ID NO: 24.

[0207] Anti-HER2 antibodies and Fab can also be antibody or antibody fragment and multispecific (for example, bispecific) antibody or antibody fragment antibody variant (comprising derivative and conjugate).Other antibody provided herein is polyclonal antibody, monoclonal antibody, multispecific antibody (multimeric antibody, for example, bispecific antibody), human antibody, chimeric antibody (for example, human mouse chimera), single-chain antibody, the antibody (that is, in vivo antibody) and its Fab of manufacturing in cell.Anti-HER2 antibodies and Fab can be any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), classification (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.In certain embodiments, antibody or its Fab is IgG antibody or its Fab.

[0208] Anti-TROP2 antibodies and antigen-binding fragments

[0209] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It shares a high structural sequence similarity with the epithelial adhesion molecule Epcam. TROP2 is a protein closely associated with tumors. It primarily promotes tumor cell growth, proliferation, and metastasis by regulating calcium ion signaling pathways, cyclin expression, and reducing fibronectin adhesion. Studies have found that TROP2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma, pancreatic cancer, and ovarian cancer. This protein can promote tumor cell proliferation, invasion, metastasis, spread, and other processes. Furthermore, in breast cancer and other cancers, elevated TROP2 expression has been found to be closely associated with more aggressive disease and poor clinical prognosis.

[0210] TROP2 is an intracellular calcium signal transducer that is differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion, and survival. It has stem cell-like properties. TROP2 is expressed in many normal tissues, but conversely, it is overexpressed in many cancers, and TROP2 overexpression has prognostic significance. Several ligands that interact with TROP2 have been proposed. TROP2 signals to cells through different pathways and is transcriptionally regulated by a complex network of several transcription factors. TROP2 expression in cancer cells is associated with drug resistance.

[0211] A detailed review of TROP2 and its overexpression in cancer can be found in Shvartsur, Anna and Benjamin Bonavida. "TROP2 and its overexpression in cancers: regulation and clinical / therapeutic implications." Genes & cancer 6.3-4 (2015): 84, which is incorporated herein by reference in its entirety.

[0212] The present disclosure provides, for example, anti-TROP2 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof. The present disclosure also provides VHHs of these antibodies. These VHHs can be used in various multispecific antibody constructs as described herein. The present disclosure also provides antigen-binding protein constructs containing antigen-binding regions derived from these anti-TROP2 antibodies.

[0213] The CDR sequences of alpaca 3H9 (3H9) and 3H9-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 25, 26, and 27, respectively. The amino acid sequence of the VHH domain of the 3H9 antibody is shown in SEQ ID NO: 2.

[0214] The CDR sequences of alpaca 4C6 (4C6) and 4C6-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 28, 29, and 30, respectively. The amino acid sequence of the VHH domain of the 4C6 antibody is shown in SEQ ID NO: 3.

[0215] The CDR sequences of Alpaca 1H11 (1H11) and 1H11-derived antibodies (eg, humanized antibodies) include the CDRs of the VHH domain as shown in SEQ ID NOs: 31, 32, and 33, respectively. The amino acid sequence of the VHH domain of the 1H11 antibody is shown in SEQ ID NO:4.

[0216] Also provided are the amino acid sequences of various modified or humanized VHHs. Because there are different ways to modify or humanize llama or alpaca antibodies (e.g., different amino acids can be substituted for the modified sequence), an antibody can have more than one version of a humanized sequence. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 2-12.

[0217] Additionally, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two, or three VHH domain CDRs selected from the group consisting of SEQ ID NOs: 25-27, SEQ ID NOs: 28-30, and SEQ ID NOs: 31-33.

[0218] In some embodiments, the antibody may have a heavy chain antibody variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR3 amino acid sequence. The selected VHH CDR1, 2, 3 amino acid sequences are Figure 34 Shown in.

[0219] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) comprising one, two, or three of the following: a VHH CDR1 having zero, one, or two amino acid insertions, deletions, or substitutions; a VHH CDR2 having zero, one, or two amino acid insertions, deletions, or substitutions; and a VHH CDR3 having zero, one, or two amino acid insertions, deletions, or substitutions, wherein VHH CDR1, VHH CDR2, and VHH CDR3 are selected from Figure 34 CDRs in .

[0220] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the following: the CDRs of SEQ ID NO: 25 with zero, one, or two amino acid insertions, deletions, or substitutions; the CDRs of SEQ ID NO: 26 with zero, one, or two amino acid insertions, deletions, or substitutions; and the CDRs of SEQ ID NO: 27 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0221] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the following: the CDRs of SEQ ID NO: 28 with zero, one, or two amino acid insertions, deletions, or substitutions; the CDRs of SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; and the CDRs of SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0222] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain antibody variable domain (VHH) containing one, two, or three of the following: the CDRs of SEQ ID NO: 31 with zero, one, or two amino acid insertions, deletions, or substitutions; the CDRs of SEQ ID NO: 32 with zero, one, or two amino acid insertions, deletions, or substitutions; and the CDRs of SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0223] Insertions, deletions and substitutions may be within a CDR sequence, or at one or both ends of a CDR sequence.In some embodiments, CDRs are determined based on the IMGT definition.

[0224] In some embodiments, the antibody or antigen-binding fragment thereof contains a heavy chain antibody variable domain (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 2. In some embodiments, the selected VHH sequence is SEQ ID NO: 3. In some embodiments, the selected VHH sequence is SEQ ID NO: 4. In some embodiments, the selected VHH sequence is SEQ ID NO: 5. In some embodiments, the selected VHH sequence is SEQ ID NO: 6. In some embodiments, the selected VHH sequence is SEQ ID NO: 7. In some embodiments, the selected VHH sequence is SEQ ID NO: 8. In some embodiments, the selected VHH sequence is SEQ ID NO: 9. In some embodiments, the selected VHH sequence is SEQ ID NO: 10. In some embodiments, the selected VHH sequence is SEQ ID NO: 11. In some embodiments, the selected VHH sequence is SEQ ID NO: 12.

[0225] In order to determine the identity percentage of two amino acid sequences or two nucleotide sequences, sequence is compared for the purpose of best comparison (for example, can introduce room for best comparison in one or both of the first and second amino acid or nucleotide sequences, and for the purpose of comparison, non-homologous sequences can be ignored).Then the amino acid residue or the nucleotide at corresponding amino acid position or nucleotide position are compared.When the position in the first sequence is occupied by the amino acid residue or nucleotide identical with the corresponding position in the second sequence, these molecules are identical at said position.The identity percentage between two sequences is the function of the quantity of the same position shared by said sequences and the length of each room, needs to introduce said function to carry out the best comparison of two sequences.For the purpose of illustration, the determination of the identity percentage between the comparison of sequence and two sequences can for example be completed using Blossum62 scoring matrix, wherein gap penalty is 12, gap extension penalty is 4 and frameshift gap penalty is 5.

[0226] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain antibody variable domains (VHH). VHH comprises Figure 34 The CDRs shown, or with Figure 35 sequence shown.

[0227] Anti-TROP2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific antibodies (multimeric antibodies, e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies manufactured in cells (i.e., in vivo antibodies), and antigen-binding fragments thereof. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0228] Anti-HER2 / TROP2 bispecific antibodies

[0229] The anti-HER2, anti-TROP2 or anti-HER2 / TROP2 antigen binding protein constructs (e.g., antibodies, bispecific antibodies, trispecific antibodies, multispecific antibodies or antibody fragments thereof) may include an antigen binding site derived from any anti-HER2 antibody, anti-TROP2 antibody or any antigen binding fragment thereof as described herein.

[0230] In certain embodiments, bispecific antibodies are designed to include VHHs targeting HER2 and VHHs targeting TROP2. In certain embodiments, the disclosure provides bispecific antibodies that are combined with both HER2 and TROP2. Bispecific antibodies can be used to treat HER2 or TROP2 positive cancers (e.g., non-small cell lung cancer) in a subject.

[0231] HER2 / TROP2 bispecific antibodies with specific structures are described below.

[0232] Bispecific V1 structure

[0233] like Figure 12F As shown, a HER2 / TROP2 bispecific antibody can be prepared to have a bispecific V1 structure. Specifically, the HER2 / TROP2 bispecific antibody comprises (a) a first polypeptide comprising, from N-terminus to C-terminus: a first heavy chain antibody variable domain (VHH1), a first hinge region, a first CH2, a first CH3, and a third heavy chain antibody variable domain (VHH2); and (b) a second polypeptide comprising, from N-terminus to C-terminus: a second heavy chain antibody variable region (VHH3), a second hinge region, a second CH2, a second CH3, and a fourth heavy chain antibody variable region (VHH4).

[0234] In some embodiments, VHH1 and VHH3 specifically bind to HER2. In some embodiments, VHH2 and VHH4 specifically bind to TROP2. In some embodiments, the sequences of VHH1 and VHH3 are identical. In some embodiments, the sequences of VHH2 and VHH4 are identical.

[0235] In some embodiments, the HER2 / TROP2 bispecific antibody comprises a knob-into-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 18. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 18.

[0236] In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 36 or 37, and SEQ ID NO: 12. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 36 or 37, and SEQ ID NO: 12.

[0237] In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise aspartic acid (Asp) at position 239. In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise glutamic acid (Glu) at position 332.

[0238] In some embodiments, the VHH2 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence. In some embodiments, the VHH4 is connected to the C-termini of the second CH2 and the second CH3 via a second linker peptide sequence.

[0239] In some embodiments, the first and / or second linker peptide sequences comprise a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 38). In some embodiments, the first and / or second linker peptide sequences comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 34) and GSGGSGGSGGSGGSG (SEQ ID NO: 35).

[0240] Bispecific V2 structure

[0241] like Figure 12E As shown in FIG, a HER2 / TROP2 bispecific antibody can be prepared to have a bispecific V2 structure. Specifically, the HER2 / TROP2 bispecific antibody comprises (a) a first polypeptide comprising, from the N-terminus to the C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from the N-terminus to the C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3.

[0242] In some embodiments, VHH1 and VHH3 specifically bind to HER2. In some embodiments, VHH2 and VHH4 specifically bind to TROP2. In some embodiments, the sequences of VHH1 and VHH3 are identical. In some embodiments, the sequences of VHH2 and VHH4 are identical.

[0243] In some embodiments, the HER2 / TROP2 bispecific antibody comprises a knob-in-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 17. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 17.

[0244] In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 34 or 35, SEQ ID NO: 12, and SEQ ID NO: 36 or 37. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 34 or 35, SEQ ID NO: 12, and SEQ ID NO: 36 or 37.

[0245] In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise aspartic acid (Asp) at position 239. In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise glutamic acid (Glu) at position 332.

[0246] In some embodiments, the VHH1 is connected to the N-terminus of the VHH2 via a first linker peptide sequence. In some embodiments, the VHH3 is connected to the N-terminus of the VHH4 via a second linker peptide sequence.

[0247] In some embodiments, the first and / or second linker peptide sequences comprise a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 38). In some embodiments, the first and / or second linker peptide sequences comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 34) and GSGGSGGSGGSGGSG (SEQ ID NO: 35).

[0248] Bispecific V3 structure

[0249] like Figure 12C As shown, a HER2 / TROP2 bispecific antibody can be prepared to have a bispecific V3 structure. Specifically, the HER2 / TROP2 bispecific antibody comprises (a) a first polypeptide comprising, from the N-terminus to the C-terminus: VHH2, VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from the N-terminus to the C-terminus: VHH4, VHH3, a second hinge region, a second CH2, and a second CH3.

[0250] In some embodiments, VHH1 and VHH3 specifically bind to HER2. In some embodiments, VHH2 and VHH4 specifically bind to TROP2. In some embodiments, the sequences of VHH1 and VHH3 are identical. In some embodiments, the sequences of VHH2 and VHH4 are identical.

[0251] In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 15 or 21. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 15 or 21.

[0252] In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12, SEQ ID NO: 34 or 35, SEQ ID NO: 1, and SEQ ID NO: 36 or 37. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12, SEQ ID NO: 34 or 35, SEQ ID NO: 1, and SEQ ID NO: 36 or 37.

[0253] In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise aspartic acid (Asp) at position 239. In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise glutamic acid (Glu) at position 332.

[0254] In some embodiments, the VHH2 is connected to the N-terminus of the VHH1 via a first linker peptide sequence. In some embodiments, the VHH4 is connected to the N-terminus of the VHH3 via a second linker peptide sequence.

[0255] In some embodiments, the first and / or second linker peptide sequences comprise a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 38). In some embodiments, the first and / or second linker peptide sequences comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 34) and GSGGSGGSGGSGGSG (SEQ ID NO: 35).

[0256] Bispecific V4 structure

[0257] like Figure 12D As shown, a HER2 / TROP2 bispecific antibody can be prepared to have a bispecific V4 structure. Specifically, the HER2 / TROP2 bispecific antibody comprises (a) a first polypeptide comprising, from the N-terminus to the C-terminus: VHH2, a first hinge region, a first CH2, a first CH3, and VHH1; and (b) a second polypeptide comprising, from the N-terminus to the C-terminus: VHH4, a second hinge region, a second CH2, a second CH3, and VHH3.

[0258] In some embodiments, VHH1 and VHH3 specifically bind to HER2. In some embodiments, VHH2 and VHH4 specifically bind to TROP2. In some embodiments, the sequences of VHH1 and VHH3 are identical. In some embodiments, the sequences of VHH2 and VHH4 are identical.

[0259] In some embodiments, the HER2 / TROP2 bispecific antibody comprises a knob-in-hole mutation. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 16 or 20. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 16 or 20.

[0260] In some embodiments, the first polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12, SEQ ID NO: 36 or 37, and SEQ ID NO: 1. In some embodiments, the second polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12, SEQ ID NO: 36 or 37, and SEQ ID NO: 1.

[0261] In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise aspartic acid (Asp) at position 239. In some embodiments, according to EU numbering, the first CH2 and the first CH3 and / or the second CH2 and the second CH3 comprise glutamic acid (Glu) at position 332.

[0262] In some embodiments, the VHH1 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence. In some embodiments, the VHH3 is connected to the C-termini of the second CH2 and the second CH3 via a second linker peptide sequence.

[0263] In some embodiments, the first and / or second linker peptide sequences comprise a sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 38). In some embodiments, the first and / or second linker peptide sequences comprise a sequence selected from GSGGSGGSGGSG (SEQ ID NO: 34) and GSGGSGGSGGSGGSG (SEQ ID NO: 35).

[0264] Antibody and ADC characteristics

[0265] The anti-HER2 antigen binding protein construct (eg, antibody, bispecific antibody, or antibody fragment thereof) or ADC derived therefrom may include an antigen binding region derived from any anti-HER2 antibody or any antigen binding fragment thereof as described herein.

[0266] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to HER2 and / or TROP2, thereby blocking the interaction of these receptors and their corresponding ligands; reducing phosphorylation of downstream signaling pathways (e.g., ERK and / or Akt pathways); and / or directly killing cancer cells through ADCC and / or CDC.

[0267] In certain embodiments, the combination with HER2 can be determined by using the whole cell binding assay of the cell (for example, CHO-h-HER2 stable cells, N87 cells or SKBR3 cells) expressing HER2. In certain embodiments, the EC50 of the antibody or its antigen-binding fragment for being combined with HER2 described herein is less than or about 100nM, less than or about 50nM, less than or about 25nM, less than or about 12.5nM, less than or about 10nM, less than or about 7.5nM, less than or about 5nM, less than or about 3nM, less than or about 2.5nM, less than or about 2.0nM, less than or about 1.5nM, less than or about 1.1nM, less than or about 1nM, less than or about 0.9nM, less than or about 0.8nM, less than or about 0.7nM.

[0268] In some embodiments, binding to different domains of the HER2 extracellular domain (ECD) can be determined by ELISA binding assays using his-tagged HER2 domain fragments. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to Domain I, Domain II, Domain III, and / or Domain IV of the HER2 ECD.

[0269] In some embodiments, the antibody described herein or its antigen-binding fragment thereof can be used to express PBMC-mediated killing of HER2-expressing breast cancer cells using PBMC and cancer cells expressing HER2 (e.g., AU-565 cells or SKBR3 cells) by in vitro cell killing assays to determine. In some embodiments, the IC50 of the antibody described herein for inducing cell killing or its antigen-binding fragment is less than or about 1 nM, less than or about 0.5 nM, less than or about 0.4 nM, less than or about 0.3 nM, less than or about 0.25 nM, less than or about 0.2 nM or less than or about 0.15 nM.

[0270] In some embodiments, the effects of the antibodies or antigen-binding fragments thereof described herein on the proliferation of cancer cells expressing HER2 (e.g., AU-565 cells or SKBR3 cells) can be determined using an in vitro proliferation assay. In some embodiments, the IC50 of the antibodies or antigen-binding fragments thereof described herein for inhibiting cell proliferation is less than or about 2 nM, less than or about 1.5 nM, less than or about 1.25 nM, less than or about 1 nM, less than or about 0.9 nM, or less than or about 0.85 nM.

[0271] In some embodiments, binding to TROP2 (e.g., human TROP2 or mouse TROP2) can be determined by whole cell binding assays using cells expressing TROP2 (e.g., CHO-h-TROP2 stable cells, 293T-m-TROP2 cells, SKBR3 cells, NCI-H1975 cells, or Colo205 cells). In some embodiments, the antibodies or antigen-binding fragments thereof described herein for binding to TROP2 have an EC50 of less than or about 1250 nM, less than or about 1000 nM, less than or about 750 nM, less than or about 500 nM, less than or about 250 nM, less than or about 100 nM, less than or about 75 nM, less than or about 50 nM, less than or about 25 nM, less than or about 12.5 nM, less than or about 10 nM, less than or about 7.5 nM, less than or about 5 nM, less than or about 3 nM, less than or about 2.5 nM, less than or about 2 nM, less than or about 1.75 nM, less than or about 1.5 nM, less than or about 1.25 nM, or less than or about 1 nM.

[0272] In some embodiments, binding to different domains of TROP2 can be determined by competitive binding assays. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to the same binding domain of TROP2. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can bind to different binding domains of TROP2.

[0273] Thermal stability can also be determined. The Tm of an antibody, antigen-binding fragment thereof, or antigen-binding protein construct (e.g., bispecific antibody) as described herein, or ADC derived therefrom can be greater than 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0274] In some embodiments, the purity of the antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., an anti-TROP2 antibody, an anti-HER2 antibody, or a bispecific antibody), or ADC derived therefrom, is greater than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, e.g., as measured by SEC-HPLC. In some embodiments, the antibody is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, e.g., as measured by SEC-HPLC.

[0275] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR).

[0276] In certain embodiments, the combination of HER2 and TROP2 can be determined by whole-cell binding assay using cells (for example, SKBR3 cells, NCI-H441 cells or HCC202 cells) expressing HER2 and TROP2. In certain embodiments, the EC50 of the antibody or its antigen-binding fragment for being combined with HER2 described herein is less than or about 125nM, less than or about 100nM, less than or about 50nM, less than or about 25nM, less than or about 12.5nM, less than or about 10nM, less than or about 7.5nM, less than or about 5nM, less than or about 3nM, less than or about 2.5nM, less than or about 2.0nM, less than or about 1.5nM, less than or about 1.1nM, less than or about 1nM, less than or about 0.9nM, less than or about 0.8nM, less than or about 0.7nM.

[0277] In some embodiments, the effect of antibodies, antigen-binding fragments thereof, or antigen-binding protein constructs (e.g., bispecific antibodies) on cell killing can be determined by using a cell killing assay using secondary Ab-MMAE. In some embodiments, the IC50 of antibodies or antigen-binding fragments thereof for inducing cell killing described herein is less than or about 1 nM, less than or about 0.5 nM, less than or about 0.4 nM, less than or about 0.3 nM, less than or about 0.25 nM, less than or about 0.2 nM, less than or about 0.15 nM, less than or about 0.125 nM, or less than or about 0.1 nM.

[0278] In some embodiments, the drug-antibody ratio (DAR) in the ADCs described herein can be determined by hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, the majority of the DAR species are D4. In some embodiments, D4 constitutes more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the DAR species. In some embodiments, the DAR in the ADCs described herein is greater than 3, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 3.6, greater than 3.7, greater than 3.8, or greater than 3.9. In some embodiments, the DAR in the ADCs described herein is less than 4.1, less than 4.2, less than 4.3, less than 4.4, less than 4.5, or less than 4.6. In some embodiments, the DAR in the ADCs described herein is about 3.7-4, about 3.8-4.1, about 3.9-4.2, about 3.9-4.1, or about 4.

[0279] In some embodiments, the effect of the ADCs described herein on cell killing can be determined using a cell killing assay using cancer cells (e.g., NCI-H441 cells, AU-565 cells, SKBR3 cells, N87 cells, SK-OV3 cells, OE-19 cells, NCI-H1975 cells, Colo205 cells, or T-47D cells). In some embodiments, the antibodies or antigen-binding fragments thereof described herein for inducing cell killing have an IC50 of less than or about 100 nM, less than or about 50 nM, less than or about 25 nM, less than or about 10 nM, less than or about 5 nM, less than or about 1 nM, less than or about 0.5 nM, less than or about 0.4 nM, less than or about 0.3 nM, less than or about 0.25 nM, less than or about 0.2 nM, less than or about 0.15 nM, less than or about 0.125 nM, less than or about 0.1 nM, less than or about 0.05 nM, less than or about 0.04 nM, less than or about 0.03 nM, or less than or about 0.02 nM.

[0280] In certain embodiments, antibodies, their antigen-binding fragments or antigen-binding protein constructs (e.g., bispecific antibodies) have functional Fc regions. In certain embodiments, the effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the effector function of a functional Fc region is phagocytosis. In certain embodiments, the effector function of a functional Fc region is ADCC and phagocytosis. In certain embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4. In certain embodiments, one or both of mutations S239D and / or I332E (S1 mutations) are introduced into the antibody Fc region to enhance the affinity of the antibody to FcγRIIIA, thereby increasing the ADCC effect. A detailed description of the S1 mutation can be found in US7662925, which is incorporated herein by reference in its entirety.

[0281] Antibodies and antigen-binding fragments

[0282] The present disclosure provides antibodies and antigen-binding fragments thereof comprising the complementarity determining regions (CDRs), VHHs, heavy chain variable regions, light chain variable regions, heavy chains, or light chains described herein.

[0283] Typically, antibodies (also known as immunoglobulins) are made of two classes of polypeptide chains: light and heavy chains. A non-limiting antibody of the present disclosure can be a complete four-immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype including IgM, IgG, IgE, IgA, or IgD, or a subisotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. The antibody can contain two identical copies of a light chain and / or two identical copies of a heavy chain. The heavy chains, each containing a variable domain (or variable region VH) and multiple constant domains (or constant regions), are bound to each other through disulfide bonds within their constant domains to form the "stem" of the antibody. The light chains, each containing a variable domain (or variable region VL) and a constant domain (or constant region), are each bound to a heavy chain through disulfide bonds. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0284] These hypervariable regions, known as complementarity determining regions (CDRs), form loops that comprise the primary antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0285] Methods for identifying the CDR regions of antibodies by analyzing their amino acid sequences are well known, and multiple definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the position of the structural loop regions. The IMGT numbering of V-domains (IG and TR) is derived from the IMGT unique numbering of the V-region.These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular Immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods in Enzymology. Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); Kontermann, R. and Dübel, S. (eds.). (2010). Antibody Engineering: Vol. 2. Springer; Lefranc M.-P., "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 7, 132-136 (1999); each of which is incorporated herein by reference in its entirety. In some embodiments, the CDRs are based on the Kabat definition.In some embodiments, the CDRs are based on the Chothia definition.In some embodiments, the CDRs are the longest CDR sequences as determined by the Kabat, Chothia, AbM, IMGT, or contact definitions.

[0286] CDR is important for recognizing the epitope of an antigen. As used herein, "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary structure of the antigen, as the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0287] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, specifically in their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Mol Immunol 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0288] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" refers to a part of the specific binding activity of an antibody that retains a complete antibody, that is, any part of an antibody that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2, VHH, and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising a binding domain as an antibody binding domain or a binding domain homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from the heavy or light chain of an intact antibody.

[0289] In some embodiments, scFV has two heavy chain variable domains and two light chain variable domains.In some embodiments, scFV has two antigen binding regions (antigen binding regions: A and B), and these two antigen binding regions can bind to corresponding target antigens with different affinities.

[0290] In some embodiments, antigen binding fragment can form a part of chimeric antigen receptor (CAR).In certain embodiments, chimeric antigen receptor is a fusion of single chain variable fragment (scFv) or VHH as described herein with CD3-ζ transmembrane domain and internal domain fusion.In certain embodiments, chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS).In certain embodiments, chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness.Therefore, on the one hand, the present disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0291] In some embodiments, the antibody or its antigen-binding fragment can bind to two different antigens or two different epitopes. In some embodiments, the antibody or its antigen-binding fragment can bind to three different antigens or three different epitopes.

[0292] Fv fragment is an antibody fragment containing complete antigen recognition and binding site. This region is composed of a dimer of a heavy chain variable domain and a light chain variable domain that are tightly associated, and the dimer can be covalent in nature, for example, in scFv. It is in this configuration that the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Together, these six CDRs or their subsets give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can also have the ability to recognize and bind antigens, but is generally carried out with an affinity lower than that of a complete binding site.

[0293] Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker positioned between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0294] In some embodiments, the scFv described herein comprises, from N-terminus to C-terminus: VH; a polypeptide linker; and VL. In some embodiments, the scFv described herein comprises, from N-terminus to C-terminus: VL; a polypeptide linker; and VH.

[0295] The Fab fragment contains the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments covalently linked by hinge cysteines between them, typically near their carboxyl termini. Other chemical couplings of antibody fragments are also known in the art.

[0296] The antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life. In some embodiments, according to EU numbering, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises aspartic acid (Asp) at position 239. In some embodiments, according to EU numbering, the Fc region in any of the antibodies or antigen-binding fragments described herein comprises glutamic acid (Glu) at position 332. In some embodiments, the Fc region described herein is any of the Fc regions described herein, comprising aspartic acid (Asp) at position 239 and / or comprising glutamic acid (Glu) at position 332 according to EU numbering. In some embodiments, Asp239 and / or Glu332 described herein can increase the effector function (e.g., ADCC or CDC) of an antibody or its antigen-binding fragment by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to a wild-type antibody or its antigen-binding fragment. Details can be found, for example, in Lazar, GA et al., "Engineered antibody Fc variants with enhanced effector function." Proceedings of the National Academy of Sciences 103.11 (2006): 4005-4010, which is incorporated herein by reference in its entirety.

[0297] In some embodiments, the Fc region of any of the antibodies or antigen-binding fragments described herein comprises a wild-type human IgG1 CH2 domain. In some embodiments, the Fc region of any of the antibodies or antigen-binding fragments described herein comprises a mutated human IgG1 CH2 domain.

[0298] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans) or extend its biological activity.

[0299] In some embodiments, the antibodies or antigen-binding fragments described herein (e.g., bispecific antibodies) can be conjugated to a therapeutic agent. Antibody drug conjugates comprising an antibody or antigen-binding fragment thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mithramycin, actinomycin D). D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and the like).

[0300] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof described herein (e.g., anti-HER2 / TROP2 bispecific antibodies) bind to an antigen (e.g., HER2) at an ability of about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, or about 200% of the binding ability of a heavy chain antibody (e.g., an anti-HER2 heavy chain antibody) comprising the same VHH of the multispecific antibody.

[0301] In some embodiments, the multispecific antibodies or antigen-binding fragments thereof (e.g., HER2 / TROP2 bispecific antibodies) described herein bind to an antigen (e.g., TROP2) at a level of about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150% or about 200% of the binding ability of an antibody or antigen-binding fragment targeting TROP2 (e.g., an anti-TROP2 heavy chain antibody) of the same VHH comprising the multispecific antibody.

[0302] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof described herein (e.g., HER2 / TROP2 bispecific antibodies) mediate complement dependent cytotoxicity (CDC) or ADC by at least or about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 30-fold, 40-fold, or 50-fold compared to complement dependent cytotoxicity (CDC) or ADC mediated by an isotype control antibody.

[0303] Antibody-drug conjugates (ADCs)

[0304] Antibodies described herein, their antigen-binding fragments or antigen-binding protein constructs (e.g., bispecific antibodies) can be conjugated to therapeutic agents (drugs). Therapeutic agents can be covalently or non-covalently bound to antibodies or antigen-binding fragments or antigen-binding protein constructs (e.g., bispecific antibodies). In certain embodiments, the bispecific antibody is an anti-HER2 / TROP2 bispecific antibody.

[0305] In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., monomethylauristatin E, monomethylauristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunomycin, dihydroxy anthracin, dendritic alkaloids such as DM-1 and DM-4, ​​diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and the like). Useful cytotoxic, cytostatic, or immunomodulatory agents include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0306] In some embodiments, the therapeutic agent may include, but is not limited to, a cytotoxic agent, such as a chemotherapeutic agent, an immunotherapeutic agent, an antiviral agent, or an antimicrobial agent. In some embodiments, the therapeutic agent to be conjugated may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0307] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of dolastatin-10) or a derivative thereof. The auristatin can be, for example, an ester formed between auristatin E and a ketoacid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in the following literature: U.S. Patent Application Publication No. 2003-0083263; International Patent Publication No. WO 04 / 010957; International Patent Publication No. WO No. 02 / 088172 and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530, 097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated herein by reference in its entirety for all purposes.

[0308] Auristatins have been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and have anti-cancer activity. Auristatins bind to tubulin and can exert cytotoxic or cytostatic effects on cancer cells. A number of different assays are known in the art that can be used to determine whether an auristatin or resulting antibody drug conjugate exerts a cytostatic or cytotoxic effect on the desired cells.

[0309] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; nitrogen mustards, such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide, and chlorambucil. hydrochloride), melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas (such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine);Antibiotics, such as aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, and mycophenolic acid acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopole; terin), methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauracil, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU;Androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; and aminolevulinic acid. acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid; 2-ethylhydrazide; procarbazine; PSK7; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2',2',2'-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel (; Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C C); mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any one of the foregoing. Also included in this definition are anti-hormonal agents for regulating or suppressing the hormone effects on tumors, such as anti-estrogen, including, for example, tamoxifen (tamoxifen), raloxifene (raloxifene), aromatase inhibition 4 (5) -imidazoles, 4-hydroxytamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone (onapristone) and toremifene (toremifene) (Fa Le Tong (Fareston));And anti-androgen, such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide) and goserelin (goserelin);And any of the above-mentioned pharmaceutically acceptable salts, acids or derivatives. A detailed description of chemotherapeutic agents can be found in, for example, US20180193477A1, which is incorporated herein by reference in its entirety.

[0310] In some embodiments, the antigen-binding construct is coupled to the drug via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is coupled to the drug via a non-cleavable linker, such as an MCC linker formed using SMCC or sulfo-SMCC. A skilled artisan with knowledge of the art and considering factors such as the site of attachment to the antigen-binding construct, any structural constraints on the drug, and the hydrophobicity of the drug can readily select an appropriate linker for a given ADC (see, for example, the review in Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (ed.), Springer-Verlag). In certain embodiments, a number of specific linker-toxin combinations have been described and can be used to prepare ADCs with the antigen-binding constructs described herein. Examples include, but are not limited to, cleavable peptide-based linkers with auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins, and PBD dimers; non-cleavable MC-based linkers with auristatins MMAF and MMAE; acid-labile hydrazone-based linkers with calicheamicin and doxorubicin; disulfide-based linkers with denstinoalkaloids such as DM1 and DM4, and bis-maleimide-trioxyethylene glycol (BMPEO)-based linkers with denstinoalkaloid DM1. Some of these therapeutic agents and linkers are described, for example, in Peters and Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; US Patent Publication Nos. US2015 / 0374847 and US20180193477A1; herein incorporated by reference in their entirety.

[0311] Depending on the desired drug and selected linker, those skilled in the art can select a suitable method for coupling it together. For example, some conventional coupling methods such as amine coupling methods can be used to form a desired drug-linker complex, which still contains a reactive group for conjugating to an antibody by a covalent bond. In certain embodiments, a drug-maleimide complex (i.e., a maleimide-connected drug) can be used for the reactive group of the present disclosure that carries a payload. In ADC preparation, the most common reactive group that can be combined with a thiol group is maleimide. Additionally, organic bromides and iodides are also frequently used.

[0312] ADCs can be prepared by one of several routes known in the art, using organic chemistry reactions, conditions, and reagents known to those skilled in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). For example, conjugation can be achieved by: (1) reacting a nucleophilic or electrophilic group of an antibody with a divalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, which is then reacted with an activated drug moiety D; or (2) reacting a nucleophilic or electrophilic group of a drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, which is then reacted with a nucleophilic or electrophilic group of an antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the ADCs described herein. Various prepared linkers, linker components, and toxins are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith and March, 2006, 6th ed., Wiley); Toki et al., (2002) J. Org. Chem. 67: 1866-1872; Frisch et al., (1997) Bioconj. Chem. 7: 180-186; Bioconjugate Technology (GT Hermanson, 2013, Academic Press); US20210379193A1 and US20180193477A1, which are incorporated herein by reference in their entirety. In addition, a number of preformed drug-linkers suitable for reaction with the selected antigen-binding construct are also commercially available, for example, linker-toxins comprising DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative BioLabs (Shirley, NY).

[0313] Several specific examples of methods for preparing ADCs are known in the art and are described in the following documents: U.S. Patent No. 8,624,003 (pot method), U.S. Patent No. 8,163,888 (one-step method) and U.S. Patent No. 5,208,020 (two-step method) and US20180193477A1, which are incorporated herein by reference in their entirety. Other methods are known in the art and include those described in the following document: Antibody Drug Conjugates: Molecular Biology Methods, 2013, Ducry (ed.), Springer-Verlag.

[0314] Drug load is represented by the number of drug moieties per antibody in the molecule of the ADC. For some antibody drug conjugates, drug load may be limited by the number of attachment sites on the antibody. For example, in the case of attachment of cysteine ​​thiols, as in certain exemplary embodiments described herein, drug load may be in the range of 0 to 8 drug moieties per antibody. In certain embodiments, higher drug loads, such as p≥5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates. In certain embodiments, the average drug load of the antibody drug conjugate ranges from 1 to about 8; about 2 to about 6; or about 3 to about 5. In fact, it has been shown that for certain antibody drug conjugates, the optimal ratio of drug moieties per antibody can be about 4. In some embodiments, the DAR is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0315] recombinant vector

[0316] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0317] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which an expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably connected within the vector or in the genome of the host cell at or near the integration site of the polynucleotide of interest or flanking the integration site to regulatory elements such as promoters, enhancers, and / or poly-A tails, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

[0318] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with a cationic condensing agent.

[0319] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective) replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation will generally only occur in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner, Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proceedings of the National Academy of Sciences of the United States of America, 91:215-219; Kass-Eisler et al., 1993, Proceedings of the National Academy of Sciences of the United States of America, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749 and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0320] For expression, a DNA insert comprising a polynucleotide encoding an antibody or polypeptide disclosed herein can be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage lambda PL promoter, E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters from retroviral LTRs, to name a few. Other suitable promoters are known to those of skill in the art. The expression construct may further contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may include translation initiated at the start and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0321] As indicated, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and noctuid Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0322] Non-limiting bacterial vectors include pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

[0323] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters from retroviral LTRs, such as the Rous sarcoma virus (RSV) promoter, and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0324] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH are available. For review, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods in Enzymology, 153:516-544 (1997).

[0325] The construct can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0326] Transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, typically about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the rear side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and adenovirus enhancers.

[0327] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or the signal may be a heterologous signal.

[0328] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST-fusion) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification, etc., is well known and conventional in the art.

[0329] The present disclosure also provides nucleic acids that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the nucleotide sequences described herein. Sequences, as well as amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein.

[0330] The present disclosure also provides nucleic acids having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology to any nucleotide sequence as described herein. Sequences, as well as amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to any of the amino acid sequences described herein.

[0331] In some embodiments, the present disclosure relates to nucleotide sequences encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0332] In some embodiments, the amino acid sequence (i) comprises; or (ii) consists of, an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0333] In some embodiments, the nucleic acid sequence (i) comprises; or (ii) consists of, a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0334] Methods for preparing antibodies

[0335] The isolated fragments of human proteins (e.g., HER2, TROP2 or cancer antigens) can be used as immunogens to produce antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous injection or intraperitoneal injection) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected together with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated with an immunogenic agent in the species to be immunized. The animal can be injected with more than one (e.g., two, three or four) antigenic peptides or proteins.

[0336] The full-length polypeptide or protein can be used as an immunogen, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of the protein and encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein.

[0337] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations can contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides. The preparations can further include an adjuvant, such as Freund's complete or incomplete adjuvant or a similar immunostimulant.

[0338] As described above, polyclonal antibodies can be prepared by immunizing a suitable subject using a polypeptide or its antigenic peptide (e.g., a portion of a protein) as an immunogen. Standard techniques can be used, such as by monitoring the antibody titer in the immunized subject over time using an enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide or peptide. If necessary, the antibody molecule can be separated from mammals (e.g., from blood) and further purified to obtain the IgG fraction by well-known techniques such as protein A of protein G chromatography. At an appropriate time after immunization, for example, when specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques such as the hybridoma technique originally described by Kohle et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0339] VHH can also be obtained from the synthetic llama VHH library of original or design. PBMC from llama can be obtained, and RNA can be isolated to produce cDNA by reverse transcription. Then, VHH gene can be amplified by PCR, and the gene is cloned on phage display vector, to build original VHH library. Synthetic (for example, humanized) VHH library can be prepared by incorporating the VHH CDR1, 2 and 3 of the reorganization produced by overlapping PCR into modified people VH scaffold, to produce enhanced diversity and keep low immunogenicity. VHH library can then be panned for antigen to obtain VHH with desired binding ability.

[0340] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding human antibodies, humanized antibodies or chimeric antibodies or their antigen-binding fragments described herein or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence of the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or their antigen-binding fragments with increased binding capacity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change antibodies or antigen-binding fragments or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell) or introducing new glycosylation sites.

[0341] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels, alpacas, and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0342] Phage display (panning) can be used to optimize the antibody sequence with desired binding capacity. In this technology, the gene encoding single-chain Fv (comprising VH or VL) or VHH can be inserted into the phage coating protein gene, so that phage " displays " scFv or VHH in its outside while containing the gene of protein in its inside, thereby causing the connection between genotype and phenotype. These display phages can then be screened for target antigen, to detect the interaction between displayed antigen binding site and target antigen. Therefore, it is possible to screen and amplify large protein libraries and obtain antibody sequences with desired binding capacity in the process referred to as in vitro selection.

[0343] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having an amino acid sequence identical to an amino acid sequence derived from human germline immunoglobulin sequences). Human antibodies may include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutation).

[0344] Humanized antibodies generally have a human framework (FR) transplanted with non-human CDRs. Therefore, humanized antibodies have one or more amino acid sequences introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domains. Humanization can be performed by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described in the following documents: for example, Jones et al., "Nature", 321: 522-525 (1986); Riechmann et al., "Nature", 332: 323-327 (1988); Verhoeyen et al., "Science", 239: 1534-1536 (1988); Each document in the document is incorporated herein by reference in its entirety. Therefore, a "humanized" antibody is a chimeric antibody in which substantially less than a complete human V domain has been replaced by a corresponding sequence from a non-human species.

[0345] More importantly, the antibody is humanized, retaining high specificity and affinity to antigen and other favorable biological properties. In order to achieve this goal, the three-dimensional model of the parent and humanized sequence can be used, and humanized antibodies are prepared by the analytical process of the parent sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is normally available and is familiar to those skilled in the art. It is available to illustrate and display the computer program of the possible three-dimensional conformational structure of selected candidate immunoglobulin sequence. The inspection of these displays allows the possible effect of the residue in the function of the candidate immunoglobulin sequence to be analyzed, that is, the residues that affect the ability of the candidate immunoglobulin and its antigen combination are analyzed. In this way, FR residues can be selected and combined from receptor sequence and input sequence, thereby realizing desired antibody characteristics, as the affinity to the target antigen is increased.

[0346] Identity or homology with respect to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to the sequence present in the human antibody, humanized antibody or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not taking into account conservative substitutions as part of the sequence identity.

[0347] In some embodiments, antibodies or their antigen-binding fragments can be covalently modified. These covalent modifications can be performed by chemical synthesis or enzymatic synthesis or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments can be introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that reacts with selected side chains or N-terminal residues or C-terminal residues.

[0348] In certain embodiments, antibody variants are provided as having a carbohydrate structure lacking the fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain relative to the sum of all sugar structures (e.g., compound, hybrid and high mannose structures) connected to Asn 297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues; or position 314 in Kabat numbering) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 can also be located at approximately ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0349] In certain embodiments, in order to promote production efficiency by avoiding Fab arm exchanges, the Fc region of antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of S228 mutations is described in the following documents, such as Silva et al. " S228P mutations prevent in vivo and in vitro IgG4 Fab arm exchanges as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015): 5462-5469, the document is incorporated to its entirety by reference.

[0350] In certain embodiments, method described herein is designed to prepare bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between antibody molecules to maximize the percentage of the heterodimer recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are substituted with larger side chains (for example, tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), a compensatory " cavity " of the same or similar size as large side chains is produced on the interface of the second antibody molecule. This provides a mechanism for improving the productive rate of heterodimers compared to the undesirable final products such as homodimers. The method is described in, for example, WO 96 / 27011, which is incorporated to its entirety by reference.

[0351] In certain embodiments, one or more amino acid residues in the hinge region of IgG are substituted.In certain embodiments, according to EU numbering, each hinge region has a C220S mutation.In certain embodiments, according to EU numbering, each hinge region has a cysteine ​​at position 220, a cysteine ​​at position 226, and a cysteine ​​at position 229. In certain embodiments, each hinge region has a serine at position 220, a cysteine ​​at position 226, and a cysteine ​​at position 229. In certain embodiments, each of a payload (e.g., a drug) and a cysteine ​​at position 220, a cysteine ​​at position 226, and a cysteine ​​at position 229 is covalently attached. In certain embodiments, each of a payload (e.g., a drug) and a cysteine ​​at position 226 and a cysteine ​​at position 229 is covalently attached. In certain embodiments, the DAR of an antibody drug conjugate is 4. In certain embodiments, the DAR of an antibody drug conjugate is 6.

[0352] In some embodiments, one or more amino acid residues in the CH3 portion of IgG are substituted. In some embodiments, one heavy chain has one or more of the following substitutions: Y349C and T366W. The other heavy chain may have one or more of the following substitutions: E356C, T366S, L368A, and Y407V. In addition, substitutions (-ppcpScp-->-ppcpPcp-) may also be introduced into the hinge region of two substituted IgGs. In some embodiments, one heavy chain has a T366Y (knob) substitution and the other heavy chain has a Y407T (hole) substitution (EU numbering).

[0353] One aspect of the present application provides a heteromultimeric (e.g., heterodimeric) protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first heavy chain constant domain 3 (CH3) domain, and the second polypeptide comprises a second CH3 domain, wherein the first CH3 domain comprises a substitution at amino acid position 354 with a bulky hydrophobic amino acid relative to a wild-type CH3 domain, and / or the second CH3 domain comprises a substitution at amino acid position 347 with a negatively charged amino acid relative to a wild-type CH3 domain, and wherein the amino acid residue numbering is based on EU numbering. In some embodiments, the bulky hydrophobic amino acid at amino acid position 354 forms a hydrophobic interaction with an amino acid residue in the second CH3 domain. In some embodiments, the second CH3 domain comprises a bulky hydrophobic residue at amino acid position 349 (e.g., Y349). In some embodiments, the negatively charged amino acid at amino acid position 347 forms an ionic bond with an amino acid residue in the first CH3 domain. In some embodiments, the first CH3 domain comprises a positively charged residue at amino acid position 360 (e.g., K360). In some embodiments, the first CH3 domain and the second CH3 domain are human CH3 domains. In some embodiments, the first CH3 domain comprises a substitution selected from the group consisting of: S354Y, S354F, and S354W. In some embodiments, the first CH3 domain comprises S354Y. In some embodiments, the second CH3 domain does not comprise a compensatory substitution (e.g., a substitution at Y349) for S354 in the first CH3 domain. In some embodiments, the second CH3 domain comprises a substitution selected from the group consisting of Q347E and Q347D. In some embodiments, the second CH3 domain comprises Q347E. In some embodiments of any of the heteromultimeric proteins described above, the first CH3 domain and the second CH3 domain further comprise a knob-to-hole (KIH) residue. In some embodiments, the knob-to-hole residues are T366Y and Y407T. In some embodiments, the first CH3 domain comprises T366Y and S354Y, and the second CH3 domain comprises Y407T and Q347E. In some embodiments, the first CH3 domain comprises Y407T and S354Y, and the second CH3 domain comprises T366Y and Q347E. Details can be found, for example, in PCT / US2020 / 025469, which is incorporated herein by reference.

[0354] Treatment

[0355] The methods described herein include methods for treating conditions associated with cancer. Typically, the methods include administering a therapeutically effective amount of an engineered antibody, an antigen-binding fragment thereof, an antigen-binding protein construct (e.g., a bispecific antibody), or an antibody drug conjugate as described herein to a subject in need or determined to be in need of such treatment.

[0356] As used in this context, "treating" means improving at least one symptom of a condition associated with cancer. Typically, cancer causes death; therefore, treatment can increase life expectancy (e.g., by at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or by at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years). Administration of a therapeutically effective amount of an agent described herein for treating a condition associated with cancer will result in a decrease in the number of cancer cells and / or relief of symptoms.

[0357] As used herein, the term "cancer" refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or cells, tissues, or organs of malignant transformation, regardless of the histopathological type or invasive stage. As used herein, the term "tumor" refers to cancer cells, such as a large number of cancer cells. The term "cancer" refers to malignant tumors of various organ systems, including malignant tumors affecting the lungs, breasts, thyroid glands, lymphoid tissues, stomachs, and urogenital tracts, and adenocarcinomas generally considered to include malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term "cancer" is generally recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including cancers of the respiratory system, gastrointestinal system, urogenital system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. In some embodiments, cancer is renal carcinoma or melanoma. Exemplary cancers include those that develop in the tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, which includes, for example, a malignant tumor composed of both cancerous and sarcomatous tissue. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin. In some embodiments, the cancer is a chemotherapy-resistant cancer.

[0358] In one aspect, the present disclosure also provides methods for treating cancer in a subject, methods for reducing the rate at which a subject's tumor volume increases over time, methods for reducing the risk of developing metastases, or methods for reducing the risk of a subject developing additional metastases. In some embodiments, the therapeutic antigen stops, slows, delays, or inhibits the progression of the cancer. In some embodiments, the treatment can reduce the number, severity, and / or duration of one or more symptoms of the subject's cancer.

[0359] In one aspect, the disclosure features methods comprising administering a therapeutically effective amount of an antibody, antigen-binding fragment thereof, antigen-binding protein construct (e.g., a bispecific antibody), or antibody drug conjugate described herein to a subject in need thereof, e.g., a subject having cancer or identified or diagnosed as having cancer, e.g., breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, gastric cancer, testicular cancer, thyroid cancer, or urothelial cancer.

[0360] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification sheets and describe the animal, human or non-human being provided for the treatment performed according to the methods of the present invention. The present invention contemplates both veterinary and non-veterinary applications. Human patients can be adults or adolescents (e.g., people under the age of 18). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine animals (e.g., pigs, minipigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals. In certain embodiments, the subject is a human. In certain embodiments, the subject is a dog.

[0361] In some embodiments, the cancer is thyroid cancer, urothelial cancer, breast cancer, colorectal cancer, renal cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.

[0362] In some embodiments, the cancer is cervical cancer, prostate cancer, thyroid cancer, urothelial cancer, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid tumor, skin cancer, liver cancer, or testicular cancer.

[0363] In some embodiments, the cancer is pancreatic cancer, lung cancer, stomach cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, skin cancer, or brain cancer.

[0364] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for developing cancer.Patients with cancer can be identified using various methods known in the art.

[0365] As used herein, an "effective amount" means an amount or dosage sufficient to produce a beneficial or desired result, including stopping, slowing, delaying, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody drug conjugate, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis.

[0366] The effective amount can be administered once or multiple times.For example, the effective amount of an antibody, an Fab or an antibody drug conjugate is enough to improve, prevent, stabilize, reverse, suppress, slow down and / or delay the amount of the progress of the patient's autoimmune disease or cancer in vitro, or is enough to improve, stop, stabilize, reverse, slow down and / or delay the amount of cell (for example, any cancer cell in biopsy cells, cancer cells described herein or cell line (for example, cancer cell line)) propagation in vitro.As understood in the art, the effective amount of an antibody, Fab or an antibody drug conjugate can vary, especially depending on the patient's medical history and other factors, such as the type (and / or dosage) of the composition used.

[0367] The effective amount and regimen for administering the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be understood by those skilled in the art that the dosage that must be administered will vary depending on, for example, the mammal to which the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, antibody drug conjugates and / or compositions disclosed herein used, and other drugs administered to the mammal.

[0368] The typical daily dose of an effective amount of an antibody, its antigen-binding fragment, antigen-binding protein construct (e.g., bispecific antibody) or antibody drug conjugate is 0.01 mg / kg to 100 mg / kg. In certain embodiments, the dosage can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In certain embodiments, the dosage can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dose is about or at least 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0369] In any of the methods described herein, at least one antibody, its antigen-binding fragment or antigen-binding protein construct (e.g., bispecific antibody), antibody drug conjugate or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, antibody drug conjugates or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered with the same composition (e.g., liquid composition). In some embodiments, at least one antibody, its antigen-binding fragment, antigen-binding protein construct (e.g., bispecific antibody) or antibody drug conjugate and at least one additional therapeutic agent are administered with the same composition (e.g., liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered with two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered in the form of a pill, tablet or capsule. In some embodiments, at least one additional therapeutic agent is administered with a sustained-release oral formulation.

[0370] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, antibody drug conjugates, or pharmaceutical compositions described herein) are administered to a subject such that the biologically active time periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) or antibody drug conjugate in the subject overlap.

[0371] In some embodiments, at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein for diagnosing or tracking the effectiveness of treatment (e.g., observing at least one symptom of cancer). As described herein, a skilled medical professional can also change (e.g., increase or decrease) the identity and amount of the antibody or antigen-binding antibody fragment, antibody drug conjugate (and / or one or more additional therapeutic agents) administered to a subject, and can also adjust (e.g., increase or decrease) the dosage or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0372] In certain embodiments, one or more other therapeutic agents can be administered to the subject.Other therapeutic agents can include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (Bruton's tyrosine kinase, BTK) inhibitors and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In certain embodiments, other therapeutic agents are inhibitors of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0373] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an activated hedgehog signaling pathway inhibitor, and an agent that selectively degrades the estrogen receptor.

[0374] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, ), everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0375] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0376] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0377] In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, or an anti-GITR antibody.

[0378] Pharmaceutical compositions and routes of administration

[0379] Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antigen-binding protein constructs, antibodies (e.g., bispecific antibodies), antigen-binding fragments, or antibody drug conjugates described herein. Two or more (e.g., two, three, or four) of any of the antigen-binding protein constructs, antibodies, antigen-binding fragments, or antibody drug conjugates described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0380] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). The composition can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvent, an antibacterial or antifungal agent, such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an isotonic agent, such as a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride) or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition can be formulated and packaged in an ampoule, a disposable syringe or a multiple-dose vial. Where necessary (e.g., in injectable formulations), suitable fluidity can be maintained, for example, by using a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0381] Compositions containing one or more of any of the antigen binding protein constructs, antibodies, antigen binding fragments, antibody drug conjugates described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound to allow for ease of administration and uniformity of dosage).

[0382] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). The LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit a high therapeutic index are preferred. In the event that an agent exhibits undesirable side effects, care should be taken to minimize potential damage (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0383] Exemplary dosages include milligram or microgram amounts of any of the antigen binding protein constructs, antibodies or antigen binding fragments, or antibody drug conjugates described herein per kilogram of body weight of the subject (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 0.1 mg / kg to about 0.5 mg / kg).

[0384] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making antibodies or antigen-binding fragments thereof or antibody drug conjugates for the various uses as described herein.

[0385] Examples

[0386] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0387] Example 1: Anti-human HER2 VHH Antibodies - Synthetic VHH Antibody Library Construction, Panning, and Hit Identification Synthetic VHH antibodies Library construction

[0388] Camel / alpaca VHH antibody is a heavy chain-only antibody that retains both the binding and function of a conventional antibody with a heavy chain (VH) and a light chain (VL). The unique feature of VHH is that it has a longer CDR3 than the VH CDR3 of a conventional antibody. A VHH synthetic library was designed. A fixed length CDR1 and CDR2 were used, each containing 8 amino acids. For CDR3, 13 different lengths ranging from 10 to 22 amino acids were designed. The designed VHH library was synthesized and cloned into the pADL-23c phagemid library. The phagemid VHH library was transformed into TG1 cells. 1.5 μg of phagemid DNA produced >2.5x 10 9 TG1 colonies. A total of 12 transformations were performed, and the combinatorial diversity of the TG1 VHH library was approximately 3 x 10 10 Afterwards, a phage library was prepared by culturing 2 L of TG1 cells and 20 times of helper phage. The phage library was purified using standard protocols. The final titer of the phage library was 2.37 x 10 at OD260. 13 / ml.

[0389] Panning and hit identification

[0390] The antigen of the recombinant human HER2 extracellular domain (h-HER2 ECD) was purchased from Sinobiological USA and biotinylated with an NHS-ester biotinylation reagent (EZ-Link sulfo-NHS-SS-biotin, Thermo Fisher). Dynabeads coupled with streptavidin coated with biotinylated h-HER2 ECD protein were used to pan the binding agents of the human HER2 antigen. After 3 rounds of panning, the h-HER2 binding agents were eluted and used to infect SS320 cells. SS320 cell colonies were selected and cultured in 2YT medium. 0.5 mM IPTG was added to induce the secretion of the VHH antibody. VHH antibodies in the supernatant were screened by ELISA using 96-well plates coated with antigens of human HER2 domain IV (Sinobiological, Pro489-Cys630 of human HER2), human HER2 domain I (homemade, Met1-Cys 195), or human HER2 domain II+III+IV (homemade, Ser196-Cys630). In total, three, ten, and fifteen 96-well plate VHH supernatant samples (2660 clones) were screened for HER2 domain IV, domain I, and domain II+III+IV antigens, respectively. 293T cells stably transfected with human HER2 were used to further screen positive binders for different HER2 domains by flow cytometry whole-cell binding assay. Cell surface h-HER2 binders were sequenced. A total of 26 clones with different sequences were obtained. CDR and framework sequences were obtained using IMGT (International Immunogenetics Information System).

[0391] Example 2: Characterization of HER2 VHH Antibodies

[0392] Construction of bivalent VHH-Fc clones

[0393] Twenty-six unique clones were constructed to produce bivalent antibodies with a human IgG1 Fc region (VHH-Fc) by adding the hinge region and constant domains (CH2 and CH3). The constructed bivalent VHH-Fc antibodies were transiently expressed in ExpiCHO cells, and the proteins in the supernatant were harvested and purified using protein A resin.

[0394] Whole-cell binding of anti-HER2 VHH-Fc clones to human HER2

[0395] Binding of bivalent anti-HER2 VHH-Fc clones to CHO cells stably transfected with human HER2 (CHO-h-HER2 stable cells) was determined by flow cytometry whole cell binding (WCB) assay. Briefly, antibodies were incubated with CHO-h-HER2 stable cells (0.2 x 106 / ml) were incubated together at different concentrations in 100 μl / well in FACS buffer (PBS with 1.0% FBS) in a 96-well plate for 30 minutes. After washing, Alexa Fluor 488-conjugated anti-human IgG Fc secondary antibody (Alexa 488AffiniPure goat anti-human IgG, Fc γ fragment specificity, Jackson labs (Jackson labs), 1:750 dilution), and incubated for 30 minutes.After washing, CytoFlex (Beckman Coulter) is used to measure median fluorescence intensity (MFI) by gating live cell colony with FITC channel.Also use N87 (gastric cancer cell line) and SKBR3 (breast cancer cell line) cell to determine the whole cell binding of VHH-Fc clone and the HER2 of endogenous expression in human cancer cell, both of which are cancer cells with high HER2 expression.The EC50 of the whole cell binding of 20 VHH-Fc clones to HER2 in CHO-h-HER2 stable cells and N87 cells, and its possible binding domain (data from ELISA screening assay) with HER2 are shown in the table below. Whole cell binding curves of the top three VHH-Fc clones 4A8, 11F6, and 11D5 and the reference antibodies Herceptin and Perjeta (from Roche) in CHO-h-HER2 stable cells, N87 cells, and SKBR3 cells. Figure 1A 、 1B and 1C.

[0396] Table 1: EC50 of whole cell binding to CHO-h-HER2 stable cells and N87 cells

[0397]

[0398]

[0399] Determining the binding domain for human HER2 against the leading anti-HER2 VHH antibody

[0400] Binding of anti-HER2 VHH-Fc clones to different domains of human HER2 ECD was determined by ELISA binding assay using his-tagged HER2 domain fragments. Human HER2 domain IV fragment (Pro489-Cys630) was purchased from Sino Biological, and domain II+III+IV fragment (Ser196-Cys630) and domain II fragment (Ser196-Asn319) were prepared. Figure 2A As shown, clone 4A8 and the reference antibody Herceptin (but not Perjeta) bind to domain IV. 11F6 and 11D5 do not bind to domain IV, suggesting that they bind to other domains. Figure 2B 11F6, but not 11D5, is shown to bind to domain II, while both 11F6 and 11D5 bind to domains II+III+IV ( Figure 2C ), indicating that 11D5 binds to domain III of human HER2.

[0401] PBMC-mediated killing of HER2-expressing breast cancer cells by a leading anti-HER2 VHH-Fc clone

[0402] One of the anti-tumor functions of anti-HER2 antibodies in vivo is antibody-dependent cell-mediated cytotoxicity (ADCC), which involves binding of the antibody to tumor cells via the variable binding domain (e.g., Fab or VHH) and binding of the Fc domain to Fcγ receptors on NK cells (e.g., FcγRIII or CD16). The bridge between tumor cells and NK cells activates NK cells, which then release proteins such as perforin and proteases to induce lysis of antibody-bound tumor cells.

[0403] Human PBMC (peripheral blood mononuclear cells, which contain NK cells) from normal donors were used. Briefly, cancer cells were seeded in 96-well plates at 15K / well and cultured in 100 μl RPMI1640 medium for one day. Fresh PBMC (0.3 x 10 6 After one day of incubation, the viable cell population in each well was determined by adding CCK-8 reagent and reading at OD450 nm. Figure 3A and 3B Anti-HER2 antibody-mediated killing of two breast cancer cell lines, AU-565 and SKBR3, in the presence of fresh PBMCs is shown. The IC50s are shown in the table below.

[0404] Table 2: IC50 (nM) of the leading anti-HER2 clones against cancer cells in the presence of PBMCs

[0405] clone IC50 (nM) in AU-565 cells IC50 (nM) in SKBR3 cells 4A8 0.19 0.14 11F6 0.22 0.21 11D5 0.38 0.29 Patjet 0.24 0.29

[0406] Effects of leading anti-HER2 VHH-Fc clones on the proliferation of HER2-expressing breast cancer cells

[0407] Activation of HER2 can cause certain cancer cells with high HER2 expression to proliferate. Anti-HER2 antibodies can inhibit proliferation by blocking HER2 activation. To determine the effect of leading anti-HER2 antibodies on proliferation, AU565 cells were incubated with different concentrations of leading anti-HER2 VHH-Fc clones or the reference antibody Herceptin. Figure 4As shown, both 4A8 and 11D5 inhibited the proliferation of AU-565 cells in a concentration-dependent manner. However, the reference antibody Herceptin showed only a very weak anti-proliferative effect, and 11F6 did not show any anti-proliferative effect in AU-565 cells. The IC50 values ​​of the anti-proliferative effects of these antibodies are shown in the table below.

[0408] Table 3: Antiproliferative effects of leading anti-HER2 clones on AU-565 cells

[0409] clone IC50 (nM) in AU-565 cells 4A8 1.02 11F6 UD 11D5 0.84 Herceptin UD

[0410] UD: Unable to determine.

[0411] The CMC profiles of 4A8, 11F6, and 11D5 were evaluated by SEC-HPLC. 11D5 showed a very good SEC-HPLC profile with >98% monomer. However, 4A8 and 11F6 showed very poor SEC profiles with less than 30% monomer (not shown). Therefore, 4A8 and 11F6 were not selected due to poor developability. The sequence of 11D5 is shown in the table below.

[0412] Table 4: Sequence of the leading anti-HER2 VHH clone 11D5 (IMGT definition)

[0413]

[0414] Example 3: Anti-human TROP2 VHH Antibodies—Immunization, Library Construction, Panning, and Hit Identification

[0415] Immunization and library construction

[0416] Recombinant human and cynomolgus monkey TROP2 extracellular domain (ECD) was purchased from Sino Biological (USA). Immunization with TROP2 antigen was performed using 2-week-old virgin female alpacas from Capralogics Inc. The titer of serum antibodies was measured by ELISA. After 4 rounds of immunization, high titers (1:50,000) of anti-human TROP2 were observed. 80 ml of whole blood was collected from the alpacas and PBMCs were isolated. Thereafter, RNA was isolated from the PBMCs.

[0417] The VHH (variable domain of the heavy chain of the heavy chain antibody) gene was amplified by PCR using 10 pairs of specially designed primers covering all alpaca germline sequences. The PCR product was purified (using DNA agarose gel and gel extraction kit) and cloned into a pADL-23c phagemid library, which was used to transform TG1 cells by electroporation. The transformed TG1 library had a diversity of 2.7 x 10 9Transformed TG1 cells were cultured in 2YT medium, and VHH-displaying phages were produced by adding helper phage and culturing overnight. Phages in the supernatant were harvested by precipitation with 4% PGE / 0.5 M NaCl and high-speed centrifugation. The phage library was purified using standard protocols. The final titer of the phage library was 1 x 10 at OD260. 13 / ml.

[0418] Panning and hit identification

[0419] Binders for human TROP2 (h-TROP2) antigen were selected using streptavidin-coupled Dynabeads coated with biotinylated h-TROP2 ECD protein. After 2-3 rounds of selection, the binders for h-TROP2 were eluted and used to infect SS320 cells. SS320 cell colonies were selected and cultured in 2YT medium. 0.5 mM IPTG was added to induce the secretion of VHH antibodies. Using CHO cells stably transfected with h-TROP2, supernatants with VHH antibodies were screened by whole-cell binding assay using flow cytometry. A total of 74 whole-cell binders were obtained. After sequencing, 25 unique sequences were obtained. CDR and framework sequences were determined using IMGT (International Immunogenetics Information System).

[0420] Example 4: Characterization of TROP2 VHH Antibodies

[0421] Construction of bivalent VHH-Fc clones

[0422] 25 unique clones from the hit identification were used to prepare bivalent antibodies with a human IgG1 Fc region (VHH-Fc) including the hinge region and constant domains (CH2 and CH3). The constructed bivalent VHH-Fc antibodies were transiently expressed in ExpiCHO cells, and the proteins in the supernatant were harvested and purified by protein A resin.

[0423] Characterization of TROP2 VHH antibodies

[0424] Binding of bivalent anti-TROP2 VHH-Fc clones to human TROP2 in CHO cells stably transfected with human TROP2 (CHO-h-TROP2 stable cells) was determined by flow cytometry whole cell binding (WCB) assay. Briefly, antibodies were incubated with CHO-h-TROP2 stable cells (0.2 x 10 6100 μl / well) were incubated together in FACS buffer at different concentrations in a 96-well plate (100 μl / well) for 30 minutes. After washing, Alexa Fluor 488-conjugated anti-human IgG Fc secondary antibody was added and incubated for 30 minutes. After washing, the MFI was measured using CytoFlex by gating the live cell population with the FITC channel. The EC50 of binding was calculated using GraphPad Prism 7.0. Representative figures are shown in Figure 5A Similarly, whole-cell binding of anti-TROP2 VHH-Fc clones to mouse TROP2 (m-TROP2) was determined using 293T cells stably transfected with mouse TROP2 (293T-m-TROP2 cells), and the results are shown in Figure 5B The EC50 for whole cell binding is shown in the table below.

[0425] Table 5: Whole cell binding of anti-TROP2 VHH-Fc clones to human or mouse TROP2

[0426]

[0427]

[0428] UD: Unable to determine

[0429] ND: Not Determined

[0430] The binding affinity of the top three clones (4C6, 3H9 and 1H11) to human cancer cells with high levels of endogenous expression of TROP2 was also determined. Figure 6 As shown in Figure 2, these leading VHH-Fc clones all bound to TROP2 endogenously expressed in the breast cancer cell line SKBR3 with high binding affinity, which was similar to or slightly higher than the binding affinity of the anti-TROP2 antibody reference antibody Immu-132 (from Gilead Sciences) in Trodelvy to TROP2. The EC50 for whole-cell binding in SKBR3 cells was Figure 6 Shown in.

[0431] The binding region on human TROP2 was also detected by competitive binding assay. Briefly, 60nM of clones 4C6, 3H9 and 1H11 were incubated with CHO-h-TROP2 stable cells in FACS buffer at 4°C in 96-well plates for 30 minutes. After washing twice, the cells were incubated with Immu-132 at different concentrations at 4°C for 30 minutes. The cells were washed twice again and resuspended in a buffer containing anti-human F(ab)2Alexa Fluor 488 (1:600 ​​dilution) and incubated at 4°C for 30 minutes. The cells were then washed once and resuspended in FACS buffer, and the MFI was measured using CytoFlex by gating the live cell population with the FITC channel. Figure 7 As shown, all three leading VHH-Fc clones competed with Immu-132 for binding to cell surface human TROP2, indicating that they share similar binding regions with Immu- 132. The sequences of the three leading VHH clones are shown in the table below.

[0432] Table 6: Sequences of anti-TROP2 VHH clones (IMGT definition)

[0433]

[0434]

[0435] Example 5: Humanization of the leading anti-TROP2 VHH clone

[0436] Humanization was performed by analyzing the germline sequences of the first three clones using Igblast. The closest human germline (IGHV-2-23*04) sequence was used to modify the alpaca framework of these three clones. Four humanized variants were prepared for 3H9, with slight differences in framework 2 and fused to human IgG1 Fc. Whole cell binding assays on CHO-h-TROP2 stable cells were performed to compare the binding activity of the humanized VHH-Fc variants with the parental alpaca clone 3H9-Fc. Figure 8 As shown, all humanized VHH-Fc variants had lower binding activity compared to the parental control, with hv3 showing the best binding among the humanized VHH-Fc variants.

[0437] The same strategy was followed for the humanization of the alpaca clone 4C6. Ten humanized VHH-Fc variants were generated for 4C6, with slight differences in framework 2 and framework 3. Whole-cell binding to CHO-h-TROP2 stable cells was performed to compare the binding affinity of these humanized VHH-Fc variants with the parental alpaca clone 4C6. The results are presented in Figures 9A-9CThe EC50 (nM) for whole cell binding is shown in the table below. The sequences of 4C6-hv4, hv9, and hv10 are shown in Table 8.

[0438] Table 7: EC50 of humanized variants of 4C6 binding to whole-cell CHO-h-TROP2 stable cells

[0439] clone EC50 (nM) of WCB against CHO-h-TROP2 stable cells 4C6 Alpaca 0.97 4C6-hv1 21.71 4C6-hv2 56.62 4C6-hv3 26.53 4C6-hv4 2.60 4C6-hv5 12.43 4C6-hv6 15.67 4C6-hv7 22.58 4C6-hv8 27.33 4C6-hv9 2.32 4C6-hv10 1.46 Immu-132 0.84

[0440] Table 8: Sequences of selected 4C6 humanized variants (IMGT definition)

[0441]

[0442]

[0443] The same strategy was used to humanize the alpaca clone 1H11. Ten humanized VHH-Fc variants were prepared, with slight differences in framework 2. The binding activity of the humanized variants to human TROP2 was tested using a whole cell binding assay. Figure 10A and 10B As shown, significantly weaker binding was observed for variants hv1, 2, 3, 4, and 5 compared to the parental alpaca clone 1H11. Figure 10B As shown in the table below, variants hv6, 7, 8, 9 and 10 retained the binding affinity of alpaca clone 1H11.

[0444] Whole-cell binding of endogenously expressed human TROP2 in tumor cells was also examined to select humanized variants with high binding affinity to TROP2. Figure 11A As shown in Figures 1 and 2, compared to the parental alpaca clone 1H11, these humanized variants all retained binding affinity to human TROP2 endogenously expressed in tumor cells NCI-H1975 (human lung cancer cells) and Colo205 (human gastric cancer cells), and the binding affinity was similar to that of the reference antibody Immu-132 for TROP2. EC50 values ​​are shown in the table below.

[0445] Table 9: EC50 and CMC of humanized variants of 1H11 binding to whole-cell CHO-h-TROP2 stable cells

[0446] Overview ND: Not Determined

[0447] Thermal shift and SEC-HPLC were performed on these potent humanized variants. As shown in the table above, 1H11-hv6 and 1H11-hv7 have lower Tm compared to other humanized variants. The SEC-HPLC profiles of most of these potent humanized variants are similar (with the exception of 1H11-hv7, which has a monomer ratio of over 95%), indicating that most potent humanized variants have good stability and developability. The sequences of selected humanized variants of 1H11 are shown in the table below.

[0448] Table 10: Sequences of selected humanized variants of 1H11 (IMGT definition)

[0449]

[0450] Example 6: Construction of anti-HER2 / TROP2 bispecific antibodies

[0451] Based on the binding affinity, potency and CMC profile of anti-HER2 VHH clone 11D5, the clone was selected to construct a bispecific antibody. Initially, anti-TROP2 VHH clone 1H11-hv10 was selected to construct a bispecific antibody to test in vitro function. In order to reduce the number of disulfide bonds in the hinge region of the VHH-Fc or BsAb construct and obtain the desired number of payload / toxin conjugates, the first cysteine ​​in the hinge region was changed to serine (C220S), resulting in a mutant construct containing only 2 cysteines in each hinge region (one heavy chain), thereby forming 2 disulfide bonds for these VHH-Fc / BsAbs. In order to detect the effects of different forms of BsAb on the binding of tumor targets HER2 and TROP2, four different forms of BsAb were prepared, such as Figures 12C-12F The whole cell binding of BsAbs to human HER2 and TROP2 was determined, and the binding curves were Figure 13A and 13B EC50 (nM) values ​​are shown in the table below. The results show that both the 1H11-hv10+11D5_Fc (tandem) and 1H11-hv10_Fc_11D5 (N-terminal and C-terminal) formats retain binding affinity to both HER2 and TROP2, with the 1H11-hv10_Fc_11D5 format having a higher binding affinity to HER2 than the 1H11-hv10+11D5_Fc format.

[0452] Table 11: Summary of WCB of BsAbs and stably transfected cells

[0453]

[0454] The sequences of these monospecific and bispecific antibodies are shown in Tables 12 and 13, all with a C220S mutation in the hinge region.

[0455] Table 12: Sequences of monospecific VHH-Fc antibodies

[0456]

[0457] Table 13: Sequences of different bispecific antibody formats

[0458]

[0459]

[0460]

[0461] Example 7: Characterization of anti-HER2 / TROP2 bispecific antibodies

[0462] Based on the whole cell binding data of different formats of BsAb, N-terminal format (anti-TROP2 in N-terminus, anti-HER2 in C-terminus) and tandem format (anti-TROP2 in top, anti-HER2 in middle) were selected to use the humanized variant 1H11-hv8 ( Figure 14A More BsAbs were prepared using the bivalent 1H11-hv8_Fc) shown in Figure 1 and the anti-HER2-VHH 11D5 fused to human IgG1-Fc, as shown in Figure 1 Figure 14B and 14C The sequences are shown in the table below, all of which have a C220S mutation in the hinge region.

[0463] Table 14: Sequences of monospecific anti-TROP2 antibodies

[0464]

[0465] Table 15: Sequences of different bispecific antibody formats

[0466]

[0467]

[0468] Using a monospecific antibody as a control, the kinetic binding of the N- and C-terminal forms of the BsAb (1H11-hv8_Fc_11D5) to the two target antigens was determined. Figures 15A-15E As shown, 1H11-hv8_Fc_11D5 bound to both human HER2 and TROP2 with good conjugation, but the monospecific antibodies 1H11-hv8 and Immu-132 bound only to human TROP2, and 11D5 and Herceptin bound only to human HER2.

[0469] Antibody drug conjugates (ADCs) have brought significant benefits to patients as anticancer drugs. Among them, four drugs using vc-MMAE (or mc-vc-PAB-MMAE) have been approved. Vc-MMAE antibody drug conjugates are composed of a monoclonal antibody (mAb) that is covalently bound to the potent anti-mitotic toxin MMAE (monomethyl auristatin E) through a lysosomal cleavable dipeptide valine-citrulline (vc) linker. Considering the escape of tumors due to the expression of different tumor-associated antigens (TAAs) that cannot be targeted by monospecific antibody drug conjugates, bispecific antibodies against HER2 / TROP2 conjugated with MMAE may provide a better treatment option.

[0470] The binding of two leading BsAbs (1H11-hv8_Fc_11D5 and 1H11-hv8+11D5_Fc) to whole-cell tumor cells expressing human HER2 or TROP2 at different levels was determined. The potential effect of ADC-mediated killing of tumor cells expressing HER2 or TROP2 at different levels was also determined in the presence of a second antibody: MMAE-conjugated anti-human IgG Fc (Fab) with a cleavable linker (designated secondary Ab-MMAE, purchased from MORADEC, AH-202-AE).

[0471] like Figure 16A As shown, whole cell binding assays showed that SKBR3 cells expressed HER2 at very high levels (MFI of about 300,000, as determined by binding to Herceptin at 100 nM) and expressed TROP2 at high levels (MFI of about 90,000, as determined by binding to Immu-132 at 100 nM). The binding affinity of the N, C-terminal form BsAb 1H11-hv8_Fc_11D5 to HER2 was higher than that of the tandem form BsAb 1H11-hv8_Fc_11D5 to HER2. The binding affinity was similar to that of Herceptin to HER2. In the presence of a secondary Ab-MMAE ratio of 1:4 or 1:6 (primary antibody relative to secondary Ab-MMAE), these antibodies were subjected to killing assays and the amount of viable cells was measured by adding cell counting-8 (CCK-8) reagent and reading at 450 nm after 1-4 hours. The results showed that Herceptin and the N- and C-terminal forms of BsAb 1H11-hv8_Fc_11D5 had similar potency in killing SKBR3 cells. However, the tandem form of BsAb 1H11-hv8+11D5_Fc was slightly less effective in killing SKBR3 cells. EC50 values ​​are shown in Table 16.

[0472] like Figure 17A As shown, whole cell binding assays showed that the human lung cancer cell line NCI-H441 cells expressed TROP2 at very high levels (MFI of approximately 200,000, as determined by binding to Immu-132 at 100 nM) and expressed HER2 at very low levels (MFI of approximately 10,000, as determined by binding to Herceptin at 100 nM). In a killing assay using the secondary Ab-MMAE at a ratio of 1:4, both forms of BsAb showed similar potency compared to the reference antibody Immu-132. The killing effect was dependent on the level of TROP2 expression ( Figure 17B ).

[0473] Whole cell binding and killing using the secondary Ab-MMAE was also performed on HCC202 cells (breast cancer cells). Figure 18A As shown, cells expressed both HER2 and TROP2 at high levels, with HER2 levels (MFI of 240,000, as determined by binding to Herceptin at 100 nM) slightly higher than TROP2 levels (MFI of 200,000, as determined by binding to Immu-132 at 100 nM). In the presence of the secondary Ab, MMAE, the potency of anti-HER2 and TROP2 mediated cell killing was also similar, as shown in Figure 2. Figure 18B and shown in the table below.

[0474] Table 16: EC50 of WCB and IC50 in killing assays of tumor cells expressing different levels of endogenous HER2 and TROP2.

[0475]

[0476] UD: Unable to determine

[0477] ND: Not Determined

[0478] Example 8: vc-MMAE conjugation for lead anti-HER2 and anti-TROP2 monospecific antibodies and anti-HER2 / TROP2 bispecific antibodies

[0479] N and C terminal form BsAb 1H11-hv8_Fc_11D5 was selected as the leading BsAb to be conjugated with vc-MMAE (or mv-vc-PAB-MMAE). Monospecific bivalent parent clones 11D5_Fc and 1H11-hv8_Fc were also conjugated. As described above, all of these VHH-Fc constructs contain a C220S mutation in the hinge region to reduce the number of cysteines from 3 to 2, resulting in only two disulfide bonds being formed in these antibodies to achieve a drug-antibody ratio (DAR) of 4 for vc-MMAE conjugation. Reference antibodies Herceptin and Immu-132 were also conjugated with a goal of achieving a DAR of about 4 for fair comparison in in vitro efficacy assays.

[0480] The final vc-MMAE conjugated ADCs of 11D5_Fc, 1H11-hv8_Fc, 1H11-hv8_Fc_11D5, Herceptin and Immu-132 were named 11D5_MMAE (full name: 11D5_Fc_MMAE), 1H11_MMAE (full name: 1H11-hv8_Fc_MMAE), BsAb-MMAE (full name: 1H11-hv8_Fc_11D5-2C_MMAE), Herceptin_MMAE and Immu-132_MMAE, respectively. The DARs of these ADCs were determined by HIC-HPLC and analyzed in Figure 19A 、 20A , 21A, 22A and 23A and shown in the table below. The purity and stability of these ADCs were determined by SEC-HPLC and the results are shown in Figure 19B 、 20B , 21B, 22B and 23B and shown in the table below. In MMAE-conjugated VHH_Fc antibodies including BsAb_MMAE, most DAR species are D4 (4 drugs conjugated) (80-100%). D3 (3 drugs conjugated) accounts for 16-18%. Almost no D2 (2 drugs conjugated) is produced. There is no D6 or D8 either, because there are only 4 cysteines in the hinge region of Fc (2 cysteines in each hinge region) to form two disulfide bonds, which is the key structure for conjugation (each cysteine ​​of the disulfide bond can be conjugated with one vc-MMAE). Therefore, the maximum amount of drug that can be conjugated to VHH_Fc is 4. A high percentage of D4 is an ideal choice for maintaining strong ADC efficacy. On the other hand, a low percentage of D2 can avoid low efficacy, and the lack of D6 / D8 can avoid potential toxicity. In contrast, in vc-MMAE-conjugated traditional antibodies (Herceptin_MMAE and Immu-132_MMAE), there is a high percentage of D2 and some D6 and D8, as shown in Figure 2. Figure 22A and 23AAs shown in the table below, although the conjugation was performed using a special DAR4 technology to minimize the production of D6 and D8.

[0481] Table 17: Analysis of vc-MMAE conjugated VHH_Fc and reference antibodies

[0482]

[0483] To understand the impact of ADCs with different DARs on in vitro potency, we first tested different DARs of MMAE-conjugated reference antibodies obtained during optimization of MMAE conjugation in an in vitro killing assay. ++++ , Figure 17A ) were used to test the killing effect of Immu-132-MMAE with DAR 3.5, 4.0 and 4.4 respectively. Figure 24A As shown in Table 18, increasing the DAR from 3.5 to 4.4 only slightly increased the killing potency of Immu-132-MMAE, from an IC50 of 0.047 nM to an IC50 of 0.033 nM, indicating that when the difference in DAR is less than 1, the effect of higher DAR on cell killing is minimal (Table 18). Similarly, in killing AU-565 cells expressing HER2 at very high levels (HER2 ++++ , Figure 25A ), the efficacy of Herceptin-MMAE with DAR 3.6, 4.0 and 4.3 was determined. Figure 24B As shown, there was no difference in the potency of Herceptin-MMAE to kill AU-565 cells at different DAR levels, indicating that slightly different DAR levels have no effect on in vitro potency if the difference in DAR is less than 1 (Table 18).

[0484] Table 18: Killing effect of reference ADCs at different DAR levels on cancer cells

[0485]

[0486] ND: Not determined.

[0487] HER2 + :MFI 10,000; HER2 ++++ : MFI 400,000, determined by binding to Herceptin at 100 nM in a flow cytometry assay in the presence of AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. TROP2 +++ :MFI 90,000;TROP2 ++++: MFI 200,000, determined by binding to Immu-132 at 100 nM in a flow cytometry assay in the presence of AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody.

[0488] Based on the above results, if the difference in DAR level is less than 1, the comparison of in vitro killing efficacy is reasonable. Therefore, a comparative study of in vitro killing of cancer cells was conducted on 11D5_MMAE, 1H11_MMAE, BsAb_MMAE, Herceptin_MMAE and Immu-132_MMAE, and the DARs were 4.0, 3.8, 3.7, 3.6 and 3.5, respectively (Table 17). Figure 16A 、 25A As shown in Figure 25B, SKBR3, AU-565, and N87 cells all expressed HER2 at very high levels (HER2 ++++ ) and expressed TROP2 at high levels (TROP2 +++ ). Anti-HER2-ADCs (Herceptin_MMAE and 11D5_MMAE) were very effective in killing these cells, with IC50 values ​​of approximately 0.017nM-0.094nM. Anti-TROP2-ADCs (Immu-132_MMAE and 1H11_MMAE) had similar killing effects on these cells as anti-HER2-ADCs, with IC50 values ​​of approximately 0.028nM-0.095nM. BsAb_MMAE was the most effective ADC in killing these cancer cells, with IC50 values ​​of approximately 0.0157nM-0.040nM ( Figure 26A 、 26B , 26C and Table 19).

[0489] The killing effects of these ADCs on the human ovarian cancer cell line SKOV3 and the human esophageal adenocarcinoma cell line OE-19 were also determined. Both SKOV3 and OE-19 cells expressed HER2 at very high or high levels, with an MFI of approximately 300,000 or 200,000 (HER2 ++++ or HER2 +++ ), as determined by binding of Herceptin, but expressed TROP2 at intermediate levels with an MFI of approximately 50,000 or 30,000 (TROP2 ++ ), as determined by binding of Immu-132 ( Figure 27A and 27B). Anti-HER2-ADC Herceptin_MMAE and 11D5_MMAE mediated effective killing of both cancer cells, with IC50 values ​​of approximately 0.072nM-0.111nM. Compared with the killing of both cancer cells mediated by anti-HER2-ADC, anti-TROP2-ADC Immu-132_MMAE and 1H11_MMAE mediated weaker killing of both cancer cells, with IC50 values ​​of approximately 0.889nM-1.441nM. Similarly, BsAb_MMAE was the most effective ADC in killing both cancer cells, with IC50 values ​​of 0.062nM and 0.069nM, respectively ( Figure 28A and 28B , and Table 19).

[0490] It was also determined that these ADCs were effective against cells expressing moderately high / very high TROP2 (TROP2 ++ / +++ , MFI of approximately 30,000-200,000, determined by binding to 100 nM Immu-132) but low HER2 (HER2 + , MFI of about 10,000-20,000, determined by binding to 100 nM Herceptin), such as NCI-H441 cells ( Figure 16A ), NCI-H1975 cells (non-small cell lung cancer cells, Figure 29A ), Colo205 cells (colorectal cancer cells, Table 29B), T-47D cells (breast cancer cells, Figure 29C ) and A431 cells (epidermoid carcinoma cells, Figure 29D ). The result is Figure 30A 、 30B , 30C, 30D, 30E and shown in Table 19. Anti-TROP2-ADC Immu-132_MMA and 1H11_MMAE, but not anti-HER2_ADC Herceptin_MMAE and 11D5_MMAE, mediated effective killing. Similarly, BsAb_MMAE was as effective as or more effective than the reference Immu-132_MMAE in killing these cancer cells (Table 19).

[0491] Therefore, BsAb_MMAE can kill cancer cells expressing HER2 and / or TROP2 at different expression levels, and monospecific ADCs such as Herceptin-MMAE and Immu-132-MMAE are more effective and have efficacy. The potential indications of BsAb_MMAE (1H11-hv8_Fc_11D5_MMAE) include, but are not limited to, many solid tumors in different stages including patients with advanced metastasis, such as breast cancer, gastric cancer, colorectal cancer, lung cancer (NSCLC and SCLC), bladder cancer, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, skin cancer (including melanoma), etc.

[0492] Table 19: Cytotoxic effects of MMAE-conjugated drugs on cancer cells expressing different levels of HER2 and TROP2

[0493]

[0494]

[0495] HER2 + :MFI,10,000-20,000;HER2 ++++ : MFI, 200,000-400,000, determined by binding to Herceptin at 100 nM in a flow cytometry assay in the presence of AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody.

[0496] TROP2 ++ :MFI, 30,000-50,000; TROP2 +++ -TROP2 ++++ MFI, 65,000-250,000, determined by binding to Immu-132 at 100 nM in a flow cytometry assay in the presence of AlexaFluor 488-conjugated anti-human IgG Fc secondary antibody. ND: Not Determined.

[0497] Example 9: In vivo efficacy of MMAE-conjugated lead BsAb in a TROP2 tumor model

[0498] To test the efficacy of MMAE-conjugated lead BsAbs in the TROP2 tumor model, the human epidermoid carcinoma cell line A431 was selected for in vivo studies because (1) A431 cancer cells express TROP2 at very high levels but express HER2 at very low levels, as Figure 29D(2) In vitro killing assays showed that both the anti-TROP2 reference ADC (Immu-132_MMAE) and the leading BsAb-ADC (BsAb_MMAE) had effective killing effects, while the non-anti-HER2 reference ADC (Herceptin_MMAE) ( Figure 30E ). Therefore, A431 is an ideal cancer model to determine the efficacy of anti-TROP2-ADCs.

[0499] In vivo efficacy studies were conducted in Nu / Nu nude mice bearing A431 epidermoid carcinoma. A431 cancer cells (0.5 x 10 6 ). When the average tumor volume reached about 175mm 3 Treatment was started at 4 pm. Drugs were administered intravenously once a week for a total of 3 times, at 3 mg / kg for the leading BsAb (1H11-hv8_Fc_11D5) conjugated or unconjugated, and at 4 mg / kg for Herceptin_MMAE or Immu-132_MMAE, with an equal molar ratio to BsAb_MMAE. Statistical analysis of tumor volume (by 2-way ANOVA and Tukey's multiple comparison test) showed that treatment with BsAb_MMAE and Immu-132_MMAE significantly inhibited tumor growth on and after day 23 after treatment compared to the negative control (PBS-treated group) ( Figure 31 In contrast, treatment with Herceptin-MMAE and unconjugated BsAb had no significant effect on tumor growth compared to the negative control. Both BsAb-MMAE and Immun-132-MMAE significantly reduced tumor growth compared to the negative control, with tumor growth inhibition (TGI) of 60.8% and 66.6%, respectively, at day 30 after treatment.

[0500] No significant changes in body weight were observed among mice from different treatment groups, indicating that the drug was well tolerated.

[0501] Example 10: In vivo efficacy of MMAE-conjugated lead BsAb in a HER2 tumor model

[0502] To test the efficacy of MMAE-conjugated lead BsAb in HER2 tumor models, the human esophageal adenocarcinoma cell line OE-19 was selected for in vivo studies. Figure 27B As shown, OE-19 cancer cells express HER2 at very high levels, but TROP2 is expressed at medium to low levels. In vitro killing assays also showed that both the anti-HER2 reference ADC (Herceptin_MMAE) and the leading BsAb-ADC had potent killing effects on OE-19 cells, but the anti-TROP2 reference ADC (Immu-132_MMAE) had a weaker killing effect ( Figure 28Band Table 19). These results suggest that OE-19 can be used as a cancer model to determine the efficacy of the anti-HER2 arm of an ADC, while the anti-TROP2 arm has limited impact.

[0503] In vivo efficacy studies were performed in immunodeficient B-NDG mice (generated at Biocytogen by deleting the IL2rg gene from NOD-scid mice, which have a severe immunodeficient phenotype) bearing OE-19 tumors. OE-19 cancer cells (0.5 x 10 in 0.1 ml) were implanted subcutaneously. 6 cells). The average tumor volume reached about 124 mm 3 Treatment was started 10 days later. For ADC, drug was administered intravenously once at 5 mg / kg, and PBS was used as a negative control. Compared with the negative control, all treatment groups significantly reduced tumor growth, as shown in Figure 2. Figure 32 As shown. However, gradual tumor growth was observed in the Immu-132_MMAE-treated group, but not in the BsAb_MMAE or Herceptin_MMAE-treated groups. The difference in tumor volume between those treated with Immu-132_MMAE and those treated with BsAb_MMAE or Herceptin_MMAE reached significance on day 22 of treatment (P < 0.05) and became more significant thereafter (P < 0.0001, by two-way ANOVA and Tukey's multiple comparison test).

[0504] No significant changes in body weight were observed among mice from different treatment groups, indicating that the drug was well tolerated.

[0505] Example 11: Conjugation of different types of payloads to anti-HER2 / TROP2 bispecific antibodies with different DAR ratios

[0506] In the above experiments, the payload selected for ADC conjugation was MMAE, a potent mitotic inhibitor by inhibiting tubulin. However, several different types of toxins / payloads with different potencies and modes of action can also be used in antibody-drug conjugates, such as SN-38 and Dxd. Both SN-38 and Dxd are topoisomerase I inhibitors whose in vitro cancer cell inhibition potency is 1 / 1000-1 / 100 of that of MMAE. The potential advantage of antibodies conjugated with SN-38 and / or Dxd is that their toxicity or unwanted side effects may be less than those of antibodies conjugated with MMAE. However, because the potency of SN-38 and Dxd is much weaker than that of MMAE, a larger amount of payload / drug is required. Therefore, by removing the C220S mutation, an anti-HER2 / TROP2 BsAb construct with three cysteines in each hinge region to form three disulfide bonds was constructed. The construct was named 1H11-hv8_Fc-11D5-3C. The sequence is shown in the table below. In the construct with two cysteines (with C220S) in each hinge region forming only two disulfide bonds, four payloads (drugs) can be conjugated ( Figure 36A ), resulting in a maximum DAR of 4.0. An example of a vc-MMAE-conjugated VHH-Fc antibody with two cysteines is Figure 19A 、 20A As shown in Figures 21A and 21A and Table 17, the DAR of these antibodies ranged from 3.87 to 4.0. This format can be used for BsAb-ADCs targeting any tumor-associated antigen. In a construct with 3 cysteines in each hinge region, 6 payloads can be conjugated ( Figure 36B ), resulting in a maximum DAR of 6.0. A similar strategy can be used for BsAb-ADCs in tandem form, such as Figure 36C and 36D As shown, or monospecific VHH_Fc-ADC, as Figure 36E and 36F As shown. Cleavable or non-cleavable linkers such as mv-vc-PABC or CL2-PABC can be used. In addition, different types of payloads such as MMAE, SN-38, and Dxd can be used.

[0507] Table 20: BsAb sequences with 3 cysteines in each hinge region

[0508]

[0509] Other embodiments

[0510] It should be understood that although the present invention has been described in conjunction with the detailed description of the present invention, the foregoing description is intended to illustrate rather than limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof, which binds to HER2 (human epidermal growth factor receptor 2), wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; The selected VHH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NO: 22, 23 and 24, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 22, 23, and 24, respectively.

3. An antibody or antigen-binding fragment thereof that binds to HER2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is shown in SEQ ID NO:

1.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment specifically binds to HER2. 5 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. 6 . An antibody or antigen-binding fragment thereof, comprising the VHH CDR1, 2, and 3 of the antibody or antigen-binding fragment thereof according to claim 1 .

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises human IgG Fc.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

9. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. An antibody or antigen-binding fragment thereof, which binds to TROP2 (tumor-associated calcium signal transducer 2), comprising: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VHH CDR3 amino acid sequence; wherein the selected VHH CDR1, 2 and 3 amino acid sequences are one of the following: (1) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 25, 26, and 27, respectively; (2) the selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, and 30, respectively; and (3) The amino acid sequences of the selected VHH CDR1, 2, and 3 are shown in SEQ ID NOs: 31, 32, and 33, respectively.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 25, 26, and 27, respectively.

12. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VHH comprises CDR1, 2, 3 whose amino acid sequences are shown in SEQ ID NOs: 28, 29, and 30, respectively.

13. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VHH comprises CDR1, 2, and 3 whose amino acid sequences are shown in SEQ ID NOs: 31, 32, and 33, respectively.

14. An antibody or antigen-binding fragment thereof, which binds to TROP2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain antibody variable domain (VHH), wherein the VHH comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NO: 2-12.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VHH comprises the sequence of SEQ ID NO:

2.

16. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VHH comprises the sequence of SEQ ID NO:

3.

17. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VHH comprises the sequence of SEQ ID NO:

4.

18. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VHH comprises the sequence of SEQ ID NO:

10.

19. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VHH comprises the sequence of SEQ ID NO:

12.

20. The antibody or antigen-binding fragment thereof according to any one of claims 10 to 19, wherein the antibody or antigen-binding fragment specifically binds to TROP2.

21. The antibody or antigen-binding fragment thereof according to any one of claims 10 to 20, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

22. An antibody or antigen-binding fragment thereof, comprising the VHH CDR1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 10 to 21.

23. The antibody or antigen-binding fragment thereof according to any one of claims 10 to 22, wherein the antibody or antigen-binding fragment comprises a human IgG Fc.

24. The antibody or antigen-binding fragment thereof according to any one of claims 10 to 23, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

25. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof according to any one of claims 10 to 24.

26. A multispecific antibody or antigen-binding fragment thereof, comprising: a first VHH (VHH1), wherein the VHH1 specifically binds to HER2; and a second VHH (VHH2), wherein the VHH2 specifically binds to TROP2. 27 . The multispecific antibody or antigen-binding fragment thereof according to claim 26 , further comprising: a third VHH (VHH3) that specifically binds to HER2; and a fourth VHH (VHH4) that specifically binds to TROP2.

28. The multispecific antibody or antigen-binding fragment thereof of claim 26 or 27, wherein the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are listed in Figure 33.

29. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 28, wherein the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is shown in SEQ ID NO:

1.

30. The multispecific antibody or antigen-binding fragment thereof of any one of claims 26 to 29, wherein the VHH2 and / or the VHH4 comprise complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are listed in Figure 34.

31. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 30, wherein the VHH2 and the VHH4 comprise an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 2-12.

32. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 31, wherein the multispecific antibody or antigen-binding fragment thereof comprises human IgG Fc.

33. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 32, wherein the VHH1 and VHH3 are linked to the N-terminus or C-terminus of the human IgG Fc.

34. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 33, wherein the VHH2 and VHH4 are linked to the N-terminus or C-terminus of the human IgG Fc.

35. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain antibody variable domain (VHH1), a first hinge region, a first CH2, a first CH3, and a second VHH (VHH2); and (b) a second polypeptide comprising, from N-terminus to C-terminus, a third VHH (VHH3), a second hinge region, a second CH2, a second CH3 and a fourth VHH (VHH4), wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

36. The polypeptide complex of claim 35, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 18; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

18. The polypeptide complex according to claim 35 or 36, wherein the VHH2 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence.

38. The polypeptide complex according to any one of claims 35 to 37, wherein the VHH4 is connected to the C-termini of the second CH2 and the second CH3 via a second linker peptide sequence.

39. The polypeptide complex according to claim 37 or 38, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

40. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3, wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2. The polypeptide complex according to claim 40 , wherein the VHH1 is connected to the N-terminus of the VHH2 via a first linker peptide sequence. The polypeptide complex according to claim 40 or 41 , wherein the VHH3 is connected to the N-terminus of the VHH4 via a second linker peptide sequence.

43. The polypeptide complex according to any one of claims 40 to 42, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 17; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

17. 44 . The polypeptide complex according to any one of claims 41 to 43 , wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

45. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH2, VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH4, VHH3, a second hinge region, a second CH2, and a second CH3, wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2. The polypeptide complex according to claim 45 , wherein the VHH2 is connected to the N-terminus of the VHH1 via a first linker peptide sequence. The polypeptide complex according to claim 45 or 46 , wherein the VHH4 is linked to the N-terminus of the VHH3 via a second linker peptide sequence.

48. The polypeptide complex of any one of claims 45 to 47, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 15 or 21; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 15 or 21.

49. The polypeptide complex according to any one of claims 46 to 48, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

50. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH2, a first hinge region, a first CH2, a first CH3, and VHH1; and (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH4, a second hinge region, a second CH2, a second CH3 and VHH3, wherein the VHH1 and the VHH3 specifically bind to HER2, and the VHH2 and the VHH4 specifically bind to TROP2.

51. The polypeptide complex of claim 50, wherein the first polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 16 or 20; and / or wherein the second polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 16 or 20. The polypeptide complex according to claim 50 or 51 , wherein the VHH1 is connected to the C-termini of the first CH2 and the first CH3 via a first linker peptide sequence.

53. The polypeptide complex according to any one of claims 50 to 52, wherein the VHH3 is connected to the C-termini of the second CH2 and the second CH3 via a second linker peptide sequence. The polypeptide complex according to claim 52 or 53, wherein the first linker peptide sequence and / or the second linker peptide sequence is at least 80% identical to SEQ ID NO: 34 or 35.

55. The polypeptide complex of any one of claims 35 to 54, wherein the VHH1 and / or the VHH3 comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are listed in Figure 33.

56. The polypeptide complex according to any one of claims 35 to 55, wherein the VHH1 and / or the VHH3 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is shown in SEQ ID NO:

1.

57. The polypeptide complex of any one of claims 35 to 56, wherein the VHH2 and / or the VHH4 comprise complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected CDR1 amino acid sequence, the CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected CDR2 amino acid sequence, and the CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected CDR3 amino acid sequence; wherein the selected CDR1, 2, and 3 amino acid sequences are listed in Figure 34.

58. The polypeptide complex according to any one of claims 35 to 57, wherein the VHH2 and / or the VHH4 comprises an amino acid sequence that is at least 80% identical to a selected VHH sequence, wherein the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 2-12.

59. A nucleic acid comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, or the polypeptide complex according to any one of claims 35 to 58.

60. The nucleic acid of claim 59, wherein the nucleic acid is DNA (eg, cDNA) or RNA (eg, mRNA).

61. A cell comprising one or more nucleic acids according to claim 59 or 60.

62. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (c) culturing the cell according to claim 61 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and (d) collecting the antibody or the antigen-binding fragment produced by the cell.

63. A T cell engager (TCE) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 25 to 34, or the polypeptide complex according to any one of claims 35 to 58.

64. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 25 to 34, or the polypeptide complex according to any one of claims 35 to 58.

65. A CAR-T, CAR-NK or CAR-NKT cell comprising the CAR according to claim 64.

66. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 25 to 34, or the polypeptide complex according to any one of claims 35 to 58, covalently bound to a therapeutic agent.

67. The antibody drug conjugate of claim 66, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

68. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, the polypeptide complex according to any one of claims 35 to 58, the TCE according to claim 63, the CAR according to claim 64, the CAR-T or CAR-NK or CAR-NKT cell according to claim 65, or the antibody drug conjugate according to claim 66 or 67.

69. The method of claim 68, wherein the subject has a cancer that expresses HER2.

70. The method of claim 68 or 69, wherein the subject has a cancer that expresses TROP2.

71. The method of any one of claims 68 to 70, wherein the cancer is gastric cancer, cervical cancer, esophageal cancer, thyroid cancer, bile duct cancer, colon cancer, rectal cancer, lung cancer, breast cancer, kidney cancer, hepatocellular carcinoma, renal cancer, endometrial cancer, pancreatic cancer, head and neck cancer, or an advanced solid tumor.

72. The method of claim 71, wherein the cancer is non-small cell lung cancer (NSCLC).

73. A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, the polypeptide complex according to any one of claims 35 to 58, the TCE according to claim 63, the CAR according to claim 64, the CAR-T or CAR-NK or CAR-NKT cell according to claim 65, or the antibody drug conjugate according to claim 66 or 67.

74. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, the polypeptide complex according to any one of claims 35 to 58, the TCE according to claim 63, the CAR according to claim 64, the CAR-T or CAR-NK or CAR-NKT cell according to claim 65, or the antibody drug conjugate according to claim 66 or 67.

75. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, the polypeptide complex according to any one of claims 35 to 58, the TCE according to claim 63, the CAR according to claim 64, the CAR-T or CAR-NK or CAR-NKT cell according to claim 65, or the antibody drug conjugate according to claim 66 or 67, and a pharmaceutically acceptable carrier.

76. An engineered antibody or antigen-binding fragment thereof comprising a serine residue at position 220 of the heavy chain according to EU numbering.

77. An antibody drug conjugate (ADC) comprising an engineered hinge region to which a therapeutic agent is covalently bound, wherein the engineered hinge region comprises a serine at position 220, wherein the therapeutic agent is bound to a cysteine ​​residue at position 226 or 229 according to EU numbering.

78. The antibody drug conjugate of claim 77, wherein the drug to antibody ratio (DAR) is 1-4.

79. The antibody drug conjugate of claim 77, wherein the ADC further comprises a VHH, wherein the VHH is linked to the engineered hinge region.

80. The antibody drug conjugate of claim 77, wherein the ADC comprises: (4) (a) a first polypeptide comprising, from the N-terminus to the C-terminus: VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from the N-terminus to the C-terminus: VHH2, second hinge region, second CH2, second CH3; (5) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3; or (6) (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH3, a second hinge region, a second CH2, a second CH3, and VHH4.

81. An antibody drug conjugate (ADC) comprising an engineered hinge region covalently bound to a therapeutic agent, wherein the therapeutic agent is bound to a cysteine ​​residue at position 220, 226, or 229 according to EU numbering.

82. The antibody drug conjugate of claim 81, wherein the drug to antibody ratio (DAR) is 4-6.

83. The antibody drug conjugate of claim 81, wherein the ADC comprises: (4) (a) a first polypeptide comprising, from the N-terminus to the C-terminus: VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from the N-terminus to the C-terminus: VHH2, second hinge region, second CH2, second CH3; (5) (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus: VHH3, VHH4, a second hinge region, a second CH2, and a second CH3; or (6) (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH3, a second hinge region, a second CH2, a second CH3, and VHH4.

84. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus, a VHH2, a second hinge region, a second CH2, and a second CH3.

85. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus, VHH1, a first hinge region, a first CH2, a first CH3, and VHH2; and (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH3, a second hinge region, a second CH2, a second CH3 and VHH4.

86. A polypeptide complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: VHH1, VHH2, a first hinge region, a first CH2, and a first CH3; and (b) a second polypeptide comprising, from N-terminus to C-terminus, VHH3, VHH4, a second hinge region, a second CH2, and a second CH3.

87. The polypeptide of any one of claims 84 to 86, wherein the VHH1 and the VHH3 specifically bind to a first tumor antigen, and the VHH2 and the VHH4 specifically bind to a second tumor antigen.

88. An antibody drug conjugate (ADC) comprising the polypeptide complex according to any one of claims 84 to 86, said polypeptide complex being covalently bound to a payload.

89. The ADC of claim 88, wherein the payload is selected from the group consisting of a cytotoxic agent, a cytostatic agent, a radionuclide, a biologically active protein, a synthetic polymer, an enzyme, a nucleic acid (e.g., DNA or RNA), and fragments thereof.

90. The ADC of claim 88, wherein each of the first hinge region and the second hinge region consists of 2 cysteines.

91. The ADC of claim 88, wherein each of the first hinge region and the second hinge region consists of 3 cysteines.

92. The ADC of claim 88, wherein each of the first hinge region and the second hinge region consists of 2 cysteines, wherein the drug to antibody ratio (DAR) is 1.0-4.

0.

93. The ADC of claim 88, wherein each of the first hinge region and the second hinge region consists of 3 cysteines, wherein the drug to antibody ratio (DAR) is 4.0-6.

0.

94. A method of diagnosing a disease or condition, wherein the method comprises incubating a sample with a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, the multispecific antibody or antigen-binding fragment thereof according to any one of claims 26 to 34, or the polypeptide complex according to any one of claims 35 to 58.

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