Methods of treating cancer using multispecific binding proteins that bind NKG2D, CD16, and tumor-associated antigens - Patent Application 20070123333

Abstract

The present disclosure relates to a method of treating cancer using a multispecific binding protein that binds to NKG2D, CD16, and a tumor-associated antigen, such as HER2. Provided is the use of the multispecific binding protein in combination with corticosteroids to reduce the risk of infusion-related reactions. Also provided is the use of the multispecific binding protein in treating cancers with low or moderate HER2 expression levels. The present disclosure also relates to pharmaceutical formulations comprising the multispecific binding protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 156,214, filed March 3, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on February 28, 2022, is named DFY-103WO_SL.txt, and is 194,985 bytes in size.

[0003] Field of the Disclosure The present disclosure relates to a method of treating cancer using a multispecific binding protein that binds to NKG2D, CD16, and a tumor-associated antigen, such as HER2. In one embodiment, the multispecific binding protein is used in combination with corticosteroids to reduce the risk of infusion-related reactions. In another embodiment, the multispecific binding protein is used to treat cancers with low or moderate HER2 expression levels. The present disclosure also relates to pharmaceutical formulations comprising the multispecific binding protein. [Background technology]

[0004] Cancer remains a major health problem, despite substantial research efforts and scientific advances reported in the literature to treat this disease. Cancer immunotherapy has been developed to promote the destruction of cancer cells using the patient's own immune system. Immune cells activated by cancer immunotherapy include T cells and natural killer (NK) cells. For example, bispecific T cell engagers are designed to target T cells against tumor cells, thereby making them cytotoxic to tumor cells. Bispecific antibodies that bind NK cells and tumor-associated antigens (TAA) have also been produced for cancer treatment (see, e.g., WO 2016 / 134371).

[0005] HER2 is a transmembrane glycoprotein in the epidermal growth factor receptor family. It is a receptor tyrosine kinase and regulates cell survival, proliferation, and growth. HER2 plays an important role in human malignancies. The ERBB2 gene is amplified or overexpressed in about 30% of human breast cancers. Patients with HER2-overexpressing breast cancer have substantially lower overall survival rates and shorter disease-free intervals than patients whose cancer does not overexpress HER2. Furthermore, overexpression of HER2 leads to increased breast cancer metastasis. Overexpression of HER2 is also known to occur in many other cancer types, including ovarian, esophageal, bladder, and gastric cancers, salivary duct cancer, lung adenocarcinoma, and invasive forms of uterine cancer, such as uterine serous endometrial cancer. Multispecific binding proteins that bind to HER2 and one or more immune cell surface proteins have been tested. For example, WO2018 / 152518 describes a multispecific binding protein that binds to HER2, NKG2D, and CD16. The present disclosure adds to these developments and provides clinical methods, including dosing regimens, for treating patients with specific HER2-targeted cancer immunotherapies with desirable safety and efficacy. Furthermore, the present disclosure adds to earlier developments in the art by providing formulations that include such cancer immunotherapies that are sufficiently stable and suitable for administration to patients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 152518 Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides, in various embodiments, a method of treating cancer using a multispecific binding protein having an antigen binding site that binds to a tumor-associated antigen, such as HER2, an antigen binding site that binds to NKG2D, and an antibody Fc domain. The multispecific binding protein can be used in combination with a corticosteroid to reduce the risk of infusion-related reactions. Alternatively, the multispecific binding protein can be formulated in a small volume at a high concentration as disclosed herein, thereby allowing subcutaneous administration and reducing the risk of infusion-related reactions. The multispecific binding protein can also be used to treat cancers with low or moderate HER2 expression levels.

[0008] Thus, in one aspect, provided herein is a method of treating cancer by administering to a subject in need thereof a therapeutically effective amount of a multispecific binding protein and a therapeutically effective amount of a corticosteroid to reduce infusion-associated reactions to the multispecific binding protein. The multispecific binding protein incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) a third antigen-binding site that binds CD16. In some embodiments, infusion-related reactions include cytokine release syndrome, anaphylaxis, chills, fever / fever, hypotension, hypertension, rigors, headache, dizziness, pruritus, sore throat, pharyngeal edema, angioedema, redness / flushing, rash / hives, bronchospasm, tachycardia, bradycardia, atrial fibrillation, hypoxia, rapid / difficulty breathing / shortness of breath / choking, chest tightness, nausea, vomiting, pain (e.g., chest pain, back pain), shivering, tremors, myalgia, fatigue, insomnia, asthenia, hypersensitivity, and / or diarrhea. In some embodiments, the corticosteroid is a glucocorticoid. In some embodiments, the glucocorticoid is selected from methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, or any pharma- ceutically acceptable salt thereof. In certain embodiments, the glucocorticoid is methylprednisolone. In some embodiments, the effective amount of methylprednisolone is 125 mg. In other particular embodiments, the glucocorticoid is dexamethasone.

[0009] In some embodiments, the corticosteroid is administered parenterally. In some embodiments, the corticosteroid is administered intravenously. In some embodiments, the corticosteroid is administered orally. In some embodiments, the corticosteroid is administered prior to administration of the multispecific binding protein. In some embodiments, the corticosteroid is administered within 6 hours prior to administration of the multispecific binding protein. In some embodiments, the corticosteroid is administered within 1 hour prior to administration of the multispecific binding protein.

[0010] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an antihistamine. In some embodiments, the antihistamine is diphenhydramine. In some embodiments, the therapeutically effective amount of diphenhydramine is 40-50 mg. In some embodiments, the antihistamine is administered intravenously. In some embodiments, the antihistamine is administered within 60 minutes or within 30 minutes prior to administration of the multispecific binding protein.

[0011] In some embodiments, the method further comprises administering a therapeutically effective amount of an antipyretic to the subject. In some embodiments, the antipyretic is acetaminophen. In some embodiments, the therapeutically effective amount of acetaminophen is between 800 and 1000 mg. In some embodiments, the antipyretic is administered intravenously. In some embodiments, the antipyretic is administered within 60 minutes or within 30 minutes prior to administration of the multispecific binding protein.

[0012] In some embodiments, the tumor associated antigen is HER2. In some embodiments, the cancer has a HER2 expression level scored as 1+ or 2+.

[0013] In another aspect, the disclosure provides a method of treating a cancer having a HER2 expression level scored as 1+ by administering to a subject in need thereof a therapeutically effective amount of a multispecific binding protein that incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) a sufficient antibody Fc domain or portion thereof that binds CD16, or a third antigen-binding site that binds CD16.

[0014] In another aspect, the disclosure provides a method of treating a cancer with a HER2 expression level scored as 2+ by administering to a subject in need thereof a therapeutically effective amount of a multispecific binding protein that incorporates (a) a first antigen-binding site that binds NKG2D, (b) a second antigen-binding site that binds HER2, and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

[0015] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a locally advanced or metastatic solid tumor. In some embodiments, the cancer is selected from breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, non-small cell lung cancer (NSCLC), glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary glands, cholangiocarcinoma, squamous cell lung, mesothelioma, liver cancer, sarcoma, bladder cancer, and gallbladder cancer. In certain embodiments, the cancer is urothelial bladder cancer or metastatic breast cancer.

[0016] In some embodiments, the multispecific binding protein is administered to the subject on days 1, 8, and 15 in an initial four-week treatment cycle. In some embodiments, the multispecific binding protein is not administered on day 22. In some embodiments, the method further comprises administering the multispecific binding protein to the subject on days 1 and 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. In some embodiments, each dose is at least 5.2×10 -5mg / kg, 1.6×10 -4 mg / kg, 5.2×10 -4 mg / kg, 1.6×10 -3 mg / kg, 5.2×10 -3 mg / kg, 1.6×10 -2 mg / kg, 5.2×10 -2 mg / kg, 1.6×10 -1 In some embodiments, the multispecific binding protein is administered in an amount selected from the group consisting of 0.52 mg / kg, 1.0 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 20 mg / kg, or 50 mg / kg. In some embodiments, the multispecific binding protein is administered by intravenous infusion. In some embodiments, the multispecific binding protein is administered subcutaneously.

[0017] In some embodiments, the multispecific binding protein is used as a monotherapy. In some embodiments, the method further comprises administering an anti-PD-1 antibody to the subject. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, 200 mg of pembrolizumab is administered on day 1 of the first treatment cycle. In some embodiments, if the subject undergoes one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles.

[0018] In some embodiments, the corticosteroid is administered to the subject on day 1 of the first four-week cycle. In some embodiments, the antihistamine is administered to the subject on day 1 of the first four-week cycle. In some embodiments, the analgesic is administered to the subject on day 1 of the first four-week cycle.

[0019] In some embodiments, the multispecific binding protein is formulated in a pharmaceutical composition comprising histidine, a sugar or sugar alcohol, and a polysorbate at a pH of 5.5 to 6.5, hi some embodiments, the pharmaceutical composition comprises greater than 50 mg / mL of the multispecific binding protein.

[0020] In another aspect, the disclosure provides a method of treating cancer by administering to a subject in need thereof a pharmaceutical composition comprising: (i) greater than 50 mg / mL of a multispecific binding protein that incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) a sufficient antibody Fc domain or portion thereof that binds CD16, or a third antigen-binding site that binds CD16; (ii) histidine; (iii) a sugar or sugar alcohol; and (iv) polysorbate, at a pH of 5.5-6.5.

[0021] In another aspect, the disclosure provides a method of treating a cancer having a HER2 expression level scored as 1+ by administering to a subject in need thereof (i) a pharmaceutical composition comprising greater than 50 mg / mL of a multispecific binding protein, histidine, a sugar or sugar alcohol, and polysorbate at a pH of 5.5-6.5, and (ii) a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein. The multispecific binding protein incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

[0022] In another aspect, the disclosure provides a method of treating a cancer having a HER2 expression level scored as 2+ by administering to a subject in need thereof (i) a pharmaceutical composition comprising greater than 50 mg / mL of a multispecific binding protein, histidine, a sugar or sugar alcohol, and polysorbate at a pH of 5.5-6.5; and (ii) a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein. The multispecific binding protein incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

[0023] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a locally advanced or metastatic solid tumor. In certain embodiments, the cancer is breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, non-small cell lung cancer (NSCLC), glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary glands, cholangiocarcinoma, squamous cell lung, mesothelioma, liver cancer, sarcoma, bladder cancer, and gallbladder cancer. In certain embodiments, the cancer is urothelial bladder cancer or metastatic breast cancer.

[0024] In certain embodiments, the pharmaceutical composition is administered to the subject on days 1, 8, and 15 in an initial four-week treatment cycle. In certain embodiments, the pharmaceutical composition is not administered on day 22. In certain embodiments, the method further comprises administering a pharmaceutical composition to the subject on days 1 and 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle.

[0025] In certain embodiments, each administered dose of the pharmaceutical composition is 5.2×10 -5 mg / kg, 1.6×10 -4 mg / kg, 5.2×10 -4 mg / kg, 1.6×10 -3 mg / kg, 5.2×10 -3 mg / kg, 1.6×10 -2 mg / kg, 5.2×10 -2 mg / kg, 1.6×10 -1 In certain embodiments, the pharmaceutical composition is administered by intravenous infusion. In certain embodiments, the multispecific binding protein is administered subcutaneously.

[0026] In certain embodiments, the pharmaceutical composition is used as a monotherapy.

[0027] In other embodiments, the method further comprises administering an anti-PD-1 antibody to the subject. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, 200 mg of pembrolizumab is administered on day 1 of the first treatment cycle. In certain embodiments, if the subject undergoes one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles.

[0028] In certain embodiments, the corticosteroid is administered to the subject on day 1 of the first four-week cycle. In certain embodiments, the antihistamine is administered to the subject on day 1 of the first four-week cycle. In certain embodiments, the antipyretic is administered to the subject on day 1 of the first four-week cycle.

[0029] In another aspect, the disclosure provides a pharmaceutical composition comprising greater than 50 mg / mL of a multispecific binding protein histidine, a sugar or sugar alcohol, and polysorbate, at a pH of 5.5-6.5. The multispecific binding protein incorporates (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds a tumor-associated antigen; and (c) a sufficient antibody Fc domain or portion thereof that binds CD16, or a third antigen-binding site that binds CD16.

[0030] In some embodiments of any one of the aforementioned pharmaceutical compositions or methods of using the pharmaceutical composition, the pharmaceutical composition comprises greater than or equal to 60 mg / mL, greater than or equal to 70 mg / mL, greater than or equal to 80 mg / mL, greater than or equal to 90 mg / mL, greater than or equal to 100 mg / mL, greater than or equal to 125 mg / mL, greater than or equal to 150 mg / mL, greater than or equal to 175 mg / mL, or greater than or equal to 200 mg / mL of multispecific binding protein. In some embodiments, the pharmaceutical composition is 60-250 mg / mL, 60-225 mg / mL, 60-200 mg / mL, 60-175 mg / mL, 50-150 mg / mL, 60-150 mg / mL, 60-125 mg / mL, 60-100 mg / mL, 60-90 mg / mL, 60-80 mg / mL, 60-70 mg / mL, 70-250 mg / mL, 70-225 mg / mL, 70-200 mg / mL, 7 ...25 mg / mL, 60-100 mg / mL, 60-90 mg / mL, 60-80 mg / mL, 60-70 mg / mL, 70-250 mg / mL, 70-225 mg / mL, 70-200 mg / mL, 70-175 mg / 75mg / mL, 70~150mg / mL, 70~150mg / mL, 70~125mg / mL, 70~100mg / mL, 70~90mg / mL, 70~80mg / mL, 80~250mg / mL, 80~ 225mg / mL, 80~200mg / mL, 80~175mg / mL, 80~150mg / mL, 80~150mg / mL, 80~125mg / mL, 80~100mg / mL, 80~90mg / mL, 9 0~250mg / mL, 90~225mg / mL, 90~200mg / mL, 90~175mg / mL, 90~150mg / mL, 90~150mg / mL, 90~125mg / mL, 90~100mg / m L, 100~250mg / mL, 100~225mg / mL, 100~200mg / mL, 100~175mg / mL, 100~150mg / mL, 100~125mg / mL, 125~250mg / mL, 125-225 mg / mL, 125-200 mg / mL, 125-175 mg / mL, 125-150 mg / mL, 150-250 mg / mL, 150-225 mg / mL, 150-200 mg / mL, 150-175 mg / mL, 175-250 mg / mL, 175-225 mg / mL, 175-200 mg / mL, 200-250 mg / mL, or 200-225 mg / mL of the multispecific binding protein.In some embodiments, the pharmaceutical composition comprises 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, or 220 mg / mL of the multispecific binding protein.

[0031] In some embodiments, the pH of the pharmaceutical composition is 5.8 to 6.2. In some embodiments, the pH of the pharmaceutical composition is 5.95 to 6.05. In some embodiments, the concentration of histidine in the pharmaceutical composition is 10 to 25 mM. In some embodiments, the concentration of histidine in the pharmaceutical composition is about 20 mM.

[0032] In some embodiments, the sugar or sugar alcohol is a disaccharide. In some embodiments, the disaccharide is sucrose. In some embodiments, the sugar or sugar alcohol is a sugar alcohol derived from a monosaccharide. In some embodiments, the sugar alcohol derived from a monosaccharide is sorbitol. In some embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical composition is 200-300 mM. In some embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM.

[0033] In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical composition is between 0.005% (w / v) and 0.05% (w / v). In some embodiments, the concentration of polysorbate 80 in the pharmaceutical composition is about 0.01% (w / v). In some embodiments, the concentration of NaCl, if present, is about 10 mM or less in the pharmaceutical formulation. In some embodiments, the concentration of NaCl, if present, is about 1 mM or less in the pharmaceutical formulation.

[0034] In some embodiments, the first antigen-binding site comprises a heavy chain variable domain with complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively, and a light chain variable domain with CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In some embodiments, the heavy chain variable domain has CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 169, respectively; and the light chain variable domain has CDR1, CDR2, CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, 101, respectively.

[0035] In some embodiments, the heavy chain variable domain of the first antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO: 94, and the light chain variable domain has an amino acid sequence at least 90% identical to SEQ ID NO: 98. In some embodiments, the heavy chain variable domain of the first antigen binding site has the amino acid sequence of SEQ ID NO: 94, and the light chain variable domain has the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen binding site that binds to NKG2D is a Fab.

[0036] In some embodiments, the second antigen-binding site comprises a heavy chain variable domain with CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively; and a light chain variable domain with CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively.

[0037] In some embodiments, the heavy chain variable domain of the second antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO: 195, and the light chain variable domain of the second antigen binding site has an amino acid sequence at least 90% identical to SEQ ID NO: 196. In some embodiments, the heavy chain variable domain of the second antigen binding site has the amino acid sequence of SEQ ID NO: 195, and the light chain variable domain of the second antigen binding site has the amino acid sequence of SEQ ID NO: 196.

[0038] In some embodiments, the second antigen-binding site is an scFv. In some embodiments, the light chain variable domain of the second antigen-binding site is linked to the heavy chain variable domain of the second antigen-binding site via a flexible linker. In some embodiments, the flexible linker has the amino acid sequence of SEQ ID NO: 143.

[0039] In some embodiments, the light chain variable domain of the second antigen-binding site is located at the N-terminus of the heavy chain variable domain of the second antigen-binding site. In some embodiments, the heavy chain variable domain of the second antigen-binding site forms a disulfide bridge with the light chain variable domain of the second antigen-binding site. In some embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, the amino acid positions being numbered under Kabat. In some embodiments, the cysteine ​​residues at position 44 of the heavy chain variable domain and at position 100 of the light chain variable domain are introduced by mutating the wild type residues at these positions to cysteine ​​residues. In some embodiments, the scFv has the amino acid sequence of SEQ ID NO: 139.

[0040] In some embodiments, the multispecific binding protein comprises an antibody Fc domain. In some embodiments, the antibody Fc domain comprises a first antibody Fc polypeptide linked to a first antigen binding site and a second antibody Fc polypeptide linked to a second antigen binding site. In some embodiments, the first antibody Fc polypeptide is linked to a heavy chain portion of the first antigen binding site. In some embodiments, the second antigen binding site is linked to the second antibody Fc polypeptide via a hinge comprising Ala-Ser. In some embodiments, the first and second antibody Fc polypeptides each comprise a hinge and a CH2 domain of a human IgG1 antibody. In some embodiments, the first and second antibody Fc polypeptides each have an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody. In some embodiments, the first and second antibody Fc polypeptides incorporate different mutations that promote heterodimerization. In some embodiments, the first antibody Fc polypeptide is a human IgG1 Fc sequence with a K360E substitution and a K409W substitution. In some embodiments, the second antibody Fc polypeptide is a human IgG1 Fc sequence with a Q347R substitution, a D399V substitution, and an F405T substitution.

[0041] In some embodiments, the multispecific binding protein binds to HER2 and comprises a first polypeptide having the amino acid sequence of SEQ ID NO:141, a second polypeptide having the amino acid sequence of SEQ ID NO:140, and a third polypeptide having the amino acid sequence of SEQ ID NO:142.

[0042] In another aspect, the disclosure provides a method of treating HER2-overexpressing gastric cancer in a subject in need thereof by administering to the subject a multispecific binding protein that incorporates (a) a first antigen-binding site that binds NKG2D, (b) a second antigen-binding site that binds HER2, and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

[0043] In certain embodiments, the gastric cancer is advanced gastric cancer or gastroesophageal junction cancer. In certain embodiments, the subject has previously received first-line treatment. In certain embodiments, the first-line treatment included a platinum salt and a fluoropyridine in combination with trastuzumab or a biosimilar to trastuzumab. In certain embodiments, the subject has progressed after first-line treatment. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., as determined by in situ hybridization).

[0044] In certain embodiments, the method of treating gastric cancer further comprises administering an anti-PD-1 antibody to the subject. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, nivolumab is administered to the subject at a dose of 120-600 mg. In certain embodiments, nivolumab is administered to the subject at a dose of 480 mg. In certain embodiments, nivolumab is administered once every four weeks. In certain embodiments, the multispecific binding protein is administered in one or more four-week treatment cycles, and nivolumab is administered on day 1 of the same treatment cycle. In certain embodiments, nivolumab is administered to the subject by intravenous infusion.

[0045] In certain embodiments, the method of treating gastric cancer further comprises administering to the subject a cytoskeleton-disrupting chemotherapeutic agent. In certain embodiments, the cytoskeleton-disrupting chemotherapeutic agent is nab-paclitaxel. In certain embodiments, the nab-paclitaxel is administered at a dose of 50-300 mg / m 2 In certain embodiments, nab-paclitaxel is administered to a subject at a dose of 100 mg / m 2In certain embodiments, nab-paclitaxel is administered to the subject three times every four weeks. In certain embodiments, the multispecific binding protein is administered in one or more four-week treatment cycles, and nab-paclitaxel is administered on days 1, 8, and 15 of the same treatment cycle. In certain embodiments, nab-paclitaxel is not administered on day 22 of the treatment cycle. In certain embodiments, nab-paclitaxel is administered to the subject by intravenous infusion.

[0046] In another aspect, the disclosure provides a method of treating triple-negative breast cancer in a subject in need thereof by administering to the subject a multispecific binding protein that incorporates (a) a first antigen-binding site that binds NKG2D, (b) a second antigen-binding site that binds HER2, and (c) a sufficient antibody Fc domain or portion thereof that binds CD16, or a third antigen-binding site that binds CD16.

[0047] In certain embodiments, the subject is not eligible for treatment in combination with anti-PD-L1 therapy, or the cancer in the subject is PD-L1 negative. In certain embodiments, the subject (a) is eligible for treatment with nab-paclitaxel after failure of combination chemotherapy for metastatic disease or recurrence within 6 months of adjuvant chemotherapy; (b) has cancer with no standard therapy or where standard therapy has failed; or (c) has advanced, possibly unresectable, recurrent, or metastatic triple-negative breast cancer. In certain embodiments, the triple-negative breast cancer has a PD-L1 score (CPS) of less than 10 as measured by immunohistochemistry. In certain embodiments, the triple-negative breast cancer is metastatic or locally advanced. In certain embodiments, the subject has not previously received chemotherapy or targeted systemic therapy.

[0048] In certain embodiments of the methods of treating gastric cancer or triple-negative breast cancer, the multispecific binding protein is administered on days 1, 8, and 15 of the initial treatment cycle. In certain embodiments, the multispecific binding protein is not administered on day 22 of the initial treatment cycle. In certain embodiments, after the initial treatment cycle, the multispecific binding protein is administered on days 1 and 15 of one or more subsequent four-week treatment cycles. In certain embodiments, the multispecific binding protein is administered at a concentration of 5.2×10 -5 mg / kg, 1.6×10 -4 mg / kg, 5.2×10 -4 mg / kg, 1.6×10 -3 mg / kg, 5.2×10 -3 mg / kg, 1.6×10 -2 mg / kg, 5.2×10 -2 mg / kg, 1.6×10 -1 In certain embodiments, the multispecific binding protein is administered in an amount selected from the group consisting of 0.52 mg / kg, 1.0 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, and 50 mg / kg. In certain embodiments, the multispecific binding protein is administered by intravenous infusion. In certain embodiments, the multispecific binding protein is used as a monotherapy.

[0049] In certain embodiments, the first antigen-binding site is in the format of a Fab having a heavy chain variable domain (VH) and a light chain variable domain (VL). The VH of the Fab has the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively. The VL of the Fab has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In certain embodiments, the VH of the Fab has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 169, respectively. In certain embodiments, the VL of the Fab has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In certain embodiments, the VH of the Fab has an amino acid sequence at least 90% identical to SEQ ID NO: 94, and the VL of the Fab has an amino acid sequence at least 90% identical to SEQ ID NO: 98. In certain embodiments, the VH of the Fab has the amino acid sequence of SEQ ID NO: 94, and the VL of the Fab has the amino acid sequence of SEQ ID NO: 98.

[0050] In certain embodiments, the second antigen-binding site is in the format of a single chain variable fragment (scFv) having a VH and a VL. The VH of the scFv has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively. The VL of the scFv has the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively. In certain embodiments, the VH of the scFv has an amino acid sequence at least 90% identical to SEQ ID NO: 195, and the VL of the scFv has an amino acid sequence at least 90% identical to SEQ ID NO: 196. In certain embodiments, the VH of the scFv has the amino acid sequence of SEQ ID NO: 195, and the VL of the scFv has the amino acid sequence of SEQ ID NO: 196. In certain embodiments, the VL of the scFv is linked to the VH of the scFv via a flexible linker, such as a flexible linker having the amino acid sequence of SEQ ID NO: 143. In certain embodiments, the VL of the scFv is positioned at the N-terminus of the VH of the scFv. In certain embodiments, the VH of the scFv forms a disulfide bridge with the VL of the scFv, such as a disulfide bridge formed between C44 of the VH and C100 of the VL. In certain embodiments, the scFv has the amino acid sequence of SEQ ID NO: 139.

[0051] In certain embodiments, the antibody Fc domain comprises a first antibody Fc sequence linked to a Fab and a second antibody Fc sequence linked to an scFv. In certain embodiments, the first antibody Fc sequence is linked to a heavy chain portion of the Fab. In certain embodiments, the scFv is linked to the second antibody Fc sequence via a hinge having the amino acid sequence Ala-Ser. In certain embodiments, the first and second antibody Fc sequences each have the hinge and CH2 domain of a human IgG1 antibody. In certain embodiments, the first and second antibody Fc sequences each have an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody. In certain embodiments, the first and second antibody Fc sequences have different mutations that promote heterodimerization. For example, in certain embodiments, the first antibody Fc sequence is a human IgG1 Fc sequence incorporating a K360E substitution and a K409W substitution, and / or the second antibody Fc sequence is a human IgG1 Fc sequence incorporating a Q347R substitution, a D399V substitution, and an F405T substitution.

[0052] In certain embodiments, the multispecific binding protein comprises (a) a first polypeptide having the amino acid sequence of SEQ ID NO:141, (b) a second polypeptide having the amino acid sequence of SEQ ID NO:140, and (c) a third polypeptide having the amino acid sequence of SEQ ID NO:142.

[0053] In certain embodiments, the multispecific binding protein is formulated in the pharmaceutical composition at a concentration of 10 mg / mL to 50 mg / mL prior to administration. In certain embodiments, the pharmaceutical composition comprises 10 mg / mL to 25 mg / mL of the multispecific binding protein. In certain embodiments, the pharmaceutical composition comprises about 15 mg / mL of the multispecific binding protein.

[0054] In certain embodiments, the pharmaceutical composition has a pH in the range of 5.5 to 6.5, for example, a pH of about 6.0. In certain embodiments, the pharmaceutical composition comprises 5 to 50 mM histidine. In certain embodiments, the pharmaceutical composition comprises 10 to 25 mM histidine. In certain embodiments, the pharmaceutical composition comprises about 20 mM histidine.

[0055] In certain embodiments, the pharmaceutical composition comprises 50-300 mM sugar or sugar alcohol (e.g., sucrose). In certain embodiments, the pharmaceutical composition comprises 150-300 mM sugar or sugar alcohol. In certain embodiments, the pharmaceutical composition comprises about 250 mM sugar or sugar alcohol.

[0056] In certain embodiments, the pharmaceutical composition comprises 0.005%-0.05% mM (w / v) polysorbate (e.g., polysorbate-80). In certain embodiments, the pharmaceutical composition comprises 0.005%-0.02% mM (w / v) polysorbate. In certain embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate.

[0057] In certain embodiments, the pharmaceutical composition comprises (i) 10 mg / mL to 50 mg / mL of the multispecific binding protein, (ii) 5 mM to 50 mM histidine, (iii) 50 mM to 300 mM sucrose, and (iv) 0.005% to 0.05% (w / v) of polysorbate 80 at a pH of 5.5 to 6.5. In certain embodiments, the pharmaceutical composition comprises (i) 10 mg / mL to 25 mg / mL of the multispecific binding protein, (ii) 10 mM to 25 mM histidine, (iii) 150 mM to 300 mM sucrose, and (iv) 0.005% to 0.02% (w / v) of polysorbate 80 at a pH of 5.5 to 6.5. In certain embodiments, the pharmaceutical composition comprises: (i) about 15 mg / mL of the multispecific binding protein; (ii) about 20 mM histidine; (iii) about 250 mM sucrose; and (iv) about 0.01% (w / v) polysorbate 80 at about pH 6.0.

[0058] Other embodiments and details of the present disclosure are presented herein below. [Brief description of the drawings]

[0059] [Figure 1]Figure 1 is a representation of a heterodimeric multispecific antibody, e.g., a trispecific binding protein (TriNKET). Each arm can represent either an NKG2D binding domain or a tumor-associated antigen binding domain. In some embodiments, the NKG2D binding domain and the tumor-associated antigen binding domain can share a common light chain.

[0060] [Figure 2A] Figures 2A-2E illustrate five exemplary formats of multispecific binding proteins, e.g., trispecific binding proteins (TriNKETs). As shown in Figure 2A, either the NKG2D binding domain or the tumor-associated antigen binding domain can take the scFv format (left arm). An antibody comprising an NKG2D targeting scFv, a tumor-associated antigen targeting Fab fragment, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. An antibody comprising a tumor-associated antigen targeting scFv, an NKG2D targeting Fab fragment, and a heterodimerized antibody constant region / domain that binds CD16 is referred to herein as F3'-TriNKET (Figure 2E). [Figure 2B] As shown in FIG. 2B, both the NKG2D-binding domain and the tumor-associated antigen-binding domain can be in an scFv format. [Figure 2C] Figures 2C-2D are illustrations of antibodies with three antigen-binding sites, including two antigen-binding sites that bind to tumor-associated antigens and an NKG2D-binding site fused to a heterodimerized antibody constant region. These antibody formats are referred to herein as F4-TriNKET. Figure 2C illustrates that the two tumor-associated antigen-binding sites are in a Fab fragment format and the NKG2D-binding site is in a scFv format. [Figure 2D] FIG. 2D illustrates that the tumor-associated antigen binding site is in an scFv format and the NKG2D binding site is in an scFv format. [Figure 2E]Figure 2E depicts a trispecific antibody (TriNKET) comprising a tumor-associated antigen-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region / domain ("CD domain") that binds CD16. The antibody format is referred to herein as F3'-TriNKET. In a particular exemplary multispecific binding protein, the heterodimerizing mutations on the antibody constant region include K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain (shown as a triangular lock-and-key shape in the CD domain). The thick bar between the heavy and light chain variable domains of the Fab fragment represents a disulfide bond.

[0061] [Diagram 3] Figure 3 is a representation of TriNKET in a Triomab format, which is a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half antibodies, each with one light and one heavy chain, that are derived from two parent antibodies. The Triomab format can be a heterodimeric construct containing one half of a rat antibody and one half of a mouse antibody.

[0062] [Figure 4] Figure 4 is a representation of TriNKET in a KiH common light chain format, including knobs-into-holes (KIH) technology. KiH is a heterodimer that includes two Fab fragments that bind targets 1 and 2, and an Fc domain stabilized by a heterodimerization mutation. TriNKET in the KiH format can be a heterodimeric construct with two Fab fragments that bind targets 1 and 2, and includes two different heavy chains and a common light chain that pairs with both heavy chains.

[0063] [Diagram 5]Figure 5 is a representation of TriNKET in a dual variable domain immunoglobulin (DVD-Ig™) format, which combines the target binding domains of two monoclonal antibodies via a flexible natural linker to produce a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the variable domain targeting antigen 2 is fused to the N-terminus of the variable domain of a Fab fragment targeting antigen 1. The DVD-Ig™ format contains a normal Fc domain.

[0064] [Figure 6] Figure 6 is a representation of TritNKET in the orthogonal Fab fragment interface (Ortho-Fab) format, which is a heterodimeric construct containing two Fab fragments that bind target 1 and target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by the orthogonal interface. Heterodimerization is ensured by mutations in the Fc domain.

[0065] [Figure 7] FIG. 7 is a representation of TriNKET in the 2-in-1 Ig format.

[0066] [Figure 8] Figure 8 is a representation of TritNKET in the ES format, which is a heterodimeric construct containing two different Fab fragments that bind target 1 and target 2 fused to an Fc domain. Heterodimerization is ensured by electrostatic steering mutations in the Fc domain.

[0067] [Figure 9] Figure 9 is a representation of TriNKET in a Fab arm swapped form: an antibody that swaps Fab fragment arms by exchanging the heavy and attached light chains (half molecules) with a heavy-light chain pair from another molecule, resulting in a bispecific antibody. The Fab arm swapped form (cFae) is a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc domain stabilized by a heterodimerization mutation.

[0068] [Figure 10] FIG. 10 is a representation of TriNKET in a SEED body format, which is a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc domain stabilized by a heterodimerization mutation.

[0069] [Figure 11] Figure 11 shows TritNKET in the LuZ-Y format, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y format is a heterodimer containing two different scFabs that bind targets 1 and 2, fused to an Fc domain. Heterodimerization is ensured through a leucine zipper motif fused to the C-terminus of the Fc domain.

[0070] [Figure 12] FIG. 12 is a representation of TriNKET in Cov-X body morphology.

[0071] [Figure 13A] 13A-13B are representations of TriNKET in a κλ-body format, which is a heterodimeric construct with two different Fab fragments fused to an Fc domain stabilized by heterodimerization mutations: one Fab fragment targeting antigen 1 contains a kappa LC and the second Fab fragment targeting antigen 2 contains a lambda LC. Figure 13A is an exemplary representation of one form of a κλ-body. [Figure 13B] FIG. 13B is an exemplary representation of another κλ body.

[0072] [Figure 14] Figure 14 is a representation of an Oasc-Fab heterodimer construct containing a Fab fragment that binds target 1 and a scFab that binds target 2, both fused to an Fc domain. Heterodimerization is ensured by mutations in the Fc domain.

[0073] [Figure 15]Figure 15 is a representation of DuetMab, which is a heterodimeric construct containing two different Fab fragments that bind antigens 1 and 2, and an Fc domain that is stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential S-S bridges that ensure correct light and heavy chain pairing.

[0074] [Figure 16] Figure 16 is a representation of CrossmAb, which is a heterodimeric construct with two different Fab fragments that bind targets 1 and 2, and an Fc domain stabilized by a heterodimerization mutation. The CL and CH1 domains, as well as the VH and VL domains, are switched, e.g., CH1 is fused in-line with the VL and CL is fused in-line with the VH.

[0075] [Figure 17] Figure 17 is a representation of Fit-Ig, which is a homodimeric construct in which a Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of a Fab fragment that binds antigen 1. The construct contains a wild-type Fc domain.

[0076] [Figure 18A] FIG. 18A is an interaction plot for the average size as measured by dynamic light scattering (DLS) after 3 weeks of incubation at 50° C. [Figure 18B] FIG. 18B is an interaction plot for the average size measured by DLS after 3 weeks of incubation at 2-8° C.

[0077] [Figure 19A] FIG. 19A is an interaction plot for monomer size as measured by DLS after 3 weeks of incubation at 50° C. [Figure 19B] FIG. 19B is an interaction plot for monomer size as measured by DLS after 3 weeks of incubation at 2-8° C.

[0078] [Figure 20A] FIG. 20A is an interaction plot for the % major species determined by size exclusion chromatography (SEC) for a 3 week incubation at 50° C. [Figure 20B] FIG. 20B is an interaction plot for the % major species determined by SEC for a 3 week incubation at 2-8° C.

[0079] [Figure 21A] FIG. 21A is an interaction plot for percent high molecular weight species (HMW%) as determined by SEC for a 3 week incubation at 50° C. [Figure 21B] FIG. 21B is an interaction plot for % HMW species determined by SEC for a 3 week incubation at 2-8° C.

[0080] [Figure 22A] FIG. 22A is an interaction plot for percent low molecular weight species (LMW%) determined by SEC for a 3 week incubation at 50° C. [Figure 22B] FIG. 22B is an interaction plot for % LMW species determined by SEC for a 3 week incubation at pH 6.0 and 2-8° C.

[0081] [Figure 23A] FIG. 23A is an interaction plot for % acidic species determined by imaging capillary isoelectric focusing (icIEF) for a 3 week incubation at 50° C. [Figure 23B] FIG. 23B is an interaction plot for % basic species for sucrose alone as determined by icIEF for a 3 week incubation at 4-8° C.

[0082] [Figure 24A]FIG. 24A is an interaction plot for the % major species determined by icIEF for a 3 week incubation at 50° C. [Figure 24B] Figures 24B-24D are interaction plots for the % major species in the formulations for sucrose alone as determined by icIEF for 3 weeks incubation at 4°C at pH 5.5 (Figure 24B), pH 6.0 (Figure 24C), and pH 6.5 (Figure 24D). [Figure 24C] Same as above. [Figure 24D] Same as above.

[0083] [Figure 25A] FIG. 25A is an interaction plot for % basic species determined by icIEF for a 3 week incubation at 50° C. [Figure 25B] FIG. 25B is an interaction plot for % basic species for sucrose alone as determined by icIEF for a 3 week incubation from 2-8° C.

[0084] [Figure 26A] FIG. 26A is an interaction plot for % purity as determined by capillary electrophoresis (CE) for a 3 week incubation at 50° C. [Figure 26B] FIG. 26B is an interaction plot for % impurities determined by CE for a 3 week incubation at 50° C.

[0085] [Figure 27A] FIG. 27A is an interaction plot for the % major species determined by capillary electrophoresis (non-reducing) (CE(NR)) for a 3 week incubation at pH 6.0 and 50° C. [Figure 27B] FIG. 27B is an interaction plot for % major species determined by CE(NR) for a 3 week incubation at pH 6.0 and 2-8° C.

[0086] [Figure 28A] FIG. 28A is an interaction plot for % HMW species as determined by CE(NR) for a 3 week incubation at 50° C. [Figure 28B] FIG. 28B shows the interaction plot for % HMW species as determined by CE(NR) for a 3 week incubation at 2-8° C.

[0087] [Figure 29A] FIG. 29A is an interaction plot for % LMW species for sucrose alone as determined by CE(NR) for a 3 week incubation at 50° C. [Figure 29B] FIG. 29B is an interaction plot for % LMW species for sucrose alone as determined by CE(NR) for a 3 week incubation at 2-8° C.

[0088] [Diagram 30] FIG. 30 is a graph of the concentration curve of A49-F3′-TriNKET-Trastuzumab over time in formulations stored at 2-8° C. or 25° C.

[0089] [Figure 31A] FIG. 31A is an image of vials containing formulations of A49-F3′-TriNKET-Trastuzumab in varying concentrations of polysorbate-80. [Figure 31B] FIG. 31B is a graph of the % monomeric species of A49-F3′-TriNKET-Trastuzumab in formulations stored at varying temperatures at specific time points, as measured by size-exclusion high performance liquid chromatography (SE-HPLC).

[0090] [Figure 32A] Figures 32A-32B are schematic diagrams of the clinical trial design. Figure 32A describes the study design for the dose escalation phase. [Figure 32B]Figure 32B describes the study design for the efficacy expansion cohort phase. Abbreviations used in the figure include: DL = dose level; Combo PD-1 = combination treatment with pembrolizumab; PK = pharmacokinetics; PD = pharmacodynamics; HER2 High = high expression of HER2 of 3+ by immunohistochemistry; MBC HER2 2+ / 1+ = metastatic breast cancer with intermediate / low expression of HER2 of 2+ / 1+ by immunohistochemistry; UBC 2L / 3L = second / third line treatment of urothelial bladder cancer.

[0091] [Figure 33A] 33A-33C are line graphs showing the cytotoxicity of A49-F3'-TriNKET-trastuzumab and trastuzumab against SKBR-3 (FIG. 33A), H661 (FIG. 33B), and 786-O (FIG. 33C) cancer cells in the presence of NK cells. [Figure 33B] Same as above. [Figure 33C] Same as above.

[0092] [Figure 34A] 34A-34B are line graphs showing the cytotoxicity of A49-F3'-TriNKET-Trastuzumab and Trastuzumab against ZR-75-1 (FIG. 34A) and MCF-7 (FIG. 34B) cancer cells in the presence of NK cells. [Figure 34B] Same as above.

[0093] [Figure 35A] 35A-35C are line graphs showing the cytotoxicity of A49-F3'-TriNKET-Trastuzumab and Trastuzumab against BT-20 (FIG. 35A), Hs578T (FIG. 35B), and DU4475 (FIG. 35C) cancer cells in the presence of KHYG-1-CD16V cells. [Figure 35B] Same as above. [Figure 35C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] definition To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0095] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0096] As used herein, the terms "Fab" and "scFv" refer to two different forms of protein fragments, each of which contains an antigen-binding site. The term "antigen-binding site" refers to the portion of an immunoglobulin molecule involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains, which are also called VH and VL, respectively. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as hypervariable regions that are inserted between more conserved adjacent stretches known as "framework regions" or "FR". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions," or "CDRs." In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single domain antibody." The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, such as a Fab, or in a recombinant polypeptide, such as an scFv, where a peptide linker may be used to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide. All amino acid positions in the heavy or light chain variable regions disclosed herein are numbered according to Kabat numbering unless otherwise indicated.

[0097] The CDRs of an antigen-binding site can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). CDRs determined under these definitions typically contain overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term "CDR" refers to the CDRs defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" refers to the CDRs defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the heavy chain CDRs and the light chain CDRs of an antibody are defined using different conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to MacCallum (supra) and the light chain CDRs are defined according to Kabat (supra), with CDRH1, CDRH2, and CDRH3 representing the heavy chain CDRs and CDRL1, CDRL2, and CDRL3 representing the light chain CDRs.

[0098] The term "tumor-associated antigen" or "TAA" as used herein means any antigen, including but not limited to, a protein, glycoprotein, ganglioside, carbohydrate, or lipid, that is associated with cancer. In certain embodiments, the tumor-associated antigen is expressed on the surface of a cell. For example, the tumor-associated antigen may be expressed on a malignant cell or in the tumor microenvironment, such as in tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrate.

[0099] The term "HER2 overexpression" as used herein to characterize a cancer means a HER2 positive cancer according to the ASCO / CAP HER2 testing guidelines (Wolff et al., (2007) J. Clin. Oncol. 25(1):118-45) and the 2018 update (Wolff et al., (2018) J. Clin. Oncol. 36(20):2105-22). A HER2 overexpressing cancer may have a HER2 expression level scored as 3+ by immunohistochemistry, or a HER2 expression level scored as 2+ by immunohistochemistry that is further supported by detection of ERBB2 gene amplification (e.g., as determined by in situ hybridization (ISH), chromogenic in situ hybridization (CISH), quantitative PCR, or DNA sequencing).

[0100] As used herein, the term "pharmaceutical formulation" refers to a combination of an active agent with a carrier, inert, or active, making the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0101] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, primate, canine, feline, and the like), and more preferably include humans.

[0102] The terms "treat", "treating", or "treatment", and other grammatical equivalents used in this disclosure, include alleviating, reducing, ameliorating, or preventing a disease, condition, or symptom, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of a symptom, inhibiting a disease or condition, e.g., halting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or arresting the symptoms of a disease or condition, and are intended to include prevention. The term further includes achieving a therapeutic benefit and / or a prophylactic benefit. The term "therapeutic benefit" refers to the eradication or amelioration of the underlying disorder being treated. Also, therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, even though the patient will still be afflicted by the underlying disorder.

[0103] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0104] The term "about" refers to any minimal variation in concentration or amount of an agent that does not alter the effectiveness of the agent in preparing a formulation and in treating a disease or disorder. In certain embodiments, the term "about" can include ±5%, ±10%, or ±15% of the specified numerical value or data point.

[0105] Ranges can be expressed in this disclosure as from one particular value and / or to another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that by use of the antecedent "about," the particular value forms another embodiment. It is further understood that each endpoint of a range is significant in relation to, and independent of, the other endpoint. It is also understood that there are a number of values ​​disclosed in this disclosure, and that each value is also disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout this application, data is provided in a number of different formats, and that this data represents endpoints as well as starting points and ranges for any combination of the data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are considered to be disclosed. It is also understood that every unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0106] Throughout this specification, when compositions are described as having, including, containing, incorporating, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is intended that the compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. In addition, it is contemplated that there are compositions of the invention that consist essentially of, or consist of, the recited components, and there are processes and methods according to the invention that consist essentially of, or consist of, the recited steps.

[0107] As a general matter, compositions specifying percentages are by weight unless otherwise indicated. Further, if a variable is not accompanied by a definition, then the variable's previous definition governs.

[0108] Multispecific Binding Proteins The present disclosure provides pharmaceutical compositions or formulations comprising a multispecific binding protein having an antigen-binding site that binds to a tumor-associated antigen, such as HER2, an antigen-binding site that binds to NKG2D, and an antibody Fc domain. Also provided are uses of the multispecific binding protein and pharmaceutical formulations in treating cancer, such as locally advanced or metastatic solid tumors. The multispecific binding protein can bind targets (e.g., HER2) on cancer cells as well as NKG2D and CD16 on natural killer cells. Such binding brings the cancer cells into close proximity with the natural killer cells, thereby facilitating direct and indirect destruction of the cancer cells by the natural killer cells.

[0109] The first component of the multispecific binding protein binds to NKG2D receptor expressing cells, including, but not limited to, NK cells, NKT cells, γδT cells, and CD8 + The multispecific binding protein may include αβT cells. Upon binding to NKG2D, the multispecific binding protein may block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NK cells. The second component of the multispecific binding protein binds to HER2-expressing cells, including, but not limited to, breast, ovarian, esophageal, bladder and gastric cancers, salivary duct cancer, adenocarcinoma of the lung, and invasive forms of uterine cancer, such as uterine serous endometrial cancer. The third component of the multispecific binding protein is an antibody Fc domain that binds to cells expressing CD16, such as NK cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0110] The multispecific binding proteins described herein can take a variety of formats. For example, one format comprises a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (Figure 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain fused, either through a linker or an antibody hinge, to a Fab fragment comprised of a heavy chain portion comprising a heavy chain variable domain and a heavy chain CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain (CL), and the heavy and light chain portions of the Fab fragment pair and bind to NKG2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain fused, either through a linker or an antibody hinge, to a single chain variable fragment (scFv) comprised of a heavy chain variable domain and a light chain variable domain that pair and bind to a tumor associated antigen.

[0111] In some embodiments, the single chain variable fragment (scFv) described above is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge has the amino acid sequence Ala-Ser. In some other embodiments, the hinge has the amino acid sequence Ala-Ser and Thr-Lys-Gly. The hinge sequence can provide flexibility in binding to the target antigen and a balance between flexibility and optimal shape.

[0112] In some embodiments, the single chain variable fragment (scFv) described above comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance the stability of the scFv. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions being numbered under Kabat. For example, in some embodiments, a disulfide bridge is formed between a cysteine ​​residue (naturally occurring or introduced by mutation) at position 44 (C44) of the VH of the scFv and a cysteine ​​residue (naturally occurring or introduced by mutation) at position 100 (C100) of the VL of the scFv, numbered under the Kabat numbering scheme. In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker can be used, for example, the (G4S)4 linker (SEQ ID NO: 143). In some scFv embodiments, the heavy chain variable domain is located N-terminal to the light chain variable domain. In some scFv embodiments, the heavy chain variable domain is located C-terminal to the light chain variable domain.

[0113] The multispecific binding proteins described herein may further comprise one or more additional antigen binding sites. The additional antigen binding site may be fused to the C-terminus of the constant region CH2 domain or to the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the additional antigen binding site optionally takes the form of a disulfide-stabilized single chain variable region (scFv), resulting in a tetravalent or trivalent multispecific binding protein. For example, the multispecific binding protein comprises an NKG2D binding site, a TAA binding site, a third antigen binding site that binds to a TAA, and a sufficient antibody constant region or portion thereof that binds to CD16, or a fourth antigen binding site that binds to CD16. Any one of these antigen binding sites may take the form of a Fab or scFv, such as the scFvs described above. In some embodiments, the third antigen binding site binds to a different TAA. In some embodiments, the third antigen binding site binds to the same TAA, with exemplary formats shown in Figures 2C and 2D. Thus, the multispecific binding protein can provide a bivalent association of TAAs. In certain embodiments, bivalent association of a TAA with a multispecific protein can stabilize the TAA on the cancer cell surface and enhance the cytotoxicity of NK cells against cancer cells. Bivalent binding of a TAA by a multispecific protein can confer stronger binding of the multispecific protein to cancer cells, thereby promoting a stronger cytotoxic response of NK cells against cancer cells, particularly against cancer cells that express low levels of the TAA.

[0114] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 in the CH2 domain, and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0115] In some embodiments, the antibody constant domain comprises the CH2 and CH3 domains of an IgG antibody, e.g., a human IgG1 antibody. In some embodiments, mutations are introduced into the antibody constant domain to allow heterodimerization with another antibody constant domain. For example, if the antibody constant domain is derived from a human IgG1 constant domain, the antibody constant domain can have an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody that differs at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0116] In some embodiments, the antibody constant domain is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, , N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0117] The individual components of the multispecific binding proteins are described in more detail below.

[0118] NKG2D binding site Upon binding to the NKG2D and CD16 receptors on natural killer cells and the TAA on cancer cells, the multispecific binding protein can associate with one or more NK activating receptors and block the binding of natural ligands to NKG2D. In certain embodiments, the multispecific binding protein can stimulate NK cells in humans. In some embodiments, the multispecific binding protein can stimulate NK cells in humans as well as in other species, such as rodents and cynomolgus monkeys.

[0119] Table 1 lists peptide sequences of heavy and light chain variable domains that can bind to NKG2D in combination. In some embodiments, the heavy and light chain variable domains are arranged in a Fab format. These NKG2D binding domains may vary in their binding affinity to NKG2D, but they all activate human NK cells. Unless otherwise indicated, the CDR sequences provided in Table 1 are determined under Kabat. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0120] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 94 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 94 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 97 or 169) sequences of SEQ ID NO: 94. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0121] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 144 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 144 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 172 or 173) sequences of SEQ ID NO: 144. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0122] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 174 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 174 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 175 or 176) sequences of SEQ ID NO: 174. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0123] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 177 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 177 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 178 or 179) sequences of SEQ ID NO: 177. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0124] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 180 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 180 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 181 or 182) sequences of SEQ ID NO: 180. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0125] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 183 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 183 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 184 or 185) sequences of SEQ ID NO: 183. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0126] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 186 and a light chain variable domain related to SEQ ID NO: 98. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 186 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 95 or 168), CDR2 (SEQ ID NO: 96), and CDR3 (SEQ ID NO: 187 or 188) sequences of SEQ ID NO: 186. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:98 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:99), CDR2 (SEQ ID NO:100), and CDR3 (SEQ ID NO:101) sequences of SEQ ID NO:98.

[0127] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 86 and a light chain variable domain related to SEQ ID NO: 90. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 87 or 166), CDR2 (SEQ ID NO: 88), and CDR3 (SEQ ID NO: 89 or 167) sequences of SEQ ID NO: 86. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:90 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:91), CDR2 (SEQ ID NO:92), and CDR3 (SEQ ID NO:93) sequences of SEQ ID NO:90.

[0128] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 102 and a light chain variable domain related to SEQ ID NO: 106. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 102 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 71 or 162), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 105 or 170) sequences of SEQ ID NO: 102. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:106 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), and CDR3 (SEQ ID NO:109) sequences of SEQ ID NO:106.

[0129] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 70 and a light chain variable domain related to SEQ ID NO: 74. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 70 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 71 or 162), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73 or 163) sequences of SEQ ID NO: 70. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:74 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:75), CDR2 (SEQ ID NO:76), and CDR3 (SEQ ID NO:77) sequences of SEQ ID NO:74.

[0130] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 70 and a light chain variable domain related to SEQ ID NO: 74. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 70 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 71 or 162), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 73 or 163) sequences of SEQ ID NO: 70. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:74 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:75), CDR2 (SEQ ID NO:76), and CDR3 (SEQ ID NO:77) sequences of SEQ ID NO:74.

[0131] In some embodiments, a Fab comprises a heavy chain variable domain related to SEQ ID NO: 78 and a light chain variable domain related to SEQ ID NO: 82. For example, the heavy chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 78 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 79 or 164), CDR2 (SEQ ID NO: 80), and CDR3 (SEQ ID NO: 81 or 165) sequences of SEQ ID NO: 78. Similarly, the light chain variable domain of the Fab can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:82 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:75), CDR2 (SEQ ID NO:76), and CDR3 (SEQ ID NO:77) sequences of SEQ ID NO:82.

[0132] The multispecific binding protein can bind to NKG2D-expressing cells, including, but not limited to, NK cells, γδ T cells, and CD8 +Upon binding to NKG2D, the multispecific binding protein can block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NK cells.

[0133] In certain embodiments, the Fab or multispecific binding protein has a concentration of from 2 nM to 120 nM, e.g., from 2 nM to 110 nM, from 2 nM to 100 nM, from 2 nM to 90 nM, from 2 nM to 80 nM, from 2 nM to 70 nM, from 2 nM to 60 nM, from 2 nM to 50 nM, from 2 nM to 40 nM, from 2 nM to 30 nM, from 2 nM to 20 nM, from 2 nM to 10 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, Approximately 3nM, approximately 2.5nM, approximately 2nM, approximately 1.5nM, approximately 1nM, approximately 0.5nM to approximately 1nM, approximately 1nM to approximately 2nM, approximately 2nM to 3nM, approximately 3nM to 4nM, approximately 4nM to approximately 5nM, approximately 5nM to approximately 6nM, approximately 6nM to approximately 7nM, approximately 7nM to approximately 8nM, approximately 8 K between nM to about 9nM, about 9nM to about 10nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, or about 8nM to about 10nM D It binds to NKG2D with an affinity of

[0134] In certain embodiments, the Fab has a K of 2 nM to 120 nM as measured by surface plasmon resonance. D In certain embodiments, the multispecific binding protein binds to NKG2D with a K of between 2 nM and 120 nM as measured by surface plasmon resonance. D In certain embodiments, the Fab binds to NKG2D with a K of 10 nM to 62 nM as measured by surface plasmon resonance. D In certain embodiments, the multispecific binding protein binds to NKG2D with a K of 10 nM to 62 nM as measured by surface plasmon resonance. D It binds to NKG2D.

[0135] In some embodiments, the Fab described above is linked to an antibody Fc polypeptide. In some embodiments, the heavy chain portion of the Fab is linked to the N-terminus of the antibody Fc polypeptide. TAA binding site

[0136] Tumor-associated antigens (TAA) that can be targeted by the multispecific binding proteins include, but are not limited to, ANO1, BCMA, EpCAM, CAIX, CEA, CCR4, CD2, CD123, CD133, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD40, CD52, CD70, CLAUDIN-18.2, DLL3, EGFR / ERBB1, GD2, IGF1R, HER2 / ERBB2, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSMA, mesothelin, MICA, MICB, TRAILR1, TR AILR2, TROP2, MAGE-A3, B7.1, B7.2, CTLA4, PD1, 5T4, GPNMB, FR-alpha, PAPP-A, FLT3, GPC3, CXCR4, ROR1, ROR2, HLA-E, PD-L1, VLA4, CD44, CD13, CD15, CD47, CLL1 , CD81, CD23, CD79a, CD79b, CD80, CRLF2, SLAMF7, CD138, CA125, NaPi2b, Nectin 4, ADAM8, ADAM9, SLC44A4, CA19-9, LILRBl, LILRB2, LILRB3, LILRB4, LILRB5, ULRA 1, LILRA2, LILRA3, ULRA4, LILRA5, and ULRA6, CCR8, CD7, CTLA4, CX3CR1, ENTPD1, ​​HAVCR2, IL-1R2, PDCD1LG2, TIGIT, TNFRSF4, TNFRSF8, TNFRSF9, GEM, NT5E, TNFRSF18, MUC1, P-cadherin, Plexin-A1, TN FRSF10B, STEAP1, CDCP1, PTK7, Axl, erbB-3, EDNRB, Tyrp1, CD14, CD163, CSF3R, Siglec-9, ITGAM, VISTA, B7-H4(VTCN1), CCR1, LRRC25, PTAFR, SIRPB1, TLR2, TLR4, CD300LB, ATP1A3, CCR5, MUC1(or MUC1-C), Plexin-A1, TNFRSF10B, STEAP1, CDCP1, PTK7, AXL, EDNRB, OLR1, and TYRP1.

[0137] In certain embodiments, the tumor-associated antigen is HER2. Table 2 lists the amino acid sequences of heavy and light chain variable domains, which, in combination, can bind to HER2. In certain embodiments, the antigen-binding site that binds to HER2 is in the format of an scFv. [Table 2-1] [Table 2-2]

[0138] Alternatively, novel antigen binding sites capable of binding to HER2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 138, or a mature extracellular fragment thereof.

[0139] In some embodiments, the scFv comprises a heavy chain variable domain related to SEQ ID NO: 195 and a light chain variable domain related to SEQ ID NO: 196. For example, the heavy chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 195 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:115), CDR2 (SEQ ID NO:116), and CDR3 (SEQ ID NO:117) sequences of SEQ ID NO:195. Similarly, the light chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 196 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO:119), CDR2 (SEQ ID NO:120), and CDR3 (SEQ ID NO:121) sequences of SEQ ID NO: 196. In some embodiments, the scFv has the amino acid sequence of SEQ ID NO:139.

[0140] In some embodiments, an scFv comprises a heavy chain variable domain related to SEQ ID NO: 197 and a light chain variable domain related to SEQ ID NO: 198. For example, the heavy chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 197 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 123), CDR2 (SEQ ID NO: 124), and CDR3 (SEQ ID NO: 125) sequences of SEQ ID NO: 197. Similarly, the light chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 198 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 127), CDR2 (SEQ ID NO: 128), and CDR3 (SEQ ID NO: 129) sequences of SEQ ID NO: 198. In some embodiments, the scFv has the amino acid sequence of SEQ ID NO: 189.

[0141] In some embodiments, an scFv comprises a heavy chain variable domain related to SEQ ID NO: 199 and a light chain variable domain related to SEQ ID NO: 200. For example, the heavy chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 199 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 131), CDR2 (SEQ ID NO: 132), and CDR3 (SEQ ID NO: 133) sequences of SEQ ID NO: 199. Similarly, the light chain variable domain of the scFv can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 200 and / or can incorporate amino acid sequences identical to the CDR1 (SEQ ID NO: 135), CDR2 (SEQ ID NO: 136), and CDR3 (SEQ ID NO: 137) sequences of SEQ ID NO: 200. In some embodiments, the scFv has the amino acid sequence of SEQ ID NO: 171.

[0142] The scFv described above comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance the stability of the scFv. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions being numbered under Kabat. For example, in some embodiments, a disulfide bridge is formed between a cysteine ​​residue (naturally occurring or introduced by mutation) at position 44 (C44) of the VH of the scFv and a cysteine ​​residue (naturally occurring or introduced by mutation) at position 100 (C100) of the VL of the scFv, numbered under the Kabat numbering scheme.

[0143] The VH and VL of the scFv can be positioned in various orientations. In certain embodiments, the VL is positioned at the N-terminus of the VH. In certain embodiments, the VL is positioned at the C-terminus of the VH.

[0144] The VH and VL of the scFv can be connected via a linker, for example, a peptide linker. In certain embodiments, the peptide linker is a flexible linker. With regard to the amino acid composition of the linker, the peptide is selected with the properties of providing flexibility, not interfering with the structure and function of other domains of the protein of the invention, and resisting cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, the VL is located at the N-terminus of the VH and is connected to the VH via a linker.

[0145] The length of a linker (e.g., a flexible linker) can be "short," e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, or "long," e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, or 20-25 amino acid residues in length.

[0146] In certain embodiments, the linker is (GS) n (SEQ ID NO: 204), (GGS) n (SEQ ID NO: 205), (GGGS) n (SEQ ID NO: 151), (GGSG) n (SEQ ID NO: 153), (GGSGG) n (SEQ ID NO: 156), and (GGGGS) n (SEQ ID NO:157), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker comprises an amino acid sequence selected from SEQ ID NO:143, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:150, SEQ ID NO:152, and SEQ ID NO:154, as listed in Table 3. [Table 3]

[0147] In certain embodiments, the light chain variable domain is linked to the N-terminus of the heavy chain variable domain via a flexible linker, such as a (G4S)4 linker (SEQ ID NO: 143).

[0148] In some embodiments, the scFv described above is linked to the antibody Fc polypeptide via a hinge sequence. In some embodiments, the hinge comprises the amino acid sequence Ala-Ser. In some other embodiments, the hinge comprises the amino acid sequences Ala-Ser and Thr-Lys-Gly. The hinge sequence can provide flexibility in binding to the target antigen, as well as a balance between flexibility and optimal shape.

[0149] Fc domain The antibody Fc domain of the multispecific binding protein comprises a first antibody Fc polypeptide linked to a Fab and a second antibody Fc polypeptide linked to an scFv. The two antibody Fc polypeptides pair to form a dimer that binds to CD16.

[0150] Within the antibody Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 in the CH2 domain, and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0151] The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can lead to the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. Promoting the preferential assembly of heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, mutations can be made based on human IgG1 by incorporating different pairs of amino acid substitutions in the CH3 domain in the first and second polypeptides that allow these two chains to selectively heterodimerize with each other. All amino acid substitution positions illustrated below are numbered according to the EU index as in Kabat.

[0152] In one scenario, an amino acid substitution in a first polypeptide replaces an original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces an original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V) such that the larger amino acid substitution (protuberance) fits into the surface of the smaller amino acid substitution (cavity). For example, one polypeptide can incorporate a T366W substitution and another can incorporate three substitutions including T366S, L368A, and Y407V.

[0153] The antibody heavy chain variable domain of the present invention may optionally be coupled, with or without a CH1 domain, to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region including the hinge, CH2 and CH3 domains. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region compared to the human IgG1 constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366J, T366K ... Includes 66L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0154] In certain embodiments, mutations that may be incorporated into the CH1 of the human IgG1 constant region may be amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be incorporated into the Cκ of the human IgG1 constant region may be amino acids E123, F116, S176, V163, S174, and / or T164.

[0155] The amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 4]

[0156] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 5. [Table 5]

[0157] Alternatively, the amino acid substitutions may be selected from the following set of substitutions shown in Table 6. [Table 6]

[0158] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 7. [Table 7]

[0159] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 8, where the position shown in the first polypeptide column is substituted with any known negatively charged amino acid and the position shown in the second polypeptide column is substituted with any known positively charged amino acid. [Table 8]

[0160] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 9, where the position shown in the first polypeptide column is substituted with any known positively charged amino acid and the position shown in the second polypeptide column is substituted with any known negatively charged amino acid. [Table 9]

[0161] Alternatively, the amino acid substitutions may be selected from the following set in Table 10. [Table 10]

[0162] When selecting an Fc substitution, one of skill in the art will understand that the first and second polypeptides in Tables 4-10 can correspond to a first and second antibody Fc polypeptide, respectively. Alternatively, the first and second polypeptides in Tables 4-10 can correspond to a second and first antibody Fc polypeptide, respectively.

[0163] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368, and Y407.

[0164] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0165] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0166] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0167] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409, and T411.

[0168] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400, and Y407.

[0169] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, Y349, K360, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399, and F405.

[0170] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399, and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, K360, Q347, and K409.

[0171] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409, and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357, and D399.

[0172] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409, and K439.

[0173] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392, and K409.

[0174] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366, and D399.

[0175] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by Q347R, D399V, and F405T substitutions.

[0176] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by Q347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions.

[0177] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366S substitution, a T368A substitution, and a Y407V substitution.

[0178] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366S substitution, a T368A substitution, and a Y407V substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution.

[0179] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L, and T394W substitutions.

[0180] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L, and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions.

[0181] Alternatively, or in addition, the structural stability of the heteromultimeric protein may be increased by introducing an S354C substitution in either the first or second polypeptide chain and a Y349C substitution in the opposing polypeptide chain, which form an artificial disulfide bridge within the interface of the two polypeptides. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by an S354C substitution and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by an Y349C substitution.

[0182] When choosing an Fc replacement, one skilled in the art will understand that the "one polypeptide chain" and the "other polypeptide chain" of the antibody constant region described above can correspond to a first antibody Fc polypeptide and a second antibody Fc polypeptide, respectively. Alternatively, the "one polypeptide chain" and the "other polypeptide chain" of the antibody constant region described above can correspond to a second antibody Fc polypeptide and a first antibody Fc polypeptide, respectively.

[0183] Exemplary Multispecific Binding Proteins Listed below are examples of TriNKETs that include a HER2-binding scFv and an NKG2D-binding Fab, each linked to an antibody constant region, and an antibody constant region incorporating a mutation that allows heterodimerization of two Fc polypeptide chains. The scFv includes a heavy chain variable domain (VH) and a light chain variable domain (VL) derived from an anti-HER2 antibody (e.g., trastuzumab), and further includes a Cys substitution for the amino acid residues at position 100 of the VL and position 44 of the VH, thereby promoting the formation of disulfide bridges between the VH and VL of the scFv. The VL is N-terminally linked to the VH via a (G4S)4 linker (SEQ ID NO: 143), and the VH is N-terminally linked to the Fc via an Ala-Ser linker. The Ala-Ser linker is included at the elbow hinge region sequence to balance flexibility and optimal shape. In certain embodiments, the additional sequence Thr-Lys-Gly can be added N-terminal or C-terminal to the Ala-Ser sequence at the hinge. As used herein to describe these exemplary TriNKETs, the Fc domain includes the antibody hinge, CH2, and CH3.

[0184] Thus, each of the TriNKETs described below has three polypeptide chains: Chain A, from N-terminus to C-terminus: comprises the VH, CH1, and antibody Fc polypeptides of the NKG2D-binding Fab; Chain B, from N-terminus to C-terminus, comprises: a VL of a HER2 binding scFv, a (G4S)4 linker (SEQ ID NO: 143), a VH of a HER2 binding scFv, an Ala-Ser linker, and an antibody Fc polypeptide; and Chain C, from N-terminus to C-terminus, comprises: the VL of an NKG2D-binding Fab, and the light chain constant domain (CL).

[0185] The amino acid sequences of exemplary TriNKETs are summarized in Table 11.

[0186] In certain embodiments, a multispecific binding protein of the disclosure comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain having the amino acid sequences of chain A, chain B, and chain C, respectively, of TriNKET disclosed in Table 11.

[0187] In an exemplary embodiment, the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates the mutations Q347R, D399V, and F405T, and the antibody Fc polypeptide linked to the HER2 scFv incorporates the matching mutations K360E and K409W to form heterodimers. In another exemplary embodiment, the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates the knob mutations T366S, L368A, and Y407V, and the antibody Fc polypeptide linked to the HER2-binding scFv incorporates the hole mutation T366W. In an exemplary embodiment, the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment comprises a S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution on the antibody Fc polypeptide linked to the HER2-binding scFv. [Table 11-1] [Table 11-2]

[0188] Certain TriNKETs and their polypeptide chains are described in more detail below. In the amino acid sequence, (G4S)4 (SEQ ID NO: 143) and the Ala-Ser linker are in bold-underlined; Cys residues in the scFv that form disulfide bridges are in bold-italic-underlined; Fc heterodimerization mutations are in bold-underlined; and CDR sequences under Kabat are underlined.

[0189] For example, a TriNKET of the present disclosure is A49-F3'-TriNKET-Trastuzumab. A49-F3'-TriNKET-Trastuzumab comprises a single chain variable fragment (scFv) (SEQ ID NO: 139) derived from HER2-binding Trastuzumab linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and an NKG2D-binding Fab fragment derived from A49 linked to a second antibody Fc polypeptide. The Fab fragment comprises a heavy chain portion having a heavy chain variable domain (SEQ ID NO: 94) and a CH1 domain, and a light chain portion having a light chain variable domain (SEQ ID NO: 98) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A49-F3'-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142.

[0190] SEQ ID NO: 140 represents the full sequence of a HER2-binding scFv linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc polypeptide comprises a Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for disulfide bond formation with a Y349C substitution in SEQ ID NO: 141, as described below. The scFv (SEQ ID NO: 139) comprises a heavy chain variable domain of trastuzumab connected to the N-terminus of the light chain variable domain of trastuzumab via a (G4S)4 linker (SEQ ID NO: 143), a scFv represented as VL-(G4S)4-VH ("(G4S)4" represented by SEQ ID NO: 143). The heavy and light chain variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH as a result of Q100C and G44C substitutions in VL and VH, respectively. Trastuzumab scFv DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT FG C GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGK C LEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDY WGQGTLVTVSS (SEQ ID NO: 139) Trastuzumab scFv-Fc (RVT) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG C GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK C LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP C RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 140)

[0191] SEQ ID NO:141 represents the heavy chain portion of a Fab fragment, which includes the heavy chain variable domain (SEQ ID NO:94) and the CH1 domain of the NKG2D binding site connected to a second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:141 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution on the Fc polypeptide in SEQ ID NO:140. In SEQ ID NO:141, the antibody Fc polypeptide also includes a K360E substitution and a K409W substitution for heterodimerization with the Fc in SEQ ID NO:140. A49 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS SSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR GAPMGAAAGWFDP WGQGTLVTVSS (SEQ ID NO: 94) A49 VH-CH1-Fc(EW) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS W LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 141)

[0192] SEQ ID NO: 142 represents the light chain portion of a Fab fragment containing the light chain variable domain (SEQ ID NO: 98) and the light chain constant domain of the NKG2D binding site. A49 VL DIQMTQSPSSVSASVGDRVTITC RASQGISSWLA WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQGVSFPRTF GGGTKVEIK (SEQ ID NO:98) A49 VL-LC DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK RTVAAPSPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 142)

[0193] Another TriNKET of the present disclosure is A49MI-F3'-TriNKET-Trastuzumab. A49MI-F3'-TriNKET-Trastuzumab comprises the same HER2-binding scFv (SEQ ID NO: 139) as A49-F3'-TriNKET-Trastuzumab linked to an antibody Fc polypeptide via a hinge containing Ala-Ser; and an NKG2D-binding Fab fragment derived from A49MI linked to a second antibody Fc polypeptide. The Fab fragment comprises a heavy chain portion comprising a heavy chain variable domain (SEQ ID NO: 144) and a CH1 domain, and a light chain portion comprising a light chain variable domain (SEQ ID NO: 98) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, which is connected to the second antibody Fc polypeptide. A49MI-F3'-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO: 140 (similar to A49-F3'-TriNKET-Trastuzumab), SEQ ID NO: 145, and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0194] SEQ ID NO: 145 represents the heavy chain portion of a Fab fragment, which comprises the heavy chain variable domain (SEQ ID NO: 144) and the CH1 domain of the NKG2D binding site connected to a first antibody Fc polypeptide. In SEQ ID NO: 144, the methionine in CDR3 of SEQ ID NO: 94 is replaced by isoleucine (M→I substitution; shown in the third bracket [ ] of SEQ ID NO: 144 and SEQ ID NO: 145). The antibody Fc polypeptide in SEQ ID NO: 145 comprises a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution in the antibody Fc polypeptide in SEQ ID NO: 140. In SEQ ID NO: 145, the antibody Fc polypeptide also comprises a K360E substitution and a K409W substitution. A49MI VH EVQLVESGGGLVKPGGSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVS SSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR GAP[I]GAAAGWFDP WGQGTLVTVSS (SEQ ID NO: 144) A49MI VH-CH1-Fc(EW) EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAP[I]GAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS WLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 145)

[0195] Another TriNKET of the present disclosure is A49-F3'-KiH-TriNKET-Trastuzumab. KiH refers to knobs-into-holes (KiH) Fc technology, which involves engineering the CH3 domain to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. The concept behind KiH Fc technology was to introduce a "knob" into one CH3 domain (CH3A) by substitution of a small residue with a bulky residue (e.g., T366W in EU numbering). CH3A To accommodate the "knob", a complementary "hole" face was created on the other CH3 domain (CH3B) by replacing the nearest neighboring residues to the knob with smaller residues (e.g., TCH366S / L368A / Y407V CH3B). The "hole" mutation was optimized by structure-guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(1):26-35). X-ray crystal structure of KiH Fc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426 (9): 1947-57; Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc domain variant with improved affinity for FcγRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored due to hydrophobic interactions driven by steric complementarity at the CH3-CH3 core interface, whereas the knob-knob and hole-hole interfaces are not favored for homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0196] A49-F3'-KiH-TriNKET-Trastuzumab contains the same HER2-binding scFv as in A49-F3'-TriNKET-Trastuzumab (SEQ ID NO: 139) linked via a hinge containing Ala-Ser to a first antibody Fc polypeptide incorporating the "hole" substitutions T366S, L368A, Y407V; and the same NKG2D-binding Fab fragment as in A49-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide incorporating the "knob" substitution T366W. A49-F3'-KiH-TriNKET-Trastuzumab contains three polypeptides having the sequences of SEQ ID NO: 146, SEQ ID NO: 147, and SEQ ID NO: 142 (as in A49-F3'-TriNKET-Trastuzumab).

[0197] SEQ ID NO: 146 represents the complete sequence of a HER2-binding scFv (SEQ ID NO: 139) linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc polypeptide comprises a T366S substitution, an L368A substitution, and a Y407V substitution for heterodimerization, as well as an S354C substitution for disulfide bond formation with a Y349C substitution in SEQ ID NO: 147, as described below. Trastuzumab scFv-Fc(KiH) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG C GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK C LEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 146)

[0198] SEQ ID NO: 147 represents a heavy chain portion of a Fab fragment having a heavy chain variable domain (SEQ ID NO: 94) and a CH1 domain of an NKG2D binding site derived from A49 connected to a second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO: 147 contains a S354C substitution, which forms a disulfide bond with a Y349C substitution in the CH3 domain of the first antibody Fc polypeptide. In SEQ ID NO: 147, the antibody Fc polypeptide also contains a T366W substitution. A49 VH-CH1-Fc(KiH) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 147)

[0199] Another TriNKET of the disclosure is A49MI-F3'-KiH-TriNKET-Trastuzumab, which comprises the same HER2-binding scFv as in A49-F3'-TriNKET-Trastuzumab (SEQ ID NO: 139) linked via a hinge containing Ala-Ser to a first antibody Fc polypeptide incorporating the "hole" substitutions T366S, L368A, Y407V; and the same NKG2D-binding Fab fragment as in A49MI-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide incorporating the "knob" substitution T366W. A49MI-F3'-KiH-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO: 146 (similar to A49-F3'-KiH-TriNKET-Trastuzumab), SEQ ID NO: 194, and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0200] SEQ ID NO: 194 represents the heavy chain portion of a Fab fragment having the heavy chain variable domain (SEQ ID NO: 144) and the CH1 domain of the NKG2D binding site derived from A49MI connected to a second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO: 194 comprises a S354C substitution, which forms a disulfide bond with a Y349C substitution in the CH3 domain of the first antibody Fc polypeptide. In SEQ ID NO: 194, the antibody Fc polypeptide also comprises a T366W substitution. A49MI VH-CH1-Fc(KiH) EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 194)

[0201] Another exemplary TriNKET of the present disclosure is A44-F3'-TriNKET-Trastuzumab. A44-F3'-TriNKET-Trastuzumab comprises the same HER2-binding scFv (SEQ ID NO: 139) as in A49-F3'-TriNKET-Trastuzumab linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and an NKG2D-binding Fab fragment derived from A44 linked to a second antibody Fc polypeptide. The Fab fragment comprises a heavy chain portion having a heavy chain variable domain (SEQ ID NO: 86) and a CH1 domain, and a light chain portion having a light chain variable domain (SEQ ID NO: 90) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A44-F3'-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO:140 (similar to A49-F3'-TriNKET-Trastuzumab), SEQ ID NO:155, and SEQ ID NO:149.

[0202] SEQ ID NO: 155 represents the heavy chain variable domain of the NKG2D binding site derived from A44 (SEQ ID NO: 86) connected to a second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO: 155 comprises a Y349C substitution in the CH3 domain, which forms a disulfide bond with a S354C substitution on the first antibody Fc polypeptide. In SEQ ID NO: 155, the antibody Fc polypeptide also comprises a K360E substitution and a K409W substitution. A44 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DGGYYDSGAGDY WGQGTLVTVSS (SEQ ID NO: 86) A44 VH-CH1-Fc(EW) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS W LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 155)

[0203] SEQ ID NO: 149 represents the light chain portion of a Fab fragment, which contains the light chain variable domain (SEQ ID NO: 90) and the light chain constant domain of the NKG2D binding site. A44VL DIQMTQSPSSVSASVGDRVTITC RASQGIDSWLA WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQGVSYPRT FGGGTKVEIK (SEQ ID NO: 90) A44 VL-CL DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 149)

[0204] Another exemplary TriNKET of the present disclosure is A44-F3'-KiH-TriNKET-Trastuzumab, which comprises the same HER2-binding scFv (SEQ ID NO: 139) as in A49-F3'-TriNKET-Trastuzumab linked via a hinge containing Ala-Ser to a first antibody Fc polypeptide incorporating the "hole" substitutions of T366S, L368A, and Y407V; and the same NKG2D-binding Fab fragment as in A44-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide incorporating the "knob" substitution of T366W. A44-F3'-KiH-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO: 146 (similar to A49-F3'-KiH-TriNKET-Trastuzumab), SEQ ID NO: 148, and SEQ ID NO: 149 (similar to A44-F3'-TriNKET-Trastuzumab).

[0205] SEQ ID NO: 148 represents the heavy chain variable domain of the NKG2D binding site derived from A44 (SEQ ID NO: 86) connected to a second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO: 148 contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with a S354C substitution on the first antibody Fc polypeptide. In SEQ ID NO: 148, the antibody Fc polypeptide also contains a T366W substitution. A44 VH-CH1-Fc(KiH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 148)

[0206] Another TriNKET of the present disclosure is A49-F3'-TriNKET-Pertuzumab. A49-F3'-TriNKET-Pertuzumab comprises an scFv derived from Pertuzumab that binds HER2 (SEQ ID NO: 189) linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A49-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49-F3'-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 190, SEQ ID NO: 141 (similar to A49-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0207] SEQ ID NO: 190 represents the full sequence of a HER2 binding scFv linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc polypeptide incorporates the Q347R, D399V, and F405T substitutions for heterodimerization and the S354C substitution for disulfide bond formation with the Y349C substitution in SEQ ID NO: 141, as described below. The scFv (SEQ ID NO: 189) comprises a heavy chain variable domain of Pertuzumab connected to the N-terminus of the light chain variable domain of Pertuzumab via a (G4S)4 linker (SEQ ID NO: 143), with the scFv represented as VL-(G4S)4-VH ("(G4S)4" represented by SEQ ID NO: 143). The heavy and light chain variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH as a result of Q100C and G44C substitutions in VL and VH, respectively. Pertuzumab scFv DIQMTQSPSSLSASVGDRVTITCKASQ DVSIGVA WYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYYIYPYT FG C GTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASG FTFTDY TMDWVRQAPGK C LEWVADV NPNSGG SIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR NLGPSFYFDY WGQGTLVTVSSA (SEQ ID NO: 189) Pertuzumab scFv-Fc DIQMTQSPSSLSASVGDRVTITCKASQ DVSIGVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYYIYPYT FGCGTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASG FTFTDY TMDWVRQAPGKCLEWVADV NPNSGG SIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR NLGPSFYFDY WGQGTLVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP C RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 190)

[0208] Another exemplary TriNKET of the present disclosure is A49MI-F3'-TriNKET-Pertuzumab. A49MI-F3'-TriNKET-Pertuzumab comprises the same HER2-binding scFv (SEQ ID NO: 189) as in A49-F3'-TriNKET-Pertuzumab linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A49MI-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49MI-F3'-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 190 (similar to A49-F3'-KiH-TriNKET-Pertuzumab), SEQ ID NO: 145 (similar to A49MI-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0209] Another exemplary TriNKET of the present disclosure is A49-F3'-KiH-TriNKET-Pertuzumab. A49-F3'-KiH-TriNKET-Pertuzumab comprises the same HER2-binding scFv (SEQ ID NO: 189) as in A49-F3'-TriNKET-Pertuzumab linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A49-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the "knob" substitution of T366W. A49-F3'-KiH-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 191, SEQ ID NO: 147 (similar to A49-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0210] SEQ ID NO: 191 represents the complete sequence of a HER2 binding scFv (SEQ ID NO: 189) linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc polypeptide incorporates a T366S substitution, an L368A substitution, and a Y407V substitution for heterodimerization, as well as an S354C substitution for disulfide bond formation with a Y349C substitution in SEQ ID NO: 191, as described below. Pertuzumab scFv-Fc(KiH) DIQMTQSPSSLSASVGDRVTITCKASQ DVSIGVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYYIYPYT FG C GTKVEIKR GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASG FTFTDY TMDWVRQAPGK C LEWVADV NPNSGG SIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR NLGPSFYFDY WGQGTLVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 191)

[0211] Another exemplary TriNKET of the present disclosure is A49MI-F3'-KiH-TriNKET-Pertuzumab. A49MI-F3'-KiH-TriNKET-Pertuzumab comprises the same HER2-binding scFv (SEQ ID NO: 189) as in A49-F3'-TriNKET-Pertuzumab linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A49MI-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the "knob" substitution of T366W. A49MI-F3'-KiH-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 191 (similar to A49-F3'-KiH-TriNKET-Pertuzumab), SEQ ID NO: 194 (similar to A49MI-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0212] Another exemplary TriNKET of the present disclosure is A44-F3'-TriNKET-Pertuzumab. A44-F3'-TriNKET-Pertuzumab comprises the same HER2-binding scFv (SEQ ID NO: 189) as in A49-F3'-TriNKET-Pertuzumab linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A44-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A44-F3'-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 190 (similar to A49-F3'-KiH-TriNKET-Pertuzumab), SEQ ID NO: 155 (similar to A44-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 149 (similar to A44-F3'-TriNKET-Trastuzumab).

[0213] Another exemplary TriNKET of the present disclosure is A44-F3'-KiH-TriNKET-Pertuzumab. A44-F3'-KiH-TriNKET-Pertuzumab comprises the same HER2-binding scFv (SEQ ID NO: 189) as in A49-F3'-TriNKET-Pertuzumab linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A44-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the "knob" substitution of T366W. A44-F3'-KiH-TriNKET-Pertuzumab comprises three polypeptides having the sequences of SEQ ID NO: 191 (similar to A49-F3'-KiH-TriNKET-Pertuzumab), SEQ ID NO: 148 (similar to A44-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 149 (similar to A44-F3'-TriNKET-Trastuzumab).

[0214] Another TriNKET of the present disclosure is A49-F3'-TriNKET-MGAH22. A49-F3'-TriNKET-MGAH22 comprises an scFv derived from MGAH22 that binds HER2 (SEQ ID NO: 171) linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A49-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide linked to the Fab fragment incorporates K360E and K409W substitutions. A49-F3'-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 192, SEQ ID NO: 141 (similar to A49-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0215] SEQ ID NO: 192 represents the complete sequence of a HER2-binding scFv linked to a primary antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc domain incorporates the Q347R, D399V, and F405T substitutions for heterodimerization and the S354C substitution for disulfide bond formation with the Y349C substitution in SEQ ID NO: 141, as described below. The scFv (SEQ ID NO: 171) comprises a heavy chain variable domain of Pertuzumab connected to the N-terminus of the light chain variable domain of Pertuzumab via a (G4S)4 linker (SEQ ID NO: 143), where the scFv is represented as VL-(G4S)4-VH ("(G4S)4" is represented by SEQ ID NO: 143). The heavy and light chain variable domains of the scFv are also connected through a disulfide bridge between C100 of VL and C44 of VH as a result of GC100 and GC44C substitutions in VL and VH, respectively. MGAH22 scFv DIVMTQSHKFMSTSVGDRVSITCKAS QDVNTAVA WYQQKPGHSPKLLIY SASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYC QQHYTTPPT FG C GTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTAS GFNIKDT YIHWVKQRPEQ C LEWIGRI YPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSR WGGDGFYAMDY WGQGASVTVSSA (SEQ ID NO: 171) MGAH22 scFv-Fc DIVMTQSHKFMSTSVGDRVSITCKAS QDVNTAVA WYQQKPGHSPKLLIY SASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYC QQHYTTPPT FG C GTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTAS GFNIKDT YIHWVKQRPEQ C LEWIGRI YPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSR WGGDGFYAMDY WGQGASVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP C RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 192)

[0216] Another TriNKET of the present disclosure is A49MI-F3'-TriNKET-MGAH22. A49MI-F3'-TriNKET-MGAH22 comprises the same HER2-binding scFv (SEQ ID NO: 171) as in A49-F3'-TriNKET-MGAH22 linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A49MI-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A49MI-F3'-KiH-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 192 (similar to A49-F3'-TriNKET-MGAH22), SEQ ID NO: 145 (similar to A49MI-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0217] Another TriNKET of the present disclosure is A49-F3'-KiH-TriNKET-MGAH22. A49-F3'-KiH-TriNKET-MGAH22 comprises the same HER2-binding scFv (SEQ ID NO: 171) as in A49-F3'-TriNKET-MGAH22 linked to a first antibody Fc polypeptide via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as in A49-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide linked to the Fab fragment incorporates the "knob" substitution of T366W. A49-F3'-KiH-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 193, SEQ ID NO: 147 (similar to A49-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0218] SEQ ID NO: 193 represents the complete sequence of a HER2 binding scFv (SEQ ID NO: 171) linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser (scFv-Fc). The first antibody Fc polypeptide incorporates a T366S substitution, an L368A substitution, and a Y407V substitution for heterodimerization and an S354C substitution for disulfide bond formation with a Y349C substitution in SEQ ID NO: 147, as described below. MGAH22 scFv-Fc(KiH) DIVMTQSHKFMSTSVGDRVSITCKAS QDVNTAVA WYQQKPGHSPKLLIY SASFRYT GVPDRFTGSRSGTDFTFTISSVQAEDLAVYYC QQHYTTPPT FG C GTKVEIKR GGGGSGGGGSGGGGSGGGGS QVQLQQSGPELVKPGASLKLSCTAS GFNIKDT YIHWVKQRPEQ C LEWIGRI YPTNGY TRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSR WGGDGFYAMDY WGQGASVTVSSA AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSRDELTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 193)

[0219] Another exemplary TriNKET of the present disclosure is A49MI-F3'-KiH-TriNKET-MGAH22. A49MI-F3'-KiH-TriNKET-MGAH22 comprises the same HER2-binding scFv (SEQ ID NO: 171) as in A49-F3'-TriNKET-MGAH22 linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A49MI-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the "knob" substitution of T366W. A49MI-F3'-KiH-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 193 (similar to A49-F3'-KiH-TriNKET-MGAH22), SEQ ID NO: 194 (similar to A49MI-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 142 (similar to A49-F3'-TriNKET-Trastuzumab).

[0220] Another exemplary TriNKET of the present disclosure is A44-F3'-TriNKET-MGAH22. A44-F3'-TriNKET-MGAH22 comprises the same HER2-binding scFv (SEQ ID NO: 171) as in A49-F3'-TriNKET-MGAH22 linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A44-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates Q347R, D399V, and F405T substitutions, and the second antibody Fc polypeptide incorporates K360E and K409W substitutions. A44-F3'-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 192 (similar to A49-F3'-TriNKET-MGAH22), SEQ ID NO: 155 (similar to A44-F3'-TriNKET-Trastuzumab), and SEQ ID NO: 149 (similar to A44-F3'-TriNKET-Trastuzumab).

[0221] Another exemplary TriNKET of the present disclosure is A44-F3'-KiH-TriNKET-MGAH22. A44-F3'-KiH-TriNKET-MGAH22 comprises the same HER2-binding scFv (SEQ ID NO: 171) as in A49-F3'-TriNKET-MGAH22 linked to a first antibody Fc polypeptide via a hinge comprising Ala-Ser; and the same NKG2D-binding Fab fragment as in A44-F3'-TriNKET-Trastuzumab, the CH1 domain of which is connected to a second antibody Fc polypeptide. The first antibody Fc polypeptide incorporates the "hole" substitutions of T366S, L368A, and Y407V, and the second antibody Fc polypeptide incorporates the "knob" substitution of T366W. A44-F3'-KiH-TriNKET-MGAH22 comprises three polypeptides having the sequences of SEQ ID NO: 193 (similar to A49-F3'-KiH-TriNKET-MGAH22), SEQ ID NO: 148 (similar to A44-F3'-KiH-TriNKET-Trastuzumab), and SEQ ID NO: 149 (similar to A44-F3'-TriNKET-Trastuzumab).

[0222] In certain embodiments, a TriNKET of the disclosure is identical to one of the exemplary TriNKETs described above that includes an EW-RVT Fc mutation, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates substitutions of Q347R, D399V, and F405T, and the antibody Fc polypeptide linked to the HER2-binding scFv incorporates the matching substitutions K360E and K409W to form heterodimers. In certain embodiments, a TriNKET of the disclosure is identical to one of the exemplary TriNKETs described above that includes a KiH Fc mutation, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment incorporates "hole" substitutions of T366S, L368A, and Y407V, and the antibody Fc polypeptide linked to the HER2-binding scFv incorporates the "knob" substitution of T366W to form heterodimers.

[0223] In certain embodiments, a TriNKET of the present disclosure is identical to one of the exemplary TriNKETs described above, except that the antibody Fc polypeptide linked to the NKG2D-binding Fab fragment contains a S354C substitution in the CH3 domain and the antibody Fc polypeptide linked to the HER2-binding scFv contains a matching Y349C substitution in the CH3 domain to form a disulfide bond.

[0224] As described in International Patent Application No. PCT / US2019 / 045561, the multispecific binding proteins disclosed herein are effective in reducing tumor growth and killing cancer cells in in vitro assays and animal models. For example, A49-F3'-TriNKET-trastuzumab is superior to trastuzumab in inducing NK cell-mediated cytotoxicity against various human cancer cell lines, such as 786-O cells expressing low levels of HER2 (HER2+), H661 cells expressing intermediate levels of HER2 (HER2++), and SkBr3 cells expressing high levels of HER2 (HER2+++). Furthermore, the multispecific binding proteins do not significantly induce NK-mediated killing of healthy non-cancer human cells (e.g., human cardiomyocytes).

[0225] Specific embodiments of multispecific binding proteins useful in any of the methods of treatment and formulations disclosed herein are described below.

[0226] In some embodiments, the first antigen binding site that binds to NKG2D comprises a heavy chain variable domain (VH) with complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) having the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively, and a light chain variable domain (VL) with CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In some embodiments, the first antigen-binding site that binds NKG2D comprises a VH with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 94, and a VL with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the first antigen-binding site that binds NKG2D is a Fab.

[0227] In some embodiments, the second antigen binding site that binds HER2 comprises a VH with CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 115, 116, 117, respectively; and a VL with CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively. In some embodiments, the second antigen binding site that binds HER2 comprises a VH with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 195, and a VL with an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 196. In some embodiments, the second antigen-binding site that binds to HER2 is a single chain variable fragment (scFv). In some embodiments, the VL of the scFv is linked to the VH of the scFv via a flexible linker. In some embodiments, the flexible linker has the amino acid sequence of SEQ ID NO: 143. In some embodiments, the VL of the scFv is positioned at the N-terminus of the VH of the scFv. In some embodiments, the VH of the scFv forms a disulfide bridge with the VL of the scFv (e.g., between residues C44 of the VH of the scFv and C100 of the VL of the scFv). In some embodiments, the scFv has the amino acid sequence of SEQ ID NO: 139.

[0228] In some embodiments, the antibody Fc domain comprises a first antibody Fc sequence linked to a Fab that binds NKG2D and a second antibody Fc sequence linked to an scFv that binds HER2. In some embodiments, the first antibody Fc sequence is linked to a heavy chain portion of the Fab. In some embodiments, the scFv is linked to the second antibody Fc sequence via a hinge that comprises Ala-Ser. In some embodiments, the first and second antibody Fc sequences each comprise a hinge and a CH2 domain of a human IgG1 antibody. In some embodiments, the first and second antibody Fc sequences each comprise an amino acid sequence that is at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody. In some embodiments, the first and second antibody Fc sequences comprise different mutations that promote heterodimerization. In some embodiments, the first antibody Fc sequence is a human IgG1 Fc sequence that comprises a K360E substitution and a K409W substitution. In some embodiments, the second antibody Fc sequence is a human IgG1 Fc sequence comprising a Q347R substitution, a D399V substitution, and a F405T substitution.

[0229] In some embodiments, the multispecific binding protein comprises a first polypeptide, a second polypeptide, and a third polypeptide having the amino acid sequences of SEQ ID NO:141, SEQ ID NO:140, and SEQ ID NO:142, respectively.

[0230] Production of multispecific binding proteins. The multispecific binding protein described above can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector; and the first, second, and third expression vectors can be stably transfected together into a host cell to produce a multimeric protein.

[0231] One of skill in the art will understand that during production and / or storage of a protein, the N-terminal glutamic acid (E) or glutamine (Q) can cyclize to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Thus, in some embodiments in which the N-terminal residue of a polypeptide's amino acid sequence is E or Q, the corresponding amino acid sequence with E or Q replaced with pyroglutamic acid is also contemplated herein.

[0232] One of skill in the art will also appreciate that during production and / or storage of a protein, the C-terminal lysine (K) of the protein can be removed (e.g., spontaneously or catalyzed by enzymes present during production and / or storage). Such removal of K is often observed in proteins that contain an Fc domain at their C-terminus. Thus, in some embodiments in which the C-terminal residue of an amino acid sequence of a polypeptide (e.g., an antibody Fc polypeptide) is a K, the corresponding amino acid sequence with the K removed is also contemplated herein.

[0233] To achieve the highest yield of multispecific binding protein, different ratios of the first, second, and third expression vectors can be explored to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

[0234] The clones can be cultured under appropriate conditions for bioreactor scale-up to maintain expression of the multispecific binding protein. The multispecific binding protein can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0235] Pharmaceutical preparations The present disclosure also provides pharmaceutical compositions and formulations comprising the multispecific binding proteins disclosed herein (e.g., A49-F3'-TriNKET-Trastuzumab) at a concentration of greater than 50 mg / mL. The pharmaceutical formulations include one or more excipients and are maintained at a particular pH. The term "excipient" as used herein means any non-therapeutic agent added to a formulation to provide a desired physical or chemical property, such as pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration.

[0236] Excipients and pH One or more excipients in the pharmaceutical composition or pharmaceutical preparation of the present invention include a buffer. The term "buffer" as used herein refers to one or more components that, when added to an aqueous solution, can protect the solution against changes in pH when acid or alkali is added, or when diluted with a solvent. In addition to phosphate buffers, glycinate, carbonate, citrate, histidine buffers, and the like can be used, in which case sodium, potassium, or ammonium ions can function as counterions.

[0237] In certain embodiments, the buffer or buffer system comprises at least one buffer having a buffer range that overlaps completely or partially with the pH range of 5.5-7.4. In certain embodiments, the buffer has a pKa of about 6.0±0.5. In certain embodiments, the buffer comprises a histidine buffer. In certain embodiments, histidine is present at a concentration of 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 5-50 mM, 10-50 mM, 15-100 mM, 20-100 mM, 5-25 mM, 10-25 mM, 15-25 mM, 20-25 mM, 5-20 mM, 10-20 mM, or 15-20 mM. In certain embodiments, histidine is present at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 50 mM. In certain embodiments, histidine is present at a concentration of 20 mM.

[0238] The pharmaceutical compositions or pharmaceutical formulations disclosed herein may have a pH of 5.5 to 6.5. For example, in certain embodiments, the pharmaceutical compositions or pharmaceutical formulations have a pH of 5.5 to 6.5 (i.e., 6.0±0.5), 5.6 to 6.4 (i.e., 6.0±0.4), 5.7 to 6.3 (i.e., 6.0±0.3), 5.8 to 6.2 (i.e., 6.0±0.2), 5.9 to 6.1 (i.e., 6.0±0.1), or 5.95 to 6.05 (i.e., 6.0±0.05). In certain embodiments, the pharmaceutical compositions or pharmaceutical formulations have a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In certain embodiments, the pharmaceutical compositions or pharmaceutical formulations have a pH of 6.0. Under scientific rounding rules, pH values ​​greater than or equal to 5.95 and less than or equal to 6.05 are rounded to 6.0.

[0239] In certain embodiments, the buffer system of the pharmaceutical composition or formulation comprises histidine at 10-25 mM and at a pH of 6.0±0.2. In certain embodiments, the buffer system of the pharmaceutical composition or formulation comprises histidine at 20 mM and at a pH of 6.0±0.2. In certain embodiments, the buffer system of the pharmaceutical composition or formulation comprises histidine at 10-25 mM and at a pH of 6.0±0.05. In certain embodiments, the buffer system of the pharmaceutical composition or formulation comprises histidine at 20 mM and at a pH of 6.0±0.05.

[0240] One or more excipients in the pharmaceutical compositions or formulations disclosed herein may further comprise a sugar or sugar alcohol. Sugars and sugar alcohols are useful in pharmaceutical compositions or formulations as heat stabilizers. In certain embodiments, the pharmaceutical composition or formulation comprises a sugar, e.g., a monosaccharide (e.g., glucose, xylose, or erythritol), a disaccharide (e.g., sucrose, trehalose, maltose, or galactose), or an oligosaccharide (e.g., stachyose). In certain embodiments, the pharmaceutical composition or formulation comprises sucrose. In certain embodiments, the pharmaceutical composition comprises a sugar alcohol, e.g., a sugar alcohol derived from a monosaccharide (e.g., mannitol, sorbitol, or xylitol), a sugar alcohol derived from a disaccharide (e.g., lactitol or maltitol), or a sugar alcohol derived from an oligosaccharide. In certain embodiments, the pharmaceutical composition or formulation comprises sorbitol.

[0241] The amount of sugar or sugar alcohol included in the formulation may vary depending on the particular context and intended purpose in which the formulation is used. In certain embodiments, the pharmaceutical composition or formulation comprises 50-300 mM, 50-250 mM, 100-300 mM, 100-250 mM, 150-300 mM, 150-250 mM, 200-300 mM, 200-250 mM, or 250-300 mM sugar or sugar alcohol. In certain embodiments, the pharmaceutical composition or formulation comprises 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 250 mM, or 300 mM sugar or sugar alcohol. In certain embodiments, the pharmaceutical composition or formulation comprises 250 mM sugar or sugar alcohol (e.g., sucrose or sorbitol).

[0242] The one or more excipients in the pharmaceutical compositions or formulations disclosed herein further comprise a surfactant. The term "surfactant", as used herein, refers to a surface-active molecule that contains both a hydrophobic portion (e.g., an alkyl chain) and a hydrophilic portion (e.g., a carboxyl group and a carboxylic acid group). Surfactants are useful in pharmaceutical compositions or formulations to reduce aggregation of therapeutic proteins. Surfactants suitable for use in pharmaceutical compositions or formulations are generally non-ionic surfactants, including, but not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; Triton; sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetadine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; ly ... The surfactants include noreyl-, myristyl-, or cetyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl-methyl taurate, or disodium oleyl methyl taurate; and MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropyl glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). In certain embodiments, the surfactant is a polysorbate. In certain embodiments, the surfactant is polysorbate 80.

[0243] The amount of non-ionic surfactant included within a pharmaceutical composition or formulation of the present invention can vary depending on the particular properties desired of the formulation, as well as the particular context and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical composition or formulation comprises 0.005%-0.5%, 0.005%-0.2%, 0.005%-0.1%, 0.005%-0.05%, 0.005%-0.02%, 0.005%-0.01%, 0.01%-0.5%, 0.01%-0.2%, 0.01%-0.1%, 0.01%-0.05%, or 0.01%-0.02% of a non-ionic surfactant (e.g., polysorbate 80). In certain embodiments, the pharmaceutical composition or formulation comprises 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% of a non-ionic surfactant, e.g., polysorbate 80. Concentrations of non-ionic surfactant are provided as % (w / v) values.

[0244] In certain embodiments, the pharmaceutical composition or formulation is isotonic. An "isotonic" formulation is a formulation that has essentially the same osmotic pressure as human blood. An isotonic formulation generally has an osmotic pressure of about 250-350 mOsmol / kgH2O. Isotonicity can be measured using a vapor pressure or ice-cooled osmometer. In certain embodiments, the osmotic pressure of the pharmaceutical composition or formulation is 250-350 mOsmol / kgH2O. In certain embodiments, the osmotic pressure of the pharmaceutical composition or formulation is 300-350 mOsmol / kgH2O.

[0245] Substances such as sugars, sugar alcohols, and NaCl can be included in pharmaceutical compositions or pharmaceutical preparations for desired osmolality.In certain embodiments, the concentration of NaCl in pharmaceutical compositions or pharmaceutical preparations, if present, is equal to or less than 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, 0.1 mM, 50 μM, 10 μM, 5 μM, or 1 μM.In certain embodiments, the concentration of NaCl in pharmaceutical compositions or pharmaceutical preparations is below the detection limit.In certain embodiments, NaCl is not added when preparing pharmaceutical compositions or pharmaceutical preparations.

[0246] The pharmaceutical compositions or formulations disclosed herein may further include one or more other substances, such as bulking agents or preservatives. Bulking agents are compounds that add mass to the lyophilization mixture and contribute to the physical structure of the lyophilized cake (e.g., promote the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may include such bulking agents. Preservatives may reduce bacterial activity and, for example, facilitate the production of multi-use (multi-dose) formulations.

[0247] The multispecific binding protein may be formulated in a pharmaceutical composition or formulation at a high concentration, for example, greater than 50 mg / mL. In certain embodiments, the pharmaceutical composition or formulation comprises greater than or equal to 60 mg / mL, greater than or equal to 70 mg / mL, greater than or equal to 80 mg / mL, greater than or equal to 90 mg / mL, greater than or equal to 100 mg / mL, greater than or equal to 125 mg / mL, greater than or equal to 150 mg / mL, greater than or equal to 175 mg / mL, or greater than or equal to 200 mg / mL of multispecific binding protein.In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is 60-250 mg / mL, 60-225 mg / mL, 60-200 mg / mL, 60-175 mg / mL, 50-150 mg / mL, 60-150 mg / mL, 60-125 mg / mL, 60-100 mg / mL, 60-90 mg / mL, 60-80 mg / mL, 60-70 mg / mL, 70-250 mg / mL, 70-225 mg / mL, 70-200 mg / mL , 70~175mg / mL, 70~150mg / mL, 70~150mg / mL, 70~125mg / mL, 70~100mg / mL, 70~90mg / mL, 70~80mg / mL, 80~250mg / mL , 80~225mg / mL, 80~200mg / mL, 80~175mg / mL, 80~150mg / mL, 80~150mg / mL, 80~125mg / mL, 80~100mg / mL, 80~90mg / m L, 90~250mg / mL, 90~225mg / mL, 90~200mg / mL, 90~175mg / mL, 90~150mg / mL, 90~150mg / mL, 90~125mg / mL, 90~100m g / mL, 100~250mg / mL, 100~225mg / mL, 100~200mg / mL, 100~175mg / mL, 100~150mg / mL, 100~125mg / mL, 125~250mg / m In certain embodiments, the pharmaceutical composition or formulation comprises 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, or 220 mg / mL of the multispecific binding protein. Exemplary Formulations

[0248] In certain embodiments, a pharmaceutical composition or formulation of the invention comprises a multispecific binding protein, histidine, a sugar or sugar alcohol (e.g., sucrose or sorbitol), and a polysorbate (e.g., polysorbate 80) at a pH of 5.5 to 6.5.

[0249] In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 10-25 mM histidine, 200-300 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.005%-0.05% of a polysorbate (e.g., polysorbate 80) at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM of a sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% of a polysorbate (e.g., polysorbate 80) at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% polysorbate (e.g., polysorbate 80) at a pH of 5.8-6.2. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.01% polysorbate (e.g., polysorbate 80) at a pH of 5.95-6.05.

[0250] In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 10-25 mM histidine, 200-300 mM sucrose, and 0.005%-0.05% polysorbate 80 at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sucrose, and 0.01% polysorbate 80 at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sucrose, and 0.01% polysorbate 80 at a pH of 5.8-6.2. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of multispecific binding protein, 20 mM histidine, 250 mM sucrose, and 0.01% polysorbate 80 at a pH of 5.95-6.05.

[0251] In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 10-25 mM histidine, 200-300 mM sorbitol, and 0.005%-0.05% polysorbate 80 at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sorbitol, and 0.01% polysorbate 80 at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sorbitol, and 0.01% polysorbate 80 at a pH of 5.8-6.2. In certain embodiments, the pharmaceutical composition or formulation comprises 10-250 mg / mL of multispecific binding protein, 20 mM histidine, 250 mM sorbitol, and 0.01% polysorbate 80 at a pH of 5.95-6.05.

[0252] In certain embodiments, the pharmaceutical composition or formulation comprises 10-50 mg / mL of the multispecific binding protein, 5 mM-50 mM histidine, 50 mM-300 mM sucrose, and about 0.005%-0.05% (w / v) polysorbate 80 at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical composition or formulation comprises 10-25 mg / mL of the multispecific binding protein, 10 mM-25 mM histidine, 150 mM-300 mM sucrose, and about 0.005%-0.02% (w / v) polysorbate 80 at a pH of 5.8-6.2. In a particular embodiment, a pharmaceutical composition or formulation comprises about 15 mg / mL of multispecific binding protein, about 20 mM histidine, about 250 mM sucrose, and about 0.01% (w / v) polysorbate 80 at about pH 6.0.

[0253] Stability of multispecific binding proteins The pharmaceutical compositions or formulations disclosed herein exhibit a high level of stability. A pharmaceutical formulation is stable if the multispecific binding protein within the formulation retains an acceptable degree of physical properties, chemical structure, and / or biological function after storage under defined conditions.

[0254] Stability can be measured by determining the percentage of the multispecific binding protein in a formulation that remains in the native conformation after storage at a defined temperature for a defined time. The percentage of protein in the native conformation can be determined, for example, by size exclusion chromatography (e.g., size-exclusion high performance liquid chromatography), where the protein in the native conformation does not aggregate (elutes in the high molecular weight fraction) or degrade (elutes in the low molecular weight fraction). In certain embodiments, more than 95%, 96%, 97%, 98%, or 99% of the multispecific binding protein has the native conformation as determined by size exclusion chromatography after incubation at 4° C. for 3 weeks. In certain embodiments, more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the multispecific binding protein has the native conformation as determined by size exclusion chromatography after incubation at 50° C. for 3 weeks. In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multispecific binding protein forms high molecular weight complexes (i.e., having a higher molecular weight than the native protein) as determined by size exclusion chromatography after 3 weeks of incubation at 4° C. In certain embodiments, less than 1%, 2%, 3%, 4%, or 5% of the multispecific binding protein forms high molecular weight complexes (i.e., having a higher molecular weight than the native protein) as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multispecific binding protein is degraded (i.e., having a lower molecular weight than the native protein) as determined by size exclusion chromatography after 3 weeks of incubation at 4° C. In certain embodiments, less than 1%, 1.5%, 2%, 2.5%, or 3% of the multispecific binding protein is degraded (i.e., has a lower molecular weight than the native protein) after incubation at 50° C. for 3 weeks as determined by size exclusion chromatography.

[0255] Stability can also be measured by determining the percentage of the multispecific binding protein present in a more acidic fraction (acidic form) compared to the major fraction (mainly charged form) of the protein. Without intending to be bound by theory, deamidation of a protein can make it more negatively charged and thus more acidic compared to the non-deamidated protein (see, e.g., Robinson, Protein Deamidation, (2002) PNAS 99(8):5283-88). The percentage of the acidic form of the protein can be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography) or imaging capillary isoelectric focusing (icIEF). In certain embodiments, at least 50%, 60%, 70%, 80%, or 90% of the multispecific binding protein in the pharmaceutical composition or formulation is in the main charged form after incubation at 4° C. for 3 weeks. In certain embodiments, at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the multispecific binding protein in the pharmaceutical composition or formulation is in the predominantly charged form after incubation at 50° C. for 3 weeks. In certain embodiments, no more than 10%, 20%, 30%, 40%, or 50% of the multispecific binding protein in the pharmaceutical composition or formulation is in the acidic form after incubation at 4° C. for 3 weeks. In certain embodiments, no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 85% of the multispecific binding protein in the pharmaceutical composition or formulation is in the acidic form after incubation at 50° C. for 3 weeks.

[0256] Stability can also be measured by determining the purity of the multispecific binding protein by electrophoresis after denaturing the protein with sodium dodecyl sulfate (SDS). Protein samples can be denatured in the presence or absence of an agent that reduces protein disulfide bonds (e.g., β-mercaptoethanol). In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical composition or formulation is at least 95%, 96%, 97%, 98%, or 99% after incubation for 3 weeks at 4° C. as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol). In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical composition or formulation, as measured by capillary electrophoresis after denaturing a protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol), is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after three weeks of incubation at 50° C. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical composition or formulation, as measured by capillary electrophoresis after denaturing a protein sample under non-reducing conditions, is at least 95%, 96%, 97%, 98%, or 99% after three weeks of incubation at 4° C. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical composition or formulation is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after incubation for 3 weeks at 50° C. as measured by capillary electrophoresis after denaturing a protein sample under non-reducing conditions.

[0257] Stability can also be measured by determining the parameters of the protein solution by dynamic light scattering. The Z-average and polydispersity index (PDI) values ​​indicate the average diameter of the particles in the solution, and these measurements increase if aggregates are present in the solution. The monomer %Pd value indicates the spread of different monomers detected, where lower values ​​indicate a monodisperse solution, which is preferred. The monomer size detected by DLS confirms that the main population is monomeric and is useful in characterizing any higher order aggregates that may be present. In certain embodiments, the Z-average of the pharmaceutical composition or pharmaceutical formulation does not increase by more than 5%, 10%, or 15% after incubation at 4° C. for 3 weeks. In certain embodiments, the Z-average of the pharmaceutical composition or pharmaceutical formulation does not increase by more than 5%, 10%, 15%, 20%, or 25% after incubation at 50° C. for 3 weeks. In certain embodiments, the PDI value of the pharmaceutical composition or formulation does not increase by more than 10%, 20%, 30%, 40%, or 50% after incubation for 3 weeks at 4° C. In certain embodiments, the PDI value of the pharmaceutical composition or formulation does not increase by more than 2-fold, 3-fold, 4-fold, or 5-fold after incubation for 3 weeks at 50° C.

[0258] Exemplary methods for determining the stability of a multispecific binding protein in a pharmaceutical composition or formulation are described in Example 1 of the present disclosure. Additionally, protein stability can be assessed by measuring the binding affinity of the multispecific binding protein to its target, or the biological activity of the multispecific binding protein in certain in vitro assays, such as the NK cell activation assay and cytotoxicity assays described in WO2018 / 152518.

[0259] Dosage Form The pharmaceutical composition or formulation can be prepared and stored as a liquid formulation or a lyophilized form. In certain embodiments, the pharmaceutical composition or formulation is a liquid formulation for storage at 2-8°C (e.g., 4°C) or a frozen formulation for storage at -20°C or below. The sugar or sugar alcohol in the formulation is used as a cryoprotectant.

[0260] Prior to pharmaceutical use, the pharmaceutical composition or pharmaceutical formulation can be diluted or reconstituted in an aqueous carrier suitable for the route of administration. Other exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. For example, when prepared for intravenous administration, the pharmaceutical composition or pharmaceutical formulation can be diluted in 0.9% sodium chloride (NaCl) solution, or 0.9% NaCl solution and 0.01% polysorbate 80. In certain embodiments, the diluted pharmaceutical composition or pharmaceutical formulation is isotonic and suitable for administration by intravenous infusion.

[0261] The pharmaceutical composition or pharmaceutical preparation contains the multispecific binding protein at a concentration appropriate for storage. In certain embodiments, the pharmaceutical composition or pharmaceutical preparation contains the multispecific binding protein at a concentration of 10-50 mg / mL, 10-40 mg / mL, 10-30 mg / mL, 10-25 mg / mL, 10-20 mg / mL, 10-15 mg / mL, 15-50 mg / mL, 15-40 mg / mL, 15-30 mg / mL, 15-25 mg / mL, 15-20 mg / mL, 20-50 mg / mL, 20-40 mg / mL, 20-30 mg / mL, 20-25 mg / mL, 30-50 mg / mL, 30-40 mg / mL, or 40-50 mg / mL. In certain embodiments, the pharmaceutical composition or formulation comprises the multispecific binding protein at a concentration of 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0262] In certain embodiments, the pharmaceutical composition or formulation is packaged in a container (e.g., a vial, bag, pen, or syringe). In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the amount of multispecific binding protein in the container is suitable for administration as a single dose. In certain embodiments, the amount of multispecific binding protein in the container is suitable for administration in multiple doses. In certain embodiments, the pharmaceutical composition or formulation comprises 0.1-2000 mg of multispecific binding protein. In certain embodiments, the pharmaceutical composition or pharmaceutical formulation has a concentration of 1-2000 mg, 10-2000 mg, 20-2000 mg, 50-2000 mg, 100-2000 mg, 200-2000 mg, 500-2000 mg, 1000-2000 mg, 0.1-1000 mg, 1-1000 mg, 10-1000 mg, 20-1000 mg, 50-1000 mg, 100-1000 mg, 200-1000 mg, 500-1000 mg, 0.1-500 mg, 1-500 mg, 10-500 mg, 20-500 mg , 50-500mg, 100-500mg, 200-500mg, 0.1-200mg, 1-200mg, 10-200mg, 20-200mg, 50-200mg, 100-200mg, 0.1-100mg, 1-100mg, 10-100mg, 20-100mg, 50-100mg, 0.1-50mg, 1-50mg, 10-50mg, 20-50mg, 0.1-20mg, 1-20mg, 10-20mg, 0.1-10mg, 1-10mg, or 0.1-1mg of multispecific binding protein. In certain embodiments, the pharmaceutical composition or formulation comprises 0.1 mg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, or 2000 mg of the multispecific binding protein.

[0263] Therapeutic Use Use with corticosteroids In one aspect, the disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific binding protein, pharmaceutical composition, or formulation disclosed herein (administered as monotherapy or in a combination therapy) and a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation. The disclosure also provides a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (administered as monotherapy or in a combination therapy) for use in a method of treating cancer in combination with a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation. In combination treatments, the multispecific binding proteins, pharmaceutical compositions, or pharmaceutical formulations disclosed herein can be combined with immunotherapy, such as anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab), and / or chemotherapy, such as cytoskeleton-disrupting chemotherapeutic agents (e.g., paclitaxel, nab-paclitaxel, docetaxel).

[0264] Corticosteroids useful in the present invention generally include steroids produced by the adrenal cortex, such as glucocorticoids and mineralocorticoids, as well as synthetic analogs and derivatives of natural corticosteroids that have anti-inflammatory activity. In certain embodiments, the corticosteroid is a glucocorticoid. Glucocorticoids bind to the glucocorticoid receptor and reduce inflammation by inhibiting the immune response. In certain embodiments, the corticosteroid is a mineralocorticoid. Mineralocorticoids bind to the mineralocorticoid receptor and reduce serum Na + / K +They act to regulate the concentration of erythrocytes. Some corticosteroids may have both glucocorticoid and mineralocorticoid functions. Examples of corticosteroids are disclosed in U.S. Patent No. 10,799,599. In certain embodiments, the corticosteroid used in the methods disclosed herein is selected from the group consisting of methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, halcinonide, icomethasone, meprednisone, mometasone, rofleponide, RPR 106541, and their respective pharma- ceutical acceptable derivatives, such as beclomethasone dipropionate (anhydrous or monohydrate), beclomethasone monopropionate, dexamethasone 21-iso- nicotinate, fluticasone propionate, icomethasone embutate, tixocortol 21-pivalate, and triamcinolone acetonide, and their pharma-ceutical acceptable salts and / or derivatives.

[0265] In certain embodiments, the glucocorticoid is methylprednisolone. Exemplary effective amounts of methylprednisolone include 8-200 mg, 20-200 mg, 25-200 mg, 50-200 mg, 75-200 mg, 100-200 mg, 125-200 mg, 150-200 mg, 175-200 mg, 25-175 mg, 50-175 mg, 75-175 mg, 100-175 mg, 125-175 mg, 150-175 mg, 20-150 mg, 25-150 mg, 50 ... The effective amount of methylprednisolone may be in the range of 0 mg, 75-150 mg, 100-150 mg, 125-150 mg, 25-125 mg, 50-125 mg, 75-125 mg, 100-125 mg, 25-100 mg, 50-100 mg, 75-100 mg, 25-75 mg, 50-75 mg, 25-50 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg. In certain embodiments, the effective amount of methylprednisolone by oral administration is about 125 mg. In certain embodiments, the effective amount of methylprednisolone by oral administration is 8 mg, 16 mg, 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg.

[0266] In certain embodiments, the glucocorticoid is dexamethasone. Exemplary effective amounts of dexamethasone may be in the range of 8-200 mg, 20-200 mg, 50-200 mg, 100-200 mg, 20-150 mg, 50-150 mg, 50-100 mg, or 100-150 mg. In certain embodiments, an effective amount of dexamethasone administered intravenously is 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, or 150 mg. In certain embodiments, an effective amount of dexamethasone administered orally is 8 mg, 16 mg, 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg.

[0267] In certain embodiments, the corticosteroid is administered parenterally. In certain embodiments, the corticosteroid is administered intravenously. In certain embodiments, the corticosteroid is administered orally.

[0268] The corticosteroid can be administered prior to, simultaneously with, or after administration of the multispecific binding protein. In certain embodiments, the corticosteroid is administered within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or immediately prior (e.g., prior to the start of administration of the multispecific binding protein) of administration of the multispecific binding protein. In certain embodiments, the corticosteroid is administered within 1 hour (e.g., prior to the start of administration of the multispecific binding protein) of administration of the multispecific binding protein. In certain embodiments, the corticosteroid is administered simultaneously with administration of the multispecific binding protein. In certain embodiments, the corticosteroid and the multispecific binding protein are diluted in a single pharmaceutical composition that is administered to the subject. In certain embodiments, the period of administration of the corticosteroid and the period of administration of the multispecific binding protein overlap, either completely or partially. In certain embodiments, the corticosteroid is administered within 2 hours, 1 hour, or 30 minutes after administration of the multispecific binding protein (eg, after the start of administration of the multispecific binding protein).

[0269] In certain embodiments, the corticosteroid is administered on day 1 of the first cycle (i.e., in combination with the first dose of the multispecific binding protein). In certain embodiments, the corticosteroid is administered only on day 1 of the first cycle (i.e., in combination with the first dose of the multispecific binding protein). In certain embodiments, the corticosteroid is further administered if the infusion-related reaction persists or recurs. In certain embodiments, the infusion-related reaction comprises a persistent rash, diarrhea, colitis, autoimmune hepatitis, arthritis, glomerulonephritis, cardiomyopathy, or uveitis, or another inflammatory eye condition.

[0270] In certain embodiments, a corticosteroid (e.g., methylprednisolone) is administered 30-90 minutes, 40-90 minutes, 50-90 minutes, 60-90 minutes, 70-90 minutes, 80-90 minutes, 30-80 minutes, 40-80 minutes, 50-80 minutes, 60-80 minutes, 70-80 minutes, 30-70 minutes, 40-70 minutes, 50-70 minutes, 60-70 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 30-50 minutes, 40-50 minutes, 30-40 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes prior to administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein.

[0271] In certain embodiments, the subject receives a premedication regimen comprising about 125 mg of methylprednisolone administered intravenously within 60 minutes of administration of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the premedication regimen further comprises 40-50 mg of diphenhydramine and 800-1000 mg of acetaminophen administered intravenously or orally 30-60 minutes prior to administration of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, about 1.6, 5.2, 10, 15, or 20 mg / kg of a multispecific binding protein (e.g., A49-F3'-TriNKET-trastuzumab) is administered to the subject following the premedication regimen.

[0272] Exemplary infusion-related reactions to the multispecific binding proteins disclosed herein include cytokine release syndrome, anaphylaxis, chills, fever / fever, hypotension, hypertension, rigors, headache, dizziness, pruritus, sore throat, pharyngeal edema, angioedema, redness / flushing, rash / hives, bronchospasm, tachycardia, bradycardia, atrial fibrillation, hypoxia, rapid breathing / dyspnea / shortness of breath / choking, chest tightness, nausea, vomiting, pain (e.g., chest pain, back pain), shivering, tremors, myalgia, fatigue, insomnia, asthenia, hypersensitivity, and diarrhea. Clinical manifestations of cytokine release syndrome are described in Shimabukuro-Vornhagen et al. and include, but are not limited to, fever (e.g., hyperthermia), fatigue, headache, rash, arthralgia, myalgia, hypotension, circulatory shock requiring vascular depression, vascular leakage, disseminated intravascular coagulation, and multiple organ system failure. In certain embodiments, co-administration of a corticosteroid reduces one or more infusion-related reactions in a subject.

[0273] Use in combination with antihistamines An antihistamine can be used to avoid or reduce an allergic reaction (e.g., anaphylaxis) to the multispecific binding protein. Thus, in certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of an antihistamine. Exemplary antihistamines are disclosed in U.S. Pat. No. 10,898,693. In certain embodiments, the antihistamine used in the methods disclosed herein is selected from crivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, cyclizine, chlorpheniramine, chlorodiphenhydramine, clemastine, cromolyn, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimethindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, nedocromil, olopatadine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, rupatadine, tripelennamine, triprolidine, and combinations thereof. In certain embodiments, the antihistamine is diphenhydramine. In certain embodiments, the therapeutically effective amount of diphenhydramine is 10-100 mg, 20-100 mg, 30-100 mg, 40-100 mg, 50-100 mg, 60-100 mg, 70-100 mg, 80-100 mg, 90-100 mg, 10-90 mg, 20-90 mg, 30-90 mg, 40-90 mg, 50-90 mg, 60-90 mg, 70-90 mg, 80-90 mg, 10-80 mg, 20-80 mg, 30-80 mg, 40-80 mg, 50-80 mg, 60-80 mg, 70-80 mg, 80-90 mg, 10-80 mg, 20-80 mg, 30-80 mg, 40-80 mg, 50-80 mg, 60-80 mg, 70-80 mg, 80-80 mg, 80-80 mg, 90-100 mg, 1 ... 0 mg, 70 to 80 mg, 10 to 70 mg, 20 to 70 mg, 30 to 70 mg, 40 to 70 mg, 50 to 70 mg, 60 to 70 mg, 10 to 60 mg, 20 to 60 mg, 30 to 60 mg, 40 to 60 mg, 50 to 60 mg, 10 to 50 mg, 20 to 50 mg, 30 to 50 mg, 40 to 50 mg, 20 to 40 mg, 30 to 40 mg, 20 to 30 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg.In certain embodiments, the therapeutically effective amount of diphenhydramine is 40-50 mg.

[0274] In certain embodiments, the antihistamine is administered parenterally. In certain embodiments, the antihistamine is administered intravenously. In certain embodiments, the antihistamine is administered orally.

[0275] The antihistamine can be administered prior to, simultaneously with, or after administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the antihistamine is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., prior to the start of administration of the multispecific binding protein). In certain embodiments, the antihistamine is administered with every dose of the multispecific binding protein.

[0276] In certain embodiments, an antihistamine (e.g., diphenhydramine) is administered 30-90 minutes, 40-90 minutes, 50-90 minutes, 60-90 minutes, 70-90 minutes, 80-90 minutes, 30-80 minutes, 40-80 minutes, 50-80 minutes, 60-80 minutes, 70-80 minutes, 30-70 minutes, 40-70 minutes, 50-70 minutes, 60-70 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 30-50 minutes, 40-50 minutes, 30-40 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes prior to administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein.

[0277] In certain embodiments, the subject is premedicated with 40-50 mg of diphenhydramine administered intravenously or orally 30-60 minutes prior to administration of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the premedication further comprises 800-100 mg of acetaminophen administered intravenously or orally prior to administration of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein.

[0278] When the methods of treatment disclosed herein include multiple doses (e.g., five or more doses) of a multispecific binding protein, in certain embodiments, an antihistamine is administered with the first dose, the first two doses, the first three doses, the first four doses, or the first five doses of the multispecific binding protein.

[0279] Use in combination with analgesics and antipyretics Analgesics can be used to alleviate pain as a result of administration of the multispecific binding proteins, pharmaceutical compositions, or pharmaceutical formulations disclosed herein, whether administered as monotherapy or in combination with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab) or a cytoskeleton-disrupting chemotherapeutic agent. Thus, in certain embodiments, the method further comprises administering a therapeutically effective amount of an analgesic to the subject. Exemplary analgesics are disclosed in U.S. Patent Application Publication No. 2015 / 0342989 and U.S. Patent No. 10,899,834.In certain embodiments, the analgesic used in the methods disclosed herein is acetaminophen, salicylamide, salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate / benorilatem and amoxiprine, acetylsalicylic acid, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexbuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen In one embodiment, the active ingredient is selected from the group consisting of morphine, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, etoricoxib, codeine, dihydrocodeine, morphine or a morphine derivative or a pharma- ceutically acceptable salt thereof, diacetylmorphine, hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene, and pentazocine, and pharma-ceutically acceptable salts thereof. In certain embodiments, the analgesic is acetaminophen.In certain embodiments, the therapeutically effective amount of acetaminophen is in the range of 325-1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000 mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg.

[0280] In certain embodiments, the analgesic is administered parenterally. In certain embodiments, the analgesic is administered intravenously. In certain embodiments, the analgesic is administered orally.

[0281] The analgesic may be administered prior to, simultaneously with, or after administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the analgesic is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to administration of the multispecific binding protein (e.g., prior to the start of administration of the multispecific binding protein). In certain embodiments, the analgesic is administered simultaneously with administration of the multispecific binding protein. In certain embodiments, the analgesic and the multispecific binding protein are diluted in a single pharmaceutical composition that is administered to the subject. In certain embodiments, the analgesic (e.g., acetaminophen) is administered with every dose of the multispecific binding protein.

[0282] In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30-90 minutes, 40-90 minutes, 50-90 minutes, 60-90 minutes, 70-90 minutes, 80-90 minutes, 30-80 minutes, 40-80 minutes, 50-80 minutes, 60-80 minutes, 70-80 minutes, 30-70 minutes, 40-70 minutes, 50-70 minutes, 60-70 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 30-50 minutes, 40-50 minutes, 30-40 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes prior to administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein.

[0283] In certain embodiments, the administration period of the analgesic agent and the administration period of the multispecific binding protein overlap completely or partially, hi certain embodiments, the analgesic agent is administered within 2 hours, 1 hour, or 30 minutes following administration of the multispecific binding protein (e.g., following the initiation of administration of the multispecific binding protein).

[0284] An antipyretic agent can be used to prevent or reduce fever as a result of administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein, whether administered as a monotherapy or as a combination treatment with an anti-PD-1 antibody (e.g., nivolumab or pembrolizumab) or a cytoskeleton-disrupting chemotherapeutic agent. Thus, in certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of an antipyretic agent. Exemplary antipyretic agents are disclosed in U.S. Patent Application Publication No. 2015 / 0342989. In certain embodiments, the antipyretic used in the methods disclosed herein is acetaminophen, salicylamide, salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate / benorilatem and amoxiprine, acetylsalicylic acid, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexiketoprofen, furunil ... The antipyretic is selected from embufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, and etoricoxib. In certain embodiments, the antipyretic is acetaminophen.In certain embodiments, the therapeutically effective amount of acetaminophen is in the range of 325-1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000 mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg.

[0285] In certain embodiments, the antipyretic is administered parenterally. In certain embodiments, the antipyretic is administered intravenously. In certain embodiments, the antipyretic is administered orally.

[0286] The antipyretic drug can be administered prior to, simultaneously with, or after administration of the multispecific binding protein. In certain embodiments, the antipyretic drug is administered within 2 hours, 1.5 hours, 1 hour (60 minutes), 45 minutes, 30 minutes, or 15 minutes prior to administration of the multispecific binding protein (e.g., prior to the initiation of administration of the multispecific binding protein). In certain embodiments, the antipyretic drug is administered simultaneously with administration of the multispecific binding protein. In certain embodiments, the antipyretic drug and the multispecific binding protein are diluted in a single pharmaceutical composition that is administered to the subject. In certain embodiments, the period of administration of the antipyretic drug and the period of administration of the multispecific binding protein overlap completely or partially. In certain embodiments, the antipyretic drug is administered within 2 hours, 1 hour, or 30 minutes following administration of the multispecific binding protein (e.g., following the initiation of administration of the multispecific binding protein). In certain embodiments, the antipyretic drug (e.g., acetaminophen) is administered with every dose of the multispecific binding protein.

[0287] The antipyretic agent can be administered prior to, simultaneously with, or following administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein. In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30-90 minutes, 40-90 minutes, 50-90 minutes, 60-90 minutes, 70-90 minutes, 80-90 minutes, 30-80 minutes, 40-80 minutes, 50-80 minutes, 60-80 minutes, 70-80 minutes, 30-70 minutes, 40-70 minutes, 50-70 minutes, 60-70 minutes, 30-60 minutes, 40-60 minutes, 50-60 minutes, 30-50 minutes, 40-50 minutes, 30-40 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes prior to administration of a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein.

[0288] In certain embodiments, the subject is premedicated with 800-1000 mg of acetaminophen administered intravenously or orally 30-60 minutes prior to administration of the disclosed multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation. In certain embodiments, the premedication further comprises 40-50 mg of diphenhydramine administered intravenously or orally prior to administration of the disclosed multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation.

[0289] Medication Regimen In certain embodiments, the method comprises administering to a subject in need thereof a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3'-TriNKET-Trastuzumab) on days 1, 8, and 15 of an initial four-week treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered to the subject only on these three days of the initial four-week treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is not administered to the subject on day 22. This regimen is a dose escalation schedule that is designed to reach maximum saturation of the target as early as possible during the course of treatment while minimizing the infusion burden on the patient.

[0290] In certain embodiments, the method further comprises administering the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation to the subject on days 1 and 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered to the subject on only these two days in each subsequent four-week treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is not administered to the subject on days 8 or 22. Subsequent treatment cycles in which the subject receives the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation once every two weeks are scheduled to maintain a particular level of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation in the subject. In certain embodiments, the subject receives at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 subsequent treatment cycles. In certain embodiments, the subject receives the subsequent treatment cycles until regression of the cancer.

[0291] In certain embodiments, the methods comprise administering a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3'-TriNKET-trastuzumab) as a monotherapy to a subject in need thereof. In certain embodiments, the methods comprise administering a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3'-TriNKET-trastuzumab) intravenously as a one-hour infusion in a four-week treatment cycle.

[0292] In certain embodiments, the method includes administering a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein (e.g., A49-F3'-TriNKET-trastuzumab) for 45-75 minutes, 50-75 minutes, 55-75 minutes, 60-75 minutes, 65-75 minutes, 70-75 minutes, 45-70 minutes, 50-70 minutes, 55-70 minutes, 60-70 minutes, 65-70 minutes, 45-65 minutes, 50-65 minutes, 55-65 minutes, 60-65 minutes, 45-60 minutes, 50-60 minutes, 55-60 minutes, 45-135 minutes, 60-135 minutes, 75-135 minutes, to a subject in need thereof over a period of about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 135 minutes, about 45 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 90 minutes, about 105 minutes, or about 120 minutes.

[0293] In certain embodiments, one or more doses of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles comprise 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 0.1-0.5 mg / kg, 0.1-0.2 mg / kg, 0.2-20 mg / kg, 0.2-10 mg / kg, 0.2-5 mg / kg, 0.2-2 mg / kg, 0.2-1 mg / kg, 0.2-0.5 mg / kg, 0.5-20 mg / kg, 0.5-10 mg / kg, 0.5-5 mg / kg, 0.5-2 mg / kg, 0.5-1 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, or 1-2 mg / kg of the multispecific binding protein relative to the subject's body weight. In certain embodiments, one or more doses of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles comprise 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein relative to the subject's body weight.

[0294] In certain embodiments, each dose of the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles comprises 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 0.1-0.5 mg / kg, 0.1-0.2 mg / kg, 0.2-20 mg / kg, 0.2-10 mg / kg, 0.2-5 mg / kg, 0.2-2 mg / kg, 0.2-1 mg / kg, 0.2-0.5 mg / kg, 0.5-20 mg / kg, 0.5-10 mg / kg, 0.5-5 mg / kg, 0.5-2 mg / kg, 0.5-1 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, or 1-2 mg / kg of the multispecific binding protein relative to the subject's body weight. In certain embodiments, each of the doses of multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation in the initial and subsequent treatment cycles comprises the same amount of multispecific binding protein in the range of 0.1-20 mg / kg, 0.1-10 mg / kg, 0.1-5 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 0.1-0.5 mg / kg, 0.1-0.2 mg / kg, 0.2-20 mg / kg, 0.2-10 mg / kg, 0.2-5 mg / kg, 0.2-2 mg / kg, 0.2-1 mg / kg, 0.2-0.5 mg / kg, 0.5-20 mg / kg, 0.5-10 mg / kg, 0.5-5 mg / kg, 0.5-2 mg / kg, 0.5-1 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, or 1-2 mg / kg of the subject's body weight.

[0295] In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein. In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein.

[0296] In certain embodiments, each of the doses in the initial and subsequent treatment cycles is 5.2×10 -5 mg / kg, 1.6×10 -4 mg / kg, 5.2×10 -4 mg / kg, 1.6×10 -3 mg / kg, 5.2×10 -3 mg / kg, 1.6×10 -2 mg / kg, 5.2×10 -2 mg / kg, 1.6×10 -1In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises 5.2×10 -5 mg / kg, 1.6×10 -4 mg / kg, 5.2×10 -4 mg / kg, 1.6×10 -3 mg / kg, 5.2×10 -3 mg / kg, 1.6×10 -2 mg / kg, 5.2×10 -2 mg / kg, 1.6×10 -1 The multispecific binding protein may be administered in an amount of 0.52 mg / kg, 0.52 mg / kg, 1 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 20 mg / kg, or 50 mg / kg.

[0297] In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered intravenously. For example, in certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by intravenous infusion, e.g., a pre-filled bag, a pre-filled pen, or a pre-filled syringe. In certain embodiments, the multispecific binding protein in the pharmaceutical composition or pharmaceutical formulation disclosed herein is diluted prior to administration. For example, in certain embodiments, the pharmaceutical composition or pharmaceutical formulation is diluted with sodium chloride and administered intravenously from a 250 ml saline bag. The intravenous infusion can last for about an hour (e.g., 50-80 minutes). In certain embodiments, the bag is connected to a channel that includes a tube and / or a needle.

[0298] In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered subcutaneously. For example, in certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by subcutaneous injection using a syringe or autoinjector. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered by subcutaneous injection in the abdomen, arm, or thigh. In certain embodiments, a suitable pharmaceutical composition or pharmaceutical formulation for subcutaneous administration comprises a multispecific binding protein at a concentration of greater than 50 mg / mL, as described in the pharmaceutical formulation section above. In certain embodiments, a pharmaceutical composition or formulation comprises greater than or equal to 60 mg / mL, greater than or equal to 70 mg / mL, greater than or equal to 80 mg / mL, greater than or equal to 90 mg / mL, greater than or equal to 100 mg / mL, greater than or equal to 125 mg / mL, greater than or equal to 150 mg / mL, greater than or equal to 175 mg / mL, or greater than or equal to 200 mg / mL of multispecific binding protein.In certain embodiments, the pharmaceutical composition or pharmaceutical formulation is 60-250 mg / mL, 60-225 mg / mL, 60-200 mg / mL, 60-175 mg / mL, 50-150 mg / mL, 60-150 mg / mL, 60-125 mg / mL, 60-100 mg / mL, 60-90 mg / mL, 60-80 mg / mL, 60-70 mg / mL, 70-250 mg / mL, 70-225 mg / mL, 70-200 mg / mL , 70~175mg / mL, 70~150mg / mL, 70~150mg / mL, 70~125mg / mL, 70~100mg / mL, 70~90mg / mL, 70~80mg / mL, 80~250mg / mL , 80~225mg / mL, 80~200mg / mL, 80~175mg / mL, 80~150mg / mL, 80~150mg / mL, 80~125mg / mL, 80~100mg / mL, 80~90mg / m L, 90~250mg / mL, 90~225mg / mL, 90~200mg / mL, 90~175mg / mL, 90~150mg / mL, 90~150mg / mL, 90~125mg / mL, 90~100m g / mL, 100~250mg / mL, 100~225mg / mL, 100~200mg / mL, 100~175mg / mL, 100~150mg / mL, 100~125mg / mL, 125~250mg / m In certain embodiments, the pharmaceutical composition or formulation comprises 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, or 220 mg / mL of the multispecific binding protein.

[0299] Cancers suitable for treatment The multispecific binding proteins, pharmaceutical compositions, or pharmaceutical preparations disclosed herein can be used to treat various types of cancer. In certain embodiments, the cancer is a solid tumor. In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer.In yet other embodiments, the cancer is angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid carcinoma, bile duct carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract carcinoma, bone cancer, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choriocarcinoma, sarcoma ... Plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, orbital cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric pyloric cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, Insulinoma, Intraepithelial neoplasia, Intraepithelial squamous cell neoplasm, Intrahepatic cholangiocarcinoma, Invasive squamous cell carcinoma, Jejunal cancer, Joint cancer, Kaposi's sarcoma, Pelvic cancer, Large cell carcinoma, Colon cancer, Leiomyosarcoma, Malignant melanoma lentigo, Lymphoma, Male genital cancer, Malignant melanoma, Malignant mesothelial tumor, Medulloblastoma, Medulloepithelioma, Meningeal cancer, Mesothelial carcinoma, Metastatic cancer, Oral cancer, Mucoepidermoid carcinoma, Multiple myeloma, Muscle cancer, Nasal passage cancer, Nervous system cancer, Neuroepithelial adenocarcinoma, Nodular melanoma, Nonepithelial skin cancer, Non-Hodgkin's lymphoma, Oat cell carcinoma, Oligodendrocyte carcinoma, Oral cancer, Osteosarcoma, Papillary serous adenocarcinoma, Penis cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, paranasal sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle carcinoma, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyocarcinoma, submesothelial carcinoma, superficial melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0300] In certain embodiments, the cancer is a hematological malignancy. In certain embodiments, the hematological malignancy is a leukemia. In certain embodiments, the leukemia is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia.

[0301] In certain embodiments, types of cancer that can be treated with the multispecific binding proteins, pharmaceutical compositions, or pharmaceutical formulations disclosed herein, where the second antigen binding site of the multispecific binding protein binds to HER2, include, but are not limited to, breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary glands, cholangiocarcinoma, squamous lung, mesothelioma, liver cancer, sarcoma, bladder cancer, and gallbladder cancer. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a locally advanced or metastatic solid tumor. In certain embodiments, the cancer is gastric cancer (e.g., HER2 high or HER2 3+). In certain embodiments, the cancer is urothelial bladder cancer (e.g., urothelial bladder cancer expressing HER2). In certain embodiments, the cancer is metastatic breast cancer (e.g., metastatic triple-negative breast cancer). In certain embodiments, the cancer is a solid tumor (e.g., HER2 high or HER2 3+).

[0302] Disclosed herein, in various embodiments, is a method of treating HER2-overexpressing gastric cancer in a subject in need thereof, comprising administering an effective amount of a multispecific binding protein or pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., as determined by in situ hybridization).

[0303] Also disclosed herein, in various embodiments, is a method of treating HER2-overexpressing gastric cancer in a subject in need thereof, comprising administering an effective amount of an anti-PD-1 antibody (e.g., nivolumab) and a multispecific binding protein or pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., as determined by in situ hybridization).

[0304] Also disclosed herein, in various embodiments, is a method of treating HER2-overexpressing gastric cancer in a subject in need thereof, comprising administering an effective amount of nab-paclitaxel and a multispecific binding protein or pharmaceutical formulation disclosed herein. In certain embodiments, the cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., as determined by in situ hybridization).

[0305] Also disclosed herein, in various embodiments, is a method of treating HER2-overexpressing gastric cancer in a subject in need thereof, comprising administering (e.g., via intravenous infusion) an effective amount of a multispecific binding protein or pharmaceutical formulation disclosed herein in combination with nivolumab, and further in combination with nab-paclitaxel. In certain embodiments, the gastric cancer has a HER2 expression level scored as 3+ by immunohistochemistry. In other embodiments, the gastric cancer has a HER2 expression level scored as 2+ by immunohistochemistry and HER2 gene amplification (e.g., as determined by in situ hybridization).

[0306] Disclosed herein, in various embodiments, is a method of treating metastatic triple-negative breast cancer (TNBC) in a subject in need thereof comprising administering an effective amount of a multispecific binding protein or pharmaceutical formulation disclosed herein.

[0307] In certain embodiments, the subject treated by the methods disclosed herein has a HER2-positive cancer. Methods for determining HER2 expression in cancer include, but are not limited to, immunohistochemistry (IHC). Anti-HER2 antibodies (e.g., Ventana 4B5 antibody and Bond Oracle CB11 antibody) have been approved by the FDA to detect HER2, and immunohistochemistry kits (e.g., HercepTest™) are commercially available. The level of HER2 expression in tumor samples, as detected by immunohistochemistry, can be quantified and scored as 1+, 2+, or 3+ according to the ASCO / CAP guidelines (Wolff et al., (2007) J. Clin. Oncol. 25(1):118-45) and the 2018 update (Wolff et al., (2018) J. Clin. Oncol. 36(20):2105-22). Under the 2018 ASCO / CAP guidelines, a cancer or tumor is scored as HER2 3+ if circumferential membrane staining of HER2 is complete, strong, and present in >10% of tumor cells in the sample, is easily seen using a low magnification objective, and is observed within a homogenous, continuous, invasive cell population. A cancer or tumor is scored as HER2 2+ if weak to moderate complete membrane staining of HER2 is observed in >10% of tumor cells in the sample. A cancer or tumor is scored as HER2 1+ if incomplete membrane staining of HER2 is faintly or barely perceptible and present in >10% of tumor cells in the sample. A cancer or tumor is scored as HER2 negative if no HER2 staining is observed or membrane staining is incomplete, faintly or barely perceptible and present in >10% of tumor cells. If the cancer or tumor is scored as HER2 2+ in this initial IHC evaluation, then reflex testing (using ISH, same specimen) or new testing (using IHC or ISH, new specimen if available) must be ordered. Based on the results of the reflex testing or new testing, the cancer or tumor may be rescored as HER2 3+ or 1+.

[0308] It is understood that ERBB2 gene amplification generally correlates with HER2 overexpression. Determining whether ERBB2 gene is amplified in a cancer tissue sample can help reduce false positive results from immunohistochemistry testing of the same sample (see, e.g., Sarode et al., (2015) Arch. Pathol. Lab. Med. 139:922-28). Thus, in certain embodiments, a cancer or tumor in a subject is evaluated for ERBB2 gene amplification. In certain embodiments, the cancer or tumor has ERBB2 gene amplification, e.g., an average ERBB2 gene copy number greater than or equal to 4.0 signals per cell (e.g., greater than or equal to 6.0 signals per cell). In certain embodiments, a cancer or tumor (e.g., a HER2 1+ cancer or tumor) has an average ERBB2 gene copy number less than 4.0 signals per cell. In certain embodiments, a cancer or tumor (e.g., a HER2 1+ cancer or tumor) does not have ERBB2 gene amplification. Methods for detecting gene amplification include, but are not limited to, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), quantitative PCR, and DNA sequencing. In certain embodiments, ERBB2 gene amplification is determined by FISH. In certain embodiments, ERBB2 gene amplification is determined by DNA sequencing (e.g., deep sequencing).

[0309] New techniques can be used to assess HER2 levels in patient samples. For example, automated quantitative analysis techniques can quantitatively assess HER2 expression by measuring the intensity of antibody-conjugated fluorophores. HERmark technology measures HER2 expression through proximity-based release of antibody-conjugated fluorescent tags. Quantitative IHC technology converts antibody / antigen complexes into red dots, which are then counted to quantify HER2 expression. Time-resolved fluorescence energy transfer technology allows assessment of HER2 expression through detection of fluorescence emitted by two fluorophores in close proximity. Quantitative real-time polymerase chain reaction technology allows quantitative measurement of the amount of HER2 mRNA in a sample. Flow cytometry technology allows measurement of the number of HER2 proteins on the surface of cells. These assays complement the results of IHC or FISH assays, thereby providing a more accurate assessment of HER2 levels in cancers or tumors.

[0310] For example, in certain embodiments, cancer or tumors can be evaluated by flow cytometry. As described in Example 5 below, a calibration curve can be generated using molecules of equivalent soluble fluorescent dye (MESF) beads with a manufacturer's predetermined amount of fluorophore. This calibration curve can be used to correlate the geometric mean fluorescence intensity (MFI) of a given cell population to a standardized number of molecules. The reagent that detects HER2 protein can be a protein that binds to HER2 (e.g., HER2 TriNKET disclosed herein) conjugated with a fluorophore. If the sample is a cell line, the geometric mean of the number of HER2 proteins on the cells can be determined. The cell line with the determined level of HER2 can then be used as a reference in, for example, an IHC assay to determine the level of HER2 in the tumor sample. In practice, SKBR3, MDA-MB-175, and MDA-MB-231 cell lines are used as references to set the level 3, level 1, and level 0 staining intensity for HercepTest.In certain embodiments, cell lines with a geometric mean of 500,000 or more (e.g., 600,000 or more, 700,000 or more, 800,000 or more, or 900,000 or more) HER2 protein on each cell are HER2 Corresponding to cells with complete and strong peri-membrane staining of HER2 in 3+ samples; 75,000–499,999 (e.g., 80,000–499,999, 90,000–499,999, 100,000–499,999, 110,000–499,999, 120,000–499,999, 85,000–399,999, 90,000–399,999, 100,000–399,999, 110,000–399,999, 115,000–399,99 HER2 9, 120,000 to 399,999, 85,000 to 299,999, 90,000 to 299,999, 95,000 to 299,999, 100,000 to 299,999, 105,000 to 299,999, 110,000 to 299,999, 85,000 to 199,999, 90,000 to 199,999, 95,000 to 199,999, 100,000 to 199,999, 105,000 to 199,999, or 110,000 to 199,999 Cell lines with a geometric mean of 10,000-74,999 (e.g., 11,000-74,999, 11,000-69,999, 11,000-64,999, 11,000-59,999, or 11,000-54,999) HER2 protein on each cell correspond to cells with faint or barely perceptible, incomplete membrane staining of HER2 in HER2 1+ samples. Cell lines with a geometric mean of HER2 protein on each cell below 10,000 (e.g., 9000 or less, 8000 or less, 7000 or less, 6000 or less, 5000 or less, 4000 or less) correspond to negative membrane staining in HER2 0 samples. According to the 2018 ASCO / CAP guidelines, more than 10% of the tumor cells in a sample must meet the required HER2 level threshold.

[0311] In certain embodiments, a subject treated by the methods disclosed herein has a cancer or tumor (e.g., any one of the cancer types disclosed in the preceding three paragraphs) with a HER2 level scored as 1+, 2+, or 3+. In certain embodiments, a subject treated by the methods disclosed herein has a cancer or tumor (e.g., any one of the cancer types disclosed in the preceding three paragraphs) with a HER2 level scored as 1+ or 2+. In certain embodiments, a subject treated by the methods disclosed herein has a cancer or tumor (e.g., any one of the cancer types disclosed in the preceding three paragraphs) with a HER2 level scored as 2+ or 3+. In certain embodiments, a subject treated by the methods disclosed herein has a cancer or tumor (e.g., any one of the cancer types disclosed in the preceding three paragraphs) with a HER2 level scored as 1+. In certain embodiments, a subject treated by the methods disclosed herein has a cancer or tumor (e.g., any one of the cancer types disclosed in the preceding three paragraphs) with a HER2 level scored as 2+. In certain embodiments, subjects treated by the methods disclosed herein have a cancer or tumor (e.g., any one of the types of cancers disclosed in the preceding three paragraphs) with HER2 levels scored as 3+. In certain embodiments, HER2 levels are determined by immunohistochemistry (e.g., HercepTest™). In certain embodiments, subjects treated by the methods disclosed herein have at least a tumor that exhibits HER2 expression as faint / barely perceptible membrane staining detected in at least or more than 10% of tumor cells. In certain embodiments, subjects treated by the methods disclosed herein have at least a tumor that exhibits HER2 expression as weak or moderate complete membrane staining detected in at least or more than 10% of tumor cells. In certain embodiments, subjects treated by the methods disclosed herein have at least a tumor that exhibits HER2 expression as strong complete membrane staining detected in at least or more than 10% of tumor cells.

[0312] In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 1+, the cancer or tumor being selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, uterine cancer, angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, bile duct cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, and the like. disease, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bone marrow cancer, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, orbital cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric pylorus Cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, malignant melanoma lentigo, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial Skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendrocyte carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer , smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle carcinoma, submesothelial carcinoma, superficial melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous cancer, VIPoma, vulvar cancer, well-differentiated cancer,or Wilms' tumor. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is a hematological malignancy, such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary gland, cholangiocarcinoma, squamous cell carcinoma of the lung, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, or metastatic breast cancer. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 1+, and the cancer or tumor is a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple-negative breast cancer with a HER2 level scored as 1+. In certain embodiments, the cancer or tumor is hormone receptor positive (i.e., positive for estrogen receptor, progesterone receptor, or both) and HER2 negative (e.g., as assessed by IHC). According to the 2018 ASCO / CAP HER2 testing guideline update, a breast cancer is considered HER2 negative in cases of IHC 0 and 1+ results, or IHC 2+ with a negative ISH assay. Thus, in certain embodiments, the cancer or tumor is a triple-negative breast cancer that is negative for estrogen receptor and progesterone receptor (e.g., as assessed by IHC) with HER2 levels scored as IHC 1+. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., as assessed by IHC) with HER2 levels scored as IHC 1+. In certain embodiments, a HER2 1+ cancer or tumor is:The HER2 1+ cancer or tumor has an average ERBB2 gene copy number of less than 4.0 signals per cell. In certain embodiments, the HER2 1+ cancer or tumor does not have ERBB2 gene amplification.

[0313] In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 2+, the cancer or tumor being selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, uterine cancer, angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, bile duct cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, and the like. disease, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract cancer, bone cancer, bone marrow cancer, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, orbital cancer, female genital tract cancer, focal nodular hyperplasia, gallbladder cancer, gastric pylorus Cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, malignant melanoma lentigo, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial Skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendrocyte carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer , smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle carcinoma, submesothelial carcinoma, superficial melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous cancer, VIPoma, vulvar cancer, well-differentiated cancer,or Wilms' tumor. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 2+, and the cancer or tumor is a hematological malignancy, such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myeloid leukemia. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 2+, the cancer or tumor being breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary glands, cholangiocarcinoma, squamous cell carcinoma of the lung, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, or metastatic breast cancer. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level scored as 2+, the cancer or tumor being a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple-negative breast cancer with a HER2 level scored as IHC 2+ in a negative ISH assay. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., as assessed by IHC) with HER2 levels scored as IHC2+ in a negative ISH assay. In certain embodiments, a HER2 2+ cancer or tumor has an average ERBB2 gene copy number of less than 4.0 signals per cell. In certain embodiments, a HER2 2+ cancer or tumor does not have ERBB2 gene amplification.

[0314] In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level that is not elevated (i.e., scored as 1+ or 2+), the cancer or tumor being selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, uterine cancer, angiogenic tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, bile duct cancer, thyroid cancer, acral lentiginous melanoma, solar plexus cancer, thyroid ... Keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, biliary tract carcinoma, bone cancer, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, orbital carcinoma, female genital tract cancer, localized nodule Hyperplasia, gallbladder cancer, gastric pyloric cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasm, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, malignant melanoma lentigo, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial adenocarcinoma, Nodal melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer , small cell cancer, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striated muscle carcinoma, submesothelial carcinoma, superficial melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous cancer, VIPoma,In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a non-elevated HER2 level (i.e., scored as 1+ or 2+), and the cancer or tumor is a hematological malignancy, such as leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelomonocytic leukemia, or myeloid blast crisis of chronic myelogenous leukemia. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level that is not elevated (i.e., scored as 1+ or 2+), the cancer or tumor being breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, adenocarcinoma of the lung, prostate cancer, cholangiocarcinoma, uterine cancer, pancreatic cancer, colorectal cancer, ovarian cancer, cervical cancer, head and neck cancer, NSCLC, glioblastoma, esophageal cancer, squamous cell carcinoma of the skin, carcinoma of the salivary glands, cholangiocarcinoma, squamous lung, mesothelioma, liver cancer, sarcoma, bladder cancer, gallbladder cancer, urothelial bladder cancer, and metastatic breast cancer. In certain embodiments, the subject treated by the methods disclosed herein has a cancer or tumor with a HER2 level that is not elevated (i.e., scored as 1+ or 2+), the cancer or tumor being a solid tumor, such as a locally advanced or metastatic solid tumor. In certain embodiments, the cancer or tumor is a triple-negative breast cancer with HER2 levels scored as IHC 1+ or IHC 2+ in a negative ISH assay. In certain embodiments, the cancer or tumor is a hormone receptor positive breast cancer (e.g., assessed by IHC) with HER2 levels scored as IHC1+ or IHC2+ in a negative ISH assay. In certain embodiments, the cancer or tumor with a non-high HER2 level score (i.e., scored as 1+ or 2+) has an average ERBB2 gene copy number of less than 4.0 signals per cell. In certain embodiments, the cancer or tumor with a non-high HER2 level score (i.e., scored as 1+ or 2+) does not have ERBB2 gene amplification.

[0315] In certain embodiments, a subject treated according to the methods disclosed herein has not undergone a prior therapy to treat cancer. In certain embodiments, a subject treated according to the methods disclosed herein has not undergone a prior chemotherapy or immunotherapy to treat cancer. In certain embodiments, a subject treated according to the methods disclosed herein has undergone a prior treatment (e.g., chemotherapy or immunotherapy), but continues to experience cancer progression despite the prior treatment. In certain embodiments, a subject treated according to the methods disclosed herein experienced cancer regression after undergoing a prior treatment (e.g., chemotherapy or immunotherapy), but later experienced cancer recurrence. In certain embodiments, a subject treated according to the methods disclosed herein is intolerant to a prior treatment (e.g., chemotherapy or immunotherapy).

[0316] In certain embodiments, the subject treated according to the methods disclosed herein meets one or more of the selection criteria for the clinical trial cohorts described in Example 3 (e.g., accelerated titration cohort, "3+3" dose escalation cohort, safety / PK / PD expansion cohort, urothelial bladder cancer (UBC) cohort, metastatic breast cancer (MBC) cohort, basket solid tumor cohort with high HER2 expression (HER2 3+), or combination treatment cohort with pembrolizumab). In certain embodiments, the subject treated according to the methods disclosed herein meets all of the selection criteria for the clinical trial cohorts described in Example 3 (e.g., accelerated titration cohort, "3+3" dose escalation cohort, safety / PK / PD expansion cohort, UBC cohort, MBC cohort, basket solid tumor cohort with high HER2 expression (HER2 3+), or combination treatment cohort with pembrolizumab).

[0317] In certain embodiments, provided herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - Histologically or cytologically proven locally advanced or metastatic solid tumors for which no standard treatment exists or for which standard treatment has failed; - Primary tumor with documented HER2 expression as detected by immunohistochemistry; - Detectable erbb2 amplification and / or erbb2 activating mutations in tumor biopsy; - Histologically or cytologically proven locally advanced or metastatic solid tumors for which no standard treatment exists or for which standard treatment has failed; - Have a HER2-expressing tumor (at least 1+ at screening) as determined by immunohistochemistry using a CLIA-certified (or equivalent) method; - Have measurable disease with at least one unidimensional measurable lesion by RECIST 1.1; - ECOG performance status of 0 or 1 and estimated life expectancy of at least 3 months at the time of study entry; - baseline left ventricular ejection fraction (LVEF) ≥ 55% as measured by echocardiogram or multi-gated acquisition (MUGA) scan; Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 / L, lymphocyte count ≥ 0.5 × 10 9 / L, platelet count ≥75×10 9 / L, and white blood cell (WBC) count ≥ 3 × 10 with hemoglobin ≥ 9 g / dL (possibly transfused). 9 adequate hematologic function defined by / L; - Adequate liver function as defined by total bilirubin level ≤ 1.5 x upper limit of normal (ULN), aspartate aminotransferase (AST) level ≤ 2.5 x ULN, and alanine aminotransferase (ALT) level ≤ 2.5 x ULN for patients with documented metastatic disease to the liver, AST and ALT levels ≤ 5 x ULN, and / or - Adequate renal function as defined by estimated creatinine clearance >50mL / min according to the Cockcroft-Gault formula.

[0318] In certain embodiments, provided herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - Eligible to receive nivolumab according to its label for a malignant tumor of epithelial origin; or have a tumor with no standard therapy or for which standard therapy has failed; and - Have received anti-PD-1 or anti-PD-L1 as a prior line of therapy; or have not received anti-PD-1 or anti-PD-L1 as a prior line of therapy and have experienced Grade 3 or 4 drug-related toxicity affecting the lungs or nervous system or Grade 2 drug-related toxicity associated with prior anti-PD-1 or anti-PD-L1 therapy.

[0319] In certain embodiments, provided herein are methods of treating cancer in a subject having one or more (e.g., all) of the following characteristics: - Eligible to receive nab-paclitaxel according to its label after failure of combination treatment for metastatic disease or recurrence within 6 months of adjuvant chemotherapy and have not been exposed to a taxane within the past 6 months; - Have a tumor with no standard treatment or for which standard treatment has failed; or -Has advanced (unresectable / recurrent / metastatic) triple-negative breast cancer (TNBC).

[0320] In certain embodiments, provided herein are methods of treating a subject having urothelial bladder cancer (UBC) and one or more (e.g., all) of the following characteristics: -Histologically or cytologically documented locally advanced or metastatic transitional cell carcinoma of the urothelium (including renal pelvis, ureter, urothelium, and urethra); - Radiographic disease progression after the subject's last line of treatment; -Has received treatment with one (and no more than one) platinum-containing regimen (e.g., platinum plus another agent, e.g., gemcitabine, methotrexate, vinblastine, doxorubicin, etc.) for inoperable locally advanced or metastatic urothelial carcinoma with radiological progression or recurrence within 6 months after the last dose of an adjuvant platinum-containing regimen that would be considered a failure of a first-line platinum-containing regimen; - Treatment with a checkpoint inhibitor (i.e., anti-PD-1 or anti-PD-L1) with radiological progression (optionally with a combination of platinum-based and PD-1 / PD-L1-based therapy); and / or - HER2 expression of at least 1+ as determined by immunohistochemistry.

[0321] In certain embodiments, provided herein are methods for treating a subject having metastatic breast cancer (MBC) and one or more (e.g., all) of the following characteristics: - histologically confirmed MBC; - 3 or fewer prior lines of cytotoxic therapy for metastatic disease; - Previous treatment with taxanes and anthracyclines, unless anthracyclines are contraindicated; - Tumor scoring of 1+ or 2+ by IHC, and if scoring is 2+, the presence of tumor amplification of ERRB2 must be determined by an FDA-approved method; and / or - Radiologic progression after last-line systemic therapy.

[0322] In certain embodiments, provided herein are methods for treating a subject having triple-negative breast cancer (TNBC) and one or more (e.g., all) of the following characteristics: - Tumor with a PD-L1 score (CPS) of less than 10, as measured by immunohistochemistry, e.g., using Agilent / Dako's PD-L1 immunohistochemistry 22C3 pharmDx assay; - Histologically documented (metastatic or locally advanced) TNBC, including HER2 status, negative ER expression, and negative PR expression that render the subject ineligible for trastuzumab, as defined by the American College of Physicians (ACP) guidelines or equivalent; o Absence of HER2 amplification as determined by in situ hybridization (HER2:CEP17 ratio less than 2.0 or single probe average HER2 gene copy number less than 4 signals / cell) or HER2 expression levels of 0, 1+, or 2+ as measured by immunohistochemistry; and o ER and PR negative (defined as <1% of cells expressing hormone receptors via immunohistochemical analysis); - Has not received prior chemotherapy or targeted systemic therapy for inoperable locally advanced or metastatic TNBC; and / or - Have measurable disease with at least one unidimensional measurable lesion by RECIST 1.1.

[0323] In certain embodiments, provided herein are methods for treating a subject having a HER2 3+ tumor and one or more (e.g., all) of the following characteristics: - A personal history of any solid tumor, except breast or gastric cancer, and erbb2 amplification within the tumor and one of the following: 1) HER2 3+ scoring by immunohistochemistry documented in the subject's most recent biopsy within 6 months, after radiological progression at the time of last line of therapy, or 2) HER2 3+ scoring by immunohistochemistry during the screening window; and - Treatment with at least one approved or established therapy of choice.

[0324] In certain embodiments, provided herein are methods for treating a subject having gastric cancer and one or more (e.g., all) of the following characteristics: -Advanced (unresectable / recurrent / metastatic) gastric cancer or cancer of the gastroesophageal junction according to the 7th American Joint Committee on Cancer (AJCC) classification; - Have HER2-positive tumors according to the American College of Physicians (ACP) guidelines and therefore eligible for trastuzumab treatment; - Previously treated with first-line therapy containing a platinum salt and a fluoropyridine in combination with trastuzumab or a biosimilar to trastuzumab; - Progressing after first-line treatment; - Having received only one line of therapy for the treatment of metastatic disease; - Have measurable disease with at least one unidimensional measurable lesion by RECIST 1.1; and / or - Has a tumor not known to be microsatellite instability (MSI) high.

[0325] In certain embodiments, provided herein are methods of treating a subject having a HER2 3+ solid tumor and one or more (e.g., all) of the following characteristics: - Have measurable disease with at least one unidimensional measurable lesion by RECIST 1.1; - A personal history of any solid tumor, except breast or gastric cancer, and erbb2 amplification within the tumor and one of the following: 1) HER2 3+ scoring by immunohistochemistry documented in the subject's most recent biopsy within 6 months, after radiological progression at the time of last line of therapy, or 2) HER2 3+ scoring by immunohistochemistry during the screening window; and / or - Undergoing treatment with at least one approved or established treatment option.

[0326] In certain embodiments, provided herein is a method for treating a subject having a malignant tumor of epithelial origin and eligible for treatment with nivolumab according to its label. In certain embodiments, provided herein is a method for treating a subject having metastatic breast cancer who has not responded to combination chemotherapy for metastatic disease or has relapsed within 6 months of adjuvant chemotherapy, has not been exposed to a taxane within the past 6 months, and is eligible for treatment with nab-paclitaxel according to its label.

[0327] In certain embodiments, provided herein is a method for treating cancer in a subject treated according to the methods disclosed herein who does not meet one or more of the exclusion criteria set forth in Example 3. In certain embodiments, a subject treated according to the methods disclosed herein does not meet any of the exclusion criteria set forth in Example 3.

[0328] In certain embodiments, provided herein is a method of treating cancer in a subject: - not currently being treated with immunotherapy, immunosuppressive drugs, such as chemotherapy or systemic corticosteroids (except for short-term treatment of allergic reactions or treatment of immunotherapy-related adverse events (irAEs) with systemic steroids, or treatment with topical or inhaled steroids with no or minimal systemic effects), or other experimental medicinal products; - not currently being treated with a tyrosine kinase inhibitor targeting HER2 or any recombinant molecule targeting HER2 or NKGHER2D; - not currently being treated with growth factors, such as granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor (excluding erythropoietin and erythropoietin analogs); - Not currently being treated with a bisphosphonate or denosumab if treatment was not initiated more than 14 days prior to the first dose of the multispecific binding protein; - Not previously treated with drugs that specifically target the HER2 pathway (except for subjects treated with a monoclonal antibody with a four-week washout period or subjects treated with a tyrosine kinase inhibitor with a two-week washout period); - Not currently treated with cytoreductive therapy, radiation therapy (except palliative bone-directed radiation therapy), cytokine therapy (except erythropoietin), major surgery (except diagnostic biopsy), or use of any investigational drug within 28 days prior to treatment with multispecific binding proteins; - Have had no malignancies within the past three years other than the target malignancy being treated with the multispecific binding protein (except for basal or squamous cell carcinoma of the skin or in situ cervical carcinoma); - Not having rapidly progressive disease; - No active or history of central nervous system (CNS) metastases; - No previous organ transplant (including autologous or allogeneic stem cell transplant); - No significant acute or chronic infection (including a positive test for human immunodeficiency virus (HIV)), or active or occult hepatitis B or active hepatitis C; - No pre-existing autoimmune disease (excluding subjects with vitiligo) or clinically relevant immunodeficiency (e.g., dysgammaglobulinemia or congenital immunodeficiency) requiring treatment with systemic immunosuppressants for more than 28 days within the past three years, or fever within the past seven days; - No known severe hypersensitivity reaction to the mAb (≥ Grade 3 NCI-CTCAE v5.0), any history of anaphylaxis, or uncontrolled asthma (e.g., three or more features of partially controlled asthma); - No persistent toxicity related to prior therapy >grade 1 NCI-CTCAE v5.0 (excluding alopecia and sensory neuropathy ≤grade 2); - Not a pregnant or lactating woman; - No known alcohol or drug abuse; and / or - No serious cardiac disease or medical conditions, including but not limited to: o History of New York Heart Association class III or IV heart failure or systolic dysfunction (LVEF <55%); o High-risk uncontrolled arrhythmias, i.e., tachycardia with a heart rate >100 beats per minute at rest; o Significant ventricular arrhythmia (ventricular tachycardia) or higher grade AV block (2nd degree AV block type 2 (Mobitz 2) or 3rd degree AV block); o Angina requiring antianginal medication; o Clinically significant valvular heart disease; o Evidence of transmural myocardial infarction on an electrocardiogram (ECG); or o Poorly controlled hypertension (defined by systolic >180mmHg or diastolic >100mmHg).

[0329] It is contemplated that the methods disclosed herein for treating patients having the above-identified cancers can be performed with or without the concomitant use of corticosteroids to reduce one or more infusion-related reactions.

[0330] As monotherapy and in combination with other cancer treatments. The multispecific binding proteins disclosed herein can be used as monotherapy or in combination with one or more therapies. In certain embodiments, the multispecific binding proteins are used as monotherapy according to the dosing regimens disclosed herein. In other embodiments, the multispecific binding proteins are used in combination with one or more therapies administered according to dosing regimens known to be suitable for treating a particular subject with a particular cancer, and the multispecific binding proteins are administered according to the dosing regimens disclosed herein. In certain embodiments, the methods of treatment disclosed herein are used as an adjunct to surgical removal of the primary lesion.

[0331] Exemplary therapeutic agents that may be used in combination with the multispecific binding proteins include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglucan, riboflavin ... amide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butosin, carmophor, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon alpha, interferon-2 alpha, interferon beta, interferon gamma (IFN-γ), colony-stimulating factor 1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor, and variations of the foregoing agents that may exhibit differential binding to their cognate receptors or increased or decreased serum half-lives.

[0332] An additional class of agents that may be used as part of a combination therapy in treating cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. Food and Drug Administration to treat melanoma and may be used as part of a combination therapy described herein.

[0333] Still other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0334] Further categories of anti-cancer agents include, for example: (i) inhibitors selected from ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors + 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0335] In certain embodiments, the methods of the invention further comprise administering an anti-PD-1 antibody to the subject. Many anti-PD-1 antibodies have been developed for therapeutic purposes and are described, for example, in Gong et al., (2018) J. ImmunoTher Cancer 6:8.

[0336] In a specific embodiment, the anti-PD-1 antibody is nivolumab. In a specific embodiment, 0.1-10 mg / kg, 0.1-3 mg / kg, 0.1-1 mg / kg, 0.1-0.3 mg / kg, 0.3-10 mg / kg, 0.3-3 mg / kg, 0.3-1 mg / kg, 1-10 mg / kg, 1-3 mg / kg, or 3-10 mg / kg of nivolumab is administered to the subject. In a specific embodiment, 120-600 mg, 120-480 mg, 120-360 mg, 120-240 mg, 240-600 mg, 240-480 mg, 240-360 mg, 360-600 mg, 360-480 mg, or 480-600 mg of nivolumab is administered to the subject. In certain embodiments, 120 mg, 240 mg, 360 mg, 480 mg, or 600 mg of nivolumab is administered to the subject. In certain embodiments, 480 mg of nivolumab is administered on day 1 of the first treatment cycle. In certain embodiments, if the subject undergoes one or more subsequent treatment cycles, 480 mg of nivolumab is administered once every four weeks in the subsequent treatment cycles, starting on day 1 of each subsequent treatment cycle. In certain embodiments, nivolumab is administered as a 30-minute, 35-minute, 45-minute, 50-minute, 60-minute, 65-minute, 70-minute, 75-minute, 80-minute, 85-minute, or 90-minute intravenous infusion in a four-week treatment cycle.

[0337] In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion and nivolumab is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion in a four week treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 minute intravenous infusion and nivolumab is administered as a 30 minute intravenous infusion in a four week treatment cycle.

[0338] In certain embodiments, a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered in combination with nivolumab in a first treatment cycle, where the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation and nivolumab are both administered on day 1, and the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered alone on day 8. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered in combination with nivolumab in subsequent weekly treatment cycles after the first cycle, where the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation and nivolumab are both administered on day 1.

[0339] In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the dose is 100-300 mg, 110-300 mg, 120-300 mg, 130-300 mg, 140-300 mg, 150-300 mg, 160-300 mg, 170-300 mg, 180-300 mg, 190-300 mg, 200-300 mg, 210-300 mg, 220-300 mg, 230-300 mg, 240-300 mg, 250-300 mg, 260-300 mg, 270-300 mg, 280-300 mg, 290-300 mg, 100-280 mg, 110-28 ... 0~280mg, 130~280mg, 140~280mg, 150~280mg, 160~280mg, 170~280mg, 180~280mg.190~280mg, 200~280mg, 210~280mg, 220~280mg, 230~280m g, 240~280mg, 250~280mg, 260~280mg, 270~280mg, 100~260mg, 110~260mg, 120~260mg, 130~260mg, 140~260mg, 150~260mg, 160~260mg, 170~ 260mg, 180~260mg, 190~260mg, 200~260mg, 210~260mg, 220~260mg, 230~260mg, 240~260mg, 250~260mg, 100~240mg, 110~240mg, 120~240mg, 130~240mg, 140~240mg, 150~240mg, 160~240mg, 170~240mg, 180~240mg, 190~240mg, 200~240mg, 210~240mg, 220~240mg, 230~240mg, 100~22 0 mg, 110-220 mg, 120-220 mg, 130-220 mg, 140-220 mg, 150-220 mg, 160-220 mg, 170-220 mg, 180-220 mg. 190-220 mg, 200-220 mg, 210-220 mg, 100-200 mg, 110-200 mg, 120-200 mg, 130-200 mg, 140-200 mg, 150-200 mg, 160-200 mg, 170-200 mg, 180-200 mg, or 190-200 mg of pembrolizumab.In certain embodiments, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg of pembrolizumab is administered to the subject. In certain embodiments, 200 mg of pembrolizumab is administered on day 1 of the first treatment cycle. In certain embodiments, if the subject undergoes one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles, starting on day 1 of the first subsequent treatment cycle. In certain embodiments, pembrolizumab is administered as a 30-minute, 35-minute, 45-minute, 50-minute, 60-minute, 65-minute, 70-minute, 75-minute, 80-minute, 85-minute, or 90-minute intravenous infusion in a three-week treatment cycle.

[0340] In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion and pembrolizumab is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion in a three week treatment cycle. In certain embodiments, the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 minute intravenous infusion and pembrolizumab is administered as a 30 minute intravenous infusion in a three week treatment cycle.

[0341] In certain embodiments, a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered in combination with pembrolizumab in a first treatment cycle, where the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation and pembrolizumab are both administered on day 1, and the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered alone on day 8. In certain embodiments, a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation is administered in combination with pembrolizumab in a subsequent three week treatment cycle after the first cycle, where the multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation and pembrolizumab are both administered on day 1.

[0342] In certain embodiments, the methods of the invention further comprise administering to the subject a cytoskeleton-disrupting chemotherapeutic agent. Many such agents have been developed for therapeutic purposes and are described, for example, in Ong et al., (2020) Cancers 12(1):238. In certain embodiments, the cytoskeleton-disrupting chemotherapeutic agent is nab-paclitaxel ... 2 , 50-200mg / m 2 , 50-150mg / m 2 , 50-100mg / m 2 , 100-300mg / m 2 , 100-200mg / m 2 , 100-150mg / m 2 , 150-300mg / m 2 , 150-200mg / m 2 , or 200 to 300 mg / m 2 of nab-paclitaxel is administered to the subject. In certain embodiments, 80 mg / m 2 , 100 mg / m 2 , 125 mg / m 2 , 200 mg / m 2 , or 260 mg / m 2 of nab-paclitaxel is administered to the subject. In certain embodiments, 100 mg / m 2 of nab-paclitaxel is administered on days 1, 8, and 15 of the first treatment cycle. In certain embodiments, when the subject undergoes one or more subsequent treatment cycles, 100 mg / m 2 of nab-paclitaxel is administered three times every four weeks in subsequent treatment cycles on days 1, 8, and 15 of each subsequent treatment cycle. In certain embodiments, nab-paclitaxel is administered as a 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, or 90 minute intravenous infusion in a four week treatment cycle.

[0343] In certain embodiments, a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion and nab-paclitaxel is administered as a 30, 45, 60, 75, or 90 minute intravenous infusion in a four week treatment cycle. In certain embodiments, a multispecific binding protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein is administered as a 60 minute intravenous infusion and nab-paclitaxel is administered as a 30 minute intravenous infusion in a four week treatment cycle.

[0344] Treatment outcomes In certain embodiments, the methods of treatment disclosed herein result in a disease response or improved survival of the subject or patient. For example, in certain embodiments, the disease response is a complete response, a partial response, or stable disease. In certain embodiments, the improved survival is improved progression-free survival (PFS) or overall survival. The improvement (e.g., in PFS) can be determined relative to a period prior to the initiation of the treatment of the present disclosure. Methods for determining disease response (e.g., complete response, partial response, or stable disease) and patient survival (e.g., PFS, overall survival) for BTC (e.g., progressive BTC, metastatic BTC) or bile duct tumor treatment are routine in the art and are contemplated herein. In some embodiments, disease response is assessed according to RECIST 1.1 after subjecting treated patients to contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) of the affected area (e.g., chest / abdomen and pelvis covering the area from the superior extent of the thoracic inlet to the pubic symphysis). EXAMPLES

[0345] The disclosure, having now been generally described, will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the disclosure and are not intended to limit the scope of the disclosure in any way.

[0346] Example 1: Formulation, packaging, and storage of A49-F3'-TriNKET-Trastuzumab. The formulations listed in Table 12 were evaluated in duplicate and randomized to evaluate the effect of pH and excipients on the stability of AA49-F3'-TriNKET-Trastuzumab (Kermit BDS Lot 7443-C3, 11.9 mg / mL). A49-F3'-TriNKET-Trastuzumab underwent buffer exchange into the respective buffer and excipient combinations by centrifugal ultrafiltration (Amicon Ultra-4 30k device MWCO) to a target concentration of 30 mg / mL. After final buffer exchange and confirmation of target concentration, each formulated sample was filtered using a 0.22 μm EMD Millipore Ultrafree-CL centrifugal filter device with Durapore membrane (Fisher Scientific catalog # UFC40GVOS). After sterile filtration, each formulation was processed aseptically in a laminar flow hood. Polysorbate 80 (PS80) was added to the formulated samples to a final concentration of 0.01%. An aliquot of each formulation was removed for time zero testing and the remaining material was divided into two equal size aliquots in 2mL x 13mm depyrogenated type 1 borosilicate glass vials (West Pharmaceuticals, catalog number 68000377), stoppered and sealed with 13mm Fluorotec stoppers (West Pharmaceuticals, catalog number 19700302). The time zero aliquot was used for the first time point testing per Table 13. One vial was stored at 2-8°C and the other was placed at 50°C for a 3 week accelerated stability study. After 3 weeks of incubation, the 2-8°C and 50°C samples were analyzed according to the test method shown in Table 13. [Table 12] [Table 13]

[0347] An accelerated stability study of A49-F3'-TriNKET-Trastuzumab was performed, where A49-F3'-TriNKET-Trastuzumab was prepared in 20 formulations, as shown in Table 12. Samples were run in duplicate and incubated at 2-8°C and 50°C for 3 weeks. At time zero and at the end of the 3 week incubation, testing of each formulated sample was performed using the assays outlined in Table 13. All formulations behaved similarly and were as expected based on appearance, concentration, pH, and osmolality.

[0348] exterior The samples were viewed at ambient laboratory conditions against a black and white background prior to opening the sample vial. No visible particles were present in all samples at both the time zero and three week conditions.

[0349] UV concentration determination Protein concentration by optical density (OD) and ultraviolet (UV) absorbance at 280 nm was determined for each sample and condition. Protein concentrations at time zero, after 3 weeks of incubation at 2-8°C, and after 3 weeks of incubation at 50°C are summarized in Table 14.

[0350] pH determination The pH was determined for each sample and condition. The pH values ​​at time zero, after 3 weeks of incubation at 2-8° C., and after 3 weeks of incubation at 50° C. are summarized in Table 15.

[0351] Dynamic Light Scattering Dynamic light scattering (DLS) samples were collected at 25° C. after 300 seconds of equilibration. Five measurements were collected for each sample. The Z-average values ​​at time zero, after 3 weeks of incubation at 2-8° C., and after 3 weeks of incubation at 50° C. are summarized in Table 16.

[0352] The average polydispersity index (%PDI) was also recorded. The %PDI values ​​at time zero, after 3 weeks of incubation at 2-8° C., and after 3 weeks of incubation at 50° C. are summarized in Table 17.

[0353] Further DLS analysis of A49-F3'-TriNKET-Trastuzumab in the samples was performed. The average percentage of monomer polydispersity (PD%) at time zero, after 3 weeks of incubation at 2-8°C, and after 3 weeks of incubation at 50°C are summarized in Table 18. The average monomer size values ​​at time zero, after 3 weeks of incubation at 2-8°C, and after 3 weeks of incubation at 50°C are summarized in Table 19. [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19]

[0354] All A49-F3'-TriNKET-Trastuzumab formulations exhibited conformational stability, with mean and monomer sizes ≦20 nm and polydispersity index (PDI) ≦0.300, as assessed by DLS. When evaluating excipient and pH combinations, sorbitol and sucrose only formulations had lower mean sizes compared to combination formulations NaCl and sorbitol, and NaCl and sucrose upon incubation at 2-8° C. and 50° C. for 3 weeks (FIGS. 18A and 18B). Mean monomer size was also lower in sorbitol and sucrose only formulations compared to combination formulations with NaCl after incubation at 2-8° C. and 50° C. for 3 weeks (FIGS. 19A and 19B).

[0355] Size Exclusion Chromatography (SEC) Size exclusion chromatography was performed to determine the percentage of high molecular weight species (%HMW), the percentage of major species (%Major), and the percentage of low molecular weight species (%LMW). Samples were diluted to 2.0 mg / mL in mobile phase buffer (containing 100 mM phosphate, 150 mM sodium chloride pH 7.3) and injected at a 100 μg load. Separation was performed using a Tosoh G3000SWxl (7.8×300 mm, catalog no. 08541) column with detection at 280 nm with an 8 nm bandwidth. Samples were analyzed at time zero, in real time, and after 3 weeks of incubation at either 2-8° C. or 50° C. The %HMW values ​​are summarized in Table 20, the %Major values ​​are summarized in Table 21, and the %LMW values ​​are summarized in Table 22. [Table 20] [Table 21] [Table 22]

[0356] After 3 weeks of incubation at 50°C, the formulated samples had main peak values ​​ranging from 91.3% to 95.2%, with formulations with only sorbitol and sucrose maintaining a greater main peak % and a lower HMW species % compared to the combined excipients NaCl and sorbitol, and NaCl and sucrose (shown in Figure 20A and Figure 20B, respectively). Importantly, both single excipients sucrose and sorbitol maintained the % main peak species over the pH range 5.5-6.5, and both exhibited only a slightly elevated % HMW species as pH increased from 5.5 to 6.5. The % LMW species tended to be lower as pH increased from 5.5 to about 6.2 for all excipients (Figure 22A).

[0357] After 3 weeks of incubation at 2-8°C, all formulated samples maintained a percentage of the main species peak above 98%. The main peak % was greater for lower pH values ​​(5.5) versus higher pH values ​​(pH 6.5), as shown in Figure 20B. The single excipients sorbitol and sucrose tended toward lower HMW % compared to the combined excipients NaCl and sorbitol, and NaCl and sucrose. For all excipients, increased pH tended toward increased HMW species % (Figure 20B). The LMW species % tended to be lower for the combined excipients, but was pH independent (Figure 22B).

[0358] Osmolality Osmolality of all samples was measured by freezing point depression. Osmolality was maintained for all samples across all conditions. Osmolality data at time zero, after 3 weeks of incubation at 2-8°C, and after 3 weeks of incubation at 50°C are summarized in Table 23. [Table 23]

[0359] Imaging capillary isoelectric focusing (icIEF) Imaging capillary isoelectric focusing (icIEF) was used to determine charge variant analysis. Charge heterogeneity was assessed by diluting starting materials and samples to 5 mg / mL in water and then combining with a master mix in a ratio of 10 μL sample to 90 μL master mix. The master mix was a combination of 1% methylcellulose, Pharmalyte 3-10, Pharmalyte 8-10.5, pI marker 5.12, pI marker 9.50, and DI water. System suitability standards were prepared and run prior to running the samples, which were run in a 96-well plate format. The method parameters utilized were as follows: focusing period #1 = 1 min, 1500 V, focusing period #2 = 8 min, 3000 V, detection = 5 exposures, sample load = 55 sec, lower pI marker = 5.12, 300 pixels, upper pI marker = 9.50, 1800 pixels. Starting material was run every 18 injections to ensure consistent readings.

[0360] The "main peak" was identified as the main peak in the formulated sample at time zero. After incubation, the peak with the same elution time would have decreased and would no longer represent the peak with the largest area under the curve, but would still have been identified as the "main peak". The percentage of protein present in the acidic fraction (Acid %) values ​​after 3 weeks of incubation at 2-8°C and after 3 weeks of incubation at 50°C are summarized in Table 24. The percentage of protein present in the main peak fraction (Main Peak %) values ​​are summarized in Table 25. The percentage of protein present in the basic fraction (Basic %) values ​​are summarized in Table 26. [Table 24] [Table 25] [Table 26]

[0361] For formulated samples after 3 weeks of incubation at 2-8°C, the % main peak value ranged from 58.3% to 62.2%, the % acidic value ranged from 34.3% to 38.4%, and the % basic value ranged from 3.3% to 3.7%. No significant model was fitted to the % main peak data, indicating that neither pH nor excipients had a significant effect on icIEF values ​​(Figures 24B-24D). Excipients were also not significant in modeling the % acidic and % basic species, with the % acidic species tending to increase with pH while the % basic species decreased (Figures 23B and 25B). Changes were however slight as observed by the narrow ranges for all values.

[0362] For formulated samples after 3 weeks of incubation at 50°C, the % main peak value ranged from 14.9% to 22.7%, the % acidic value ranged from 70.6% to 81.6%, and the % basic value ranged from 2.4% to 8.8%. The data show a shift from the % main peak species to the % acidic species, with the % basic remaining relatively consistent with the results from the 3 weeks of 2-8°C incubation. In evaluating the 3 weeks of 50°C formulated samples, the sample with sucrose as the only excipient had the highest % acidic species (Figure 23A) as well as the lowest % main peak and % basic species (Figures 24A and 25A). This was consistent across all pH values ​​(5.5-6.5), while formulations with the other three excipients trended toward lower % acidic species at lower pH values ​​that increased with increasing pH.

[0363] Capillary electrophoresis (CE) Reduced capillary gel electrophoresis was performed to assess purity. SDS-CGE was assessed using a Sciex PA800+ with UV detection at 220 nm. Samples were prepared by diluting 100 μg of sample in Beckman SDS sample buffer and adding 5 μL of β-mercaptoethanol. Samples were heated at 70° C. for 10 minutes. Separation occurred for 20 minutes using normal polarity, a 1 minute ramp, 15 kV voltage, and 20 psi pressure. The capillary length was 30.2 cm and the detector length was 10.2 cm. Starting material was used as a reference. A summary of sample purity percentages is shown in Table 27. A summary of sample impurity percentages is shown in Table 28. [Table 27] [Table 28]

[0364] Non-reducing capillary gel electrophoresis was also performed to assess purity. SDS-CGE was assessed using a Sciex PA800+ with UV detection at 220 nm. Samples were prepared by diluting 100 μg of sample in Beckman SDS sample buffer and adding 5 μL of 250 mM iodoacetamide. Samples were heated at 70° C. for 10 minutes. Separation occurred for 20 minutes using normal polarity, a 1 minute ramp, 15 kV voltage, and 20 psi pressure. The capillary length was 30.2 cm and the detector length was 10.2 cm. Starting material was used as a reference. A summary of the % HMW CE (NR) data is shown in Table 29. A summary of the % Main Peak CE (NR) data is shown in Table 30. A summary of the % LMW CE (NR) data is shown in Table 31. [Table 29] [Table 30] [Table 31]

[0365] As assessed by reduced CE, % purity values ​​for sorbitol-only and sucrose-only formulations among the 3 week 50° C. samples were maintained across the pH range (pH 5.5-6.5) (FIG. 26A), while the combination excipients exhibited more variability with respect to pH in that the % purity decreased at lower pH values ​​(5.5 vs. 6.5) (FIG. 26B).

[0366] As assessed by non-reduced CE, formulations containing only sorbitol or only sucrose among the 3 week 50°C samples only had lower % HMW species compared to the combined excipients NaCl and sorbitol and NaCl and sucrose (Figures 27A and 28A). pH level had no significant effect on the % main peak value.

[0367] As assessed by reduced and non-reduced CE, for the 3 week 2-8 °C samples, there were no significant models that fit the reduced CE data, and for the non-reduced CE data, the sorbitol and sucrose only formulations had a greater % of the main peak species (Figure 27B).

[0368] statistical analysis Trends were analyzed using Design Expert v9 software. A summary of the analysis is shown in Table 32. Models in bold were not included in the final optimization evaluation. [Table 32]

[0369] Excipient Selection The performance of formulations containing 250 mM sorbitol or 250 mM sucrose as excipients is more favorable than formulations containing a combination of sorbitol and NaCl or a combination of sucrose and NaCl. Thus, the optimal formulation for A49-F3'-TriNKET-Trastuzumab was determined to be 20 mM histidine, 250 mM sucrose or sorbitol, and 0.01% PS80 at pH 6.0.

[0370] Example 2. High concentration formulation of A49-F3'-TriNKET-Trastuzumab. The feasibility of a high concentration A49-F3'-TriNKET-Trastuzumab formulation was evaluated in two phases: evaluation of the solubility limits and short-term stability of the formulation.

[0371] To assess solubility, A49-F3'-TriNKET-Trastuzumab drug substance from the ultrafiltration / diafiltration (UF / DF) load was exchanged into a pH 6 buffer of 20 mM histidine and 250 mM sucrose used in the clinical trial described in Example 3, identical to the pharmaceutical formulation selected in Example 1, but without polysorbate-80. A49-F3'-TriNKET-Trastuzumab was then concentrated to approximately 233 mg / mL and held at both 5°C and 25°C with intermittent checks on A49-F3'-TriNKET-Trastuzumab protein concentration. Table 33 shows the average concentration of A49-F3'-TriNKET-Trastuzumab at each storage condition at all time points. [Table 33]

[0372] The A49-F3'-TriNKET-Trastuzumab concentration settled to approximately 222-223 mg / mL after 48 hours in each condition and remained at 222-223 mg / mL after 120 hours (Figure 30), suggesting that the solubility limit of A49-F3'-TriNKET-Trastuzumab in this formulation is approximately 222 mg / mL.

[0373] Short-term stability was evaluated by preparing samples as described above up to protein concentrations of 150 mg / mL, 175 mg / mL, and 200 mg / mL. Solutions at each concentration were spiked with 0.01% (w / v) polysorbate-80, the same as the pharmaceutical formulation selected in Example 1 and used in the clinical trial described in Example 3. Two additional solutions at a concentration of 150 mg / mL were prepared with 0.3% (w / v) polysorbate-80 and 0.05% (w / v) polysorbate-80, respectively, to evaluate the effect of polysorbate-80 concentration on the stability of high-concentration A49-F3'-TriNKET-Trastuzumab. Each condition was stored at 5° C., 25° C., and 40° C. and sampled weekly for limited product quality testing consisting of appearance (FIG. 31A), A49-F3′-TriNKET-Trastuzumab concentration, and purity by size-exclusion high performance liquid chromatography (SE-HPLC) (FIG. 31B). The results demonstrate that A49-F3′-TriNKET-Trastuzumab remains stable in these formulations at both 5° C. and 25° C. for over two weeks.

[0374] Example 3: Treatment of locally advanced or metastatic solid tumors with A49-F3'-TriNKET-Trastuzumab the purpose This clinical trial is designed with two phases: a dose escalation phase and a efficacy, followed by an efficacy expansion cohort phase. The primary objectives of the dose escalation phase of the study are to evaluate the safety and tolerability of A49-F3'-TriNKET-trastuzumab, as well as to determine the maximum tolerated dose of A49-F3'-TriNKET-trastuzumab in patients with advanced (unresectable, recurrent, or metastatic) solid tumors for which no effective standard therapy exists or who have recurrence or are intolerant to standard therapy. The primary objective of the efficacy expansion cohort phase of the study is to evaluate the overall response rate (ORR) according to the modified Response Evaluation Criteria in Solid Tumors version 1.1 (mRECIST 1.1).

[0375] Secondary objectives of this clinical trial: - Characterize the pharmacokinetics of A49-F3'-TriNKET-trastuzumab; -To evaluate the immunogenicity of A49-F3'-TriNKET-trastuzumab and correlate its immunogenicity with its exposure and clinical activity; -Evaluate the duration of response (DOR) of A49-F3'-TriNKET-trastuzumab; -Evaluate best overall response (BOR); -To evaluate progression-free survival (PFS) for A49-F3'-TriNKET-trastuzumab; -Evaluate overall survival (OS) time; and -To evaluate the safety of A49-F3'-TriNKET-trastuzumab in combination with pembrolizumab.

[0376] Study design This is a Phase I / II, open-label, dose-escalation study with sequential parallel-group efficacy extension designed to determine the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of A49-F3'-TriNKET-trastuzumab alone and in combination with pembrolizumab. The study consists of two parts: (1) The dose escalation part (Phase I) is divided into three stages: (A) Accelerated titration; (B) "3+3" dose escalation; and (C) Safety / Pharmacokinetic (PK) / Pharmacodynamic (PD) Expansion Cohort (2) The efficacy expansion cohort part (Phase II) will be divided into the following four cohorts: (A) Urothelial bladder cancer (UBC) (B) Metastatic breast cancer (MBC) (C) Basket solid tumor with high HER2 expression (HER2 3+) (D) Combination treatment with pembrolizumab.

[0377] In an exemplary embodiment, patients enrolled during the dose escalation and efficacy expansion parts (UBC, MBC, or basket [HER2 3+] cohorts) receive A49-F3'-TriNKET-trastuzumab as monotherapy intravenously as a 1-hour infusion in 4-week treatment cycles. For treatment cycle 1, patients receive A49-F3'-TriNKET-trastuzumab on days 1, 8, and 15. For treatment cycle 2 and subsequent cycles, patients receive A49-F3'-TriNKET-trastuzumab once every 2 weeks (e.g., days 1 and 15) until confirmed progression, unacceptable toxicity (as described in this example under the "Dose-Limiting Toxicity (DLT)" section), or any reason for withdrawal from the study or investigational medicinal product (IMP) accrual. Patients enrolled in the combination treatment with the pembrolizumab cohort of the efficacy expansion cohort part will receive A49-F3'-TriNKET-trastuzumab as a 1 hour IV infusion and pembrolizumab as a 30 minute IV infusion in a 3 week treatment cycle. In an exemplary embodiment, 200 mg of pembrolizumab will be administered with A49-F3'-TriNKET-trastuzumab according to its label.

[0378] For treatment cycle 1, patients receive A49-F3'-TriNKET-trastuzumab and pembrolizumab on day 1 and A49-F3'-TriNKET-trastuzumab alone on day 8. For treatment cycle 2 and subsequent cycles, patients receive A49-F3'-TriNKET-trastuzumab and pembrolizumab once every 3 weeks on day 1 of every cycle until confirmed progression, unacceptable toxicity (as described in this example under the section "Dose-Limiting Toxicity (DLT)"), or any reason for withdrawal from the study or occurrence of IMP.

[0379] Patients who experience a confirmed complete response (CR) will be treated for up to 12 months after confirmation, at the discretion of the investigator. Treatment beyond 12 months is acceptable if the patient would benefit from continued treatment.

[0380] Figures 32A and 32B are schematic diagrams of the clinical trial plan. Figure 32A describes the trial design for the dose escalation phase. Figure 32B describes the trial design for the efficacy expansion cohort phase.

[0381] Selection Criteria General inclusion criteria for patients enrolled in any of the cohorts in this clinical trial include: - Signed written informed consent; Age ≥ 18 years (including male and female patients); - Histologically or cytologically proven locally advanced or metastatic solid tumors for which no standard treatment exists or for which standard treatment has failed; - Primary tumor must have documented HER2 expression by immunohistochemistry; - ECOG performance status of 0 or 1 and estimated life expectancy of at least 3 months at the time of study entry; - baseline left ventricular ejection fraction (LVEF) ≥ 55% as measured by echocardiogram (preferred) or multi-gated acquisition (MUGA) scan; Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 / L, lymphocyte count ≥ 0.5 × 10 9 / L, platelet count ≥75×10 9 / L, and white blood cell (WBC) count ≥ 3 × 10 with hemoglobin ≥ 9 g / dL (possibly transfused). 9 adequate hematologic function defined by / L; - Adequate liver function defined by total bilirubin level ≤ 1.5 x upper limit of normal (ULN), aspartate aminotransferase (AST) level ≤ 2.5 x ULN, and alanine aminotransferase (ALT) level ≤ 2.5 x ULN for patients with documented metastatic disease to the liver, AST and ALT levels ≤ 5 x ULN; Adequate renal function as defined by estimated creatinine clearance >50 mL / min according to the Cockcroft-Gault formula, and - Effective contraceptive methods for women of childbearing potential (WOCBP) patients, as defined by WHO guidelines as one "highly effective" method or two "effective" methods.

[0382] Additional inclusion criteria for patients enrolled in the accelerated titration or "3+3" dose escalation phase of the dose escalation part described in this Example include: - Evidence of disease of interest, but measurable lesions are not required; and Availability of an archival tumor biopsy (≤6 months old, at least 8 slides) or fresh biopsy (at least 10 slides and 3 cores) obtained within the screening window.

[0383] Additional inclusion criteria for patients enrolled in the safety / PK / PD expansion cohort phase of the dose escalation part described in this Example included: - A fresh tumor biopsy obtained during the screening window to have a formalin-fixed paraffin-embedded (SIP) paraffin block or sufficient unstained slides to perform IHC with at least 12 whole slides and with at least 3 fresh cores (at least 3 slides unstained); and - HER2 by IHC of at least 1+ at screening.

[0384] Additional inclusion criteria for patients enrolled in the UBC expansion cohort described in this Example include: -Histologically or cytologically documented locally advanced or metastatic transitional cell carcinoma of the urothelium (including renal pelvis, ureter, urothelium, and urethra); - Radiographic disease progression after the subject's last line of treatment; Treatment with one (and not more than one) platinum-containing regimen (e.g., platinum plus another agent, e.g., gemcitabine, methotrexate, vinblastine, doxorubicin, etc.) for inoperable locally advanced or metastatic urothelial carcinoma with radiological progression or recurrence within 6 months after the last dose of an adjuvant platinum-containing regimen, which would be considered a failure of the first-line platinum-containing regimen; - Treatment with checkpoint inhibitors (i.e., anti-PD-1 or anti-PD-L1) with radiological progression (optionally treatment with a combination of platinum-based and PD-1 / PD-L1-based therapy); - HER2 expression of at least 1+ by IHC; and - Availability of fresh tumor biopsies obtained during the screening window to have formalin-fixed paraffin-embedded (FFPE) paraffin blocks or sufficient unstained slides to perform IHC with at least 12 whole slides and with at least 3 fresh cores (at least 3 slides unstained).

[0385] Additional inclusion criteria for patients enrolled in the MBC expansion cohort described in this Example include: - histologically confirmed MBC; - 3 or fewer prior lines of cytotoxic therapy for metastatic disease; - Prior treatment with a taxane and an anthracycline, unless an anthracycline is contraindicated; -Tumor scoring of 1+ or 2+ by IHC, and if scoring is 2+, the presence of tumor amplification of ERRB2 must be determined by an FDA-approved method; - Radiologic progression after last line of systemic therapy; and - Availability of fresh tumor biopsies obtained during the screening window to have formalin-fixed paraffin-embedded (FFPE) paraffin blocks or sufficient unstained slides to perform IHC with at least 12 whole slides and with at least 3 fresh cores (at least 3 slides unstained).

[0386] Additional inclusion criteria for patients enrolled in the basket (HER2 3+) cohort described in this example include: - History of any solid tumor except breast or gastric cancer and ERBB2 amplification within the tumor and one of the following: 1) HER2 3+ scoring by IHC documented on recent biopsy within 6 months, after radiological progression at time of last line of treatment, or 2) HER 3+ scoring by IHC during the screening window; Treatment with at least one approved or established therapy of choice; and - Availability of fresh tumor biopsies obtained during the screening window to have formalin-fixed paraffin-embedded (FFPE) paraffin blocks or sufficient unstained slides to perform IHC with at least 12 whole slides and with at least 3 fre...

Claims

1. A composition comprising a multispecific binding protein for use in treating breast cancer in a subject in need thereof, wherein the breast cancer is positive for a hormone receptor, (i) has a HER2 expression level scored as 2+ as determined by immunohistochemistry (IHC) but does not have ERBB2 gene amplification as determined by an in situ hybridization (ISH) assay; or (ii) has a HER2 expression level scored as 1+ as determined by IHC; wherein the multispecific binding protein is (a) a first antigen-binding site that binds to NKG2D; (b) a second antigen-binding site that binds to HER2; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen-binding site that binds to CD16; A composition comprising:

2. The composition for use described in claim 1, wherein the hormone receptor is an estrogen receptor, a progesterone receptor, or both.

3. A composition for use as described in claim 1 or 2, characterized in that the multispecific binding protein is administered to the subject on days 1, 8, and 15, but not on day 22, of an initial four-week treatment cycle.

4. The composition for use according to claim 3, wherein after the initial treatment cycle, the multispecific binding protein is further administered to the subject in one or more subsequent four-week treatment cycles on days 1 and 15 of each subsequent treatment cycle.

5. The composition for use according to claim 3 or 4, wherein each dose comprises the multispecific binding protein in an amount selected from the group consisting of 5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg administered by intravenous infusion.

6. The composition for use according to any one of claims 3 to 5, further comprising administering to the subject on day 1 of the initial four-week treatment cycle a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein.

7. The composition for use according to claim 6, wherein the corticosteroid is a glucocorticoid selected from the group consisting of methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, and pharma- ceutically acceptable salts thereof.

8. The composition for use according to claim 6 or 7, wherein the infusion-related reactions include cytokine release syndrome, anaphylaxis, chills, fever, hypotension, hypertension, rigors, headache, dizziness, pruritus, sore throat, pharyngeal edema, angioedema, flushing, rash, bronchospasm, tachycardia, bradycardia, atrial fibrillation, hypoxia, dyspnea, shortness of breath, chest tightness, nausea, vomiting, shivering, tremors, pain, fatigue, insomnia, asthenia, hypersensitivity, and diarrhea.

9. A composition for use according to any one of claims 1 to 8, characterized in that chemotherapy is further administered to the subject.

10. The first antigen-binding site that binds to NKG2D, (a) a heavy chain variable domain (VH) comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 188, respectively; and (b) a light chain variable domain (VL) comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively; A composition for use according to any one of claims 1 to 9, comprising: (a) the VH comprises CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96, and 169, respectively; and (b) the VL comprises CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively.

12. The composition for use described in claim 10 or 11, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:

98.

13. The second antigen-binding site that binds to HER2, (a) a VH comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116, and 117, respectively; and (b) a VL comprising the CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120, and 121, respectively; A composition for use according to any one of claims 1 to 12, comprising:

14. The composition for use described in claim 13, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:

196.

15. A composition for use according to any one of claims 1 to 14, wherein the first antigen-binding site that binds to NKG2D is a Fab and the second antigen-binding site that binds to HER2 is a scFv.

16. The composition for use of claim 15, wherein element (c) of the multispecific binding protein is an antibody Fc domain comprising a first antibody Fc sequence linked to the C-terminus of a heavy chain portion of the first antigen-binding site and a second antibody Fc sequence linked to the C-terminus of the second antigen-binding site via a hinge comprising Ala-Ser, and wherein the first and second antibody Fc sequences comprise different mutations that promote heterodimerization.

17. The composition for use of claim 16, wherein the first antibody Fc sequence is a human IgG1 Fc sequence comprising a K360E substitution and a K409W substitution, and the second antibody Fc sequence is a human IgG1 Fc sequence comprising a Q347R substitution, a D399V substitution, and an F405T substitution according to the EU numbering system.

18. The multispecific binding protein, (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:141; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 140; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142.

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