Pharmaceutical formulations and dosage regimens of multi-specific binding protein that binds to her2, NKG2d and CD16 for cancer treatment

Abstract

To provide pharmaceutical formulations and administration regimens for treating cancer comprising a multi-specific binding protein that binds to HER2, NKG2D and CD16.SOLUTION: The disclosure relates to pharmaceutical formulations for multispecific binding proteins having an scFv that binds to epidermal growth factor receptor 2 (ErbB2 or HER2), a Fab that binds to NKG2D, and an antibody Fc domain. Dosage regimens for such multispecific binding proteins and pharmaceutical formulations for the treatment of cancer, such as locally advanced or metastatic solid tumors, are also provided. The disclosure provides pharmaceutical formulations comprising multispecific binding proteins comprising a HER2-binding scFv, a Fab that binds to NKG2D and an antibody Fc domain, of which ingredients are optimized for the stability of the multispecific protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 894,047, filed August 30, 2019, U.S. Provisional Patent Application No. 62 / 895,320, filed September 3, 2019, and U.S. Provisional Patent Application No. 62 / 916,935, filed October 18, 2019, the disclosures of each of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 27, 2020, is named DFY-078WO_SL.txt and is 194,972 bytes in size.

[0003] Field of Disclosure The present disclosure relates generally to pharmaceutical formulations of multispecific binding proteins having an epidermal growth factor receptor 2 (ErbB2 or HER2)-binding single chain variable fragment (scFv) and an NKG2D-binding Fab and an antibody Fc domain, and dosage regimens of such multispecific binding proteins and pharmaceutical formulations for use in treating cancer, such as locally advanced or metastatic solid tumors. [Background technology]

[0004] background Cancer remains a major health problem, despite considerable research efforts and scientific advances reported in the literature for the treatment of this disease. Cancer immunotherapy is being developed to use the patient's own immune system to promote the destruction of cancer cells. Immune cells activated by cancer immunotherapy include T cells and natural killer (NK) cells. For example, bispecific T cell engagers are designed to direct T cells to tumor cells, making them cytotoxic to tumor cells. Bispecific antibodies that bind to NK cells and tumor-associated antigens (TAA) have also been created for cancer treatment (see, for example, WO2016 / 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 approximately 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. In addition, 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 stomach cancers, salivary gland ductal carcinomas, adenocarcinomas of the lung, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. Multispecific binding proteins that bind to HER2 and one or more immune cell surface proteins have been studied. For example, WO2018 / 152518 describes multispecific binding proteins that bind to HER2, NKG2D and CD16. The present disclosure contributes to these developments and provides clinical methods that include dosage regimens for treating patients with specific HER2-targeted cancer immunotherapy with desired stability and efficacy. Furthermore, the present disclosure contributes to the prior development in the field by providing formulations that include such cancer immunotherapy drugs that are sufficiently stable and suitable for administration to patients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 134371 [Patent Document 2] International Publication No. 2018 / 152518 Summary of the Invention [Means for solving the problem]

[0007] Disclosure Summary The present disclosure provides a pharmaceutical formulation comprising a multispecific binding protein having a HER2-binding scFv, an NKG2D-binding Fab and an antibody Fc domain, and the components in the formulation are optimized for the stability of the multispecific binding protein.Dosage regimens for using the multispecific binding protein and pharmaceutical formulation in the treatment of cancer, such as locally advanced or metastatic solid tumors, are also provided.

[0008] Additionally, in one aspect, the disclosure provides a pharmaceutical formulation comprising histidine, polysorbate, a sugar or sugar alcohol, and a multispecific binding protein comprising an antibody Fc domain, a Fab that binds to NKG2D, and a single chain variable fragment (scFv) that binds to HER2, at a pH of 5.5 to 6.5.

[0009] In certain embodiments, the concentration of histidine in the pharmaceutical formulation is 10 to 25 mM. In certain embodiments, the concentration of histidine in the pharmaceutical formulation is about 20 mM.

[0010] In certain embodiments, the sugar or sugar alcohol is a disaccharide. In certain embodiments, the disaccharide is sucrose. In certain embodiments, the sugar or sugar alcohol is a sugar alcohol derived from a monosaccharide. In certain embodiments, the sugar alcohol derived from a monosaccharide is sorbitol. In certain embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is 200-300 mM. In certain embodiments, the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM.

[0011] In certain embodiments, the polysorbate is polysorbate 80. In certain embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is 0.005% to 0.05%. In certain embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01%.

[0012] In certain embodiments, the concentration of NaCl, if present, is about 10 mM or less in the pharmaceutical formulation. In certain embodiments, the concentration of NaCl, if present, is about 1 mM or less in the pharmaceutical formulation.

[0013] In certain embodiments, the pH of the pharmaceutical formulation is 5.8 to 6.2. In certain embodiments, the pH of the pharmaceutical formulation is 5.95 to 6.05.

[0014] In certain embodiments, the concentration of the multispecific binding protein in the pharmaceutical formulation is about 10 to about 20 mg / mL.

[0015] In certain embodiments, greater than 94% of the multispecific binding proteins have a native conformation as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. In certain embodiments, less than 4% of the multispecific binding proteins form high molecular weight complexes as determined by size exclusion chromatography after 3 weeks of incubation at 50° C.

[0016] In another aspect, the present disclosure provides the use of the pharmaceutical preparation disclosed herein in the treatment of cancer.In certain embodiments, the pharmaceutical preparation is diluted with 0.9% NaCl solution before use.

[0017] In another aspect, the disclosure provides a method for treating cancer, comprising administering to a subject in need thereof a multispecific binding protein on days 1, 8, and 15 of an initial 4 week treatment cycle, wherein the multispecific binding protein comprises: (a) a Fab that binds NKG2D; (b) an scFv that binds HER2; and (c) an antibody Fc domain.

[0018] In certain embodiments, the method further comprises administering to the subject the multispecific binding protein in one or more subsequent 4 week treatment cycles after the initial treatment cycle, wherein the multispecific binding protein is administered on days 1 and 15 in each subsequent treatment cycle. In certain embodiments, each of the doses contains 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 by intravenous infusion in an amount selected from the group consisting of 0.52 mg / kg, 1 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 20 mg / kg, and 50 mg / kg.

[0019] In certain embodiments, the multispecific binding protein is used as a monotherapy.

[0020] In certain 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 3 weeks in the subsequent treatment cycles.

[0021] In certain embodiments, cancer is HER2 positive as determined by immunohistochemistry.In certain embodiments, the HER2 level in cancer is scored as at least 1+ as determined by immunohistochemistry.In certain embodiments, the HER2 level in cancer is scored as 2+ or 3+.In certain embodiments, the HER2 level in cancer is scored as 3+.

[0022] In certain embodiments, the cancer has an amplification of the ERBB2 gene. In certain embodiments, the ERBB2 gene amplification is determined by fluorescent in situ hybridization. In certain embodiments, the ERBB2 gene amplification is determined by DNA sequencing.

[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 selected from the group consisting of gastric cancer, colorectal cancer, non-small cell lung cancer (NSCLC), head and neck cancer, biliary tract cancer, glioblastoma, sarcoma, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, squamous cell carcinoma of the skin, prostate cancer, salivary gland cancer, breast cancer, pancreatic cancer, and gallbladder cancer. In certain embodiments, the cancer is urothelial bladder cancer or metastatic breast cancer.

[0024] The following features may be incorporated into any of the above-described embodiments.

[0025] In certain embodiments, the Fab comprises a heavy chain variable domain and a light chain variable domain, where (a) the heavy chain variable domain comprises 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) the light chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100 and 101, respectively.

[0026] In certain embodiments, (a) the heavy chain variable domain comprises the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96 and 169, respectively, and (b) the light chain variable domain comprises 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 heavy chain variable domain of a Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94, and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. In certain embodiments, the heavy chain variable domain of a Fab comprises the amino acid sequence of SEQ ID NO: 94, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 98.

[0027] In certain embodiments, the scFv comprises a heavy chain variable domain and a light chain variable domain, where (a) the heavy chain variable domain comprises the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116 and 117, respectively, and (b) the light chain variable domain comprises 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 heavy chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195, and the light chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 196. In certain embodiments, the heavy chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 195, and the light chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 196.

[0028] In certain embodiments, the light chain variable domain of the scFv is linked to the heavy chain variable domain of the scFv via a flexible linker. In certain embodiments, the flexible linker comprises the amino acid sequence of SEQ ID NO: 143. In certain embodiments, the flexible linker consists of the amino acid sequence of SEQ ID NO: 143. In certain embodiments, the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv.

[0029] In certain embodiments, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In certain embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain.

[0030] In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:139.

[0031] 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 the heavy chain portion of the Fab. In certain embodiments, the scFv is linked to the second antibody Fc sequence via a hinge comprising Ala-Ser.

[0032] In certain embodiments, the first and second antibody Fc sequences each comprise the hinge and CH2 domains of a human IgG1 antibody. In certain embodiments, the first and second antibody Fc sequences each comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a wild-type human IgG1 antibody.

[0033] In certain embodiments, the first and second antibody Fc sequences comprise different mutations that promote heterodimerization.In certain embodiments, the first antibody Fc sequence is a human IgG1 Fc sequence that comprises K360E and K409W substitutions.In certain embodiments, the second antibody Fc sequence is a human IgG1 Fc sequence that comprises Q347R, D399V and F405T substitutions.

[0034] In certain embodiments, the multispecific binding protein comprises (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.

[0035] Other embodiments and details of the present disclosure are provided herein below. [Brief description of the drawings]

[0036] [Figure 1] Figure 1 shows a trispecific antibody (TriNKET) containing a HER2-binding scFv, an NKG2D-targeting Fab, and a heterodimerizing antibody-constant Fc domain that binds CD16 ("F3'-TriNKET" format). In an exemplary embodiment, the Fc domain linked to the Fab fragment contains the mutations K360E and K409W, and the Fc domain linked to the scFv contains the matching mutations Q347R, D399V, and F405T, to form the Fc heterodimer (shown in Figure 1 as a triangular lock and key format for the Fc domains). In another exemplary embodiment, the Fc domain linked to the Fab fragment contains the mutations Q347R, D399V, and F405T, and the Fc domain linked to the scFv contains the matching mutations K360E and K409W, to form the heterodimer.

[0037] [Diagram 2]Figure 2A is an interaction plot of the average size as measured by dynamic light scattering (DLS) after 3 weeks of incubation at 50° C. Figure 2B is an interaction plot of the average size as measured by DLS after 3 weeks of incubation at 2-8° C.

[0038] [Diagram 3] Figure 3A is an interaction plot of monomer size as measured by DLS after 3 weeks of incubation at 50° C. Figure 3B is an interaction plot of monomer size as measured by DLS after 3 weeks of incubation at 2-8° C.

[0039] [Figure 4] Figure 4A is an interaction plot of the % major species as determined by size exclusion chromatography (SEC) for a 3 week incubation at 50° C. Figure 4B is an interaction plot of the % major species as determined by SEC for a 3 week incubation at 2-8° C.

[0040] [Diagram 5] Figure 5A is an interaction plot of percent high molecular weight species (HMW%) as determined by SEC for a 3 week incubation at 50° C. Figure 5B is an interaction plot of % HMW species as determined by SEC for a 3 week incubation at 2-8° C.

[0041] [Figure 6] Figure 6A is an interaction plot of percent low molecular weight species (LMW%) as determined by SEC for a 3 week incubation at 50° C. Figure 6B is an interaction plot of % LMW species as determined by SEC for a 3 week incubation at pH 6.0 at 2-8° C.

[0042] [Figure 7]Figure 7A is an interaction plot of % acidic species as determined by image capillary isoelectric focusing (icIEF) for a 3 week incubation at 50° C. Figure 7B is an interaction plot of % basic species for sucrose alone as determined by icIEF for a 3 week incubation at 4-8° C.

[0043] [Figure 8A-B] Figure 8A is an interaction plot of the % major species as determined by icIEF for 3 weeks incubation at 50° C. Figures 8B-8D are interaction plots of the % major species in sucrose-only formulations as determined by icIEF for 3 weeks incubation at 4° C. at pH 5.5 (Figure 8B), pH 6.0 (Figure 8C) and pH 6.5 (Figure 8D). [Figure 8C-D] Figure 8A is an interaction plot of the % major species as determined by icIEF for 3 weeks incubation at 50° C. Figures 8B-8D are interaction plots of the % major species in sucrose-only formulations as determined by icIEF for 3 weeks incubation at 4° C. at pH 5.5 (Figure 8B), pH 6.0 (Figure 8C) and pH 6.5 (Figure 8D).

[0044] [Figure 9] Figure 9A is an interaction plot of % basic species as determined by icIEF for 3 weeks incubation at 50° C. Figure 9B is an interaction plot of % basic species for sucrose alone as determined by icIEF for 3 weeks incubation at 2-8° C.

[0045] [Figure 10] Figure 10A is an interaction plot of % purity as determined by capillary electrophoresis (CE) for 3 weeks incubation at 50° C. Figure 10B is an interaction plot of % impurities as determined by CE for 3 weeks incubation at 50° C.

[0046] [Figure 11]Figure 11A is an interaction plot of the % major species as determined by capillary electrophoresis (non-reduced) (CE(NR)) for a 3 week incubation at pH 6.0 at 50° C. Figure 11B is an interaction plot of the % major species as determined by CE(NR) for a 3 week incubation at pH 6.0 at 2-8° C.

[0047] [Figure 12] Figure 12A is an interaction plot of % HMW species as determined by CE(NR) for 3 weeks incubation at 50° C. Figure 12B shows an interaction plot of % HMW species as determined by CE(NR) for 3 weeks incubation at 2-8° C.

[0048] [Figure 13] Figure 13A is an interaction plot of % LMW species with sucrose alone as determined by CE(NR) for 3 weeks incubation at 50° C. Figure 13B is an interaction plot of % LMW species with sucrose alone as determined by CE(NR) for 3 weeks incubation at 2-8° C.

[0049] [Figure 14A] Figures 14A-14B are schematic diagrams of the clinical trial design. Figure 14A describes the study design for the dose escalation phase. Figure 14B describes the study design for the efficacy expansion cohort phase. Abbreviations used in the figures include: DL = dose level; Combo PD-1 = combination therapy with pembrolizumab; PK = pharmacokinetics; PD = pharmacodynamics; Her2 HIGH = high expression of HER2 by immunohistochemistry of 3+; MBC HER2 2+ / 1+ = metastatic breast cancer with intermediate / low expression of HER2 by immunohistochemistry of 2+ / 1+; UBC 2L / 3L = second / third line treatment of urothelial bladder cancer. [Figure 14B]Figures 14A-14B are schematic diagrams of the clinical trial design. Figure 14A describes the study design for the dose escalation phase. Figure 14B describes the study design for the efficacy expansion cohort phase. Abbreviations used in the figures include: DL = dose level; Combo PD-1 = combination therapy with pembrolizumab; PK = pharmacokinetics; PD = pharmacodynamics; Her2 HIGH = high expression of HER2 by immunohistochemistry of 3+; MBC HER2 2+ / 1+ = metastatic breast cancer with intermediate / low expression of HER2 by immunohistochemistry of 2+ / 1+; UBC 2L / 3L = second / third line treatment of urothelial bladder cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description definition To facilitate the understanding of this invention, several terms and phrases are defined below.

[0051] As used herein, the terms "a" and "an" mean "one or more" and include the plural forms unless the context is inappropriate.

[0052] As used herein, the terms "Fab" and "scFv" refer to two different forms of protein fragments, each of which contains an antigen-binding site. As used herein, the term "antigen-binding site" refers to the portion of an immunoglobulin molecule that participates 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, also called "VH" and "VL", respectively. Three highly variable regions within the V regions of the heavy and light chains are called "hypervariable regions", which are interposed between more conserved adjacent regions 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 of immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged 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 called "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, resulting in 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, that uses a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide. All amino acid positions of the heavy or light chain variable regions disclosed herein are numbered according to the Kabat numbering system.

[0053] 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 described in MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin et al., J. Mol. Biol. 262:732-745 (1996). 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 protein of immunological interest. (1991). In certain embodiments, the heavy and 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). CDRH1, CDRH2 and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 represent the light chain CDRs.

[0054] As used herein, the term "pharmaceutical formulation" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0055] 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, ape, equine, bovine, porcine, primate, canine, feline, etc.), and more preferably include humans.

[0056] The terms "treat", "treating" or "treatment" as used in this disclosure, and other grammatical equivalents, are intended to include alleviating, relieving, relieving, or preventing a disease, condition, or symptom, preventing further symptoms, relieving or preventing the underlying metabolic cause of a symptom, inhibiting a disease or condition, such as preventing the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by a disease or condition, or arresting the symptoms of a disease or condition, including prophylaxis. These terms further include obtaining therapeutic and / or prophylactic benefits. Therapeutic benefit refers to the eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, such that the patient experiences improvement even though the patient may still be afflicted by the underlying disorder.

[0057] The term "about" refers to any minimal change in concentration or amount of an agent that does not alter the efficacy of the agent during preparation of a formulation and during treatment of a disease or disorder. In certain embodiments, the term "about" can include ±5%, ±10%, or ±15% of a specified numerical value or data point.

[0058] Ranges may be expressed in this disclosure as "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints for each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It will also be understood that there are several values ​​disclosed in this disclosure, and that each value is also disclosed as "about" that particular value in addition to the value itself. It will also be understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points and ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it will be understood that data points greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are also disclosed, and data points between 10 and 15 are also considered disclosed. It will also be understood that each unit between the two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0059] Throughout this specification, when compositions are described as having, including, or comprising certain components, or when processes and methods are described as having, including, or comprising certain steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0060] In general, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the variable's preceding definition controls.

[0061] Multispecific Binding Proteins The present disclosure provides a pharmaceutical formulation comprising a multispecific binding protein having a HER2-binding scFv, an NKG2D-binding Fab, and an antibody Fc domain, which are components in a formulation optimized for the stability of the multispecific binding protein. Also provided is a dosage regimen for using the multispecific binding protein and pharmaceutical formulation in the treatment of cancer, such as locally advanced or metastatic solid tumors. The multispecific binding protein can bind to HER2 on cancer cells and to NKG2D and CD16 on natural killer cells. Such binding brings cancer cells closer to natural killer cells, which promotes the direct and indirect destruction of cancer cells by natural killer cells.

[0062] The first component of the multispecific binding protein is capable of binding to, but is not limited to, NK cells, NKT cells, γδ T cells and CD8 + The multispecific binding protein binds to NKG2D receptor expressing cells, which may include αβ T cells. When bound to NKG2D, the multispecific binding protein can 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, which may include, but are not limited to, breast cancer, ovarian cancer, esophageal cancer, bladder cancer and gastric cancer, salivary gland ductal carcinoma, lung adenocarcinoma, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. 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.

[0063] 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 antibody Fc sequence (hinge-CH2-CH3) fused via either a linker or an antibody hinge to a heavy chain portion of a Fab comprising a heavy chain variable domain and a CH1 domain. The immunoglobulin light chain comprises a light chain portion of a Fab 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 antibody Fc sequence (hinge-CH2-CH3) fused via either 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 HER2.

[0064] The individual components of the multispecific binding proteins are described in more detail below.

[0065] NKG2D binding site Upon binding to the NKG2D and CD16 receptors on natural killer cells and tumor-associated antigens on cancer cells, the multispecific binding protein can bind to more than one NK activating receptor and block the binding of natural ligands to NKG2D.In certain embodiments, the protein can act as an agonist to NK cells in humans.In some embodiments, the protein can act as an agonist to NK cells in humans and in other species, such as rodents and cynomolgus monkeys.

[0066] Table 1 lists the peptide sequences of heavy and light chain variable domains that can be combined to bind to NKG2D. In some embodiments, the heavy and light chain variable domains are arranged in Fab format. These NKG2D binding domains may differ in terms of their binding affinity to NKG2D, but nevertheless, they all activate human NK cells. Unless otherwise indicated, the CDR sequences provided in Table 1 are determined based on Kabat. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0067] 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 may 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 may 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 may 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 may 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.

[0068] 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 may 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 may 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 may 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 may 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.

[0069] 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 may 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 may 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 may 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 may 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.

[0070] 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 may 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 may 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 may 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 may 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.

[0071] 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 may 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 may 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 may 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 may 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.

[0072] 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 may 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 may 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 may 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 may 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.

[0073] 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 may 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 may 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 may 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 may 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.

[0074] 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 may 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 may 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 may 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 may 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.

[0075] In some embodiments, the 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 may 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 may 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 may 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 may 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.

[0076] 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 may 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 may 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 may 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 may 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.

[0077] 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 may 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 may 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 may 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 may 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.

[0078] 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 may 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 may 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 may 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 may 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.

[0079] Multispecific binding proteins include, but are not limited to, NK cells, γδ T cells and CD8 +It can bind to NKG2D-expressing cells, including αβT cells. 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.

[0080] In certain embodiments, the Fab or multispecific binding protein binds to NKG2D at a concentration of between 2 nM and 120 nM, e.g., between 2 nM and 110 nM, between 2 nM and 100 nM, between 2 nM and 90 nM, between 2 nM and 80 nM, between 2 nM and 70 nM, between 2 nM and 60 nM, between 2 nM and 50 nM, between 2 nM and 40 nM, between 2 nM and 30 nM, between 2 nM and 20 nM, between 2 nM and 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 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 0.5nM to about 1nM, about 1nM to about 2nM, about 2nM to 3nM, about 3nM to 4nM, about 4nM to about 5nM, about 5nM to about 6nM, about 6nM to about 7nM, about 7nM to about 8nM , about 8nM 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 with an affinity of

[0081] 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.

[0082] In some embodiments, the Fab is linked to an antibody Fc sequence. In some embodiments, the heavy chain portion of the Fab is linked to the N-terminus of the antibody Fc sequence.

[0083] HER2 binding site The HER2 binding site of the multispecific binding protein disclosed herein comprises a heavy chain variable domain and a light chain variable domain fused together to form an scFv. Table 2 lists peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to HER2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0084] 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. [ka]

[0085] 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 may 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 may 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 may 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 may 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 comprises the amino acid sequence of SEQ ID NO: 139.

[0086] In some embodiments, the 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 may 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 may 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 may 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 may 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 comprises the amino acid sequence of SEQ ID NO: 189.

[0087] In some embodiments, the 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 may 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 may 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 may 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 may 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 comprises the amino acid sequence of SEQ ID NO: 171.

[0088] Said scFv comprises heavy chain variable domain and light chain variable domain.In some embodiments, heavy chain variable domain and light chain variable domain form disulfide bridge to enhance the stability of scFv.For example, disulfide bridge can be formed between C44 residue of heavy chain variable domain and C100 residue of light chain variable domain, and amino acid position is numbered according to Kabat.

[0089] The VH and VL of the scFv can be positioned in various orientations. In certain embodiments, the VL is located at the N-terminus of the VH. In certain embodiments, the VL is located at the C-terminus of the VH.

[0090] The VH and VL of scFv can be connected via a linker, for example, a peptide linker. In certain embodiments, the peptide linker is a flexible linker. For the amino acid composition of the linker, the peptide is selected to provide flexibility, not interfere with the structure and function of other domains of the protein of the present invention, and have the property of resisting cleavage by proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, VL is located at the N-terminus of VH and is connected to VH via a linker.

[0091] The length of the linker (e.g., a flexible linker) may 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.

[0092] 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) sequence, 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 or consists of 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. In certain embodiments, the linker is a (G4S)4 (SEQ ID NO:203) linker consisting of the sequence of SEQ ID NO:143. [Table 3-1] [Table 3-2]

[0093] 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: 203).

[0094] In some embodiments, the above scFv is linked to the antibody Fc sequence via a hinge sequence. In some embodiments, the hinge comprises the amino acids Ala-Ser. In some other embodiments, the hinge comprises the amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence provides flexibility in binding to the target antigen and can balance flexibility with optimal geometry.

[0095] Fc domain The antibody Fc domain of the multispecific binding protein comprises a first antibody Fc sequence linked to a Fab and a second antibody Fc sequence linked to an scFv. The two antibody Fc sequences pair to form a dimer that binds to CD16.

[0096] In antibody Fc domain, CD16 binding is mediated by hinge region and CH2 domain.For example, in human IgG1, interaction with CD16 is mainly concentrated on amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and carbohydrate residue N-acetyl-D-glucosamine in CH2 domain (see Sondermann et al., Nature, 406 (6793):267-273).Based on known domains, mutations can be selected to enhance or reduce binding affinity to CD16, for example, by using phage display library or yeast surface display cDNA library, or mutations can be designed based on known three-dimensional structure of interaction.

[0097] The assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. The promotion of 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, in the CH3 domain based on human IgG1, mutations can be introduced by incorporating different pairs of amino acid substitutions in these two chains that allow the first polypeptide and the second polypeptide to selectively heterodimerize with each other. All amino acid substitution positions exemplified below are numbered according to the EU index as in Kabat.

[0098] In one scenario, the amino acid substitutions in a first polypeptide replace the 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 the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T) or valine (V), resulting in the larger amino acid substitution (protrusion) fitting into the surface of the smaller amino acid substitution (pocket). For example, one polypeptide can incorporate a T366W substitution and the other can incorporate three substitutions including T366S, L368A and Y407V.

[0099] The antibody heavy chain variable domain of the present invention can be linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region, including hinge, CH2 and CH3 domains, with or without a CH1 domain. 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, IgG2, IgG3 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 of another mammal, such as a rabbit, dog, cat, mouse or horse. One or more mutations can be incorporated into the constant region relative 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, T366L, 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.

[0100] In certain embodiments, mutations that can be incorporated into the CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171 and / or V173. In certain embodiments, mutations that can be incorporated into the Cκ of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174 and / or T164.

[0101] The amino acid substitutions may be selected from the following set of substitutions shown in Table 4. [Table 4]

[0102] Alternatively, the amino acid substitutions can be selected from the set of substitutions shown in Table 5: [Table 5]

[0103] Alternatively, the amino acid substitutions can be selected from the set of substitutions shown in Table 6 below. [Table 6]

[0104] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 7. [Table 7-1] [Table 7-2]

[0105] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 8, in which the position(s) shown in the column for the first polypeptide are replaced by any known negatively charged amino acid, and the position(s) shown in the column for the second polypeptide are replaced by any known positively charged amino acid. [Table 8]

[0106] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 9, in which the position(s) shown in the example first polypeptide are replaced by any known positively charged amino acid, and the position(s) shown in the example second polypeptide column are replaced by any known negatively charged amino acid. [Table 9]

[0107] Alternatively, the amino acid substitutions can be selected from the following set in Table 10. [Table 10]

[0108] When selecting Fc substitutions, one of skill in the art will understand that the first and second polypeptides in Tables 4-10 can correspond to the first and second antibody Fc sequences, respectively. Alternatively, one of skill in the art will understand that the first and second polypeptides in Tables 4-10 can correspond to the second and first antibody Fc sequences, respectively.

[0109] 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 the group consisting of T366, L368, and Y407.

[0110] 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 the group consisting of 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.

[0111] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of 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 an IgG1 constant region at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411.

[0112] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of 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 an IgG1 constant region at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405 and T411.

[0113] 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 the group consisting of 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 the group consisting of T366, N390, K392, K409 and T411.

[0114] 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 the group consisting of 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 the group consisting of L351, D399, S400 and Y407.

[0115] 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 the group consisting of 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 the group consisting of Q347, E357, D399 and F405.

[0116] 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 the group consisting of 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 the group consisting of Y349, K360, Q347 and K409.

[0117] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of 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 an IgG1 constant region at one or more positions selected from the group consisting of D356, E357 and D399.

[0118] 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 the group consisting of 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 the group consisting of K370, K392, K409, and K439.

[0119] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of 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 an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409.

[0120] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of 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 an IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399.

[0121] 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.

[0122] 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.

[0123] 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 T366S, T368A and Y407V substitutions.

[0124] 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, T368A and 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.

[0125] 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.

[0126] 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.

[0127] Alternatively, or in addition, S354C can be introduced into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain to form an artificial disulfide bridge within the interface of the two polypeptides, thereby increasing the structural stability of the heteromultimeric protein. In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that 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 that of the IgG1 constant region by an Y349C substitution.

[0128] When selecting Fc substitutions, one skilled in the art will understand that the "one polypeptide chain" and the "other polypeptide chain" of the above antibody constant region may correspond to a first antibody Fc sequence and a second antibody Fc sequence, respectively. Alternatively, the "one polypeptide chain" and the "other polypeptide chain" of the above antibody constant region may correspond to a second antibody Fc sequence and a first antibody Fc sequence, respectively.

[0129] Exemplary Multispecific Binding Proteins Listed below are examples of TriNKETs that each include a HER2-binding scFv and an NKG2D-binding Fab linked to an antibody constant region, which includes a mutation that allows the heterodimerization of two Fc 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 VL and position 44 of VH, thereby promoting the formation of disulfide bridges between the VH and VL of the scFv. The VL is linked to the N-terminus of the VH via a (G4S)4 linker (SEQ ID NO: 203), and the VH is linked to the N-terminus of the Fc via an Ala-Ser linker. The Ala-Ser linker is included in the elbow hinge region sequence, which balances flexibility with optimal geometry. In certain embodiments, the additional sequence Thr-Lys-Gly can be added to the N-terminus or C-terminus of the Ala-Ser sequence of the hinge. As used herein to describe these exemplary TriNKETs, Fc includes the antibody hinge, CH2 and CH3.

[0130] Thus, each of the TriNKETs described below comprises three polypeptide chains: N-terminus to C-terminus: Chain A, containing the VH, CH1, and Fc of the NKG2D-binding Fab; N-terminus to C-terminus: Chain B comprises a VL of a HER2 binding scFv, a (G4S)4 linker (SEQ ID NO: 203), a VH of a HER2 binding scFv, an Ala-Ser linker, and an Fc; and N-terminus to C-terminus: Chain C, including VL and CL of NKG2D-binding Fab Includes.

[0131] The amino acid sequences of exemplary TriNKETs are summarized in Table 11.

[0132] In certain embodiments, a multispecific binding protein of the disclosure comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first, second, and third polypeptide chains comprise the amino acid sequence of chain A, chain B, and chain C, respectively, of TriNKET disclosed in Table 11. In certain embodiments, the first, second, and third polypeptide chains consist of the amino acid sequence of chain A, chain B, and chain C, respectively, of TriNKET disclosed in Table 11.

[0133] In an exemplary embodiment, to form a heterodimer, the Fc domain linked to the NKG2D-binding Fab fragment comprises the mutations Q347R, D399V and F405T, and the Fc domain linked to the HER2 scFv comprises the matching mutations K360E and K409W. In another exemplary embodiment, the Fc domain linked to the NKG2D-binding Fab fragment comprises the knob mutations T366S, L368A and Y407V, and the Fc domain linked to the HER2-binding scFv comprises the "hole" mutation T366W. In an exemplary embodiment, the Fc domain linked to the NKG2D-binding Fab fragment comprises the S354C substitution in the CH3 domain, which forms a disulfide bond with the Y349C substitution of the Fc linked to the HER2-binding scFv. [Table 11-1] [Table 11-2]

[0134] Specific TriNKETs and their polypeptide chains are described in more detail below. In the amino acid sequence, (G4S)4 (SEQ ID NO:203) and the Ala-Ser linker are shown in bold underline; the Cys residues of the scFv that form the disulfide bridge are shown in bold italics underline; the Fc heterodimerization mutations are shown in bold underline; the CDR sequences according to Kabat are underlined.

[0135] For example, the TriNKET of the present disclosure is A49-F3'-TriNKET-Trastuzumab. A49-F3'-TriNKET-Trastuzumab comprises a single chain variable region fragment (scFv) (SEQ ID NO: 139) from Trastuzumab that binds to HER2, linked to an Fc domain via a hinge that includes Ala-Ser; and an NKG2D-binding Fab fragment from A49, comprising a heavy chain portion that includes a heavy chain variable domain (SEQ ID NO: 94) and a CH1 domain, and a light chain portion that includes a light chain variable domain (SEQ ID NO: 98) and a light chain constant domain, wherein the heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the Fc domain. A49-F3'-TriNKET-Trastuzumab comprises three polypeptides having the sequences of SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142.

[0136] SEQ ID NO: 140 represents the full sequence of a HER2-binding scFv (scFv-Fc) linked to an Fc domain via a hinge containing Ala-Ser. The Fc domain linked to the scFv contains Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution for disulfide bond formation with the Y349C substitution of SEQ ID NO: 141 described below. The scFv (SEQ ID NO: 139) comprises a heavy chain variable domain of trastuzumab connected to the N-terminus of a light chain variable domain of trastuzumab via a (G4S)4 linker (SEQ ID NO: 203), and the scFv is represented as VL-(G4S)4-VH ("(G4S)4" is represented by SEQ ID NO: 203 or SEQ ID NO: 143). Additionally, the heavy and light chain variable domains of the scFv are 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 [ka] Trastuzumab scFv-Fc (RVT) [ka]

[0137] SEQ ID NO: 141 represents the heavy chain portion of a Fab fragment, which contains the heavy chain variable domain of the NKG2D binding site (SEQ ID NO: 94) and the CH1 domain connected to the Fc domain. The Fc domain of SEQ ID NO: 141 contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution of the Fc (SEQ ID NO: 140) linked to the HER2-binding scFv. In SEQ ID NO: 141, the Fc domain also contains K360E and K409W substitutions for heterodimerization with the Fc of SEQ ID NO: 140. A49 VH [ka] A49 VH-CH1-Fc(EW) [ka]

[0138] 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 [ka] A49 VL-LC [ka]

[0139] Another TriNKET of the present disclosure is A49MI-F3'-TriNKET-Trastuzumab, which comprises the same Her2-binding scFv (SEQ ID NO: 139) as A49-F3'-TriNKET-Trastuzumab linked to the Fc domain via a hinge containing Ala-Ser; and an NKG2D-binding Fab fragment from A49MI, comprising 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, wherein the heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the Fc domain. 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).

[0140] SEQ ID NO: 145 represents the heavy chain portion of a Fab fragment, comprising the heavy chain variable domain of the NKG2D binding site (SEQ ID NO: 144) and the CH1 domain connected to the Fc domain. 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 [ ] in SEQ ID NO: 144 and SEQ ID NO: 145). The Fc domain of SEQ ID NO: 145 comprises a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution of the Fc (SEQ ID NO: 140) linked to the HER2-binding scFv. In SEQ ID NO: 145, the Fc domain also comprises K360E and K409W substitutions. A49MI VH [ka] A49MI VH-CH1-Fc(EW) [ka]

[0141] Another TriNKET of the present disclosure is A49-F3'-KiH-TriNKET-Trastuzumab. KiH refers to knob-into-hole (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 is the replacement of small residues with bulky residues (e.g., T366W in EU numbering). CH3A The first step was to introduce a "knob" into one of the CH3 domains (CH3A) by replacing the adjacent residues closest to the knob with smaller residues (e.g., T366S / L368A / Y407V CH3B ) to generate a complementary "hole" face on the other CH3 domain (CH3B). The "hole" mutations were 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 variant with improved affinity for FcγRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the core interface between the CH3 domains, while the knob-knob and hole-hole interfaces disfavor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

[0142] A49-F3'-KiH-TriNKET-Trastuzumab contains the same Her2-binding scFv (SEQ ID NO: 139) as A49-F3'-TriNKET-Trastuzumab, linked via a hinge containing Ala-Ser to an Fc domain containing the "hole" substitutions T366S, L368A and Y407V; and the same NKG2D-binding Fab fragment as A49-F3'-TriNKET-Trastuzumab, in which the CH1 domain is connected to an Fc domain containing the "knob" substitution T366W. A49-F3'-KiH-TriNKET-Trastuzumab contains three polypeptides having the sequences SEQ ID NO: 146, SEQ ID NO: 147 and SEQ ID NO: 142 (as in A49-F3'-TriNKET-Trastuzumab).

[0143] SEQ ID NO: 146 represents the full sequence of a HER2-binding scFv (SEQ ID NO: 139) linked to an Fc domain via a hinge containing Ala-Ser (scFv-Fc). The Fc domain linked to the scFv contains T366S, L368A and Y407V substitutions for heterodimerization, and an S354C substitution to form a disulfide bond with the Y349C substitution of SEQ ID NO: 147 described below. Trastuzumab scFv-Fc(KiH) [ka]

[0144] SEQ ID NO: 147 represents the heavy chain portion of a Fab fragment, comprising the heavy chain variable domain of the NKG2D binding site from A49 (SEQ ID NO: 94) and the CH1 domain connected to the Fc domain. The Fc domain of SEQ ID NO: 147 comprises an S354C substitution, which forms a disulfide bond with the Y349C substitution in the CH3 domain of the Fc (SEQ ID NO: 146) linked to the HER2-binding scFv. In SEQ ID NO: 147, the Fc domain also comprises a T366W substitution. A49 VH-CH1-Fc(KiH) [ka]

[0145] Another TriNKET of the present disclosure is A49MI-F3'-KiH-TriNKET-Trastuzumab, which comprises the same Her2-binding scFv (SEQ ID NO: 139) as A49-F3'-TriNKET-Trastuzumab linked via a hinge containing Ala-Ser to an Fc domain containing the "hole" substitutions T366S, L368A and Y407V; and the same NKG2D-binding Fab fragment as A49MI-F3'-TriNKET-Trastuzumab, in which the CH1 domain is connected to an Fc domain containing 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).

[0146] SEQ ID NO: 194 represents the heavy chain portion of a Fab fragment, comprising the heavy chain variable domain of the NKG2D binding site from A49MI (SEQ ID NO: 144) and the CH1 domain connected to the Fc domain. The Fc domain of SEQ ID NO: 194 comprises an S354C substitution, which forms a disulfide bond with the Y349C substitution in the CH3 domain of the Fc (SEQ ID NO: 146) linked to the HER2-binding scFv. In SEQ ID NO: 194, the Fc domain also comprises a T366W substitution. A49MI VH-CH1-Fc(KiH) [ka]

[0147] 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 A49-F3'-TriNKET-Trastuzumab linked to the Fc domain via a hinge comprising Ala-Ser; and an NKG2D-binding Fab fragment from A44, comprising a heavy chain portion comprising a heavy chain variable domain (SEQ ID NO: 86) and a CH1 domain, and a light chain portion comprising a light chain variable domain (SEQ ID NO: 90) and a light chain constant domain, wherein the heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the Fc domain. 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.

[0148] SEQ ID NO: 155 represents the heavy chain variable domain of the NKG2D binding site from A44 (SEQ ID NO: 86) connected to the Fc domain. The Fc domain of SEQ ID NO: 155 contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution in the Fc (SEQ ID NO: 140) linked to the HER2-binding scFv. In SEQ ID NO: 155, the Fc domain also contains K360E and K409W substitutions. A44 VH [ka] A44 VH-CH1-Fc(EW) [ka]

[0149] 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. A44 VL [ka] A44 VL-CL [ka]

[0150] 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 A49-F3'-TriNKET-Trastuzumab linked via a hinge containing Ala-Ser to an Fc domain containing the "hole" substitutions T366S, L368A and Y407V; and the same NKG2D-binding Fab fragment as A44-F3'-TriNKET-Trastuzumab, in which the CH1 domain is connected to an Fc domain containing the "knob" substitution 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).

[0151] SEQ ID NO: 148 represents the heavy chain variable domain of the NKG2D binding site from A44 (SEQ ID NO: 86) connected to an Fc domain. The Fc domain of SEQ ID NO: 148 contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with the S354C substitution in the Fc (SEQ ID NO: 146) linked to the HER2-binding scFv. In SEQ ID NO: 148, the Fc domain also contains a T366W substitution. A44 VH-CH1-Fc(KiH) [ka]

[0152] Another TriNKET of the present disclosure is A49-F3'-TriNKET-Pertuzumab. A49-F3'-TriNKET-Pertuzumab comprises a HER2-binding scFv (SEQ ID NO: 189) from Pertuzumab linked to an Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment comprises 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).

[0153] SEQ ID NO: 190 represents the full sequence of a HER2 binding scFv (scFv-Fc) linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv comprises Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution for forming a disulfide bond with the Y349C substitution of SEQ ID NO: 141 above. 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: 203), and the scFv is represented as VL-(G4S)4-VH ("(G4S)4" is represented by SEQ ID NO: 203 or SEQ ID NO: 143). Additionally, the heavy and light chain variable domains of the scFv are 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 [ka] Pertuzumab scFv-Fc [ka]

[0154] 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 A49-F3'-TriNKET-Pertuzumab, linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49MI-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment comprises 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).

[0155] 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 A49-F3'-TriNKET-Pertuzumab linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises the "hole" substitutions of T366S, L368A and Y407V, and the Fc domain linked to the Fab fragment comprises 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).

[0156] SEQ ID NO: 191 represents the full sequence of a HER2 binding scFv (SEQ ID NO: 189) linked to an Fc domain via a hinge containing Ala-Ser (scFv-Fc). The Fc domain linked to the scFv contains T366S, L368A and Y407V substitutions for heterodimerization, and an S354C substitution to form a disulfide bond with the Y349C substitution of SEQ ID NO: 191 above. Pertuzumab scFv-Fc(KiH) [ka]

[0157] 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 A49-F3'-TriNKET-Pertuzumab linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49MI-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises the "hole" substitutions of T366S, L368A and Y407V, and the Fc domain linked to the Fab fragment comprises 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).

[0158] 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 A49-F3'-TriNKET-Pertuzumab, linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A44-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment comprises 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).

[0159] 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 A49-F3'-TriNKET-Pertuzumab linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A44-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv comprises the "hole" substitutions of T366S, L368A and Y407V, and the Fc domain linked to the Fab fragment comprises 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).

[0160] Another TriNKET of the present disclosure is A49-F3'-TriNKET-MGAH22. A49-F3'-TriNKET-MGAH22 comprises a scFv (SEQ ID NO: 171) from MGAH22 that binds HER2 linked to an Fc domain via a hinge that includes Ala-Ser; and the same NKG2D-binding Fab fragment as A49-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment contains 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).

[0161] SEQ ID NO: 192 represents the full sequence of a HER2 binding scFv (scFv-Fc) linked to an Fc domain via a hinge comprising Ala-Ser. The Fc domain linked to the scFv comprises Q347R, D399V and F405T substitutions for heterodimerization, and an S354C substitution for disulfide bond formation with the Y349C substitution of SEQ ID NO: 141 above. 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: 203), and the scFv is represented as VL-(G4S)4-VH ("(G4S)4" is represented by SEQ ID NO: 203 or SEQ ID NO: 143). Additionally, the heavy and light chain variable domains of the scFv are connected through a disulfide bridge between C100 of VL and C44 of VH as a result of G100C and G44C substitutions in VL and VH, respectively. MGAH22 scFv [ka] MGAH22 scFv-Fc [ka]

[0162] Another TriNKET of the present disclosure is A49MI-F3'-TriNKET-MGAH22. A49MI-F3'-TriNKET-MGAH22 contains the same Her2-binding scFv (SEQ ID NO: 171) as A49-F3'-TriNKET-MGAH22 linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49MI-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment contains 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).

[0163] Another TriNKET of the present disclosure is A49-F3'-KiH-TriNKET-MGAH22. A49-F3'-KiH-TriNKET-MGAH22 contains the same Her2-binding scFv (SEQ ID NO: 171) as A49-F3'-TriNKET-MGAH22 linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains the "hole" substitutions of T366S, L368A, and Y407V, and the Fc domain linked to the Fab fragment contains 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).

[0164] SEQ ID NO: 193 represents the full sequence of a HER2-binding scFv (SEQ ID NO: 171) linked to an Fc domain via a hinge containing Ala-Ser (scFv-Fc). The Fc domain linked to the scFv contains T366S, L368A and Y407V substitutions for heterodimerization, and an S354C substitution to form a disulfide bond with the Y349C substitution of SEQ ID NO: 147 above. MGAH22 scFv-Fc(KiH) [ka]

[0165] Another exemplary TriNKET of the present disclosure is A49MI-F3'-KiH-TriNKET-MGAH22. A49MI-F3'-KiH-TriNKET-MGAH22 contains the same Her2-binding scFv (SEQ ID NO: 171) as A49-F3'-TriNKET-MGAH22 linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A49MI-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains the "hole" substitutions of T366S, L368A, and Y407V, and the Fc domain linked to the Fab fragment contains 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).

[0166] Another exemplary TriNKET of the present disclosure is A44-F3'-TriNKET-MGAH22. A44-F3'-TriNKET-MGAH22 contains the same Her2-binding scFv (SEQ ID NO: 171) as A49-F3'-TriNKET-MGAH22 linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A44-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains Q347R, D399V and F405T substitutions, and the Fc domain linked to the Fab fragment contains 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).

[0167] Another exemplary TriNKET of the present disclosure is A44-F3'-KiH-TriNKET-MGAH22. A44-F3'-KiH-TriNKET-MGAH22 contains the same Her2-binding scFv (SEQ ID NO: 171) as A49-F3'-TriNKET-MGAH22 linked to the Fc domain via a hinge containing Ala-Ser; and the same NKG2D-binding Fab fragment as A44-F3'-TriNKET-Trastuzumab, with the CH1 domain connected to the Fc domain. The Fc domain linked to the scFv contains the "hole" substitutions of T366S, L368A, and Y407V, and the Fc domain linked to the Fab fragment contains 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).

[0168] In certain embodiments, the TriNKET of the present disclosure is identical to one of the above exemplary TriNKETs containing the EW-RVT Fc mutation, except that the Fc domain linked to the NKG2D-binding Fab fragment to form a heterodimer contains the substitutions Q347R, D399V, and F405T, and the Fc domain linked to the HER2-binding scFv contains the matching substitutions K360E and K409W. In certain embodiments, the TriNKET of the present disclosure is identical to one of the above exemplary TriNKETs containing the KiH Fc mutation, except that the Fc domain linked to the NKG2D-binding Fab fragment to form a heterodimer contains the "hole" substitutions T366S, L368A, and Y407V, and the Fc domain linked to the HER2-binding scFv contains the "knob" substitution T366W.

[0169] In certain embodiments, a TriNKET of the present disclosure is identical to one of the exemplary TriNKETs described above, except that the Fc domain linked to the NKG2D-binding Fab fragment contains a S354C substitution in the CH3 domain and the Fc domain linked to the HER2-binding scFv contains a matching Y349C substitution in the CH3 domain to form a disulfide bond.

[0170] As described in International 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 moderate 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-cancerous human cells (e.g., human cardiomyocytes).

[0171] Production of multispecific binding proteins The above multispecific binding protein can be produced by using recombinant DNA technology 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; the first, second and third expression vectors can be stably transfected together into host cells to produce a multimeric protein.

[0172] One of skill in the art will appreciate that during production and / or storage of a protein, the N-terminal glutamic acid (E) or glutamine (Q) may 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 having E or Q replaced with pyroglutamate is also contemplated herein.

[0173] One of skill in the art will also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein may be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed in proteins that contain an Fc domain at the C-terminus. Thus, in some embodiments where the C-terminal residue of a polypeptide's amino acid sequence (eg, an Fc domain sequence) is K, the corresponding amino acid sequence in which the K is removed is also contemplated herein.

[0174] To achieve the highest yield of multispecific proteins, different ratios of the first, second and third expression vectors can be investigated 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.

[0175] Clones can be cultured under conditions suitable for bioreactor scale-up and sustained expression of the multispecific binding proteins. Multispecific binding proteins 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.

[0176] Pharmaceutical preparations The present disclosure also provides a pharmaceutical formulation containing a therapeutically effective amount of the multispecific binding protein disclosed herein (e.g., A49-F3'-TriNKET-Trastuzumab). The pharmaceutical formulation includes one or more excipients and is maintained at a certain pH. As used herein, the term "excipient" refers to any non-therapeutic agent added to the formulation to provide desired physical or chemical properties, such as pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability.

[0177] Excipients and pH One or more excipients in the pharmaceutical formulation of the present invention include buffering agent.As used herein, the term "buffering agent" refers to one or more components that, when added to an aqueous solution, can protect the solution from pH fluctuations when adding acid or alkali or diluting with a solvent.In addition to phosphate buffer, glycine buffer, carbonate buffer, citrate buffer, histidine buffer, etc. can be used, in which sodium, potassium or ammonium ion can serve as counterion.

[0178] In certain embodiments, the buffer or buffer system comprises at least one buffer having a buffering 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, the 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.

[0179] The pharmaceutical formulation of the present invention may have a pH of 5.5 to 6.5. For example, in certain embodiments, the pharmaceutical formulation has 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 formulation has 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 formulation has a pH of 6.0. According to scientific rounding rules, a pH greater than or equal to 5.95 and less than or equal to 6.05 is rounded as 6.0.

[0180] In certain embodiments, the buffer system of the pharmaceutical formulation comprises 10-25 mM histidine at a pH of 6.0±0.2. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 20 mM histidine at a pH of 6.0±0.2. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 10-25 mM histidine at a pH of 6.0±0.05. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 20 mM histidine at a pH of 6.0±0.05.

[0181] One or more excipients in the pharmaceutical formulation of the present invention further comprises a sugar or sugar alcohol. Sugars and sugar alcohols are useful as heat stabilizers in pharmaceutical formulations. In certain embodiments, the pharmaceutical formulation comprises a sugar, such as a monosaccharide (glucose, xylose, or erythritol), a disaccharide (e.g., sucrose, trehalose, maltose, or galactose), or an oligosaccharide (e.g., stachyose). In certain embodiments, the pharmaceutical formulation comprises sucrose. In certain embodiments, the pharmaceutical formulation comprises a sugar alcohol, such as 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 formulation comprises sorbitol.

[0182] The amount of sugar or sugar alcohol contained in the formulation may vary depending on the particular situation and intended purpose in which the formulation is used. In certain embodiments, the pharmaceutical formulation contains 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 of sugar or sugar alcohol. In certain embodiments, the pharmaceutical formulation contains 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 250 mM, or 300 mM of sugar or sugar alcohol. In certain embodiments, the pharmaceutical formulation contains 250 mM of sugar or sugar alcohol (e.g., sucrose or sorbitol).

[0183] One or more excipients in the pharmaceutical formulation disclosed herein further comprise a surfactant. As used herein, the term "surfactant" 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 formulations to reduce aggregation of therapeutic proteins. Surfactants suitable for use in pharmaceutical 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 laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetadine; lauryl-, myristyl-, Linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate or disodium oleyl methyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene 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.

[0184] The amount of non-ionic surfactant contained in the pharmaceutical formulation of the present invention can vary depending on the particular properties desired for the formulation and the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation contains 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 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% non-ionic surfactant (e.g., polysorbate 80).

[0185] In certain embodiments, the pharmaceutical formulation is isotonic. An "isotonic" formulation is one that has essentially the same osmolarity as human blood. An isotonic formulation generally has an osmolarity of about 250-350 mOsmol / kg H2O. Isotonicity can be measured using a vapor pressure or ice osmometer. In certain embodiments, the osmolarity of the pharmaceutical formulation is 250-350 mOsmol / kg H2O. In certain embodiments, the osmolarity of the pharmaceutical formulation is 300-350 mOsmol / kg H2O.

[0186] Substances such as sugar, sugar alcohol and NaCl can be included in pharmaceutical formulations for desired osmolality.In certain embodiments, when NaCl is present in pharmaceutical formulations, its concentration is equal to or less than 10mM, 9mM, 8mM, 7mM, 6mM, 5mM, 4mM, 3mM, 2mM, 1mM, 0.5mM, 0.1mM, 50μM, 10μM, 5μM or 1μM.In certain embodiments, the concentration of NaCl in pharmaceutical formulations is below detection limit.In certain embodiments, NaCl salt is not added when preparing pharmaceutical formulations.

[0187] The pharmaceutical formulation of the present invention may further comprise one or more other substances, such as bulking agents or preservatives. A "bulking agent" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates 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 formulation of the present invention may contain such bulking agents. Preservatives may reduce bacterial action, for example, facilitating the production of multi-use (multiple dose) formulations.

[0188] Exemplary Formulations In certain embodiments, a pharmaceutical 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 5.5-6.5.

[0189] In certain embodiments, the pharmaceutical formulation comprises 10-50 mg / mL of the multispecific binding protein, 10-25 mM histidine, 200-300 mM sugar or sugar alcohol (e.g., sucrose or sorbitol), and 0.005%-0.05% polysorbate (e.g., polysorbate 80) at a pH of 5.5-6.5. In certain embodiments, the pharmaceutical formulation comprises 10-50 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 5.5-6.5. In certain embodiments, the pharmaceutical formulation comprises 10-50 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 formulation comprises 10-50 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.

[0190] In certain embodiments, the pharmaceutical formulation comprises 10-50 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 formulation comprises 10-50 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 formulation comprises 10-50 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 formulation comprises 10-50 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sucrose, and 0.01% polysorbate 80 at a pH of 5.95-6.05.

[0191] In certain embodiments, the pharmaceutical formulation comprises 10-50 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 formulation comprises 10-50 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 formulation comprises 10-50 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 formulation comprises 10-50 mg / mL of the multispecific binding protein, 20 mM histidine, 250 mM sorbitol, and 0.01% polysorbate 80 at a pH of 5.95-6.05.

[0192] Stability of multispecific binding proteins The pharmaceutical formulations of the present invention exhibit a high level of stability. A pharmaceutical formulation is stable if the multispecific binding protein in the formulation retains an acceptable degree of physical properties, chemical structure and / or biological function after storage under defined conditions.

[0193] Stability can be measured by determining the percentage of the multispecific binding protein in the formulation that remains in a native conformation after storage at a defined temperature for a defined time. The percentage of protein in native conformation can be determined, for example, by size exclusion chromatography (e.g., size-exclusion high performance liquid chromatography), where the protein in native conformation is not aggregated (not eluted in the high molecular weight fraction) or degraded (not eluted in the low molecular weight fraction). In certain embodiments, more than 95%, 96%, 97%, 98% or 99% of the multispecific binding protein has a native conformation as determined by size exclusion chromatography after 3 weeks of incubation at 4°C. In certain embodiments, more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the multispecific binding protein has a native conformation 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 forms high molecular weight complexes (i.e., has 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., has 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 degrades (i.e., has 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) as determined by size exclusion chromatography after 3 weeks of incubation at 50° C.

[0194] Stability can also be measured by determining the percentage of the multispecific binding protein that is present in a more acidic fraction ("acidic form") compared to the major fraction of the protein ("mainly charged form"). Without wishing to be bound by theory, deamidation of a protein can cause the protein to become more negatively charged and therefore more acidic compared to the non-deamidated protein (see, e.g., Robinson, Protein Deamidation, (2002) PNAS 99(8):5283-88). Percentage of protein in acidic form The acidic form can be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography) or image capillary isoelectric focusing (icIEF). In certain embodiments, at least 50%, 60%, 70%, 80% or 90% of the multispecific binding protein in the pharmaceutical formulation is in the predominantly charged form after 3 weeks of incubation at 4° C. In certain embodiments, at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the multispecific binding protein in the pharmaceutical formulation is in the predominantly charged form after 3 weeks of incubation at 50° C. In certain embodiments, no more than 10%, 20%, 30%, 40% or 50% of the multispecific binding protein in the pharmaceutical formulation is in the acidic form after 3 weeks of incubation at 4° C. 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 formulation is in the acidic form after incubation at 50° C. for 3 weeks.

[0195] 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). The protein sample can be denatured in the presence or absence of an agent that reduces disulfide bonds in the protein (e.g., β-mercaptoethanol). In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation is at least 95%, 96%, 97%, 98% or 99% after 3 weeks of incubation 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 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 3 weeks of incubation at 50° C. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical 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 3 weeks of incubation at 4° C. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% after 3 weeks of incubation at 50° C. as measured by capillary electrophoresis after denaturing a protein sample under non-reducing conditions.

[0196] Stability can also be measured by determining parameters of protein dissolution by dynamic light scattering. Z-average and polydispersity index (PDI) values ​​indicate the average diameter of particles in solution, and these measurements increase when aggregates are present in the solution. Monomer Pd% values ​​indicate the spread of different monomers detected, with lower values ​​indicating a monodisperse solution, which is preferred. Monomer size detected by DLS is useful in confirming that the predominant population is monomeric, and in characterizing any higher order aggregates that may be present. In certain embodiments, the Z-average value of the pharmaceutical formulation does not increase by more than 5%, 10%, or 15% after 3 weeks of incubation at 4° C. In certain embodiments, the Z-average value of the pharmaceutical formulation does not increase by more than 5%, 10%, 15%, 20%, or 25% after 3 weeks of incubation at 50° C. In certain embodiments, the PDI value of the pharmaceutical formulation does not increase by more than 10%, 20%, 30%, 40%, or 50% after 3 weeks of incubation at 4° C. In certain embodiments, the PDI value of the pharmaceutical formulation does not increase by more than 2-fold, 3-fold, 4-fold, or 5-fold after incubation at 50° C. for 3 weeks.

[0197] An exemplary method for determining the stability of a multispecific binding protein in a pharmaceutical formulation is 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.

[0198] Dosage form The pharmaceutical formulation can be prepared and stored as a liquid formulation or a lyophilized formulation. In certain embodiments, the pharmaceutical formulation is a liquid formulation for storage at 2-8°C (e.g., 4°C) or a frozen formulation for storage at or below -20°C. The sugar or sugar alcohol in the formulation is used as a lyoprotectant.

[0199] Before pharmaceutical use, pharmaceutical formulations can be diluted or reconstituted with aqueous carriers 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 solution, Ringer's solution, or dextrose solution.For example, when pharmaceutical formulations are prepared for intravenous administration, they can be diluted with 0.9% sodium chloride (NaCl) solution.In certain embodiments, the diluted pharmaceutical formulations are isotonic and suitable for administration by intravenous infusion.

[0200] The pharmaceutical formulation comprises the multispecific binding protein at a concentration suitable for storage. In certain embodiments, the pharmaceutical formulation comprises 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 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.

[0201] In certain embodiments, the pharmaceutical 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 the multispecific binding protein in the container is suitable for administration as a single dose. In certain embodiments, the amount of the multispecific binding protein in the container is suitable for administration in multiple doses. In certain embodiments, the pharmaceutical formulation comprises the multispecific binding protein in an amount of 0.1-2000 mg. In certain embodiments, the pharmaceutical formulation comprises 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, Contains amounts of 20-500mg, 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. In certain embodiments, the pharmaceutical formulation comprises the multispecific binding protein in an amount of 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.

[0202] Dosage Regimen and Therapeutic Use In another aspect, the disclosure provides a method for treating cancer, comprising administering to a subject in need thereof a multispecific binding protein disclosed herein (e.g., A49-F3'-TriNKET-Trastuzumab) on days 1, 8, and 15 of an initial 4-week treatment cycle. In certain embodiments, the multispecific binding protein is administered to the subject only on these 3 days of the initial 4-week treatment cycle. In certain embodiments, the multispecific binding protein is not administered to the subject on day 22. This regimen is a dose-intensified schedule designed to reach target maximum saturation as early as possible during the course of treatment while minimizing the infusion burden on the patient.

[0203] In certain embodiments, the method further comprises administering the multispecific binding protein to the subject in one or more subsequent 4-week treatment cycles after the first treatment cycle, where the multispecific binding protein is administered on days 1 and 15 in each subsequent treatment cycle. In certain embodiments, the multispecific binding protein is administered to the subject only on these two days in the subsequent 4-week treatment cycle. In certain embodiments, the multispecific binding protein is not administered to the subject on days 8 or 22. The subsequent treatment cycles in which the subject receives the multispecific binding protein once every two weeks are designed to maintain a certain level of the multispecific binding protein 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 the cancer regresses.

[0204] In certain embodiments, one or more doses in the initial and subsequent treatment cycles comprise the multispecific binding protein in an amount 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. In certain embodiments, one or more doses in the initial and subsequent treatment cycles comprise the multispecific binding protein in an amount selected from the group consisting of 0.1 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, and 20 mg / kg.

[0205] In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises the multispecific binding protein in an amount selected from the group consisting 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, and 1-2 mg / kg. In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises the multispecific binding protein in the same amount selected from the group consisting 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, and 1-2 mg / kg.

[0206] In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises the multispecific binding protein in an amount selected from the group consisting of 0.1 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, and 20 mg / kg. In certain embodiments, each of the doses in the initial and subsequent treatment cycles comprises the multispecific binding protein in the same amount selected from the group consisting of 0.1 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, and 20 mg / kg.

[0207] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 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 compound may be administered in an amount selected from the group consisting of 0.52 mg / kg, 1 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 20 mg / kg, and 50 mg / kg.

[0208] In certain embodiments, the multispecific binding protein is administered intravenously. For example, in certain embodiments, the multispecific binding protein is administered by intravenous infusion, e.g., using a pre-filled bag, a pre-filled pen, or a pre-filled syringe. In certain embodiments, the multispecific binding protein in a pharmaceutical formulation disclosed herein is diluted prior to administration. For example, in certain embodiments, the pharmaceutical formulation is diluted with sodium chloride and administered intravenously from a 250 ml saline bag. The intravenous infusion can be an infusion over about 1 hour (e.g., 50-80 minutes). In certain embodiments, the bag is connected to a channel that includes a tube and / or a needle.

[0209] The types of cancer that can be treated with the multispecific binding protein or pharmaceutical preparation disclosed herein include, but are not limited to, breast cancer, thyroid cancer, gastric cancer, renal cell carcinoma, lung adenocarcinoma, 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, salivary gland cancer, biliary tract cancer, lung squamous cell carcinoma, 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 urothelial bladder cancer or metastatic breast cancer.

[0210] 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. Anti-HER2 antibodies (e.g., Ventana 4B5 and Bond Oracle CB11 antibodies) have been approved by the FDA for the detection of HER2, and immunohistochemistry kits (e.g., HercepTest™) are commercially available. HER2 expression levels in tumor samples, as detected by immunohistochemistry, are quantified and assessed according to ASCO / CAP guidelines (Wolff et al., 2011). al. (2007) J. Clin. Oncol. 25(1):118-45) In certain embodiments, subjects treated by the methods disclosed herein have tumors with HER2 levels scored as 1+, 2+, or 3+. In certain embodiments, subjects treated by the methods disclosed herein have tumors with HER2 levels scored as 2+ or 3+. In certain embodiments, subjects treated by the methods disclosed herein have tumors 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 tumors that exhibit HER2 expression at least as faint / barely perceptible membrane staining detected in at least 10% or more than 10% of tumor cells. In certain embodiments, subjects treated by the methods disclosed herein have tumors that exhibit HER2 expression at least as weak to moderate complete membrane staining detected in at least 10% or more than 10% of tumor cells. In certain embodiments, subjects treated by the methods disclosed herein have tumors that exhibit HER2 expression as strong, complete membrane staining detected in at least 10% or more than 10% of tumor cells.

[0211] In certain embodiments, the subject treated by the method disclosed herein has a cancer with ERBB2 gene amplification. ERBB2 gene amplification is generally correlated with HER2 overexpression, and determining whether ERBB2 gene is amplified in cancer tissue samples can help reduce false positive results from immunohistochemical testing of the same sample (see, for example, Sarode et al., (2015) Arch. Pathol. Lab. Med. 139:922-28). 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).

[0212] In certain embodiments, the subject treated according to the methods disclosed herein has not previously undergone a treatment to treat cancer. In certain embodiments, the subject treated according to the methods disclosed herein has not previously undergone chemotherapy or immunotherapy to treat cancer. In certain embodiments, the subject treated according to the methods disclosed herein has previously undergone a treatment (e.g., chemotherapy or immunotherapy) but continues to experience cancer progression despite the previous treatment. In certain embodiments, the subject treated according to the methods disclosed herein has experienced cancer regression after previous treatment (e.g., chemotherapy or immunotherapy) but has subsequently experienced cancer recurrence. In certain embodiments, the subject treated according to the methods disclosed herein is intolerant to previous treatment (e.g., chemotherapy or immunotherapy).

[0213] In certain embodiments, all subjects treated according to the methods disclosed herein meet the inclusion criteria for the clinical trial cohorts described in Example 3 (e.g., accelerated escalation cohorts, "3+3" dose escalation cohorts, safety / PK / PD expansion cohorts, urothelial bladder cancer (UBC) cohorts, metastatic breast cancer (MBC) cohorts, basket solid tumor cohorts with high HER2 expression (HER2 3+), or combination therapy cohorts with pembrolizumab). In certain embodiments, subjects treated according to the methods disclosed herein do not meet any of the exclusion criteria described in Example 3.

[0214] The multispecific binding protein disclosed herein can be used as a monotherapy or can be used in combination with one or more therapies.In certain embodiments, the multispecific binding protein is used as a monotherapy according to the dosage regimen disclosed herein.In other embodiments, the multispecific binding protein is used in combination with one or more therapies, the multispecific binding protein is administered according to the dosage regimen disclosed herein, and the one or more therapies are administered according to the dosage regimen known to be suitable for treating a particular subject with a particular cancer.In certain embodiments, the method of treatment disclosed herein is used as an adjunct to surgical removal of primary tumor.

[0215] 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, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogaine, and the like. genil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2alpha, 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 above agents that may exhibit differential binding to their cognate receptors or increased or decreased serum half-lives.

[0216] Another class of drugs that can be used as part of combination therapy in treating cancer is immune checkpoint inhibitors.Exemplary immune checkpoint inhibitors include drugs 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.CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for treating melanoma.

[0217] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0218] Further categories of anti-cancer drugs include, for example, (i) 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, (ii) an inhibitor selected from a cytotoxic agent, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) 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.

[0219] In certain embodiments, the methods of the invention further comprise administering to the subject an anti-PD-1 antibody. Many anti-PD-1 antibodies have been developed for therapeutic purposes, see, for example, Gong et al. al., (2018) J. ImmunoTher Cancer (2018) 6:8. In an embodiment, the anti-PD-1 antibody is pembrolizumab. In a particular embodiment, 200 mg of pembrolizumab is administered on day 1 of the first treatment cycle. In a particular embodiment, when the subject undergoes one or more subsequent treatment cycles, starting on day 1 of the first subsequent treatment cycle, 200 mg of pembrolizumab is administered once every 3 weeks in the subsequent treatment cycle.

[0220] In certain embodiments, the methods of treatment disclosed herein result in an improvement in disease response or survival of the subject or patient. For example, in certain embodiments, the disease response is a complete response, partial response, or stable disease. In certain embodiments, the improvement in survival is an improvement in progression-free survival (PFS) or overall survival. The improvement (e.g., of PFS) can be determined relative to the period before the start of the treatment of the present disclosure. Methods of determining disease response (e.g., complete response, partial response, or stable disease) and patient survival (e.g., PFS, overall survival) for BTC (e.g., advanced BTC, metastatic BTC), or biliary tumor therapy are conventional in the art and contemplated herein. In some embodiments, disease response is evaluated according to RECIST 1.1 after the treated patient undergoes contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) of the affected area (e.g., chest / abdomen and pelvis, including the area from the top of the thoracic inlet to the pubic symphysis). EXAMPLES

[0221] The disclosure, having been generally described herein, will be more readily understood by reference to the following examples, which are included merely 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.

[0222] Example 1 Formulation, packaging and storage of A49-F3'-TriNKET-Trastuzumab To evaluate the effect of pH and excipients on the stability of A49-F3'-TriNKET-Trastuzumab (Kermit BDS Lot 7443-C3, 11.9 mg / mL), the formulations listed in Table 12 were evaluated in duplicate and randomized fashion. A49-F3'-TriNKET-Trastuzumab was buffer exchanged into each buffer and excipient combination 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 sterile filtered using a 0.22 μm EMD Millipore Ultrafree-CL centrifugal filter device with a Durapore membrane (Fisher Scientific Cat. No. UFC40GVOS). After sterile filtration, each formulation was handled aseptically in a laminar flow hood. Formulated samples were spiked with polysorbate 80 (PS80) 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 into depyrogenated type 1 borosilicate glass vials, 2 mL x 13 mm (West Pharmaceuticals Catalog No. 68000377), stoppered with 13 mm Fluorotec stoppers (West Pharmaceuticals Catalog No. 19700302) and sealed. 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 the 3 week incubation, the 2-8°C and 50°C samples were analyzed according to the test method shown in Table 13. [Table 12] [Table 13]

[0223] 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, each formulated sample was tested using the assays as outlined in Table 13. All formulations behaved similarly and within expected ranges as assessed by appearance, concentration, pH and osmolality.

[0224] exterior Samples were viewed under a black and white background at ambient laboratory conditions prior to opening the sample vials. In all samples, no visible particulates were present at either the time zero or the three week conditions.

[0225] Determining UV concentration Protein concentration was determined for each sample and condition by ultraviolet (UV) absorbance at optical density (OD) 280 nm. 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.

[0226] 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.

[0227] Dynamic Light Scattering Dynamic light scattering (DLS) samples were collected at 25° C. after equilibration for 300 seconds. Five measurements were collected per 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.

[0228] 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.

[0229] 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 15-1] [Table 15-2] [Table 16] [Table 17] [Table 18-1] [Table 18-2] [Table 19]

[0230] All A49-F3'-TriNKET-Trastuzumab formulations demonstrated conformational stability, with mean and monomer sizes ≦20 nm and polydispersity (PDI) ≦0.300, as assessed by DLS. When excipient and pH combinations were evaluated, sorbitol-only and sucrose-only formulations had lower mean sizes compared to NaCl and sorbitol combinations and NaCl and sucrose combinations at 2-8°C and 50°C incubation for 3 weeks (comparative modeling shown in Figure 2A and Figure 2B). Mean monomer size was also smaller for sorbitol-only and sucrose-only formulations compared to NaCl combinations after 2-8°C and 50°C incubation for 3 weeks (Figures 3A and 3B).

[0231] Size Exclusion Chromatography (SEC) Size exclusion chromatography was performed according to the draft method 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 loading amount of 100 μg. Separation was performed on a Tosoh Corporation G3000SW×1 (7.8×300 mm, Catalog No. 08541) column with detection at 280 nm and a bandwidth of 8 nm. Samples were analyzed in real time, at time zero, and after 3 weeks of incubation at either 2-8°C or 50°C. HMW% values ​​are summarized in Table 20, Major% values ​​are summarized in Table 21, and LMW% values ​​are summarized in Table 22. [Table 20] [Table 21] [Table 22-1] [Table 22-2]

[0232] 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 formulations with only sucrose maintaining higher Main Peak % and lower HMW species % compared to the combined excipients NaCl and sorbitol and NaCl and sucrose (shown in Figures 4A and 5A, respectively). Importantly, both the single excipient sucrose and single excipient sorbitol maintained their Main Peak % over the pH range of 5.5 to 6.5, and both showed only a slight increase in HMW species % as the pH increased from 5.5 to 6.5. The LMW species % tended to decrease as the pH increased from 5.5 to approximately 6.2 for all excipients (Figure 6A).

[0233] After 3 weeks of incubation at 2-8 °C, all formulated samples maintained a percentage of the main species peak greater than 98%. The % main peak was higher at lower pH values ​​(5.5) compared to higher pH values ​​(pH 6.5) as shown in Figure 4B. Single excipient sorbitol and single excipient sucrose tended to have lower HMW% compared to the combined excipients NaCl and sorbitol and NaCl and sucrose. For all excipients, there was a trend for the % HMW species to increase with increasing pH (Figure 4B). The % LMW species tended to be lower for the combined excipients but was pH dependent (Figure 6B).

[0234] Osmolality Osmolality (osmo) 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]

[0235] Image Capillary Isoelectric Focusing (icIEF) Image capillary isoelectric focusing (icIEF) was used to determine charge variant analysis. Charge heterogeneity was assessed using the Protein Simple-Maurice draft method. Starting materials and samples were diluted to 5mg / mL in water and then combined with the master mix at 10μL sample to 90μL 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 sample run, which were run in a 96-well plate format. The method parameters used for Maurice were: electrophoresis time #1 = 1 min, 1500 V, electrophoresis time #2 = 8 min, 3000 V, detection = 5 exposures, sample load = 55 sec, lower pI marker = 5.12, 300 pixels, higher pI marker = 9.50, 1800 pixels. Starting material was run every 18 injections to ensure consistent readings.

[0236] The "main peak" was defined as the main peak in the formulated sample at time zero. After incubation, a peak with the same elution time may have decreased and no longer represent the peak with the largest area under the curve, but was still defined as the "main peak". The values ​​for the percentage of protein present in the acidic fraction (% acidic) after 3 weeks of incubation at 2-8°C and after 3 weeks of incubation at 50°C are summarized in Table 24. The values ​​for the percentage of protein present in the main peak fraction (% main peak) are summarized in Table 25. The values ​​for the percentage of protein present in the basic fraction (% basic) are summarized in Table 26. [Table 24] [Table 25-1] [Table 25-2] [Table 26]

[0237] For formulated samples after 3 weeks of incubation at 2-8 °C, the %Main Peak values ​​ranged from 58.3% to 62.2%, the %Acidic values ​​ranged from 34.3% to 38.4%, and the %Basic values ​​ranged from 3.3% to 3.7%. No significant models were fitted to the %Main Peak data, indicating that neither pH nor excipients had a significant effect on the icIEF values ​​(Figures 8B-8D). Excipients were also not important in modeling the %Acidic and %Basic species, with the %Acidic species tending to increase with pH, ​​whereas the %Basic species decreased (Figures 7B and 9B). Changes were small but observed within a narrow range for all values.

[0238] For formulated samples after 3 weeks incubation at 50°C, the %MainPeak values ​​ranged from 14.9% to 22.7%, the %Acidic values ​​ranged from 70.6% to 81.6%, and the %Basic values ​​ranged from 2.4% to 8.8%. This data shows a shift from the %MainPeak to the %Acidic and %Basic still relatively consistent with the 3 week 2-8°C incubation results. In evaluating the 3 week 50°C formulated samples, the sample with sucrose as the only excipient had the highest %Acidic species (Figure 7A) and the lowest %MainPeak and %Basic species (Figures 8A and 9A). This was consistent across all pH values ​​(5.5-6.5), while formulations with the other three excipients had %Acidic species that were lower at lower pH values ​​and tended to increase with increasing pH.

[0239] 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 according to draft ATM. 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 min. Separation was performed over 20 min using positive polarity, 1 min temperature ramp, 15 kV voltage, and 20 psi pressure. The capillary length was 30.2 cm with a 10.2 cm length to the detector. The starting material was used as a reference. Sample purity percentages are summarized in Table 27. Sample impurity percentages are summarized in Table 28. [Table 27-1] [Table 27-2] [Table 28]

[0240] Non-reduced capillary gel electrophoresis was also performed to assess purity. SDS-CGE was assessed using a Sciex PA800+ with UV detection at 220 nm according to draft ATM. 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 min. Separation was performed over 20 min using positive polarity, 1 min temperature ramp, 15 kV voltage, and 20 psi pressure. The capillary length was 30.2 cm with a 10.2 cm length to the detector. The 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 30-1] [Table 30-2] [Table 31]

[0241] As assessed by reduced CE, % purity values ​​for sorbitol-only and sucrose-only formulations were maintained across the pH range (pH 5.5-6.5) among the 3 week 50°C samples (Figure 10A), while the combination excipients showed greater variability with pH as the % purity decreased at lower pH values ​​(5.5 vs. 6.5) (Figure 10B).

[0242] As assessed by non-reduced CE, formulations containing only sorbitol or only sucrose had lower % HMW species compared to the combined NaCl and sorbitol excipients and the combined NaCl and sucrose excipients among the 3 week 50° C. samples (FIGS. 11A and 12A). pH level had no significant effect on the % Main Peak values.

[0243] As assessed by reduced and non-reduced CE, there was no significant model that fit the reduced CE data for the 3-week 2-8 °C samples, and for the non-reduced CE data, the sorbitol-only and sucrose-only formulations had a higher % of main peak species (Figure 11B).

[0244] statistical analysis Trends were analyzed using Design Expert v9 software and a summary of the analysis is shown in Table 32. Models in bold were not included in the final optimization evaluation. [Table 32]

[0245] Excipient Selection The performance of formulations containing 250 mM sorbitol or 250 mM sucrose as excipients was more favorable than those containing combinations of sorbitol and NaCl or sucrose and NaCl. Therefore, the optimal formulation of A49-F3'-TriNKET-Trastuzumab was determined to be 20 mM histidine, 250 mM sucrose or sorbitol, and 0.01% PS80 at pH 6.0.

[0246] Example 2 Pharmacokinetic (PK) analysis of A49-F3'-TriNKET-Trastuzumab This study was designed to determine the pharmacokinetic (PK) profile of A49-F3'-TriNKET-Trastuzumab when administered as a 30-minute IV infusion at 1 mg / kg, 10 mg / kg or 50 mg / kg to cynomolgus monkeys on days 1 and 15 of the study, followed by observation periods of 13 and 6 days, respectively. A summary of the key PK parameters following the initial intravenous infusion of A49-F3'-TriNKET-Trastuzumab on day 1 to male and female cynomolgus monkeys is presented in Tables 33 and 34, respectively. [Table 33] [Table 34]

[0247] A summary of the main PK parameters following a second intravenous infusion of A49-F3'-TriNKET-Trastuzumab to male and female cynomolgus monkeys on day 15 is presented in Tables 35 and 36, respectively. [Table 35] [Table 36]

[0248] t maxThe time when t occurred was generally 15 minutes after the end of infusion (EOI). However, as shown in Tables 33 and 35, max also occurred at EOI on day 1 in male cynomolgus monkeys receiving 10 mg / kg and 30 minutes after EOI on day 15 in male cynomolgus monkeys receiving 50 mg / kg. As shown in Tables 34 and 36, t max also occurred 1 hour after EOI on day 15 in the females receiving cynomolgus monkeys 1 mg / kg or 50 mg / kg, respectively. These data indicate that A49-F3'-TriNKET-Trastuzumab has a long terminal half-life (t 1 / 2 ) and decreased slowly. These data also showed low serum clearance and a volume of distribution similar to blood volume (73.4 mL / kg) and smaller than total body water volume (693 mL / kg).

[0249] Maximum serum concentrations of A49-F3'-TriNKET-Trastuzumab for dose levels (C max ) and area under the concentration-time curve (AUC 0~144時間 The ratio of C to A49-F3'-TriNKET-Trastuzumab was also evaluated. max Ratios and AUC ratios are presented by dose level. [Table 37]

[0250] As shown in Table 37, C max and AUC 0-144時間 The C values ​​increased approximately proportionally with increasing dose over the dose range of 1 to 50 mg / kg. max and AUC 0-144時間The values ​​were similar to the exposure index in male cynomolgus monkeys. At the second injection on day 15, the C max and AUC 0-144時間 Values ​​were similar after the first dose on day 1 at the 1 and 10 mg / kg dose levels, but generally slightly higher at the 50 mg / kg dose level. AUC 0-144時間 The accumulation ratio based on values ​​was slightly higher than that at the 50 mg / kg dose level, indicating that some accumulation occurred following repeated IV infusion administration of A49-F3'-TriNKET-Trastuzumab at this dose level.

[0251] In addition, no anti-drug antibody positivity was observed in any samples from animals treated with A49-F3'-TriNKET-Trastuzumab, therefore, it was concluded that no test article-related anti-drug antibodies were found in this study.

[0252] Example 3 Treatment of locally advanced or metastatic solid tumors with A49-F3'-TriNKET-Trastuzumab the purpose This clinical study is designed in two phases: a dose escalation phase and, following efficacy, an efficacy expansion cohort phase. The primary objective of the dose escalation phase of the study is to evaluate the safety and tolerability of A49-F3'-TriNKET-trastuzumab in patients with advanced (unresectable, recurrent or metastatic) solid tumors for which no effective standard treatment exists or who have relapsed or are intolerant to standard treatment, and to determine the maximum tolerated dose of A49-F3'-TriNKET-trastuzumab. The primary objective of the efficacy expansion cohort phase of the study is to evaluate the overall response rate (ORR) according to the revised Response Evaluation Criteria in Solid Tumors version 1.1 (mRECIST 1.1) by an Independent Endpoint Review Committee (IERC).

[0253] The secondary objectives of this clinical study are: - To characterize the pharmacokinetics of A49-F3'-TriNKET-trastuzumab; - To evaluate the immunogenicity of A49-F3'-TriNKET-Trastuzumab and correlate it with its exposure and clinical activity; - To evaluate the duration of response (DOR) of A49-F3'-TriNKET-Trastuzumab by IERC; - To assess the Best Overall Response (BOR) by the IERC; - To evaluate the progression-free survival (PFS) of A49-F3'-TriNKET-Trastuzumab by IERC; - To assess overall survival (OS); and - To evaluate the safety of A49-F3'-TriNKET-trastuzumab in combination with pembrolizumab.

[0254] Study design This is a Phase I / II, open-label, dose-escalation study with an ongoing parallel-group efficacy extension study 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 the following three phases: (A) Accelerated increase, (B) "3+3" dose escalation, and (C) Safety / Pharmacokinetic (PK) / Pharmacodynamic (PD) Expansion Cohort (2) The efficacy expansion cohort part (Phase II) is divided into the following four cohorts: (A) Urothelial bladder cancer (UBC) (B) Metastatic breast cancer (MBC) (C) Basket-type solid tumor with high HER2 expression (HER2 3+) (D) Combination therapy with pembrolizumab.

[0255] In one exemplary embodiment, patients enrolled in the dose escalation and efficacy expansion (UBC, MBC, or basket [HER2 3+] cohort) parts 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 progression, unacceptable toxicity (as described in the "Dose-Limiting Toxicity (DLT)" section of this Example), or any reason for withdrawal from the study or investigational medicinal product (IMP) occurs. Patients enrolled in the combination therapy with 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 one exemplary embodiment, 200 mg of pembrolizumab is administered as per its label with A49-F3'-TriNKET-trastuzumab.

[0256] For treatment cycle 1, patients will 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 will receive A49-F3'-TriNKET-trastuzumab and pembrolizumab on day 1 of each cycle once every 3 weeks until progression, unacceptable toxicity (as described in the "Dose-Limiting Toxicity (DLT)" section of this Example), or any reason for withdrawal from the study or IMP.

[0257] Patients who achieve a confirmed complete response (CR) will be administered treatment for up to 12 months after confirmation, at the discretion of the investigator. Treatment beyond 12 months will be permitted if the investigator, after discussion with the Sponsor Medical Monitor, believes such patients would benefit from continued treatment.

[0258] Figures 14A-B are schematic diagrams of the clinical trial design. Figure 14A describes the study design for the dose escalation phase. Figure 14B describes the study design for the efficacy expansion cohort phase.

[0259] Inclusion criteria General inclusion criteria for patients enrolled in any of the cohorts in this example clinical study include: - Patients who signed written informed consent; - Patients ≥ 18 years of age (including male and female patients); - Patients with histopathologically and cytologically proven locally advanced or metastatic solid tumors for which no standard treatment exists or for which standard treatment has failed. Primary tumors must have demonstrated HER2 expression by immunohistochemistry; - Patients with an ECOG performance status of 0 or 1 at study entry and an expected life expectancy of at least 3 months; - Patients with baseline left ventricular ejection fraction (LVEF) ≥ 55% as measured by echocardiography (preferred) or multi-gated acquisition (MUGA) scan; - White blood cell (WBC) count ≧3×10 9 / L and absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L, lymphocyte count ≥ 0.5 × 10 9 / L, platelet count ≥75×10 9 / L and hemoglobin ≥ 9 g / dL (may have received a blood transfusion); - Patients with 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, or for patients with documented metastatic disease to the liver, AST and ALT levels ≤ 5 x ULN; - Patients with adequate renal function, as defined by an estimated creatinine clearance >50 mL / min according to the Cockcroft-Gault formula; and - For women of childbearing potential (WOCBP) patients, patients using effective contraception as defined by WHO guidelines for 1 "highly effective" methods or 2 "effective" methods.

[0260] Additional inclusion criteria for patients enrolled in the accelerated escalation or "3+3" dose escalation phase of the dose escalation part described in this Example include: - Patients with evidence of the target disease, but measurable disease is not required for participation; and - Patients with available archived tumor biopsy specimens (≤6 months old, at least 8 slides) or fresh biopsy specimens obtained within the screening period (at least 10 slides and 3 cores).

[0261] Additional inclusion criteria for patients enrolled in the safety / PK / PD expansion cohort phase of the dose escalation part described in this Example include: - Patients with fresh tumor biopsy specimens obtained during the screening period to obtain formalin-fixed paraffin-embedded (FFPE) paraffin blocks, or patients with sufficient unstained slides to perform IHC (at least 3 unstained slides) with at least 12 slides overall and at least 3 fresh cores; and - Patients have at least 1+ HER2 by IHC at screening.

[0262] Additional inclusion criteria for patients enrolled in the UBC expansion cohort described in this Example include: - Patients with histopathologically or cytologically proven locally advanced or metastatic transitional cell carcinoma of the urothelium (including the renal pelvis, ureter, urothelium, and urethra); - Patients with radiographic disease progression after last line of therapy; - Patients who have received one (and a maximum of 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 who have radiographic progression or who have relapsed within 6 months after the last dose of an adjuvant platinum-containing regimen and are considered to have failed a first-line platinum-containing regimen; - Patients who have been treated with a checkpoint inhibitor (i.e., anti-PD-1 or anti-PD-L1) and have radiographic progression (receiving a combination of platinum-based and PD-1 / PD-L1-based therapy, if appropriate); - Patients with at least 1+ HER2 expression by IHC; and - Patients with fresh tumor biopsy specimens obtained during the screening period to obtain formalin-fixed paraffin-embedded (FFPE) paraffin blocks, or patients with sufficient unstained slides to perform IHC (at least three unstained slides) with at least 12 slides overall and at least three fresh cores.

[0263] Additional inclusion criteria for patients enrolled in the MBC expansion cohort described in this Example include: - Patients with histopathologically confirmed MBC; - Patients who have received ≤3 lines of cytotoxic therapy for metastatic disease; - Patients who have received a taxane and an anthracycline unless an anthracycline is contraindicated; - Patients with tumors scored 1+ or 2+ by IHC. For scores of 2+, the presence of tumor amplification of ERRB2 must be defined by an FDA-approved method; - Patients who have progressed (radiographically) after last line of systemic therapy; and - Patients with fresh tumor biopsy specimens obtained during the screening period to obtain formalin-fixed paraffin-embedded (FFPE) paraffin blocks, or patients with sufficient unstained slides to perform IHC (at least three unstained slides) with at least 12 slides overall and at least three fresh cores.

[0264] Additional inclusion criteria for patients enrolled in the basket (HER2 3+) cohort described in this example include: - Patients with any solid tumor, except breast or gastric cancer, with a history of intratumoral ERRB2 amplification and one of the following: 1) HER2 3+ by IHC documented on most recent biopsy within 6 months after last-line radiographic progression, or 2) HER 3+ by IHC during the screening period; - Patients have received at least one line of approved or established therapy; and - Patients with fresh tumor biopsy specimens obtained during the screening period to obtain formalin-fixed paraffin-embedded (FFPE) paraffin blocks, or patients with sufficient unstained slides to perform IHC (at least three unstained slides) with at least 12 slides overall and at least three fresh cores.

[0265] Additional inclusion criteria for patients enrolled in the combination cohort phase with pembrolizumab in the efficacy expansion part described in this Example include: - Patients who are eligible to receive pembrolizumab according to its label for malignancies of epithelial origin; and - Patients with fresh tumor biopsy specimens obtained during the screening period to obtain formalin-fixed paraffin-embedded (FFPE) paraffin blocks, or patients with sufficient unstained slides to perform IHC (at least three unstained slides) with at least 12 slides overall and at least three fresh cores.

[0266] Exclusion criteria Exclusion criteria for patients enrolled in the clinical study of this example included the following: - Patients taking concomitant medications with unauthorized drugs, including: o Immunotherapy, immunosuppressants (including chemotherapy or systemic corticosteroids except for short-term treatment of allergic reactions or treatment of irAEs), or other experimental pharmaceutical products. · Exception: ● Short-term administration of systemic steroids (e.g., for allergic reactions or to manage irAEs) is permitted. • Steroids with no or minimal systemic effects (topical or inhaled) are permitted; o HER2-targeted TKIs or any recombinant molecule targeting HER2 or NKG2D; o Growth factors (granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor). · Exception: • Erythropoietin and erythropoietin analogues may be prescribed at the discretion of the investigator; Bisphosphonate or denosumab treatment is not permitted. · Exception: Bisphosphonates or denosumab are permitted as long as they are not started more than 14 days after the first dose of A49-F3'-TriNKET-trastuzumab. - Patients have previously been treated with agents that specifically target the HER2 pathway. · Exception: mAbs or tyrosine kinase inhibitors (TKIs) may be tolerated, provided there is a drug holiday (4 weeks for mAbs or protein therapeutics and 2 weeks for TKIs); - Patients with concomitant anticancer treatment (e.g., cytoreductive therapy, radiation therapy (excluding palliative bone-directed radiation therapy), immunotherapy, or cytokine therapy excluding erythropoietin), major surgery (excluding previous diagnostic biopsy), concomitant systemic therapy with steroids or other immunosuppressants, or use of an investigational drug within 28 days prior to the start of study treatment. Short-term administration of systemic steroids (e.g., for allergic reactions or management of irAEs) is permitted. Patients receiving bisphosphonates are eligible, provided that treatment was initiated at least 14 days prior to the first dose of A49-F3'-TriNKET-trastuzumab. - Patients with a previous malignancy other than the target malignancies investigated in this study, except for basal or squamous cell carcinoma of the skin or cervical intraepithelial neoplasia, within the past 3 years; - Patients with rapidly progressive disease; - Patients with active central nervous system (CNS) metastases or a history of central nervous system (CNS) disease; - Patients who have undergone any organ transplant, including autologous or allogeneic stem cell transplant; - Patients with significant acute or chronic infections (including positive medical history test for human immunodeficiency virus (HIV), or active or latent hepatitis B, or active hepatitis C, tested during the screening period); - Patients with a pre-existing autoimmune disease (excluding vitiligo patients) that required treatment with systemic immunosuppressants for more than 28 days within the past 3 years, or a clinically significant immune deficiency (e.g., congenital gammopathy or congenital immune deficiency), or fever within days 1 to 7; - Patients with any history of severe hypersensitivity reaction to mAb (≥ grade 3 NCI-CTCAE v5.0), anaphylaxis, or uncontrolled asthma (e.g., 3 or more features of asthma with some degree of control); - Patients with residual toxicity related to previous therapy > grade 1 NCI-CTCAE v5.0, but alopecia and sensory neuropathy ≤ grade 2, are acceptable; - Female patients who are pregnant or breastfeeding during the study; - Patients with alcohol or drug abuse; - Patients with serious cardiac or medical conditions, including but not limited to: ○ History of New York Heart Association class III or IV heart failure or systolic dysfunction (LVEF <55%); High-risk uncontrolled arrhythmias, i.e., tachycardia with a heart rate >100 beats per minute at rest; ○ Significant ventricular arrhythmia (ventricular tachycardia) or more severe AV block (second-degree AV block type 2 (Mobitz 2) or third-degree AV block); Angina requiring antianginal medication; o Clinically significant valvular heart disease; o Evidence of transmural infarction on ECG; o Uncontrolled hypertension (defined by systolic >180mmHg or diastolic >100mmHg); o Clinically significant uncontrolled cardiac risk factors, clinically significant pulmonary disease, or any clinically significant medical condition that, in the opinion of the investigator, may limit participation in this study; - Patients with any other significant disease that, in the opinion of the investigator, may impair their ability to participate (e.g. inflammatory bowel disease); - Patients with any psychiatric condition that would prevent them from understanding or interpreting the informed consent; - Patients who are legally incompetent or have limited legal capacity; or - Patients unable to sign and provide informed consent, including compliance with the requirements and restrictions listed in the Informed Consent Form (ICF) and this protocol.

[0267] Dose-limiting toxicity (DLT) Safety and tolerability will be assessed for each cohort. Dose-limiting toxicities (DLTs) will be assessed for the first 21 days for patients enrolled in the dose escalation part and for patients enrolled in the pembrolizumab combination cohort. A DLT is an adverse drug reaction of ≥ grade 3 according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0 occurring during the DLT evaluation period of the dose escalation cohort. An adverse drug reaction may be an adverse event suspected by the investigator and / or sponsor to be related to A49-F3'-TriNKET-Trastuzumab. A DLT is defined as any of the following occurring within the first 21 days of treatment for patients enrolled in the dose escalation part and for patients enrolled in the pembrolizumab combination cohort: - Any grade 3-4 non-hematologic toxicity except: i. Grade 3 nausea, vomiting, and diarrhea lasting <72 hours in the absence of maximal medical therapy; ii. Grade 4 vomiting and diarrhea lasting <72 hours in the absence of maximal medical therapy; iii. Grade 3 fatigue for <5 days; iv. Grade 3 hypertension in the absence of maximal medical therapy. - Any of the following hematological toxicities: i. Grade 4 neutropenia >5 days; ii. Grade 3 thrombocytopenia with bleeding; iii. Grade 4 thrombocytopenia. - and excluding: i. A single laboratory value outside the normal range that, according to the investigator, is unlikely to be related to study treatment, is not accompanied by any clinical symptoms, and resolves to ≤ Grade 1 within 7 days with appropriate medical management.

[0268] The DLT observation period may include the first 3 weeks of treatment with the investigational drug in the dose escalation part for all dose cohorts of all patients with data to use in implementing the dose escalation algorithm for the determination of the maximum tolerated dose (MTD). Additional patients may be enrolled in the dose escalation phase, and these patients may have adverse events collected; if necessary, these patients may not have a specific DLT observation period. The safety monitoring committee may take a conservative approach in considering treatment-related toxicity as drug-related. Treatment-related serious adverse events are considered drug-related unless a clear relationship to the underlying disease or commonly accepted comorbidities is evident.

[0269] Safety will be assessed by recording, reporting and analysis of baseline medical status, adverse events (AEs), vital signs and physical examination findings including determination of left ventricular ejection fraction, electrocardiograms, and laboratory tests.

[0270] Dosage and Administration A49-F3'-TriNKET-Trastuzumab dose escalation Dose levels are assigned to each patient upon study entry. Dose levels are adapted to body weight changes as necessary. The decision to escalate to the next dose level is based on safety assessment after all patients in the cohort reach day 21 (DLT evaluation period). In certain embodiments, patients are administered an IV infusion of A49-F3'-TriNKET-Trastuzumab over 1 hour (e.g., 50-70 minutes) once every 2 weeks. Dosage of A49-F3'-TriNKET-Trastuzumab is calculated based on the patient's body weight, determined the day before or the day of each drug administration. In an exemplary embodiment, the starting dose of A49-F3'-TriNKET-Trastuzumab is 5.2 x 10 -5 The initial 8 dose levels (DL) will follow an accelerated dose escalation design, consisting of single patient cohorts with escalation steps of 3.3-fold or less. If a DLT is observed, 5 more patients at the dose level where the DLT is observed will be accrued and the dose escalation will be switched to a "3+3" design.

[0271] Similar to the accelerated titration phase, dose escalation will proceed with no more than a 3.3-fold increase between dose levels in the "3+3" titration phase. Table 38 outlines the starting dose (mg / kg) and dose levels (DL) by body weight for the titration scheme. [Table 38]

[0272] In an exemplary embodiment, three patients are initially enrolled in a given dose cohort during the "3+3" phase. After enrolling the first patient, the second patient is enrolled immediately two days after the first patient's second injection of A49-F3'-TriNKET-Trastuzumab. After at least 48 hours of follow-up after administration of A49-F3'-TriNKET-Trastuzumab to the second patient, the third patient is administered the first dose of A49-F3'-TriNKET-Trastuzumab. More than three patients can be enrolled in a particular dose cohort (e.g., in the case of DLT observed in the first three patients of a particular cohort or after three patients are enrolled in DL 7) without any predefined interval between treatment initiation, as long as no infusion reaction, cytokine release syndrome, or treatment-related toxicity of grade 3 or higher is observed during treatment of the first three patients. In such cases, the same predefined interval as for the first 3 patients is repeated. If no DLT is observed in any of these patients, the study proceeds to enroll 3 additional patients in the next higher dose cohort. If one patient develops a DLT at a particular dose, 3 additional patients are enrolled in that same dose cohort. The occurrence of DLT in more than 1 of 6 patients in a particular dose cohort suggests that the MTD has been exceeded and that further dose escalation will not be pursued (see the Dose-Limiting Toxicity (DLT) section in this Example).

[0273] In an exemplary embodiment, once the safety of DL 10 (1.6 mg / kg) is established by the Safety Monitoring Committee, up to 10 additional patients (up to a total of 16 patients per DL) are treated at DL 9 to increase the safety, PK and PD database at that DL, but continue accrual at DL 11 according to the "3+3" rule. A similar process applies to DLs 10-13. Accrual in the Safety / PK / PD Expansion Cohort phase continues to proceed without a predefined observation period. Mandatory tumor biopsies are performed at screening (within 30 days prior to the first investigational drug dose) and within 1-7 days prior to the sixth investigational drug dose. Safety information is generated during treatment of these patients and communicated to the Safety Monitoring Committee. The same process is performed for subsequent DLs of the Safety / PK / PD Expansion Cohort.

[0274] Efficacy Expansion Cohort Dosing As previously described in this example, there are four efficacy expansion cohorts: the UBC cohort; the MBC cohort; the basket (HER2 3+) cohort, which includes patients with solid tumors that highly express HER2 and have received at least one first-line treatment consisting of an established or approved therapy; and the combination therapy cohort with pembrolizumab. Accumulation in these cohorts will begin as follows: - Three monotherapy cohorts will begin following dose- and schedule-finding of A49-F3'-TriNKET-trastuzumab in the first three cohorts (UBC, MBC, and basket disease (HER2 3+)) - Once safety of DL11 has been established by the Safety Monitoring Board, begin accrual of the safety run-in period for the combination of A49-F3'-TriNKET-trastuzumab and pembrolizumab. The dose of A49-F3'-TriNKET-trastuzumab in combination with pembrolizumab is declared safe during the "3+3" dose escalation of A49-F3'-TriNKET-trastuzumab as monotherapy prior to testing in combination with pembrolizumab (subsequent DLs are approved as safe by the Safety Monitoring Board during dose escalation).

[0275] In the first three efficacy expansion cohorts, patients will receive A49-F3'-TriNKET-trastuzumab as monotherapy. Up to 40 patients may be enrolled in each of these three expansion cohorts, with the first 20 patients in each cohort being observed for at least 3 months before a futility analysis. Mandatory tumor biopsies will be performed at screening (within 30 days prior to the first study drug dose) and within 1-7 days prior to the sixth study drug dose.

[0276] In the efficacy expansion cohort of combination therapy with pembrolizumab, patients will receive A49-F3'-TriNKET-trastuzumab (as a 1 hour IV infusion) at DL 10 and the approved dose of pembrolizumab at 200 mg (as a 30 minute IV infusion) in a 3 week treatment cycle. This safety run-in period will be followed by the same "3+3" design as previously described. Patients in this study will meet the patient inclusion criteria described in the "Inclusion Criteria" section.

[0277] Safety in the efficacy expansion cohort (A49-F3'-TriNKET-Trastuzumab monotherapy cohort): All safety information from participating patients is continuously monitored by the safety monitoring committee. In an exemplary embodiment, a group of 20 patients is enrolled and followed by the safety monitoring committee for 4 weeks. Then, within 4 weeks after treating 40 patients and following for at least 4 weeks, the above safety review is performed. Then, the same process is performed, each time enrolling 40 patients and following for at least 4 weeks.

[0278] Safety during the Efficacy Expansion Cohort (Pembrolizumab and A49-F3'-TriNKET-Trastuzumab Combination Therapy Cohort): All safety information from participating patients will be continuously monitored by the Safety Monitoring Committee, similar to that described for the "3+3" dose escalation part. For each patient, safety and tolerability data will be reviewed by the Safety Monitoring Committee over a 21-day DLT evaluation period, and progression to further doses will depend on the Safety Monitoring Committee. If the combination treatment is safe to progress (as determined by the Safety Monitoring Committee), a group of 20 patients will be enrolled and followed for 3 weeks.

[0279] Evaluation items The study is designed to evaluate the clinical benefit of A49-F3'-TriNKET-trastuzumab, optionally in combination with pembrolizumab, as a treatment for patients with locally advanced or metastatic solid tumors, by assessing primary and secondary endpoints.

[0280] Primary and secondary endpoint analysis The occurrence of DLT during the first 3 weeks of treatment is measured as the primary endpoint in the dose escalation part. The maximum tolerated dose (MTD) is determined during the dose escalation part and is defined as the highest dose level (DL) at which no more than 1 of 6 treated patients experience a DLT event. The maximum tolerated dose is determined by individual patient data from the dose escalation part. In addition, for the final statistical analysis, the following can be analyzed: - The number and proportion of patients in the DLT population who experience a DLT during the first DLT evaluation period at each dose level; - The number and proportion of treatment-emergent adverse events experienced by patients in the DLT population during the first DLT evaluation period at each dose level.

[0281] Confirmed overall response rate according to mRECIST 1.1, adjudicated by an Independent Endpoint Review Committee (IERC), will be measured as the primary endpoint for the efficacy expansion cohort. Overall response rate will be defined as the best response achieved during all tumor assessment visits after initiation of study treatment until documented disease progression, taking into account the following requirements for confirmation: For complete and partial responses, responses must be confirmed according to mRECIST 1.1. Confirmation may be assessed at regularly scheduled 6-week assessment intervals, but no sooner than 4 weeks after first demonstration of a complete or partial response. Confirmed partial responses may be confirmed at any assessment later than the next assessment after first demonstration of a partial response.

[0282] Best overall response of stable disease may require that the overall response of stable disease be assessed at least 37 days after the start of study treatment. Responses at each scheduled tumor evaluation, and best overall response, will be listed for each patient.

[0283] Secondary and secondary endpoints analysis Secondary endpoints of the study may include: - Number, severity, and duration of treatment-emergent adverse events across all dose groups / indications per NCI-CTCAE v5.0; - Number, severity, and duration of treatment-related adverse events according to NCI-CTCAE v5.0; - Duration of response per investigator assessment by mRECIST 1.1; - Pharmacokinetic profile; - Best overall response per mRECIST 1.1 as assessed by the investigator; - Progression-free survival per investigator assessment by mRECIST 1.1; - overall survival; - Progressive disease profile; - serum titers of anti-A49-F3'-TriNKET-Trastuzumab antibodies; - HER2 expression on tumor tissue; - ERBB2 status (amplified / unamplified, mutated / nonmutated); - Undetermined efficacy at week 13 by mRECIST 1.1 (for safety / PK / PD expansion cohort); - Progression-free survival by IERC mRECIST 1.1; duration of response by IERC mRECIST 1.1 (for efficacy expansion cohort).

[0284] Efficacy parameters: The primary efficacy parameter in the expansion part was mRECIST The best overall response is best overall response by mRECIST 1.1. ORR according to investigator assessment will be determined according to mRECIST 1. Overall response rate will be assessed throughout the entire study period. For best overall responses of partial and complete response, confirmation of response by mRECIST 1.1 is required. Responses at each scheduled tumor assessment and best overall response will be listed for each patient. Overall response rate (defined as complete response + partial response) values ​​and percentages will be tabulated by cohort. For the HER2 high basket cohort, overall phase response values ​​and percentages will be tabulated for each tumor type with >5 patients enrolled and treated for 4 weeks. Tumor types represented by <5 patients (1-4 patients) will be presented as a single subgroup. Duration of response by mRECIST 1.1 will be calculated for each patient with confirmed response in the expansion cohort and analyzed using Kaplan-Meier methods in all cohorts. Progression-free survival and overall survival will be presented in patient listings and analyzed using the Kaplan-Meier method in the full analysis set of the expansion cohort enrolling all planned patients.

[0285] Pharmacokinetic Profile: Serum concentrations of A49-F3'-TriNKET-Trastuzumab will be determined by validated methods. The following PK parameters will be estimated and reported: - AUC0→t: area under the concentration-time curve from time of administration to the time of last observation (calculated by linear trapezoidal summation); - ● AUC0→∞: area under the curve from the time of administration extrapolated to infinity (calculated by linear trapezoidal summation and extrapolated to infinity using Clast / λz); - λz: terminal elimination rate constant. The value of λz is determined from the slope of the regression line of log(concentration) versus time with the following constraints: (i) there must be at least three consecutive measurable concentrations, (ii) all concentrations must decrease with time, and (iii) the correlation coefficient (r) of the regression must be ≥ 0.95; - Cmax: maximum serum concentration observed after administration; - tmax: the time when Cmax occurs; and - Estimated half-life determined as t1 / 2: 0.693 / λz.

[0286] PK parameters will be summarized using descriptive statistics. Mean concentration-time plots as well as individual concentration-time plots will be depicted. Unexplained missing data may be imputed if the consistency of the analysis will be affected. A conservative principle will be used for data imputation.

[0287] Serum titers of anti-drug antibodies: A safety immunogenicity testing strategy will be implemented and performed as follows: - Immunogenicity Assessment of Biotechnology-Derived Therapeutic Proteins(Guideline on Immunogenicity Assessment of Therapeutic Proteins. 18 May 2017 EMEA / CHMP / BMWP / 14327 / 2006 Rev 1 Committee for Medicinal Products for Human Use (CHMP); European Medicines Agency); - Immunogenicity assessment of mAbs intended for in vivo clinical use(Guideline on Immunogenicity Assessment of Monoclonal Antibodies Intended for In Vivo Clinical Use. 24 May 2012 EMA / CHMP / BMWP / 86289 / 2010 Committee for Medicinal Products for Human Use (CHMP); see European Medicines Agency); - FDA (2009, draft) Guidance for Industry: Assay Development for Immunogenicity Testing of Therapeutic Proteins.

[0288] Apply a qualified method that uses an acid dissociation step to detect anti-drug (i.e., anti-A49-F3'-TriNKET-Trastuzumab) antibodies in the presence of excess drug in human serum. There is no need to remove the drug after acid treatment. Determine the ADA titer of positive samples.

[0289] Biomarkers: Summary statistics for biomarkers will be provided for all pre-planned time points separately for each DL or cohort. Changes to baseline levels will be presented where applicable. Profiles over time will be presented for each patient.

[0290] Safety Analysis: The extent of exposure to A49-F3'-TriNKET-Trastuzumab will be characterized by duration (weeks), number of doses, cumulative dose (mg / kg), dose intensity (mg / kg / week), relative dose intensity (actual dose administered / planned dose), number of dose reductions, and number of dose delays. Safety analyses will be performed in the safety population. Descriptive statistics will be used to tabulate safety endpoints by DL and cohort. Safety assessments will be based on review of the occurrence of adverse events, including adverse events of special interest, adverse drug reactions, and changes in vital signs, electrocardiograms, weight, and laboratory values ​​(hematology and serum chemistry). The on-treatment period will be defined as the period from the first dose of study treatment to the last dose of study treatment +30 days or the first date of new anticancer therapy -1 day, whichever occurs first.

[0291] Adverse Events (AEs): Adverse events will be coded according to the Medical Dictionary for Regulatory Activities (MedDRA). The severity of AEs will be graded using the NCI-CTCAE v5.0 Toxicity Grading Scale. Treatment-emergent adverse events (TEAEs) are AEs on the start date of the on-treatment period or if there is a progression of the event during the on-treatment period. The occurrence of TEAEs from any cause and AEs defined as potentially related to A49-F3'-TriNKET-Trastuzumab will be summarized by preferred term and system organ class and described in terms of intensity and relationship to A49-F3'-TriNKET-Trastuzumab. All premature / permanent discontinuations will be summarized by primary reason for withdrawal from the study. The duration of a TEAE will be defined as the time between onset and resolution to baseline. The duration of grades 3 and 4 will be defined by the time a particular TEAE reaches grade 3 or 4 severity over its course. Descriptive statistics will be examined to indicate dose-related ADRs.

[0292] Test variables: Test results are categorized by grade according to NCI-CTCAE. Worst on-trial grade after initial study treatment is summarized. Change in toxicity grading from initial treatment to highest grade is shown. Results for variables not part of NCI-CTCAE are presented as below, within, or above normal range. Only patients with post-baseline test values ​​are included in this analysis.

[0293] PE (including vital signs, 12-lead ECG, and transthoracic echocardiography (TT-ECHO) / MUGA): Record PE data, including vital signs (temperature, respiratory rate, heart rate, and blood pressure) and 12-lead ECG.

[0294] Incorporation by Reference The entire disclosures of each of the patent documents and scientific articles referenced herein are incorporated by reference for all purposes.

[0295] Equivalent The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all respects as illustrative rather than limiting the present disclosure described herein. The various structural elements of the different embodiments and the various method steps disclosed may be used in various combinations and permutations, and all such variations are considered to be possible forms of the present disclosure. Therefore, the scope of the present disclosure is indicated by the appended claims, rather than by the above description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein. In certain embodiments, for example, the following are provided: (Item 1) (a)(i) a Fab that binds to NKG2D, (ii) a single chain variable fragment (scFv) that binds to HER2, and (iii) a multispecific binding protein comprising an antibody Fc domain; (b) histidine; (c) with a sugar or sugar alcohol; (d) Polysorbate and A pharmaceutical formulation having a pH of 5.5 to 6.5. (Item 2) 2. The pharmaceutical preparation according to item 1, wherein the concentration of histidine in the pharmaceutical preparation is 10 to 25 mM. (Item 3) Item 4. The pharmaceutical preparation according to item 2, wherein the concentration of histidine in the pharmaceutical preparation is about 20 mM. 4. The pharmaceutical formulation according to any one of items 1 to 3, wherein the sugar or sugar alcohol is a disaccharide. (Item 5) 5. The pharmaceutical formulation according to item 4, wherein the disaccharide is sucrose. (Item 6) 4. The pharmaceutical formulation according to any one of items 1 to 3, wherein the sugar or sugar alcohol is a sugar alcohol derived from a monosaccharide. (Item 7) The pharmaceutical formulation according to item 6, wherein the sugar alcohol derived from a monosaccharide is sorbitol. 8. The pharmaceutical formulation according to any one of items 4 to 7, wherein the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is 200 to 300 mM. (Item 9) 9. The pharmaceutical formulation according to item 8, wherein the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM. (Item 10) 10. The pharmaceutical formulation according to any one of items 1 to 9, wherein the polysorbate is polysorbate 80. (Item 11) Item 11. The pharmaceutical formulation according to item 10, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is 0.005% to 0.05%. (Item 12) 12. The pharmaceutical formulation according to item 11, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01%. (Item 13) 13. The pharmaceutical formulation according to any one of items 1 to 12, wherein the concentration of NaCl, if present, in the pharmaceutical formulation is about 10 mM or less. (Item 14) 14. The pharmaceutical formulation according to item 13, wherein the concentration of NaCl, if present, in the pharmaceutical formulation is about 1 mM or less. (Item 15) 15. The pharmaceutical formulation according to any one of items 1 to 14, wherein the pH is 5.8 to 6.2. (Item 16) 16. The pharmaceutical formulation according to any one of items 1 to 15, wherein the pH is 5.95 to 6.05. (Item 17) the Fab comprises a heavy chain variable domain and a light chain variable domain; (a) the heavy chain variable domain comprises 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; (b) the light chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100 and 101, respectively; 17. The pharmaceutical formulation according to any one of items 1 to 16. (Item 18) (a) the heavy chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96 and 169, respectively; (b) the light chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100 and 101, respectively; 18. The pharmaceutical formulation according to item 17. (Item 19) 19. The pharmaceutical formulation of any one of items 1 to 18, wherein the heavy chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 20) 20. The pharmaceutical formulation of any one of items 1 to 19, wherein the heavy chain variable domain of the Fab comprises the amino acid sequence of SEQ ID NO: 94 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 98. (Item 21) the scFv comprises a heavy chain variable domain and a light chain variable domain; (a) the heavy chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116 and 117, respectively; (b) the light chain variable domain comprises the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120 and 121, respectively; 21. The pharmaceutical formulation according to any one of items 1 to 20. (Item 22) 22. The pharmaceutical formulation of claim 21, wherein the heavy chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195 and the light chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 196. (Item 23) 23. The pharmaceutical formulation of claim 21, wherein the heavy chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 195 and the light chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 196. (Item 24) 24. The pharmaceutical formulation of any one of items 21 to 23, wherein the light chain variable domain of the scFv is linked to the heavy chain variable domain of the scFv via a flexible linker. (Item 25) 25. The pharmaceutical formulation of item 24, wherein the flexible linker comprises the amino acid sequence of SEQ ID NO: 143. (Item 26) 26. The pharmaceutical formulation according to item 24 or 25, wherein the flexible linker consists of the amino acid sequence of SEQ ID NO: 143. (Item 27) 27. The pharmaceutical formulation of any one of items 21 to 26, wherein the light chain variable domain of the scFv is located N-terminal to the heavy chain variable domain of the scFv. (Item 28) 28. The pharmaceutical formulation according to any one of items 21 to 27, wherein the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. (Item 29) 29. The pharmaceutical formulation of item 28, wherein the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain. (Item 30) 30. The pharmaceutical formulation according to any one of items 21 to 29, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 139. (Item 31) 31. The pharmaceutical formulation of any one of items 1 to 30, wherein the antibody Fc domain comprises a first antibody Fc sequence linked to the Fab and a second antibody Fc sequence linked to the scFv. (Item 32) 32. The pharmaceutical formulation of claim 31, wherein the first antibody Fc sequence is linked to the heavy chain portion of the Fab. (Item 33) 33. The pharmaceutical formulation of claim 31 or 32, wherein the scFv is linked to the second antibody Fc sequence via a hinge comprising Ala-Ser. (Item 34) 34. The pharmaceutical formulation of any one of items 31 to 33, wherein the first and second antibody Fc sequences each comprise the hinge and CH2 domains of a human IgG1 antibody. (Item 35) Item 36. The pharmaceutical formulation according to Item 34, wherein the first and second antibody Fc sequences each comprise an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a wild-type human IgG1 antibody. 36. The pharmaceutical formulation of any one of items 31 to 35, wherein the first and second antibody Fc sequences contain different mutations that promote heterodimerization. (Item 37) 37. The pharmaceutical formulation of item 36, wherein the first antibody Fc sequence is a human IgG1 Fc sequence comprising a K360E and K409W substitution. (Item 38) 38. The pharmaceutical formulation of item 36 or 37, wherein the second antibody Fc sequence is a human IgG1 Fc sequence comprising Q347R, D399V and F405T substitutions. (Item 39) the multispecific binding protein comprising: (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. 39. The pharmaceutical formulation according to any one of the preceding items, comprising: (Item 40) 40. The pharmaceutical formulation according to any one of items 1 to 39, wherein the concentration of the multispecific binding protein in the pharmaceutical formulation is about 10 to about 20 mg / mL. (Item 41) 41. The pharmaceutical formulation of any one of items 1 to 40, wherein more than 94% of the multispecific binding protein has a native conformation as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. (Item 42) 42. The pharmaceutical formulation of any one of items 1 to 41, wherein less than 4% of the multispecific binding protein forms high molecular weight complexes as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. (Item 43) 43. A pharmaceutical formulation according to any one of items 1 to 42 for use in the treatment of cancer. (Item 44) 44. The pharmaceutical formulation according to item 43, which is diluted with 0.9% NaCl solution before said use. (Item 45) 1. A method for treating cancer, comprising administering to a subject in need thereof a multispecific binding protein on days 1, 8 and 15 of an initial 4 week treatment cycle, wherein the multispecific binding protein comprises: (a) Fab that binds to NKG2D; (b) an scFv that binds to HER2; and (c) Antibody Fc domain A method comprising: (Item 46) 46. ​​The method of claim 45, further comprising administering to the subject the multispecific binding protein in one or more subsequent 4 week treatment cycles after the initial treatment cycle, wherein the multispecific binding protein is administered on days 1 and 15 in each subsequent treatment cycle. (Item 47) Each dose contained 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 47. The method of claim 45 or 46, comprising administering to said patient 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, and 50 mg / kg. (Item 48) 48. The method of any one of items 45 to 47, wherein the multispecific binding protein is administered by intravenous infusion. (Item 49) 49. The method of any one of items 45 to 48, wherein the multispecific binding protein is used as a monotherapy. (Item 50) 49. The method of any one of items 45 to 48, further comprising administering to the subject an anti-PD-1 antibody. (Item 51) 51. The method of claim 50, wherein the anti-PD-1 antibody is pembrolizumab. (Item 52) 52. The method of item 51, wherein 200 mg of pembrolizumab is administered on day 1 of the first treatment cycle. (Item 53) 53. The method of item 51 or 52, wherein, if the subject receives one or more subsequent treatment cycles, 200 mg of pembrolizumab is administered once every three weeks in the subsequent treatment cycles. (Item 54) 54. The method of any one of items 45 to 53, wherein the cancer is HER2 positive as determined by immunohistochemistry. (Item 55) 54. The method of any one of items 45 to 53, wherein the HER2 level in the cancer is scored as at least 1+ as determined by immunohistochemistry. (Item 56) 56. The method of item 54 or 55, wherein the HER2 level in the cancer is scored as 2+ or 3+. (Item 57) 56. The method of item 54 or 55, wherein the HER2 level in the cancer is scored as 3+. (Item 58) 58. The method of any one of items 45 to 57, wherein the cancer has an amplification of the ERBB2 gene. (Item 59) 59. The method of claim 58, wherein ERBB2 gene amplification is determined by fluorescent in situ hybridization. (Item 60) 59. The method of claim 58, wherein ERBB2 gene amplification is determined by DNA sequencing. (Item 61) 61. The method of any one of items 45 to 60, wherein the cancer is a solid tumor. (Item 62) 62. The method of claim 61, wherein the cancer is a locally advanced or metastatic solid tumor. (Item 63) 63. The method of claim 62, wherein the cancer is urothelial bladder cancer or metastatic breast cancer. (Item 64) 63. The method of claim 61 or 62, wherein the cancer is selected from the group consisting of gastric cancer, colorectal cancer, non-small cell lung cancer (NSCLC), head and neck cancer, biliary tract cancer, glioblastoma, sarcoma, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, squamous cell carcinoma of the skin, prostate cancer, salivary gland cancer, breast cancer, pancreatic cancer, and gallbladder cancer. (Item 65) the Fab comprises a heavy chain variable domain and a light chain variable domain; (a) the heavy chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96 and 188, respectively; (b) the light chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100 and 101, respectively; 65. The method according to any one of items 45 to 64. (Item 66) (a) the heavy chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 168, 96 and 169, respectively; (b) the light chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100 and 101, respectively; Item 66. The method according to item 65. (Item 67) 67. The method of any one of items 45 to 66, wherein the heavy chain variable domain of the Fab comprises an amino acid sequence at least 90% identical to SEQ ID NO: 94 and the light chain variable domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 98. (Item 68) 68. The method of any one of items 45 to 67, wherein the heavy chain variable domain of the Fab comprises the amino acid sequence of SEQ ID NO: 94 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 98. (Item 69) the scFv comprises a heavy chain variable domain and a light chain variable domain; (a) the heavy chain variable domain comprises CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 115, 116 and 117, respectively; (b) the light chain variable domain comprises the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 119, 120 and 121, respectively; 69. The method according to any one of items 45 to 68. (Item 70) 70. The method of claim 69, wherein the heavy chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 195 and the light chain variable domain of the scFv comprises an amino acid sequence at least 90% identical to SEQ ID NO: 196. (Item 71) 71. The method of claim 69 or 70, wherein the heavy chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 195 and the light chain variable domain of the scFv comprises the amino acid sequence of SEQ ID NO: 196. (Item 72) 72. The method of any one of items 69 to 71, wherein the light chain variable domain of the scFv is linked to the heavy chain variable domain of the scFv via a flexible linker. (Item 73) 73. The method of claim 72, wherein the flexible linker comprises the amino acid sequence of SEQ ID NO: 143. (Item 74) 74. The method according to item 72 or 73, wherein the flexible linker consists of the amino acid sequence of SEQ ID NO: 143. (Item 75) 75. The method of any one of items 69 to 74, wherein the light chain variable domain of the scFv is located N-terminal to the heavy chain variable domain of the scFv. (Item 76) 76. The method of any one of items 69 to 75, wherein the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. (Item 77) 77. The method of claim 76, wherein the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain. (Item 78) 78. The method according to any one of items 69 to 77, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 139. (Item 79) 79. The method of any one of items 45 to 78, wherein the antibody Fc domain comprises a first antibody Fc sequence linked to the Fab and a second antibody Fc sequence linked to the scFv. (Item 80) 80. The method of claim 79, wherein the first antibody Fc sequence is linked to the heavy chain portion of the Fab. (Item 81) 81. The method of claim 79 or 80, wherein the scFv is linked to the second antibody Fc sequence via a hinge comprising Ala-Ser. (Item 82) 82. The method of any one of items 79 to 81, wherein the first and second antibody Fc sequences each comprise the hinge and CH2 domains of a human IgG1 antibody. (Item 83) 83. The method of item 82, wherein the first and second antibody Fc sequences each comprise an amino acid sequence that is at least 90% identical to amino acids 234 to 332 of a wild-type human IgG1 antibody. (Item 84) 84. The method of any one of items 79 to 83, wherein the first and second antibody Fc sequences contain different mutations that promote heterodimerization. (Item 85) 85. The method of claim 84, wherein the first antibody Fc sequence is a human IgG1 Fc sequence comprising a K360E and K409W substitution. (Item 86) 86. The method of claim 84 or 85, wherein the second antibody Fc sequence is a human IgG1 Fc sequence comprising Q347R, D399V and F405T substitutions. (Item 87) the multispecific binding protein comprising: (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. 87. The method according to any one of items 45 to 86, comprising: (Item 88) 88. The method of any one of items 45 to 87, wherein the multispecific binding protein is in a pharmaceutical formulation comprising histidine, a sugar or sugar alcohol, and a polysorbate, at a pH of 5.5 to 6.5. (Item 89) Item 89. The method according to Item 88, wherein the concentration of histidine in the pharmaceutical preparation is 10 to 25 mM. (Item 90) 90. The method of claim 89, wherein the concentration of histidine in the pharmaceutical formulation is about 20 mM. (Item 91) 91. The method according to any one of items 88 to 90, wherein the sugar or sugar alcohol in the pharmaceutical formulation is a disaccharide. (Item 92) Item 92. The method of item 91, wherein the disaccharide is sucrose. (Item 93) 93. The method according to any one of items 88 to 92, wherein the sugar or sugar alcohol in the pharmaceutical formulation is a sugar alcohol derived from a monosaccharide. (Item 94) Item 94. The method of item 93, wherein the sugar alcohol derived from a monosaccharide is sorbitol. (Item 95) 95. The method according to any one of items 88 to 94, wherein the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is 200 to 300 mM. (Item 96) 96. The method of claim 95, wherein the concentration of the sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM. (Item 97) 97. The method of any one of items 88 to 96, wherein the polysorbate in the pharmaceutical formulation is polysorbate 80. (Item 98) Item 98. The method according to item 97, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is 0.005% to 0.05%. (Item 99) 99. The method of claim 98, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01%. (Item 100) 99. The method of any one of items 88 to 99, wherein the concentration of NaCl, if present, is about 10 mM or less in the pharmaceutical formulation. (Item 101) 101. The method of claim 100, wherein the concentration of NaCl, if present, is about 1 mM or less in the pharmaceutical formulation. (Item 102) 102. The method according to any one of items 88 to 101, wherein the pH of the pharmaceutical formulation is 5.8 to 6.2. (Item 103) 103. The method according to any one of items 88 to 102, wherein the pH of the pharmaceutical formulation is 5.95 to 6.05. (Item 104) 104. The method of any one of items 88 to 103, wherein the concentration of the multispecific binding protein in the pharmaceutical formulation is about 10 to about 20 mg / mL. (Item 105) 105. The method of any one of items 88 to 104, wherein more than 94% of the multispecific binding protein in the pharmaceutical formulation has a native conformation as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. (Item 106) 106. The method of any one of items 88 to 105, wherein less than 4% of the multispecific binding protein in the pharmaceutical formulation forms high molecular weight complexes as determined by size exclusion chromatography after 3 weeks of incubation at 50° C. (Item 107) 107. The method according to any one of items 88 to 106, wherein the pharmaceutical formulation is diluted with 0.9% NaCl solution before administration to a subject in need thereof.

Claims

[Claim 1] The invention described in the specification.

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