Methods of treating cancer using multispecific binding proteins that bind NKG2d, CD16, and her2

Abstract

The present disclosure provides methods of treating cancer using multispecific binding proteins that bind NKG2D, CD16, and HER2, as a monotherapy or in combination with other cancer therapies such as targeted therapeutic or immunotherapeutic agents.

Description

Attorney Docket No.: DFY-135WO METHODS OF TREATING CANCER USING MULTISPECIFIC BINDING PROTEINS THAT BIND NKG2D, CD16, AND HER2 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 598,377, filed November 13, 2023, the entire contents of each of which are incorporated by reference herein for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file copy, created on November 01, 2024, is named DFY-135WO_SL.xml and 168,482 bytes in size. FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to methods of treating cancer using multispecific binding proteins that bind NKG2D, CD16, and HER2, as a monotherapy or in combination with other cancer therapies such as targeted therapeutic or immunotherapeutic agents. BACKGROUND

[0004] Cancer continues to be a significant health problem despite the substantial research efforts and scientific advances reported in the literature for treating this disease. Cancer immunotherapies are being developed to facilitate destruction of cancer cells using the patient’s own immune system. The immune cells activated by cancer immunotherapies include T cells and natural killer (NK) cells. For example, bispecific T-cell engagers are designed to direct T cells against tumor cells, thereby rendering cytotoxicity against the tumor cells. Bispecific antibodies that bind NK cells and a tumor-associated antigen (TAA) have also been created for cancer treatment.

[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, which is correlated with substantially lower overall survival rates and shorter disease-free intervals than patientsAttorney Docket No.: DFY-135WO whose cancer does not overexpress HER2. Moreover, overexpression of HER2 leads to increased breast cancer metastasis. Over-expression of HER2 is also known to occur in many other cancer types, including ovarian, esophageal, bladder and gastric cancer, salivary duct carcinoma, adenocarcinoma of the lung, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma.

[0006] Despite advances that have been made to date in treating cancers associated with HER2, there is still a need for additional methods and compositions for effectively treating subjects with HER2 associated cancers. SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides methods of treating cancer using multispecific binding proteins that bind NKG2D, CD16, and HER2. Such a multispecific binding protein, also called a “TriNKET,” redirects an NK cell, which is an innate immune lymphocyte, to target aberrant cells that expresses HER2. Co-engagement of NKG2D and CD16 synergistically enhances cytotoxicity of the NK cells towards the HER2-expressing target cells. Such TriNKETs may be used to treat patients having HER2-expressing cancer. More specifically, the present provides clinical methods, including dosage regimens, to treat patients with specific HER2-targeting cancer immunotherapies with desired safety and efficacy. Furthermore, the present disclosure provides formulations containing such cancer immunotherapies that are sufficiently stable and suitable for administration to patients.

[0008] Accordingly, in one aspect, the present disclosure provides a method of treating cancer, the method includes administering to a subject in need thereof an effective amount of a multispecific binding protein including (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) a first antibody Fc region and a second antibody Fc region that together form a dimer that binds CD16, wherein the subject also receives an effective amount of an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor.

[0009] The anti-TROP2 antibody can be sacituzumab. The topoisomerase I inhibitor can be SN-38. The anti-TROP2 antibody can be conjugated to the topoisomerase I inhibitor through a hydrolysable linker. In a specific embodiment, the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor is sacituzumab govitecan-hziy.

[0010] The cancer treated by this method can be a breast cancer, for example, a hormone receptor (HR) positive and HER2 negative metastatic breast cancer or a HER2Attorney Docket No.: DFY-135WO positive metastatic breast cancer. The cancer treated by this method can also be a lung cancer, for example, a HER2 activated non-small cell lung cancer (NSCLC).

[0011] The anti-TROP2 antibody conjugated to the topoisomerase I inhibitor (e.g., sacituzumab govitecan-hziy) can be administered intravenously at a dose of 10 mg / kg on Day 1 and Day 8 of one or more three-week treatment cycles. To conform to this treatment schedule, the multispecific binding protein can be administered intravenously at a dose of 7.5 mg / kg on Day 1 and Day 8 of the one or more three-week treatment cycles.

[0012] In another aspect, the present disclosure provides a method of treating a HER2 activated non-small cell lung cancer (NSCLC), the method includes administering to a subject in need thereof an effective amount of a multispecific binding protein including (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) a first antibody Fc region and a second antibody Fc region that together form a dimer that binds CD16.

[0013] The multispecific binding protein can be used as a monotherapy, which can be administered intravenously at a dose of 10 mg / kg on Day 1, Day 8, and Day 15 of an initial four-week treatment cycle, and on Day 1 and Day 15 of one or more subsequent four-week treatment cycles.

[0014] Alternatively, the subject may also receive an effective amount of an anti- TROP2 antibody conjugated to a toposiomerase I inhibitor. The anti-TROP2 antibody can be sacituzumab. The topoisomerase I inhibitor can be SN-38. The anti-TROP2 antibody can be conjugated to the topoisomerase I inhibitor through a hydrolysable linker. In a specific embodiment, the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor is sacituzumab govitecan-hziy. The anti-TROP2 antibody conjugated to the topoisomerase I inhibitor (e.g., sacituzumab govitecan-hziy) can be administered intravenously at a dose of 10 mg / kg on Day 1 and Day 8 of one or more three-week treatment cycles. To conform to this treatment schedule, the multispecific binding protein can be administered intravenously at a dose of 7.5 mg / kg on Day 1 and Day 8 of the one or more three-week treatment cycles.

[0015] The multispecific binding protein used in any of the methods above can include the first antigen-binding site having (a) a heavy chain variable domain (VH) including complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences including the amino acid sequences of SEQ ID Nos: 95, 96, and 121, respectively; and (b) a light chain variable domain (VL) including CDR1, CDR2, and CDR3 sequences including the amino acid sequences of SEQ ID Nos: 99, 100, and 101, respectively. In a specific example, the VH ofAttorney Docket No.: DFY-135WO the first antigen-binding site includes CDR1, CDR2, and CDR3 sequences including the amino acid sequences of SEQ ID NOs: 95, 96, and 97, respectively; and the VL of the first antigen-binding site includes CDR1, CDR2, and CDR3 sequences including the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively. In certain embodiments, the VH of the first antigen-binding site includes an amino acid sequence at least 90% identical to SEQ ID NO:94, and the VL of the first antigen-binding site includes an amino acid sequence at least 90% identical to SEQ ID NO:98.

[0016] The multispecific binding protein used in any of the methods above can include the second antigen-binding site having (a) a VH including CDR1, CDR2, and CDR3 sequences including g the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively; and (b) a VL including CDR1, CDR2, and CDR3 sequences including the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively. The VH of the second antigen-binding site can include an amino acid sequence at least 90% identical to SEQ ID NO:156, and the VL of the second antigen-binding site can include an amino acid sequence at least 90% identical to SEQ ID NO:160.

[0017] The first antigen-binding site can be included in an antibody Fab fragment linked to an N-terminus of the first antibody Fc region, and the second antigen-binding site can be included in a single chain variable fragment (scFv) linked to an N-terminus of the second antibody Fc region.

[0018] The first antibody Fc region and the second antibody Fc region can each be a human IgG1 Fc region. The first antibody Fc region and the second antibody Fc region can each have an amino acid sequence at least 90% identical to SEQ ID NO: 141 and optionally each include one or more mutations, relative to SEQ ID NO: 141, to promote heterodimerization. For example, the first antibody Fc region includes K360E and K409W substitutions relative to SEQ ID NO: 141, and the second antibody Fc region can include Q347R, D399V and F405T substitutions relative to SEQ ID NO: 141, numbered according to the EU numbering system. Alternatively or in addition, the first antibody Fc region can include a Y349C substitution relative to SEQ ID NO: 141, and the second antibody Fc region can include an S354C substitution relative to SEQ ID NO: 141, numbered according to the EU numbering system.

[0019] In a specific example, the multispecific binding protein includes (a) a first polypeptide having the amino acid sequence of SEQ ID NO:184; (b) a second polypeptide having the amino acid sequence of SEQ ID NO:183; and (c) a third polypeptide having the amino acid sequence of SEQ ID NO:185.Attorney Docket No.: DFY-135WO

[0020] Other embodiments and details of the disclosure are presented herein below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG.1 illustrates a multispecific binding protein that contains a HER2-binding scFv, a NKG2D-targeting Fab, and a first antibody Fc region and a second antibody Fc region that form a dimer that binds CD16. In an exemplary embodiment, the Fc region linked to the Fab fragment comprises mutations of K360E and K409W and the Fc region linked to the scFv comprises matching mutations Q347R, D399V, and F405T for forming an Fc heterodimer (shown as a triangular lock-and-key format in the Fc domains in FIG.1). The antibody format is referred herein as F3’-TriNKET. In another exemplary embodiment, the Fc region linked to the Fab fragment can comprises the mutations of Q347R, D399V, and F405T, and the Fc region linked to the scFv comprises matching mutations K360E and K409W for forming a heterodimer. DETAILED DESCRIPTION

[0022] The present disclosure provides methods of treating cancer using multispecific binding proteins that bind NKG2D, CD16, and HER2. Such a multispecific binding protein, also called a “TriNKET,” redirects an NK cell, which is an innate immune lymphocyte, to target aberrant cells that expresses HER2. Co-engagement of NKG2D and CD16 synergistically enhances cytotoxicity of the NK cells towards the HER2-expressing target cells. Such TriNKETs may be used to treat patients having HER2-expressing cancer. More specifically, the present provides clinical methods, including dosage regimens, to treat patients with specific HER2-targeting cancer immunotherapies with desired safety and efficacy. Furthermore, the present disclosure provides formulations containing such cancer immunotherapies that are sufficiently stable and suitable for administration to patients. Definitions

[0023] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0024] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0025] As used herein, the term “antigen-binding site” refers to the part of the immunoglobulin molecule that participates in antigen binding. In certain embodiments (e.g., human antibodies), the antigen-binding site is formed by amino acid residues of the N-Attorney Docket No.: DFY-135WO terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains, which are also called “VH” and “VL,” respectively. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as “hypervariable regions” which are interposed between more conserved flanking stretches known as “framework regions,” or “FR.” Thus, the term “FR” refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. The three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed 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 a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” Exemplary forms of antigen-binding sites that include a VH and a VL include but are not limited to an antigen-binding fragment of an antibody that retains the antigen-binding surface, such as a Fab fragment, and a recombinant polypeptide using a peptide linker to connect the VH to the VL in a single polypeptide, such as a single-chain variable fragment (scFv). In certain embodiments, an antigen-binding site is formed by a single antibody chain providing a “single domain antibody” or “sdAb” (e.g., “VHH” in camelid or “VNAR” in cartilaginous fish). A “single domain antibody” is a small polypeptide or protein (generally ~12-15 kDa) including a single monomeric heavy chain variable domain or a single monomeric or light chain variable domain, but not both. For example, a VHH generally includes four framework regions flanking three complementarity determining regions (CDRs), represented as FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. A VNARgenerally includes only two CDRs, namely, CDR1 and CDR3, but hypervariable regions HV2 and HV4 also contribute to antibody diversity. Single domain antibodies can also be synthetically produced using techniques well-known in the art. For example, methods of designing and producing single domain antibodies are described in Hoey et al., Exp. Biol. Med.244:1568- 76 (2019), Sonneson et al., Biochemistry 48:6693–95 (2009), Saerens et al. J. Mol. Biol. 352:597–607 (2005), Wagner et al. Int. J. Mol. Sci.19:pii:E3444 (2018), Rizk et al. Nat. Struct. Mol. Biol.18:437–42 (2011). A single domain antibody can also be synthetic, e.g., a synethtic fully human antibody. An exemplary method of generating synthetic fully human VH antibodies based on human germline sequences is described in Mindrebo et al., (2023) Proc. Natl. Acad. Sci. U.S.A.120(24): e2216612120. An exemplary method of generating synthetic fully human VL antibodies based on human germline sequences is described in Kim et al., (2014) Mabs.6(1): 219–35. As used herein, the term “single domain antibody” doesAttorney Docket No.: DFY-135WO not include a constant domain. To reduce immunogenecity in humans, antigen-binding sites can be humanized (see, e.g., Safdari et al., Biotechnol. Genet. Eng. Rev. (2013) 29:175-86; Sulea, Methods Mol. Biol. (2022) 2446:299-312) or fully human. All the amino acid positions in heavy or light chain variable regions disclosed herein are numbered according to Kabat numbering, unless otherwise indicated.

[0026] The CDRs of an antigen-binding site derived from a conventional antibody can be determined by various methods known in the art, for example, as described in Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991) (the Kabat definition), Chothia et al., J. Mol. Biol.196:901- 917 (1987) (the Chothia definition), MacCallum et al., J. Mol. Biol.262:732-745 (1996) (the Contact definition), or Lefranc, The Immunologist, 7, 132-136 (1999) (the IMGT definition). As a result, once the heavy and / or light chain variable regions have been elucidated, then it is possible to identify the CDRs in such heavy and / or light chain variable regions using one of the foregoing approaches. The CDRs determined under these definitions typically include overlapping or subsets of amino acid residues when compared against each other. In certain embodiments, the term “CDR” is a CDR under the Contact definition. In certain embodiments, the term “CDR” is a CDR under the Kabat definition. In certain embodiments, the term “CDR” is a CDR under the Chothia definition. In certain embodiments, the term “CDR” is a CDR under the IMGT definition. In certain embodiments, heavy chain CDRs and light chain CDRs of an antibody are identified under different definitions, for example, the heavy chain CDRs under the Contact definition and the light CDRs under the Kabat definition.

[0027] In the context of a single domain antibody, CDR sequences can be determined using methods well-known in the art. For example, the disclosures of Vattekatte et al. PeerJ 8:e8408 (2020), Muyldermans et al. Protein Engineering 7:1129-35 (1994), Sircar et al. J. Immunol.186:6357-67 (2011), Vu et al. Mol. Immunol.34:1121-31 (1997), and Zuo et al. BMC Genomics 18:797 (2017) provide details on the determination of CDR sequences in single domain antibodies.

[0028] The term “tumor-associated antigen” or “TAA,” as used herein, means any antigen, including but not limited to a protein, glycoprotein, ganglioside, carbohydrate, or lipid, that is present (e.g., expressed) on an outer surface of a cell in a cancer or tumor, either at a primary site or at a metastatic site (e.g., a tumor-draining lymph node). In a solid tumor, a tumor-associated antigen can be expressed on malignant cells or other cells in the tumor microenvironment, such as stromal cells (e.g., fibroblasts, mesenchymal stromal cells), localAttorney Docket No.: DFY-135WO or tumor-infiltrating immune cells (e.g., tumor-associated myeloid cells (e.g., tumor- associated macrophages, myeloid-derived suppressor cells, tumor-associated neutrophils, tumor-associated dendritic cells) or regulatory T cells), and tumor endothelial cells.

[0029] As used herein, the term “pathogen-associated antigen” or “PAA” means any antigen, including but not limited to a protein (e.g., glycoprotein or lipid-modified protein), ganglioside, carbohydrate, or lipid, that is present (e.g., expressed) on an outer surface a cell infected by a pathogen (e.g., a virus or another intracellular pathogen). A pathogen- associated antigen can be a protein encoded by a gene of the pathogen and / or by a gene of the cell. In certain embodiments, a PAA encoded by a gene of the cell is expressed at a higher level on an outer surface of the pathogen-infected cell than on an outer surface of a cell of the same type but not infected by the pathogen.

[0030] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0031] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, primates, canines, felines, and the like), and more preferably include humans.

[0032] The terms “treat,” “treating,” or “treatment,” and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. The term “therapeutic benefit” refers to eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0033] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a multispecific binding protein of the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations,Attorney Docket No.: DFY-135WO applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0034] As used herein, the term “monotherapy” means a single active agent is used to treat a disease or disorder in a subject as a line of therapy, in the absence of any other therapeutic agent administered to the subject to treat the same disease or disorder in the same line of therapy. It is understood that the subject may receive one or more other therapeutic agents to treat another disease, disorder, or condition. For example, when receiving a monotherapy, the subject may also receive one or more therapeutic agents to treat symptoms associated with the disease or disorder (e.g., inflammation, pain, weight loss, and / or general malaise associated with cancer) but not the underlying disease or disorder itself. In another example, when receiving a monotherapy, the subject may also receive one or more therapeutic agents to reduce adverse effects (e.g., infusion-related reactions) of the monotherapy.

[0035] As used herein, the term “combination” in the context of therapies means that two or more different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0036] The term “about” refers to any minimal alteration to a given value, including ±5%, ±10%, or ±15% of a specified numerical value or data point. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular valueAttorney Docket No.: DFY-135WO and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as “about” that particular value in addition to the value itself.

[0037] Throughout the description, where compositions are described as having, including, containing, incorporating, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is intended that compositions and methods are inclusive or open-ended and do not exclude additional, unrecited components or steps. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0038] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls. Multispecific Binding Proteins

[0039] The present disclosure provides multispecific binding proteins having an antigen-binding site that bind a tumor-associated antigen such as HER2, an antigen-binding site that binds NKG2D, and a first antibody Fc region and a second antibody Fc region that form a dimer that binds CD16. NKG2D is an activating receptor expressed on NK cells and a costimulatory receptor expressed on certain T cells (e.g., NKT cells, CD8+αβ T cells, and γδ T cells). CD16 is expressed on NK cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. The multispecific binding proteins are capable of binding HER2 on cancer cells and NKG2D and CD16 on natural killer cells. Such binding brings the cancer cell into proximity with the natural killer (NK) cell, which facilitates direct and indirect destruction of the cancer cell by the NK cells. Moreover, co-engagement of NKG2D and CD16 synergistically enhances cytotoxicity of the NK cells towards the target cells. Also provided herein are uses of the multispecific binding proteins and pharmaceutical compositions in treating a cancer.

[0040] The multispecific binding proteins described herein can take various formats. For example, one format involves a heterodimeric, multispecific antibody including a firstAttorney Docket No.: DFY-135WO immunoglobulin heavy chain, a second immunoglobulin heavy chain and an immunoglobulin light chain (FIG.1). The first immunoglobulin heavy chain includes a first Fc (hinge-CH2- CH3) domain fused via a linker and / or an antibody hinge to a heavy chain portion of a Fab fragment, which includes a heavy chain variable domain and a heavy chain CH1 domain. This heavy chain portion pairs with the immunoglobulin light chain, composed of a light chain variable domain and a light chain constant domain (CL), to form a Fab fragment that binds NKG2D. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2- CH3) domain fused via a linker and / or an antibody hinge (e.g., Ala-Ser sequence) to a single- chain variable fragment (scFv), composed of a heavy chain variable domain and light chain variable domain which pair and bind HER2.

[0041] Individual components of the multispecific binding proteins are described in more detail below. NKG2D-binding site

[0042] The multispecific binding proteins disclosed herein can engage NK cells via an antigen-binding site that binds NKG2D. In certain embodiments, the antigen-binding site can block one or more natural ligands, such as ULBP6 and MICA, from binding NKG2D. In certain embodiments, the antigen-binding site can bind human NKG2D and an NKG2D in other species such as cynomolgus monkey.

[0043] Table 1 lists various peptide sequences of VH and VL domains that, in combination, can bind to NKG2D. These NKG2D binding domains can vary in their binding affinity to NKG2D, but they are all expected to activate human NK cells when incorporated into a multispecific binding protein disclosed herein. Unless indicated otherwise, the CDR sequences provided in Table 1 are determined under the Kabat definition. The CDR sequences of these antibodies under the Chothia, Contact, or IMGT definitions can be determined by methods known in the art. Table 1. Exemplary NKG2D-binding sitesAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WO

[0044] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49. ForAttorney Docket No.: DFY-135WO example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 94, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 94 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 94 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 97, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0045] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49MI. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 83, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 83 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 83 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 84, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1 , CDR2 , and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0046] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49MQ. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at leastAttorney Docket No.: DFY-135WO 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 103, and / or incorporate amino acid sequences identical to the CDR1, CDR2 , and CDR3 sequences of SEQ ID NO: 103 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 104, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1 , CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0047] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49ML. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 114, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 114 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 114 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 115, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0048] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49MF. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 116, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences ofAttorney Docket No.: DFY-135WO SEQ ID NO: 116 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 116 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 117, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0049] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49MV. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 118, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 118 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 118 under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 119, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0050] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A49 consensus sequences. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 120, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 120 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 120Attorney Docket No.: DFY-135WO under the Kabat definition are set forth in SEQ ID NOs: 95, 96, and 121, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 98, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 98 under the Kabat definition are set forth in SEQ ID NOs: 99, 100, and 101, respectively.

[0051] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from A44. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 86, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 86 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 86 under the Kabat definition are set forth in SEQ ID NOs: 87, 88, and 89, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 90, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 90 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 90 under the Kabat definition are set forth in SEQ ID NOs: 91, 92, and 93, respectively.

[0052] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from E78. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 102, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 102 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 102 under the Kabat definition are set forth in SEQ ID NOs: 71, 72, and 105, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%,Attorney Docket No.: DFY-135WO at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 106, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106 under the Kabat definition are set forth in SEQ ID NOs: 107, 108, and 109, respectively.

[0053] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from E79. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 70, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70 under the Kabat definition are set forth in SEQ ID NOs: 71, 72, and 73, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 74, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 74 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 74 under the Kabat definition are set forth in SEQ ID NOs: 75, 76, and 77, respectively.

[0054] In some embodiments, the antigen-binding site that binds NKG2D includes a heavy chain variable domain and a light chain variable domain derived from F63. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 78, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 78 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 78 under the Kabat definition are set forth in SEQ ID NOs: 79, 80, and 81, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 82, and / or incorporate amino acid sequencesAttorney Docket No.: DFY-135WO identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 82 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 82 under the Kabat definition are set forth in SEQ ID NOs: 75, 76, and 85, respectively.

[0055] In certain embodiments, the antigen-binding site binds NKG2D with an affinity of KDof 20 nM to 1,000 nM, for example, 50 nM to 1,000 nM, 100 nM to 1,000 nM, 200 nM to 1,000 nM, 300 nM to 1,000 nM, 400 nM to 1,000 nM, 500 nM to 1,000 nM, 600 nM to 1000 nM, 700 nM to 1,000 nM, 800 nM to 1,000 nM, 100 nM to 900 nM, 200 nM to 900 nM, 300 nM to 900 nM, 400 nM to 900 nM, 500 nM to 900 nM, 600 nM to 900 nM, 700 nM to 900 nM, 800 nM to 900 nM, 100 nM to 800 nM, 200 nM to 800 nM, 300 nM to 800 nM, 400 nM to 800 nM, 500 nM to 800 nM, 600 nM to 800 nM, 700 nM to 800 nM, 700-800 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, or about 1,000 nM, as measured by surface plasmon resonance (SPR) or bio-layer inferometry (BLI). HER2-binding site

[0056] The multispecific binding proteins of the disclosure are capable of binding HER2 expressed on the surface of target cells (e.g., cancer cells). Table 2 lists amino acid sequences of heavy chain variable domains and light chain variable domains that can pair and bind HER2. Additional antigen-binding sites can be identified by screening for binding to HER2 or a mature extracellular fragment thereof. Table 2. Exemplary HER2-binding sitesAttorney Docket No.: DFY-135WOAttorney Docket No.: DFY-135WO

[0057] In some embodiments, the antigen-binding site that binds HER2 includes a heavy chain variable domain and a light chain variable domain derived from trastuzumab. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 156, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 156 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 156 under the Chothia definition are set forth in SEQ ID NOs: 157, 158, and 159, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 160, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 160 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 160 or SEQ ID NO: 165 under the Chothia definition are set forth in SEQ ID NOs: 161, 162, and 163, respectively.

[0058] In some embodiments, the antigen-binding site that binds HER2 includes a heavy chain variable domain and a light chain variable domain derived from pertuzumab. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 167, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 167 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 167 under the Chothia definition are set forth in SEQ ID NOs: 168, 169, and 170, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 171, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 171Attorney Docket No.: DFY-135WO under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 171 under the Chothia definition are set forth in SEQ ID NOs: 172, 173, and 174, respectively.

[0059] In some embodiments , the antigen-binding site that binds HER2 includes a heavy chain variable domain and a light chain variable domain derived from MGAH22. For example, the heavy chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 175, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 175 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 175 under the Chothia definition are set forth in SEQ ID NOs: 176, 177, and 178, respectively. Similarly, the light chain variable domain can comprise an amino acid sequence at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 179, and / or incorporate amino acid sequences identical to the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 179 under any suitable CDR definition (e.g., Kabat, Chothia, Contact, or IMGT). The CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 179 under the Chothia definition are set forth in SEQ ID NOs: 180, 181, and 182, respectively.

[0060] The antigen-binding site that binds HER2 can be present in an scFv. In some embodiments, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv to enhance stability. For example, a disulfide bridge can be formed between the C44 residue of the heavy chain variable domain and the C100 residue of the light chain variable domain, the amino acid positions numbered under Kabat. In some embodiments, the amino acids at position 44 of the VH and position 100 of the VL, numbered under the Kabat numbering scheme, are mutated to cysteine residues to form the disulfide bridge. For example, in some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 164, and the VL comprises the amino acid sequence of SEQ ID NO: 165.

[0061] The VH and VL of the scFv can be positioned in various orientations. In certain embodiments, the VL is positioned N-terminal to the VH. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 166. In certain embodiments, the VL is positioned C-terminal to the VH.Attorney Docket No.: DFY-135WO

[0062] The VH and VL of the scFv can be connected via a linker, e.g., a peptide linker. In certain embodiments, the peptide linker is a flexible linker. Regarding the amino acid composition of the linker, peptides are selected with properties that confer flexibility, do not interfere with the structure and function of the other domains of the proteins of the present invention, and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, the VL is positioned N- terminal to the VH and is connected to the VH via a linker. The linkers in the “Linkers” subsections below are useful in the scFv. In some embodiments, the HER2-binding scFv comprises a (G4S)4linker (SEQ ID NO:128). Linkers

[0063] Components of the multispecific binding proteins of the disclosure can be connected via a linker, e.g., a peptide linker. For example, the VH and VL domains of an scFv can be connected by a linker, e.g., a peptide linker. In certain embodiments, the VL is positioned N-terminal to the VH and is connected to the VH via a linker. In another example, a Fab fragment and an Fc region disclosed herein can be connected by a linker, e.g., a peptide linker. In another example, an scFv and an Fc region disclosed herein can be linked by a linker, e.g., a peptide linker. In another example, a single domain antibody and an Fc region disclosed herein can be linked by a linker, e.g., a peptide linker. In yet another example, a first single domain antibody and a second single domain antibody of the disclosure can be linked by a linker, e.g., a peptide linker.

[0064] In certain embodiments, the peptide linker is a flexible linker. The amino acid composition of the linker can be selected with properties that confer flexibility, do not interfere with the structure and function of the other domains of the proteins of the present disclosure, and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance.

[0065] The length of the linker (e.g., flexible linker) can be short, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues, or long, e.g., at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30, 20-25, or about 20 amino acids in length.

[0066] In certain embodiments, the linker includes a (GS)n(SEQ ID NO: 122), (GGS)n(SEQ ID NO: 123), (GGGS)n(SEQ ID NO: 124), (GGSG)n (SEQ ID NO: 125), (GGSGG)n (SEQ ID NO: 126), and (GGGGS)n(SEQ ID NO: 127) sequence, in which n is 1, 2, 3, 4, 5, 6,Attorney Docket No.: DFY-135WO 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker includes an amino acid sequence selected from SEQ ID NOs: 128-140, as listed in Table 3. Table 3. Exemplary linkers

[0067] In specific embodiments, the light chain variable domain of an scFv is linked to the N-terminus of the heavy chain variable domain of the scFv via a flexible linker, e.g., a (G4S)4linker (SEQ ID NO: 128). In some embodiments, an scFv of the present disclosure is linked to the C-terminus of an antibody Fc region via a flexible linker, e.g., a (G4S)4 linker (SEQ ID NO: 128). In some embodiments, an scFv of the present disclosure is linked to the N-terminus of an antibody Fc region via a flexible linker, e.g., a Gly-Ser linker or an Ala-Ser linker. In some embodiments, a Fab domain of the disclosure is linked to an antibody Fc polypeptide via an antibody hinge sequence (the lower hinge being part of the Fc polypeptide).Attorney Docket No.: DFY-135WO Fc regions

[0068] The multispecific binding proteins of the present disclosure includes a first antibody Fc region and a second antibody Fc region that form a dimer that binds CD16. In certain embodiments, the dimer binds CD16A (also known as FcγRIIIA). In certain embodiments, the antibody Fc regions are IgG1, IgG2, IgG3, or IgG4 Fc regions from human or another mammal, such as rabbit, dog, cat, mouse, or horse. In certain embodiments, the antibody Fc regions are human IgG1, IgG2, IgG3, or IgG4 Fc regions. Amino acid sequences of wild-type human IgG1, IgG2, IgG3, and IgG4 Fc regions are provided in Table 4 below. Table 4. Sequences of wild-type human IgG1, IgG2, IgG3, and IgG4 Fc regions

[0069] It is understood that the first and second antibody Fc regions in the multispecific binding protein disclosed herein can have amino acid mutations (e.g., substitutions, insertions, or deletions) relative to a wild-type antibody Fc sequence. In certain embodiments, the first and second antibody Fc regions in the multispecific binding proteinAttorney Docket No.: DFY-135WO disclosed herein each include an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a wild-type antibody Fc sequence. In certain embodiments, the first and second antibody Fc regions in the multispecific binding protein disclosed herein are each a human IgG1 Fc region including an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:141. In certain embodiments, the first and second antibody Fc regions in the multispecific binding protein disclosed herein are each a human IgG2 Fc region including an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:142. In certain embodiments, the first and second antibody Fc regions in the multispecific binding protein disclosed herein are each a human IgG3 Fc region including an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:143. In certain embodiments, the first and second antibody Fc regions in the multispecific binding protein disclosed herein are each a human IgG4 Fc region including an amino acid sequence at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:144.

[0070] Within a human IgG1 or IgG3 Fc region, CD16A (also known as FcγRIIIA) binding is mediated by the hinge and the CH2 domain. For example, within human IgG1, the interaction with CD16 is primarily focused on amino acid residues Asp 265 – Glu 269, Asn 297 – Thr 299, Ala 327 – Ile 332, Leu 234 – Ser 239, and carbohydrate residue N-acetyl-D- glucosamine in the CH2 domain (see, Sondermann et al., Nature 406 (6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using phage-displayed libraries or yeast surface-displayed cDNA libraries, or can be designed based on the known three-dimensional structure of the interaction.

[0071] In some embodiments, the first and / or second antibody Fc regions differ from the wild-type human IgG1 Fc region at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439. In some embodiments, the first and / or second antibody Fc regions differ from the wild-type human IgG1 Fc region at one or more positions selected from R292, S298, L234, L235, G237, P329, A330, P331, and K414. InAttorney Docket No.: DFY-135WO some embodiments, the first and / or second antibody Fc regions differ from the human IgG1 Fc region at one or more positions selected from L234, L235, G237, P329, A330, and P331. In some embodiments, the first and / or second antibody Fc regions differ from the human IgG1 Fc region at one or more positions selected from R292, S298, and K414. In some embodiments, the first and / or second antibody Fc regions differ from the human IgG1 Fc region at: (a) positions L234 and L235; (b) positions L234, L235, and P329; or (c) positions L234, L235, G237, A330, and P331.

[0072] In some embodiments, the first and / or second antibody Fc domain regions have reduced FcγRIIA-binding function relative to a corresponding wild type antibody Fc region (e.g., a human IgG1 Fc region). In some embodiments, the first and / or second antibody Fc regions include an S298A substitution relative to a human IgG1 Fc region.

[0073] In some embodiments, the first and / or second antibody Fc regions have reduced FcγRIIB-binding function and / or improved FcγRIIIA-binding function relative to a corresponding wild type antibody Fc region (e.g., a human IgG1 Fc region). In some embodiments, the mutations are at one or more of positions P329, A330, and P331.

[0074] In certain embodiments, the first and / or second antibody Fc regions bind FcγRI, also known as CD64.

[0075] An antibody Fc region may also confer complement-dependent cytotoxicity (CDC) which is typically mediated by a complement component (e.g., C1q). In certain embodiments, the first and / or second antibody Fc regions disclosed herein incorporate one or more mutations that reduce binding to a complement component (e.g., C1q). In certain embodiments, the one or more mutations abrogate the binding of the antibody Fc region to a complement component (e.g., C1q). Fc mutations that reduce binding to C1q are known in the art. For example, as disclosed in U.S. Patent Nos.5,648,260 and 5,624,821, the amino acid residues of Fc at positions 234, 235, 236, 237, 297, 318, 320, and 322 are implicated in C1q binding. As disclosed in Tao et al., J. Exp. Med. (1993) 178:661-667 and Brekke et al., Eur. J. Immunol. (1994) 24:2542-47, residue Pro at position 331 is implicated in C1q binding. As disclosed in Idusogie et al., J. Immunol. (2000) 164:4178-84, mutations of Fc at positions 270 (e.g., D270A), 322 (K322A), 329 (e.g., P329A), and 331 (e.g., P331A, P331S, or P331G) reduced C1q binding.

[0076] Accordingly, in certain embodiments, the first and / or second antibody Fc regions incorporate a mutation (e.g., substitution relative to wild-type human IgG1 Fc) at one or more of positions selected from 234, 235, 236, 237, 270, 297, 318, 320, 322, 329, and 331. In certain embodiments, the first and second antibody Fc regions are human IgG1 antibody FcAttorney Docket No.: DFY-135WO regions incorporating mutation(s) G237A, A330S, P331S, and / or P329A. In certain embodiments, the first and second antibody Fc regions are human IgG1 antibody Fc regions incorporating mutations G237A, A330S, and P331S. In certain embodiments, the first and second antibody Fc regions are human IgG1 antibody Fc regions incorporating mutation P329A.

[0077] The assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which may lead to the assembly of homodimers of each antibody heavy chain as well as assembly of heterodimers. Promoting the preferential assembly of heterodimers can be accomplished by incorporating heterodimerization mutations, e.g., different mutations in the CH3 domain of each antibody heavy chain constant region as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1 and incorporating distinct pairs of amino acid substitutions within a first Fc region and a second Fc region that allow these two chains to selectively heterodimerize with each other. The positions of amino acid substitutions illustrated below are all numbered according to the EU index.

[0078] One or more mutations can be incorporated into the constant region as compared to 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.

[0079] In one scenario, an amino acid substitution in the first Fc region replaces 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 the second Fc region replaces the original amino acid(s) with a smaller amino acid(s), chosen from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (a protuberance) fits into the surface of the smaller amino acid substitutions (a cavity). ForAttorney Docket No.: DFY-135WO example, one Fc region can incorporate a T366W substitution, and the other Fc region can incorporate three substitutions including T366S, L368A, and Y407V.

[0080] Alternatively, amino acid substitutions could be selected from the following sets of substitutions shown in Table 5, below. Table 5. Fc heterodimerization mutationsIn this table, the symbol “ / ” between mutations means “and.”

[0081] Alternatively, at least one amino acid substitution can be made at K392, K370, K409, and / or K439 of the first Fc region, and at least one amino acid substitution can be made at D399, E356, and / or E357 of the second Fc region, where the amino acid residues inAttorney Docket No.: DFY-135WO the first Fc region is replaced by any known negatively-charged amino acid, and the amino acid residue in the second Fc region is replaced by any known positively-charged amino acid.

[0082] Alternatively, or in addition, the structural stability of a hetero-multimeric protein may be increased by introducing S354C on either of the first or second Fc region, and Y349C on the other Fc region. The two Cys residues form an artificial disulfide bridge within the interface of the two Fc regions.

[0083] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an IgG1 constant region at position T366, and the amino acid sequence of the other Fc region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, L368 and Y407.

[0084] In some embodiments, the amino acid sequence of one Fc 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 Fc 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.

[0085] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an 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 Fc region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411.

[0086] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an 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 Fc region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405.

[0087] In some embodiments, the amino acid sequence of one Fc 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 Fc 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.

[0088] In some embodiments, the amino acid sequence of one Fc 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 FcAttorney Docket No.: DFY-135WO 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.

[0089] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an IgG1 constant region by an S354C substitution, and the amino acid sequence of the other Fc region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution.

[0090] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other Fc region differs from the amino acid sequence of an IgG1 constant region by O347R, D399V and F405T substitutions.

[0091] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an IgG1 constant region by a T366W substitution, and the amino acid sequence of the other Fc region differs from the amino acid sequence of an IgG1 constant region by T366S, T368A, and Y407V substitutions.

[0092] In some embodiments, the amino acid sequence of one Fc region differs from the amino acid sequence of an IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the other Fc region differs from the amino acid sequence of an IgG1 constant region by T350V, T366L, K392L, and T394W substitutions.

[0093] When selecting Fc substitutions, a skilled person would appreciate that the “one Fc region” and “the other Fc region” of an Fc dimer described above may correspond to the first antibody Fc region and the second antibody Fc region, respectively. Alternatively, the “one Fc region” and “the other Fc region” of an Fc dimer described above may correspond to the second antibody Fc region and the first antibody Fc region, respectively.

[0094] It is understood that where the two Fc regions incorporate different amino acid sequences or mutations, either chain can be linked to any other domain of the multispecific binding proteins disclosed herein. Where an exemplary multispecific binding protein is described to incorporate a first set of mutations in a given first Fc region and a second set of mutations in the second Fc region, the reverse construct, in which the first Fc region incorporates the second set of mutations and the second Fc region incorporates the first set of mutations, is also contemplated.

[0095] Unless indicated otherwise, the amino acid substitutions described above are identified in the context of human IgG1. It is understood that corresponding substitutions are also contemplated in the context of human IgG2, IgG3, and IgG4. For example, the amino acid residue at position 234 of human IgG4 is F, whereas the amino acid residue at positionAttorney Docket No.: DFY-135WO 234 of human IgG1 is L. Where an L234A substitution is described in the context of human IgG1, an F234A substitution in the context of human IgG4 is also contemplated. All the amino acid positions in antibody Fc domains in this application are numbered according to the EU numbering system. Exemplary multispecific binding protein

[0096] Provided below is an example of a TriNKET called “A49-F3’-TriNKET- Trastuzumab.” It includes a HER2-binding scFv and an NKG2D-binding Fab each linked to an antibody Fc region, wherein antibody constant regions incorporate mutations that promote their heterodimerization. The scFv includes a heavy chain variable domain (VH) and a light chain variable domain (VL) derived from trastuzumab, and further includes substitution of cysteine for the amino acid residues at position 100 of the VL and position 44 of the VH, thereby facilitating formation of a disulfide bridge between the VH and VL of the scFv. The VL is linked N-terminal to the VH via a (G4S)4linker (SEQ ID NO:128), and the VH is linked N-terminal to an Fc via an Ala-Ser hinge. The Ala-Ser hinge is included at the elbow hinge region sequence to balance between flexibility and optimal geometry.

[0097] A49-F3’-TriNKET-Trastuzumab includes a single-chain variable fragment (scFv) (SEQ ID NO:166) derived from trastuzumab that binds HER2, linked via a hinge including Ala-Ser to a first antibody Fc polypeptide; and an NKG2D-binding Fab fragment derived from A49 linked to a second antibody Fc polypeptide. The Fab fragment includes a heavy chain portion having a heavy chain variable domain (SEQ ID NO:94) and a CH1 domain, and a light chain portion having a light chain variable domain (SEQ ID NO:98) and a light chain constant domain. The heavy chain variable domain is connected to the CH1 domain, and the CH1 domain is connected to the second antibody Fc polypeptide. A49-F3’- TriNKET-Trastuzumab includes three polypeptides having the sequences of SEQ ID NO:183, SEQ ID NO:184, and SEQ ID NO:185.

[0098] SEQ ID NO:183 represents the full sequence of the HER2-binding scFv linked to the first antibody Fc polypeptide via a hinge including Ala-Ser (scFv-Fc). The first antibody Fc polypeptide includes Q347R, D399V, and F405T substitutions for heterodimerization and an S354C substitution for forming a disulfide bond with a Y349C substitution in SEQ ID NO:184 as described below. The scFv (SEQ ID NO:166) includes a heavy chain variable domain of trastuzumab connected to the N-terminus of a light chain variable domain of trastuzumab via a (G4S)4linker (SEQ ID NO:128), the scFv represented as VL-(G4S)4-VH. The heavy and the light variable domains of the scFv are also connectedAttorney Docket No.: DFY-135WO through a disulfide bridge between C100 of VL and C44 of VH, as a result of Q100C and G44C substitutions in the VL and VH, respectively. Trastuzumab scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS (SEQ ID NO:166) Trastuzumab scFv-Fc (RVT) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW GQGTLVTVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:183)

[0099] SEQ ID NO:184 represents the heavy chain portion of the Fab fragment, which includes a heavy chain variable domain (SEQ ID NO:94) of an NKG2D-binding site and a CH1 domain, connected to the second antibody Fc polypeptide. The antibody Fc polypeptide in SEQ ID NO:184 includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the Fc polypeptide in SEQ ID NO:183. In SEQ ID NO:184, the antibody Fc polypeptide also includes K360E and K409W substitutions for heterodimerization with the Fc in SEQ ID NO:183. A49 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSS (SEQ ID NO:94) A49 VH-CH1-Fc (EW) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKAttorney Docket No.: DFY-135WO TISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:184)

[0100] SEQ ID NO:185 represents the light chain portion of the Fab fragment, including a light chain variable domain (SEQ ID NO:98) of an NKG2D-binding site and a light chain constant domain. A49 VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK (SEQ ID NO:98) A49 VL-LC DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK RTVAAPSPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:185) Production of Multispecific Binding Proteins

[0101] The multispecific binding proteins disclosed herein can be made using recombinant DNA technology well known to a skilled person in the art. For example, where the TriNKET includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (see the exemplary multispecific binding protein above), a first nucleic acid sequence encoding the first immunoglobulin heavy chain can be cloned into a first expression vector, a second nucleic acid sequence encoding the second immunoglobulin heavy chain can be cloned into a second expression vector, and a third nucleic acid sequence encoding the immunoglobulin light chain can be cloned into a third expression vector. In some embodiments, the first, second, and third expression vectors can be stably transfected together into host cells to produce the multimeric proteins.

[0102] A signal peptide can be fused to an N-terminus of any of three polypeptides to allow secretion of the mature protein into the culture medium of the host cells. To produce such mature proteins recombinantly, the nucleic acid sequences can encode a corresponding immature polypeptide sequence that includes the signal peptide.

[0103] The expression vectors can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, HeLa cells, babyAttorney Docket No.: DFY-135WO hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. It is understood that Fc affinity chromatography may be used for protein purification when the polypeptide comprises an IgG Fc region. Accordingly, the host cells above, which do not otherwise produce IgG proteins, are particularly suitable for recombinantly producing polypeptides comprising an IgG Fc region.

[0104] Specific expression and purification conditions will vary depending upon the expression system employed. If the engineered genes are to be expressed in eukaryotic host cells, e.g., CHO cells, they are first inserted into an expression vector containing a suitable eukaryotic promoter, a nucleotide sequence encoding a signal peptide, a stop codon, and a polyadenylation sequence. Optionally, the vector or gene constructs may contain enhancers and introns. The gene constructs can be introduced into eukaryotic host cells using conventional techniques. In some embodiments, the engineered genes in the expression vectors encode a signal peptide fused to the N-terminus of the polypeptide to be produced. The secretion signal is typically cleaved when the polypeptide is transported to the extracellular space, resulting in mature polypeptides secreted to the culture medium. In some embodiments, the signal peptide comprises a signal sequence from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, or prolactin.

[0105] In some embodiments, the expression vectors can be stably inserted into the genome of eukaryotic cells, resulting in stably transfected cells that can be used for protein production over more passages than transiently transfected cells. The stable transfection allows isolation of single clones of host cells that produce the recombinant protein at high yield. Following transfection, single clones can be isolated and high-yield clones selected using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[0106] The polypeptides can be produced by growing (culturing) a host cell transfected with expression vectors under conditions that permit expression of the polypeptide(s) of the protein. For example, high-yield clones can be cultured under conditions suitable for bio- reactor scale-up and maintained expression of the proteins of the present disclosure. Following expression, the proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification (e.g., for binding to an IgG Fc region or an affinity tag such as glutathione-S-transferase (GST) or histidine tag fused to the protein), gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed- mode chromatographyAttorney Docket No.: DFY-135WO

[0107] To achieve the highest yield of the multispecific binding protein, different ratios of the first, second, and third expression vector can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[0108] The multispecific binding proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze- thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography. For example, a multispecific binding protein may be purified by affinity protein A capture chromatography, such as with a MabSelect SuRe resin (Cytiva), followed by a cation exchange chromatography polishing step, such as with a POROS XS strong cation exchange resin (Thermo Fisher). If higher purity is required, size exclusion chromatography may be used in addition to or in lieu of cation exchange chromatography to polish the protein further and remove unwanted homodimers of various molecular weights. Such size exclusion chromatography can, for example, be performed with a Superdex 200 prep grade resin (Cytiva).

[0109] A skilled person in the art would appreciate that during production and / or storage of proteins, N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Accordingly, in some embodiments where the N-terminal residue of an amino acid sequence of a polypeptide is E or Q, a corresponding amino acid sequence with the E or Q replaced with pyroglutamate is also contemplated herein.

[0110] A skilled person in the art would also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed with proteins that include an Fc domain at their C-termini. Accordingly, in some embodiments where the C-terminal residue of an amino acid sequence of a polypeptide (e.g., an antibody Fc polypeptide) is K, a corresponding amino acid sequence with the K removed is also contemplated herein. Pharmaceutical Compositions

[0111] The present disclosure also provides pharmaceutical compositions or formulations that contain a multispecific binding protein described herein. TheAttorney Docket No.: DFY-135WO pharmaceutical composition can be formulated for use in a variety of drug delivery systems. One or more pharmaceutically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23d ed. 2020).

[0112] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23d ed.2020)).

[0113] Pharmaceutical compositions containing a multispecific binding protein disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectalAttorney Docket No.: DFY-135WO administration. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0114] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof. An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and / or a needle.

[0115] In certain embodiments, the formulation is a liquid formulation. In certain embodiments, an aqueous formulation is prepared including the protein of the present disclosure in a pH-buffered solution. The pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide. In certain embodiments, a salt or buffer components may be added in an amount of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion. Intravenous formulations can be diluted with 0.9% Sodium Chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0116] In certain embodiments, the formulation is a lyophilized formulation including a multspecific binding protein disclosed herein and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative. The amount of sucrose or maltose usefulAttorney Docket No.: DFY-135WO for stabilization of the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1:2 to 1:5. Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8. In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents.

[0117] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In certain embodiments, the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0118] The pharmaceutical compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[0119] In certain embodiments, the present disclosure provides a formulation with an extended shelf life including the protein of the present disclosure, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.Attorney Docket No.: DFY-135WO

[0120] A polyol, which acts as a tonicifier and may stabilize the antibody, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) may be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol may be about 7.5 to about 15 mg / mL. In certain embodiments, the concentration of mannitol may be about 10 to about 14 mg / mL. In certain embodiments, the concentration of mannitol may be about 12 mg / mL. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0121] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4thed., 1996). In certain embodiments, the formulation may contain polysorbate 80 between about 0.1 mg / mL and about 10 mg / mL, or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.

[0122] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.

[0123] In certain embodiments, the liquid formulation of the disclosure may be prepared in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable orAttorney Docket No.: DFY-135WO unsuitable for intravenous administration. In certain embodiments, the sugar may be disaccharides, e.g., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0124] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0125] In certain embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED.1: 10-29).

[0126] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(−)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. Therapeutic Uses

[0127] The present disclosure provides methods of treating cancer, the method comprising administering a multispecific binding protein disclosed herein, or a pharmaceutical composition containing the multispecific binding protein, to a subject in need thereof. In certain embodiments, the subject is an adult (e.g., at least 18 years of age). In certain embodiments, the method of the present disclosure is used as a first-line therapy. In certain embodiments, the method of the present disclosure is used as an adjunct to surgical removal of the primary lesion. HER2 status

[0128] A subject in need of the treatment can have a cancer that expresses HER2. Methods of characterizing HER2 expression and genetic changes to the ERBB2 gene, which encodes the HER2 protein, are known in the art. In particular, the ASCO / CAP guidelines (e.g., Wolff et al., (2007) J. Clin. Oncol.25(1):118-45), including applicable updates (e.g., the 2018 update according to Wolff et al., (2018) J. Clin. Oncol.36(20):2105-22; the 2023Attorney Docket No.: DFY-135WO update according to Wolff et al., (2023) J. Clin. Oncol.41(22):3867-72), provide diagnostic methods well recognized in the field.

[0129] The HER2 expression level can be determined by immunohistochemistry (IHC). Anti-HER2 antibodies (e.g., Ventana 4B5 antibody and Bond Oracle CB11 antibody) have been approved by the FDA for detecting HER2, and immunohistochemistry kits (e.g., HercepTestTM) are commercially available. The level of HER2 expression in a tumor sample, as detected by immunohistochemistry, can be quantified and scored as 0, 1+, 2+, or 3+ according to the ASCO / CAP guidelines. It is understood that a tumor with a HER2 level scored as 0 by IHC can nevertheless express HER2. For example, HercepTest 0 patients may have a small number of tumor cells with a faint staining for HER2, or the expression level of HER2 may be lower than the detection limit of HercepTest but nevertheless detectable by other methods (e.g., immunohistochemistry or flow cytometry using a more sensitive anti- HER2 antibody). It is also understood that HER2 expression level may change over time, and a patient with no detectable HER2 expression may be found to have detectable HER2 expression (e.g., HER21+ by HercepTest) at a later time, either naturally (e.g., as a result of hormone level fluctuation) or in response to a concomitant therapy. As a result, HER20 patients are not excluded from the method of the present disclosure.

[0130] With respect to breast cancer, under the 2018 ASCO / CAP guideline, a cancer or tumor is scored as HER23+ if in a sample, circumferential membrane staining of HER2 is complete, intense and in >10% of tumor cells, which is readily appreciated using a low power objective and observed within a homogeneous and contiguous invasive cell population. A cancer or tumor is scored as HER22+ if weak to moderate complete membrane staining of HER2 is observed in >10% of tumor cells in a sample. A cancer or tumor is scored as HER2 1+ if incomplete membrane staining of HER2 is faint or barely perceptible and in >10% of tumor cells in a sample. A cancer or tumor is scored as HER20 if no HER2 staining is observed or membrane staining is incomplete and is faint or barely perceptible and in ≤10% of tumor cells.

[0131] New technologies can be employed to assess HER2 levels in patient samples. For example, the automated quantitative analysis technology can quantitatively assess HER2 expression by measuring the intensity of antibody-conjugated fluorophores. The HERmark technology measures HER2 expression through a proximity-based release of antibody-bound fluorescent tags. The quantitative IHC technology converts antibody / antigen complexes into red dots, subsequently counted to quantify HER2 expression. The time-resolved fluorescence energy transfer technology enables assessment of HER2 expression through the detection ofAttorney Docket No.: DFY-135WO fluorescence emitted by two fluorophores in close proximity. The quantitative real-time polymerase chain reaction technology enables quantitative measurement of the amount of HER2 mRNA in a sample. The flow cytometry technology enables measurement of the number of HER2 proteins on the surface of a cell. These assays can complement the results of the IHC assay, thereby obtaining more accurate assessment of the HER2 level in the cancer or tumor.

[0132] ERBB2 gene amplification is the main mechanism of HER2 overexpression. Determining whether ERBB2 gene is amplified in a cancer tissue sample may help reduce false-positive results from immunohistochemistry of the same sample (see, e.g., Sarode et al., (2015) Arch. Pathol. Lab. Med.139:922–28). Accordingly, in certain embodiments, the cancer or tumor in the subject has been assessed by ERBB2 gene amplification. ERBB2 gene amplification can be determined by in situ hybridization (ISH) (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization, or silver in situ hybridization), quantitative PCR, or DNA sequencing (e.g., next generation sequencing). When measured by ISH, two parameters are usually considered: the average HER2 signals per cell and the HER2 to chromosome 17 enumeration probe (CEP17) ratio. A positive ISH result can be an average HER2 signals per cell greater than or equal to 6.0, or a combination of an average HER2 signals per cell greater than or equal to 4.0 and a HER2 to CEP17 ratio greater than or equal to 2.0. A negative ISH result can be an average HER2 signals per cell smaller than 4.0. Additional algorithms for determining ERBB2 gene amplification are provided in the ASCO / CAP guidelines.

[0133] Mutations in the coding sequence of the ERBB2 gene can change the HER2 amino acid sequence (e.g., in the tyrosine kinase domain), resulting in higher activity of the HER2 protein. Exemplary HER2 mutations in cancer patients are described in Connell and Doherty, ESMO Open (2017) 2(5): e000279; Gaibar et al., J Oncol. (2020) 6375956. Specific HER2 activating mutations include but are not limited to insYVMA (duplication of Y772 to A775), insGSP (duplication of G778 to P780), insTGT (deletion of G776 and insertion of amino acid sequence VC at the deletion site), L755S substitution, V777L substitution, and S310F substitution. The mutations that change the HER2 amino acid sequence can be identified by DNA sequencing (e.g., next generation sequencing), using samples such as a tumor biopsy, a tumor draining lymph node biopsy, or a blood sample to test circulating tumor DNA.Attorney Docket No.: DFY-135WO

[0134] The HER2 status of various cancers can be determined based on HER2 expression level, ERBB2 gene amplification, mutations in the ERBB2 gene, or a combination thereof.

[0135] With respect to breast cancer, under the 2018 ASCO / CAP guideline, HER23+ IHC cancer or tumor is classified as HER2 positive, and HER21+ or 0 IHC cancer or tumor is classified as HER2 negative. Where a cancer or tumor is scored as HER22+ in the initial IHC assessment, a reflex test (same specimen using ISH) or a new test (new specimen if available, using IHC or ISH) must be ordered. Based on the result of the reflex test or the new test, the cancer or tumor may be classified as HER2 positive or HER2 negative, according to the ASCO / CAP guidelines. In certain embodiments, the subject treated by the method disclosed herein has a HER2 positive breast cancer. In certain embodiments, the HER2 positive breast cancer is eligible for treatment with trastuzumab. In certain embodiments, the subject treated by the method disclosed herein has a HER2 negative breast cancer. In certain embodiments, the HER2 negative breast cancer is ineligible for treatment with trastuzumab.

[0136] Medical guidelines may not be readily available to classify HER2 status as “positive” vs. “negative” in certain other types of cancer (e.g., non-small cell lung cancer, colorectal cancer). With respect to these cancers, HER2 status required by certain patient populations are described in more detail in connection with specific patient populations and can be determined by persons of ordinary skill in the medical field. Patient populations

[0137] The present disclosure provides a method of treating a subject or patient having cancer, the method comprising administering to the subject or patient a multispecific binding protein disclosed herein, either alone or in combination with one or more other therapeutic agents. The subject or patient can fall within any one of the following patient populations. Breast cancer

[0138] In certain embodiments, the subject has breast cancer. Breast cancer can be characterized by their status of HER2 and hormone receptors (including estrogen receptor and progesterone receptor). A breast cancer positive in estrogen receptor, progesterone receptor, or both is called hormone receptor (HR) positive breast cancer. A breast cancer negative in all three receptors—HER2, estrogen receptor, and progesterone receptor—are called triple negative breast cancer.Attorney Docket No.: DFY-135WO

[0139] In certain embodiments, the breast cancer is a hormone receptor (HR) positive and HER2 negative metastatic breast cancer. In certain embodiments, the HR positive status is confirmed with a tumor biopsy from a locally recurrent or metastatic site. In certain embodiments, the HER2 negative status is determined according to the 2018 ASCO / CAP guidelines for HER2 testing, including IHC 2+ / FISH negative, ICH 1+, and IHC 0. In certain embodiments, the subject has disease progression on two or more previous lines of endocrine therapy (ET), with or without a targeted therapy in the metastatic setting. In certain embodiments, the subject has received at least one line of ET in the metastatic setting followed by an adjuvant ET, and has recurrence of the breast cancer while on the first 24 months of starting the adjuvant ET. In certain embodiments, the subject has disease progression within 6 months of starting a first-line ET, with or without a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in the metastatic setting. In certain embodiments, the subject has disease recurrence while on the first 24 months of starting adjuvant ET with a CDK 4 / 6 inhibitor, and the subject is no longer a candidate for additional ET in the metastatic setting. In certain embodiments, the subject has progression of unresectable locally advanced or metastatic breast cancer after the last systemic therapy or is intolerant of the last systemic therapy. In certain embodiments, the subject has received one or more prior targeted therapies, including but not limited to phosphatidylinositol 3-kinase (PI3K) inhibitors (for those with PIK3CA mutations), mammalian target of rapamycin (mTOR) inhibitors, or selective estrogen receptor degraders (SERD) for ESR1 mutations. In certain embodiments, the subject is no longer a candidate for an additional endocrine treatment, with or without targeted therapies. In certain embodiments, the subject has disease measurable by RECIST 1.1.

[0140] In certain embodiments, the breast cancer is a HER2 positive metastatic breast cancer. In certain embodiments, the HER2 positive status is determined according to the 2018 ASCO / CAP guidelines for HER2 testing, including IHC 3+ and IHC2+ / ISH+. In some embodiments, the subject has received prior treatment with any one of trastuzumab, pertuzumab, ado-trastuzumab emtansine (T-DM1), or trastuzumab deruxtecan (T-DXd). In certain embodiments, the subject has progression of unresectable locally advanced or metastatic breast cancer after the last systemic therapy or is intolerant of the last systemic therapy. In certain embodiments, the subject has disease measurable by RECIST 1.1. In certain embodiments, the subject has a combination of all the characteristics described above in this paragraph.Attorney Docket No.: DFY-135WO

[0141] In certain embodiments, the subject having breast cancer (e.g., HR positive and HER2 negative metastatic breast cancer or HER2 positive metastatic breast cancer) is treated with an effective amount of the multispecific binding protein disclosed herein, in combination with an effective amount of an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor. In certain embodiments, the subject having breast cancer (e.g., HR positive and HER2 negative metastatic breast cancer, or HER2 positive metastatic breast cancer) is treated with an effective amount of the multispecific binding protein disclosed herein as a monotherapy. Non-small cell lung cancer

[0142] In certain embodiments, the subject has non-small cell lung cancer (NSCLC). NSCLC is a main type of lung cancer and includes, without limitation, adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0143] In certain embodiments, the NSCLC overexpresses HER2, has ERBB2 gene amplification, and / or has a HER2 activating mutation. In certain embodiments, HER2 overexpression means a score of 2+ or 3+ as assessed by IHC. In certain embodiments, the subject has an activating HER2 mutation selected from insYVMA (duplication of Y772 to A775), insGSP (duplication of G778 to P780), insTGT (deletion of G776 and insertion of amino acid sequence VC at the deletion site), L755S substitution, V777L substitution, and S310F substitution. In certain embodiments, the HER2 overexpression, ERBB2 gene amplification, or activating HER2 mutation is assessed with a tumor biopsy from a locally recurrent or metastatic site. In certain embodiments, the subject has recurrent or progressive disease during or after platinum doublet-based chemotherapy or has recurrent or progressive disease within 6 months after completing platinum-based chemotherapy for local disease, including those with actionable genetic alterations. In certain embodiments, the subject has disease progression while on or after anti-PD-(L)1 therapy. In certain embodiments, the subject has an actionable mutation (e.g., in EGFR, ALK, ROS1, or RET) has had disease progression on, has been intolerant to, or has not been a candidate for receiving standard tyrosine kinase inhibitors (TKIs). In certain embodiments, the subject has progression of unresectable locally advanced or metastatic NSCLC after the last systemic therapy or is intolerant of the last systemic therapy. In certain embodiments, the subject has a combination of all the characteristics described above in this paragraph with respect to recurrence or progression during or after prior treatments.Attorney Docket No.: DFY-135WO

[0144] In certain embodiments, the subject having NSCLC is treated with an effective amount of the multispecific binding protein disclosed herein in combination with an effective amount of an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor. In certain embodiments, the subject having NSCLC is treated with an effective amount of the multispecific binding protein disclosed herein as a monotherapy. TriNKET and combination therapies

[0145] The present disclosure provides treating a subject having cancer (e.g., the specific types of cancer disclosed herein) by administering an effective amount of the multispecific binding protein disclosed herein (e.g., A49-F3’-TriNKET-Trastuzumab), either as a monotherapy or in combination with effective amounts of one or more additional therapeutic agents. The multispecific binding protein and / or the additional therapeutic agents can be provided in a pharmaceutical composition (e.g., one disclosed herein). TriNKET dosage regimen

[0146] In certain embodiments, the method includes administering to a subject in need thereof a multispecific binding protein or pharmaceutical composition disclosed herein in an initial four-week treatment cycle on Day 1, Day 8, and Day 15. In certain embodiments, the multispecific binding protein or pharmaceutical composition is administered to the subject only on these three days in the initial four-week treatment cycle. In specific embodiments, the multispecific binding protein or pharmaceutical composition is not administered to the subject on Day 22. This regimen is a dose intensification schedule, which is designed to reach maximal saturation of the target as early as possible during the course of the treatment while minimizing the infusion burden for the patient.

[0147] In certain embodiments, the method further includes administering to the subject the multispecific binding protein or pharmaceutical composition on Day 1 and Day 15 in each of one or more subsequent four-week treatment cycles after the initial treatment cycle. In certain embodiments, the multispecific binding protein or pharmaceutical composition is administered to the subject only on these two days in each subsequent four-week treatment cycle. In specific embodiments, the multispecific binding protein or pharmaceutical composition is not administered to the subject on Day 8 or Day 22. The subsequent treatment cycles, in which the subject receives administration of the multispecific binding protein or pharmaceutical composition 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, 15, 16, 17, or 18 subsequentAttorney Docket No.: DFY-135WO treatment cycles. In certain embodiments, the subject receives at least 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 7-24, 8-24, 9- 24, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 16-24, 17-24, or 18-24 subsequent treatment cycles. In certain embodiments, the subject receives subsequent treatment cycles until regression of the cancer.

[0148] Routes of administration of the multispecific binding protein can be intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection.

[0149] In certain embodiments, one or more doses of the multispecific binding protein in the initial and subsequent treatment cycles contain 0.5-20 mg / kg, 0.5-15 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, 1-2 mg / kg, 5-20 mg / kg, 5-15 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 10-15 mg / kg, or 15-20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, one or more doses of the multispecific binding protein or pharmaceutical composition in the initial and subsequent treatment cycles contain 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, the one or more doses are administered intravenously.

[0150] In certain embodiments, each of the doses of the multispecific binding protein or pharmaceutical composition in the initial and subsequent treatment cycles contains 0.5-20 mg / kg, 0.5-15 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, 1-2 mg / kg, 5-20 mg / kg, 5-15 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 10-15 mg / kg, or 15-20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses of the multispecific binding protein or pharmaceutical composition in the initial and subsequent treatment cycles contains a same amount in the range of 0.5-20 mg / kg, 0.5-15 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, 1-2 mg / kg, 5-20 mg / kg, 5-15 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 10-15 mg / kg, or 15-20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each dose is administered intravenously.Attorney Docket No.: DFY-135WO

[0151] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains a same amount of 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each dose is administered intravenously.

[0152] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 5.2 × 10-5mg / kg, 1.6 × 10-4mg / kg, 5.2 × 10-4mg / kg, 1.6 × 10-3mg / kg, 5.2 × 10-3mg / kg, 1.6 × 10-2mg / kg, 5.2 × 10-2mg / kg, 1.6 × 10-1mg / kg, 0.52 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains a same amount of 5.2 × 10-5mg / kg, 1.6 × 10-4mg / kg, 5.2 × 10-4mg / kg, 1.6 × 10-3mg / kg, 5.2 × 10-3mg / kg, 1.6 × 10-2mg / kg, 5.2 × 10-2mg / kg, 1.6 × 10-1mg / kg, 0.52 mg / kg, 1 mg / kg, 1.6 mg / kg, 5.2 mg / kg, 10 mg / kg, 20 mg / kg, or 50 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each dose is administered intravenously.

[0153] In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 5 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 10 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 15 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the initial and subsequent treatment cycles contains 20 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each dose is administered intravenously.

[0154] Where the multispecific binding protein is used in combination with an additional therapeutic agent that has a different treatment cycle, the regimen of theAttorney Docket No.: DFY-135WO multispecific binding protein can be adjusted to synchronize the treatment cycles. For example, where the additional therapeutic agent has a three-week treatment cycle (optionally with administration on Day 1 and Day 8 of each cycle), the multispecific binding protein can be administered also on Day 1 and Day 8 of three-week treatment cycles. In certain embodiments, the multispecific binding protein and the additional therapeutic agent are both administered on Day 1 and Day 8 of three-week treatment cycles. Given the increased administration frequency of the multispecific binding protein relative to that of the four-week treatment cycles, the dose of the multispecific binding protein can be reduced, e.g., by 25%. Accordingly, in certain embodiments, each of the doses in the three-week treatment cycles contains 3.75 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the three-week treatment cycles contains 7.5 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the three-week treatment cycles contains 11.25 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each of the doses in the three-week treatment cycles contains 15 mg / kg of the multispecific binding protein relative to the body weight of the subject. In certain embodiments, each dose is administered intravenously.

[0155] It is contemplated that a priming dose can be used to reduce adverse effects (e.g., infusion-related reactions) of the multispecific binding protein. It has been observed in a clinical study that the majority of infusion-related reactions occurred at the first dose, whereas subsequent doses were much better tolerated. It has also been observed that 5 mg / kg of the multispecific binding protein was pharmacodynamically active in many tumors. Accordingly, in certain embodiments, where a subject is scheduled to receive greater than 5 mg / kg of the multispecific binding protein in initial and subsequent treatment cycles (e.g., 7.5 mg / kg, 10 mg / kg, 11.25 mg / kg, 15 mg / kg, or 20 mg / kg), the first dose can be reduced to 5 mg / kg, notwithstanding the embodiments above specifying the amount of multispecific binding protein of each dose. The initially-scheduled higher dose can be given in subsequent doses (e.g., starting from Day 8 of the initial treatment cycle).

[0156] In certain embodiments, the multispecific binding protein or pharmaceutical composition is administered intravenously by intravenous infusion, e.g., with a prefilled bag, a prefilled pen, or a prefilled syringe. In certain embodiments, the bag is connected to a channel including a tube and / or a needle. In certain embodiments, the multispecific binding protein, in a pharmaceutical composition disclosed herein, is diluted prior to administration. For example, in certain embodiments, the pharmaceutical composition is diluted with sodiumAttorney Docket No.: DFY-135WO chloride and is administered intravenously from a 250 ml saline bag. The intravenous infusion may be for about one hour (e.g., 50 to 80 minutes), 1-4 hours, 1-3 hours, 1-2 hours, 2-4 hours, 2-3 hours, or 3-4 hours. In certain embodiments, the multispecific binding protein is administered by intravenous infusion over a period of 1-2 hours.

[0157] It is contemplated that slower intravenous infusion may reduce adverse effects (e.g., infusion-related reactions) of the multispecific binding protein. Accordingly, in certain embodiments, administration of early doses (e.g., the doses of an initial four-week treatment cycle) can be slower than administration of subsequent doses. This safety measure may be particularly helpful to subjects who receive high-dose therapies. In certain embodiments, where a subject is scheduled to receive 5 mg / kg or higher dose of the multispecific binding protein in initial and subsequent treatment cycles (e.g., 5 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg), the multispecific binding protein is administered to the subject by intravenous infusion over a period of 3-4 hours in one or more doses of the initial treatment cycle (e.g., in the first dose, in the first two doses, or in the first three doses). The period of intravenous infusion can be reduced to 1-2 hours in subsequent doses. Monotherapy and combination therapies

[0158] It is understood that the multispecific binding proteins of the present disclosure may be used as a monotherapy or in combination with one or more other therapies that can be used to treat the disease or condition. Accordingly, in certain embodiments, the method of the present disclosure includes administering to a subject in need thereof a multispecific binding protein (e.g., A49-F3’-TriNKET-Trastuzumab) or pharmaceutical composition disclosed herein as a monotherapy. In certain embodiments, the subject has received, is receiving, or is scheduled to receive one or more additional therapies suitable for use in treating the disease or condition.

[0159] In certain embodiments, the subject has received, is receiving, or is scheduled to receive an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor. Exemplary anti- TROP2 antibodies are described in King et al. Invest New Drugs.2018 Oct;36(5):836-847, including but not limited to sacituzumab, datopotamab, and RN926. Exemplary topoisomerase I inhibitors are described in Thomas et al., Clin Cancer Res.2019 Nov 15;25(22):6581-6589, including but not limited to SN-38, LMP400, LMP776, and etirinotecan. In certain embodiments, the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor through a linker degradable in physiological conditions (e.g., a hydrolysable linker). Such linkers are described in Kang et al., Chem Sci.2021 OctAttorney Docket No.: DFY-135WO 6;12(41):13613-13647, including but not limited to CL2A, MC, MC-GGFG-AM, and AcBut. In certain embodiments, the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor via a degradable (e.g., hydrolysable) linker at a ratio of 1:5 to 1:9. In certain embodiments, the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor via a degradable (e.g., hydrolysable) linker at a ratio of 1:7 to 1:8. In certain embodiments, the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor via a degradable (e.g., hydrolysable) linker at an average ratio of 1.76.

[0160] In certain embodiments, the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor comprises sacituzumab govitecan-hziy. In certain embodiments, sacituzumab govitecan-hziy is administered to the subject intravenously at a dose of 10 mg / kg on Day 1 and Day 8 of one or more three-week treatment cycles. In certain embodiments, the dosage regimen of the multispecific binding protein is adjusted to three- week treatment cycles, as disclosed herein. Prophylactic Premedication

[0161] Prophylactic premedications are contemplated for reducing one or more adverse effects of (e.g., infusion-related reactions to) the multispecific binding protein administered according to the methods disclosed herein. It is understood that the prophylactic premedications can be used when the multispecific binding protein is provided as either a monotherapy or in combination with another cancer treatment. Corticosteroids

[0162] In certain embodiments, the method of the present disclosure includes administering to the subject a therapeutically effective amount of a corticosteroid to reduce one or more infusion-related reactions to the multispecific binding protein. Corticosteroids that are useful in the present invention generally include steroids produced by the adrenocortex, such as glucocorticoids and mineralocorticoids, and synthetic analogs and derivatives of naturally occurring corticosteroids having anti-inflammatory activity. In certain embodiments, the corticosteroid is a glucocorticoid. Glucocorticoids bind the glucocorticoid receptor and reduce inflammation by inhibiting the immune response. In certain embodiments, the corticosteroid is a mineralocorticoid. Mineral corticoids bind the mineralocorticoid receptor and act to regulate Na+ / K+concentrations in the serum. Some corticosteroids can have both glucocorticoid and mineralocorticoid functions. Examples of corticosteroids are disclosed in U.S. Patent No.10,799,599. In certain embodiments, the corticosteroid used in the method disclosed herein is selected from methylprednisolone,Attorney Docket No.: DFY-135WO dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, paramethasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, halcinonide, icomethasone, meprednisone, mometasone, rofleponide, RPR 106541, and their respective pharmaceutically acceptable derivatives, such as beclomethasone dipropionate (anhydrous or monohydrate), beclomethasone monopropionate, dexamethasone 21-isonicotinate, fluticasone propionate, icomethasone enbutate, tixocortol 21-pivalate, and triamcinolone acetonide, and pharmaceutically acceptable salts and / or derivatives thereof.

[0163] In certain embodiments, the glucocorticoid is methylprednisolone. Exemplary effective amounts of methylprednisolone can be in the range of 8 to 200 mg, 20 to 200 mg, 25 to 200 mg, 50 to 200 mg, 75 to 200 mg, 100 to 200 mg, 125 to 200 mg, 150 to 200 mg, 175 to 200 mg, 25 to 175 mg, 50 to 175 mg, 75 to 175 mg, 100 to 175 mg, 125 to 175 mg, 150 to 175 mg, 20 to 150 mg, 25 to 150 mg, 50 to 150 mg, 75 to 150 mg, 100 to 150 mg, 125 to 150 mg, 25 to 125 mg, 50 to 125 mg, 75 to 125 mg, 100 to 125 mg, 25 to 100 mg, 50 to 100 mg, 75 to 100 mg, 25 to 75 mg, 50 to 75 mg, 25 to 50 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg. In certain embodiments, the effective amount of methylprednisolone is about 125 mg. In certain embodiments, the effective amount of methylprednisolone by oral administration is 8 mg, 16 mg 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg.

[0164] In certain embodiments, the glucocorticoid is dexamethasone. Exemplary effective amounts of dexamethasone can be in the range of 8-200 mg, 20-200 mg, 50-200 mg, 100-200 mg, 8-100 mg, 20-100 mg, 50-100 mg, 8-50 mg, 20-50 mg, 8-20 mg, 20-150 mg, 50-150 mg, 50-100 mg, or 100-150 mg. In certain embodiments, the effective amount of dexamethasone by intravenous administration is 8-20 mg. In certain embodiments, the effective amount of dexamethasone by intravenous administration is 4 mg, 8 mg, 12 mg, 16 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, or 150 mg. In certain embodiments, the effective amount of dexamethasone by oral administration is 8 mg, 16 mg, 32 mg, 48 mg, 64 mg, 80 mg, 96 mg, or 120 mg.

[0165] In certain embodiments, the corticosteroid is administered parenterally. In certain embodiments, the corticosteroid is administered intravenously. In certain embodiments, the corticosteroid is administered orally.Attorney Docket No.: DFY-135WO

[0166] The corticosteroid can be administered prior to, simultaneously with, or subsequent to the administration of the multispecific binding protein. In certain embodiments, the corticosteroid is administered within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multispecific binding protein (e.g., prior to the beginning of the administration of the multispecific binding protein). In certain embodiments, the corticosteroid is administered within 1 hour prior to the administration of the multispecific binding protein (e.g., prior to the beginning of the administration of the multispecific binding protein). In certain embodiments, the corticosteroid is administered simultaneously with the administration of the multispecific binding protein. In certain embodiments, the corticosteroid and the multispecific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the duration of administration of the corticosteroid and the duration of administration of the multispecific binding protein completely or partially overlap. In certain embodiments, the corticosteroid is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multispecific binding protein (e.g., subsequent to the beginning of the administration of the multispecific binding protein).

[0167] In certain embodiments, the corticosteroid is administered on day 1 of the first cycle (i.e., in combination with the first dose of the multispecific binding protein). In certain embodiments, the corticosteroid is administered only on day 1 of the first cycle (i.e., in combination with the first dose of the multispecific binding protein). In certain embodiments, the corticosteroid is further administered if an infusion-related reaction persists or recurs. In certain embodiments, infusion-related reactions include a persistent rash, diarrhea, colitis, autoimmune hepatitis, arthritis, glomerulonephritis, cardiomyopathy, or uveitis or another inflammatory eye conditions.

[0168] In certain embodiments, the corticosteroid (e.g., methylprednisolone, dexamethasone) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.Attorney Docket No.: DFY-135WO

[0169] In certain embodiments, subjects receive premedication treatment including about 125 mg of methylprednisolone administered intravenously, or 8-20 mg of dexamethasone intravenously (e.g., where the subject receives a combination therapy with sacituzumab govitecan-hziy), within 60 minutes of administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, premedication treatment further includes intravenous or oral administration of 25 to 50 mg diphenhydramine and 800 to 1000 mg of acetaminophen 30 to 60 minutes prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.

[0170] Exemplary infusion-related reactions to a multispecific binding protein disclosed herein include cytokine release syndrome, anaphylaxis, chills, fever / pyrexia, hypotension, hypertension, rigors, headache, dizziness, itching, sore throat, laryngeal edema, angioedema, redness / flushing, rash / urticaria, bronchospasm, tachycardia, bradycardia, auricular fibrillation, hypoxia, respiratory distress / dyspnea / shortness of breath / breathless sensation, chest tightness, nausea, vomiting, pain (e.g., chest pain, back pain), shivering, tremors, myalgia, tiredness, insomnia, asthenia, hypersensitivity, and diarrhea. Clinical presentations of cytokine release syndrome are described in Shimabukuro-Vornhagen et al., include but are not limited to fever (e.g., high fever), fatigue, headache, rash, arthralgia, myalgia, hypotension, vasopressor-requiring circulatory shock, vascular leakage, disseminated intravascular coagulation, and multi-organ system failure. In certain embodiments, the co- administration of the corticosteroid reduces one or more of the infusion-related reactions in the subject. Antihistamines

[0171] An antihistamine can be used to avoid or mitigate an allergic response (e.g., anaphylaxis) to the multispecific binding protein. Accordingly, in certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an antihistamine. Exemplary antihistamines are disclosed in U.S. Patent No.10,898,693. In certain embodiments, the antihistamine used in the method disclosed herein is selected from crivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, cyclizine, chlorpheniramine, chlorodiphenhydramine, clemastine, cromolyn, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, nedocromil, olopatadine,Attorney Docket No.: DFY-135WO phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, rupatadine, tripelennamine, triprolidine, and combinations thereof. In certain embodiments, the antihistamine is diphenhydramine. In certain embodiments, the therapeutically effective amount of diphenhydramine is 10 to 100 mg, 20 to 100 mg, 25 to 100 mg, 30 to 100 mg, 40 to 100 mg, 50 to 100 mg, 60 to 100 mg, 70 to 100 mg, 80 to 100 mg, 90 to 100 mg, 10 to 90 mg, 20 to 90 mg, 30 to 90 mg, 40 to 90 mg, 50 to 90 mg, 60 to 90 mg, 70 to 90 mg, 80 to 90 mg, 10 to 80 mg, 20 to 80 mg, 30 to 80 mg, 40 to 80 mg, 50 to 80 mg, 60 to 80 mg, 70 to 80 mg, 10 to 70 mg, 20 to 70 mg, 30 to 70 mg, 40 to 70 mg, 50 to 70 mg, 60 to 70 mg, 10 to 60 mg, 20 to 60 mg, 30 to 60 mg, 40 to 60 mg, 50 to 60 mg, 10 to 50 mg, 20 to 50 mg, 25 to 50 mg, 30 to 50 mg, 40 to 50 mg, 20 to 40 mg, 30 to 40 mg, 20 to 30 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg. In certain embodiments, the therapeutically effective amount of diphenhydramine is 40 to 50 mg. In certain embodiments, the therapeutically effective amount of diphenhydramine is 25 to 50 mg.

[0172] In certain embodiments, the antihistamine is administered parenterally. In certain embodiments, the antihistamine is administered intravenously. In certain embodiments, the antihistamine is administered orally.

[0173] The antihistamine can be administered prior to, simultaneously with, or subsequent to the administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, the antihistamine is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multispecific binding protein or pharmaceutical composition disclosed herein (e.g., prior to the beginning of the administration of the multispecific binding protein or pharmaceutical composition). In certain embodiments, the antihistamine is administered with every dose of the multispecific binding protein.

[0174] In certain embodiments, the antihistamine (e.g., diphenhydramine) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.Attorney Docket No.: DFY-135WO

[0175] In certain embodiments, a subject receives premedication treatment including 25 to 50 mg of diphenhydramine administered intravenously or orally 30 to 60 minutes prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, premedication treatment further includes intravenous or oral administration of 800 to 100 mg acetaminophen prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.

[0176] Where the method of treatment disclosed herein includes multiple doses (e.g., five or more doses) of the multispecific binding protein, in certain embodiments, the antihistamine is administered with the first dose, the first two doses, the first three doses, the first four doses, or the first five doses of the multispecific binding protein. Analgesics and Antipyretics

[0177] An analgesic can be used to relieve pain as a result of the administration of the multispecific binding protein or pharmaceutical composition disclosed herein, whether administered as a monotherapy or as a combination therapy. Accordingly, in certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an analgesic. Exemplary analgesics are disclosed in U.S. Patent Application Publication No.2015 / 0342989 and U.S. Patent No.10,899,834. In certain embodiments, the analgesic used in the method disclosed herein is selected from acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate / benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, and tenoxicam, mpyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, etoricoxib, codeine, dihydrocodeine, morphine or a morphine derivative or pharmaceutically acceptable salt thereof, diacetylmorphine, hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene, and pentazocine, and pharmaceutically acceptable salts thereof. In certain embodiments, the analgesic is acetaminophen. In certainAttorney Docket No.: DFY-135WO embodiments, the therapeutically effective amount of acetaminophen is in the range of 325- 1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg.

[0178] In certain embodiments, the analgesic is administered parenterally. In certain embodiments, the analgesic is administered intravenously. In certain embodiments, the analgesic is administered orally.

[0179] The analgesic can be administered prior to, simultaneously with, or subsequent to the administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, the analgesic is administered within 2 hours, within 1.5 hours, within 1 hour (60 minutes), within 45 minutes, within 30 minutes, within 15 minutes, or immediately prior to the administration of the multispecific binding protein (e.g., prior to the beginning of the administration of the multispecific binding protein). In certain embodiments, the analgesic is administered simultaneously with the administration of the multispecific binding protein. In certain embodiments, the analgesic and the multispecific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the analgesic (e.g., acetaminophen) is administered with every dose of the multispecific binding protein.

[0180] In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.

[0181] In certain embodiments, the duration of administration of the analgesic and the duration of administration of the multispecific binding protein completely or partially overlap. In certain embodiments, the analgesic is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multispecific binding protein (e.g., subsequent to the beginning of the administration of the multispecific binding protein).

[0182] An antipyretic can be used to prevent or reduce fever as a result of the administration of the multispecific binding protein or pharmaceutical composition disclosed herein, whether administered as a monotherapy or as a combination therapy. Accordingly, inAttorney Docket No.: DFY-135WO certain embodiments, the method further includes administering to the subject a therapeutically effective amount of an antipyretic. Exemplary antipyretics are disclosed in U.S. Patent Application Publication No.2015 / 0342989. In certain embodiments, the antipyretic used in the method disclosed herein is selected from acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate / benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, and tenoxicam, mpyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, and etoricoxib. In certain embodiments, the antipyretic is acetaminophen. In certain embodiments, the therapeutically effective amount of acetaminophen is in the range of 325-1000 mg, 400-1000 mg, 500-1000 mg, 600-1000 mg, 700-1000mg, 800-1000 mg, 900-1000 mg, 325-800 mg, 400-800 mg, 500-800 mg, 600-800 mg, 700-800 mg, 325-600 mg, 400-600 mg, or 500-600 mg. In certain embodiments, the effective amount of acetaminophen is 325 mg, 500 mg, 650 mg, 700 mg, 800 mg, 900 mg, or 1000 mg.

[0183] In certain embodiments, the antipyretic is administered parenterally. In certain embodiments, the antipyretic is administered intravenously. In certain embodiments, the antipyretic is administered orally.

[0184] The antipyretic can be administered prior to, simultaneously with, or subsequent to the administration of the multispecific binding protein. In certain embodiments, the antipyretic is administered within 2 hours, 1.5 hours, 1 hour (60 minutes), 45 minutes, 30 minutes, or 15 minutes prior to the administration of the multispecific binding protein (e.g., prior to the beginning of the administration of the multispecific binding protein). In certain embodiments, the antipyretic is administered simultaneously with the administration of the multispecific binding protein. In certain embodiments, the antipyretic and the multispecific binding protein are diluted into a single pharmaceutical composition administered to the subject. In certain embodiments, the duration of administration of the antipyretic and the duration of administration of the multispecific binding protein completely or partiallyAttorney Docket No.: DFY-135WO overlap. In certain embodiments, the antipyretic is administered within 2 hours, 1 hour, or 30 minutes subsequent to the administration of the multispecific binding protein (e.g., subsequent to the beginning of the administration of the multispecific binding protein). In certain embodiments, the antipyretic (e.g., acetaminophen) is administered with every dose of the multispecific binding protein.

[0185] The antipyretic can be administered prior to, simultaneously with, or subsequent to the administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, the analgesic (e.g., acetaminophen) is administered 30 to 90 min., 40 to 90 min., 50 to 90 min., 60 to 90 min., 70 to 90 min., 80 to 90 min., 30 to 80 min., 40 to 80 min., 50 to 80 min., 60 to 80 min., 70 to 80 min., 30 to 70 min., 40 to 70 min., 50 to 70 min., 60 to 70 min., 30 to 60 min., 40 to 60 min., 50 to 60 min., 30 to 50 min., 40 to 50 min., 30 to 40 min., about 30 min., about 40 min., about 50 min., about 60 min., about 70 min., about 80 min., or about 90 min., prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.

[0186] In certain embodiments, subjects receive premedication treatment including 800 to 1000 mg of acetaminophen administered intravenously or orally 30 to 60 minutes prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, premedication treatment further includes intravenous or oral administration of 25 to 50 mg diphenhydramine prior to administration of the multispecific binding protein or pharmaceutical composition disclosed herein.

[0187] The description above describes multiple aspects and embodiments of the disclosure. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments. INCORPORATION BY REFERENCE

[0188] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0189] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Various structural elements of the different embodiments and various disclosed method stepsAttorney Docket No.: DFY-135WO may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. The scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

Attorney Docket No.: DFY-135WO WHAT IS CLAIMED IS:

1. A method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of a multispecific binding protein comprising: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) a first antibody Fc region and a second antibody Fc region that together form a dimer that binds CD16, wherein the subject also receives an effective amount of an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor.

2. The method of claim 1, wherein the anti-TROP2 antibody comprises sacituzumab.

3. The method of claim 1 or 2, wherein the topoisomerase I inhibitor comprises SN-38.

4. The method of any one of claims 1-3, wherein the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor through a hydrolysable linker.

5. The method of any one of claims 1-4, wherein the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor comprises sacituzumab govitecan-hziy.

6. The method of any one of claims 1-5, wherein the cancer is a breast cancer.

7. The method of claim 6, wherein the cancer is a hormone receptor (HR) positive and HER2 negative metastatic breast cancer.

8. The method of claim 6, wherein the cancer is a HER2 positive metastatic breast cancer.

9. The method of any one of claims 1-5, wherein the cancer is a lung cancer.

10. The method of claim 9, wherein the cancer is a HER2 activated non-small cell lung cancer (NSCLC).

11. The method of any one of claims 1-10, wherein the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor is administered intravenously at a dose of 10 mg / kg on Day 1 and Day 8 of one or more three-week treatment cycles.Attorney Docket No.: DFY-135WO 12. The method of claim 11, wherein the multispecific binding protein is administered intravenously at a dose of 7.5 mg / kg on Day 1 and Day 8 of the one or more three-week treatment cycles.

13. A method of treating a HER2 activated non-small cell lung cancer (NSCLC), the method comprising administering to a subject in need thereof an effective amount of a multispecific binding protein comprising: (a) a first antigen-binding site that binds NKG2D; (b) a second antigen-binding site that binds HER2; and (c) a first antibody Fc region and a second antibody Fc region that together form a dimer that binds CD16.

14. The method of claim 13, wherein subject receives the multispecific binding protein as a monotherapy.

15. The method of claim 14, wherein the multispecific binding protein is administered intravenously at a dose of 10 mg / kg on Day 1, Day 8, and Day 15 of an initial four-week treatment cycle, and on Day 1 and Day 15 of one or more subsequent four-week treatment cycles.

16. The method of claim 13, wherein the subject also receives an effective amount of an anti-TROP2 antibody conjugated to a topoisomerase I inhibitor.

17. The method of claim 16, wherein the anti-TROP2 antibody comprises sacituzumab.

18. The method of claim 16 or 17, wherein the topoisomerase I inhibitor comprises SN- 38.

19. The method of any one of claims 16-18, wherein the anti-TROP2 antibody is conjugated to the topoisomerase I inhibitor through a hydrolysable linker.

20. The method of any one of claims 16-19, wherein the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor comprises sacituzumab govitecan-hziy.

21. The method of any one of claims 16-20, wherein the anti-TROP2 antibody conjugated to the topoisomerase I inhibitor is administered intravenously at a dose of 10 mg / kg on Day 1 and Day 8 of one or more three-week treatment cycles.Attorney Docket No.: DFY-135WO 22. The method of any one of claims 16-21, wherein the multispecific binding protein is administered intravenously at a dose of 7.5 mg / kg on Day 1 and Day 8 of the one or more three-week treatment cycles.

23. The method of any one of claims 1-22, wherein the first antigen-binding site comprises: (a) a heavy chain variable domain (VH) comprising complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity- determining region 3 (CDR3) sequences comprising the amino acid sequences of SEQ ID NOs: 95, 96, and 121, respectively; and (b) a light chain variable domain (VL) comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively.

24. The method of claim 23, wherein: (a) the VH of the first antigen-binding site comprises CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 95, 96, and 97, respectively; and (b) the VL of the first antigen-binding site comprises CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively.

25. The method of claim 23 or 24, wherein the VH of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:94, and the VL of the first antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

98.

26. The method of any one of claims 1-25, wherein the second antigen-binding site comprises: (a) a VH comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively.

27. The method of claim 26, wherein the VH of the second antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:156, and the VL of the secondAttorney Docket No.: DFY-135WO antigen-binding site comprises an amino acid sequence at least 90% identical to SEQ ID NO:

160.

28. The method of any one of claims 1-27, wherein the first antigen-binding site is comprised in an antibody Fab fragment linked to an N-terminus of the first antibody Fc region, and the second antigen-binding site is comprised in a single chain variable fragment (scFv) linked to an N-terminus of the second antibody Fc region.

29. The method of any one of claims 1-28, wherein the first antibody Fc region and the second antibody Fc region are each a human IgG1 Fc region.

30. The method of any one of claims 1-29, wherein the first antibody Fc region and the second antibody Fc region each comprise an amino acid sequence at least 90% identical to SEQ ID NO:

141.

31. The method of claim 29 or 30, wherein the first antibody Fc region and the second antibody Fc region each comprise one or more mutations, relative to SEQ ID NO: 141, to promote heterodimerization.

32. The method of claim 31, wherein the first antibody Fc region comprises K360E and K409W substitutions relative to SEQ ID NO: 141, and the second antibody Fc region comprises Q347R, D399V and F405T substitutions relative to SEQ ID NO: 141, numbered according to the EU numbering system.

33. The method of claim 31 or 32, wherein the first antibody Fc region comprises a Y349C substitution relative to SEQ ID NO: 141, and the second antibody Fc region comprises an S354C substitution relative to SEQ ID NO: 141, numbered according to the EU numbering system.

34. The method of any one of claims 1-33, wherein the multispecific binding protein comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:184; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:183; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:185.

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