T cell and NK cell engagers

Abstract

The present disclosure relates to T cell and / or NK cell engaging agents and their uses. In some embodiments, the present disclosure provides a bispecific antibody molecule or antigen-binding fragment thereof comprising (i) a first antigen-binding domain that selectively binds to transmembrane and immunoglobulin domain containing 2 (TMIGD2) and (ii) a second antigen-binding domain that selectively binds to at least one tumor-associated antigen (TAA). Such agents (e.g., bispecific antibody molecules or antigen-binding fragments thereof) disclosed herein can be used in methods of treating a subject having a disease, disorder, or condition and / or in methods of modulating the immune response of a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 308,800, filed February 10, 2022, the contents of which are incorporated by reference in their entirety herein. [Background technology]

[0002] Immunotherapy has been explored for many diseases and disorders, including cancer, but has encountered functional limitations that still need to be addressed. A network of co-stimulatory and co-inhibitory ligands and receptors tightly controls the immune system. Immune checkpoints negatively regulate the progression of immune responses based on complex interactions. Currently available immunotherapies can regulate immune responses in some patients, but the expression of immune checkpoints and their interactions with natural binding partners can vary between patients.

[0003] Thus, there is a need for the development of new and improved therapeutic modalities optimized to target tumor sites, particularly in the tumor microenvironment. Summary of the Invention

[0004] The present disclosure provides, inter alia, bispecific antibody molecules or antigen-binding fragments thereof that interact with specific targets, and methods of using such bispecific antibody molecules or antigen-binding fragments thereof. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise a first antigen-binding domain that selectively binds to transmembrane and immunoglobulin domain-containing 2 (TMIGD2). In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise a second antigen-binding domain that selectively binds to at least one tumor-associated antigen (TAA). HERV-H LTR-associated 2 (HHLA2) is an exemplary TAA that is a B7 gene family member that is widely expressed in a variety of tumors and antigen-presenting cells. HHLA2 interacts with both inhibitory and stimulatory receptors to regulate T cell and NK cell function. TMIGD2 is an activating receptor for HHLA2 that is expressed in both T cells and NK cells, and interacts with HHLA2 to costimulate T cell and NK cell activation.

[0005] While various TAA-binding agents have been investigated as immunotherapeutic agents, the present disclosure encompasses, inter alia, the discovery of bispecific antibody molecules or antigen-binding fragments thereof with novel activity that can selectively bind at least one TAA expressed on cancer cells and selectively bind TMIGD2 expressed on immune effector cells, particularly T cells (e.g., naive and / or effector T cells) and / or NK cells. Combining such tumor-directed approaches to enhance immune regulation in patients can provide a powerful strategy to generate and shape immune responses at tumor sites, particularly in the tumor microenvironment (TME). The bispecific antibody molecules or antigen-binding fragments thereof described herein can activate both the innate and adaptive immune systems. Thus, the present disclosure provides several examples of bispecific antibody molecules or antigen-binding fragments thereof that are particularly useful for the treatment of various cancers, including solid or hematological tumors, as well as for modulating immune responses in subjects.

[0006] In one aspect, the present disclosure provides a bispecific antibody molecule or antigen-binding fragment thereof, comprising: (i) a first antigen-binding domain that selectively binds to TMIGD2; and (ii) a second antigen-binding domain that selectively binds to at least one TAA. In some embodiments, the first antigen-binding domain activates immune effector cells. In some embodiments, the first antigen-binding domain recruits immune effector cells to the tumor microenvironment. In some embodiments, the immune effector cells include or are T cells (e.g., naive T cells and / or effector T cells) and / or NK cells. In some embodiments, the at least one TAA includes or is HHLA2, CD123, B7-H3, B7-H4, CD33, CD30, HER2, CD22, CD79b, Nectin-4, Trop-2, BCMA, CD19, folate receptor alpha, EGFR, or a combination thereof. In certain embodiments, at least one TAA comprises or is HHLA2. In certain embodiments, at least one TAA comprises or is CD123. In certain embodiments, at least one TAA comprises or is B7-H3. In certain embodiments, at least one TAA comprises or is B7-H4. In certain embodiments, at least one TAA comprises or is CD33. In certain embodiments, at least one TAA comprises or is CD30. In certain embodiments, at least one TAA comprises or is HER2. In certain embodiments, at least one TAA comprises or is CD22. In certain embodiments, at least one TAA comprises or is CD79b. In certain embodiments, at least one TAA comprises or is Nectin-4. In certain embodiments, at least one TAA comprises or is Trop-2. In certain embodiments, at least one TAA comprises or is BCMA, hi certain embodiments, at least one TAA comprises or is CD19.In certain embodiments, at least one TAA comprises or is folate receptor alpha. In certain embodiments, at least one TAA comprises or is EGFR.

[0007] In some embodiments, the first antigen-binding domain comprises or is an antibody or antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain comprises or is an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment comprises or is an scFv, Fab, Fab', F(ab')2, nanobody, or camelid antibody. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises (i) a chimeric, human, or humanized antibody or antigen-binding fragment thereof, and / or (iii) a monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises (i) a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, and / or (ii) a light chain constant region selected from a kappa or lambda light chain constant region.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises an Fc region with reduced effector function. In some embodiments, the Fc region comprises one or more amino acid substitutions of L234A and / or L235A, numbered according to the EU index. In some embodiments, the modification comprises N297A, numbered according to the EU index. In some embodiments, the first antigen-binding domain comprises or is an scFv and the second antigen-binding domain comprises or is an antibody. In some embodiments, the scFv and the antibody are covalently linked by a peptide linker. In some embodiments, the peptide linker is at least 5 amino acids in length.

[0009] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one bispecific antibody molecule or antigen-binding fragment thereof of any aspect or embodiment described herein and a pharma- ceutically acceptable carrier.

[0010] In another aspect, the disclosure provides a method of treating a subject having a disease, disorder, or condition comprising administering a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein. In another aspect, the disclosure provides a method of modulating an immune response in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0011] In some embodiments, the subject has or is at risk of developing cancer. In some embodiments, the subject has a solid tumor or a blood cancer. In some embodiments, the solid tumor is or includes one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer. In some embodiments, the blood cancer includes or is a leukemia or lymphoma. In some embodiments, the leukemia includes or is acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the lymphoma includes or is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

[0012] In some embodiments, at least one bispecific antibody molecule or antigen-binding fragment thereof of any aspect or embodiment described herein is administered in combination with at least one additional agent.

[0013] In another aspect, the disclosure provides a nucleic acid molecule (e.g., one or more of the nucleic acid molecules in Tables 1-5) encoding at least one bispecific antibody molecule or antigen-binding fragment thereof of any aspect or embodiment described herein.

[0014] In another aspect, the disclosure provides an expression vector comprising a nucleic acid molecule of any aspect or embodiment described herein.

[0015] In another aspect, the disclosure provides a host cell comprising or expressing at least one bispecific antibody molecule or antigen-binding fragment of any aspect or embodiment described herein, a nucleic acid molecule of any aspect or embodiment described herein, or an expression vector of any aspect or embodiment described herein.

[0016] In another aspect, the disclosure provides a method of making at least one bispecific antibody molecule or antigen-binding fragment thereof, the method comprising: (i) culturing a host cell comprising a nucleic acid molecule of any aspect or embodiment described herein or an expression vector of any aspect or embodiment described herein under conditions suitable for expression of the at least one bispecific antibody molecule or antigen-binding fragment thereof; and (ii) recovering the at least one bispecific antibody molecule or antigen-binding fragment thereof.

[0017] In another aspect, the present disclosure provides a method for detecting the presence or level of a TMIGD2 polypeptide and / or a TAA in a sample, comprising detecting the TMIGD2 polypeptide and / or a TAA in the sample using at least one bispecific antibody molecule or antigen-binding fragment thereof of any aspect or embodiment described herein.

[0018] In another aspect, the present disclosure provides a kit comprising at least one bispecific antibody molecule or antigen-binding fragment thereof of any aspect or embodiment described herein and instructions for use.

[0019] The figures described below, which together constitute a drawing, are for illustration purposes only and not for limitation. [Brief description of the drawings]

[0020] [Figure 1] 1 is a schematic diagram showing an exemplary format of a bispecific antibody molecule using a scFv targeting TMIGD2 linked to the C-terminus of the heavy chain of an IgG antibody targeting a TAA. The linkers within the scFv domain (between the VH and VL domains) and between the scFv region and the Fc region are both (G4S)4 repeats.

[0021] [Figure 2A] Graphs showing fold change in T cell activation in TMIGD2-Jurkat NFAT reporter cells with plate-bound (FIG. 2A) and soluble (FIG. 2B) anti-TMIGD2 / anti-CD20 bispecific antibody molecule (TankeC4S2), anti-TMIGD2 IgG4 (Amp4.5), or human IgG4 isotype control relative to anti-CD3 stimulation alone. [Figure 2B] Graphs showing fold change in T cell activation in TMIGD2-Jurkat NFAT reporter cells with plate-bound (FIG. 2A) and soluble (FIG. 2B) anti-TMIGD2 / anti-CD20 bispecific antibody molecule (TankeC4S2), anti-TMIGD2 IgG4 (Amp4.5), or human IgG4 isotype control relative to anti-CD3 stimulation alone.

[0022] [Diagram 3] Graph showing fold change in T cell activation of anti-TMIGD2 / anti-CD20 bispecific antibody molecule (Tanke) in combination with anti-CD3 or human IgG4 isotype control relative to anti-CD3 stimulation alone in an NFAT TMIGD2-jurkat reporter assay in the presence of TAA (CD20)-expressing Raji target cells.

[0023] [Figure 4]FIG. 13 is a graph showing the fold change in T cell activation relative to anti-CD3 stimulation of anti-TMIGD2 / anti-CD20 bispecific antibody molecules (TK20-17C7-01 and TK20-39C9-2134) alone in combination with anti-CD3 or anti-CD20 (IgG1 LALA) controls in an NFAT TMIGD2-jurkat reporter assay in the presence of TAA (CD20)-expressing Raji target cells.

[0024] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, and other terms, are set forth throughout the specification. Publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional details regarding its practice are incorporated herein by reference in their entireties.

[0025] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to encompass the listed components or steps, whether presented by themselves or with one or more additional components or steps, and (iv) when ranges are provided, the endpoints are included.

[0026] About: As used herein, the term "about" refers to a value similar to a stated reference value when applied to one or more subject values. In some embodiments, the term "about" refers to a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise clear from the context (except when such a value exceeds 100% of the possible values).

[0027] Affinity maturation: As used herein, the term "affinity maturation" refers to an antibody molecule having one or more modifications in one or more CDRs thereof that result in an improvement in the affinity of the antibody molecule for an antigen compared to a parent antibody molecule that does not have those one or more modifications. In some embodiments, an affinity matured antibody molecule has nanomolar or even picomolar affinity for a target antigen. Affinity matured antibody molecules may be produced by any of a variety of procedures known in the art. H and V L Affinity maturation by domain shuffling is described in Marks et al., BioTechnology 10:779-783 (1992). Random mutagenesis of CDR and / or framework residues is described in Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier et al., Gene 169:147-155 (1995), Yelton et al., J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0028] Agent: As used herein, the term "agent" refers to a form of treatment, e.g., biological, chemical, or other treatment. For example, a biologic may be or may include a biological entity and / or compound, including, for example, an antibody molecule or antigen-binding fragment thereof, an organic molecule (e.g., a small molecule), a peptide (e.g., a fusion protein), an aptamer, a nucleic acid, a chimeric antigen receptor, a glycoprotein, a sugar, a lipid, a growth factor, an enzyme, a synthetic molecule, a carbohydrate, a lipid, a hormone, a polymer, or a derivative, variant, complex, or any combination thereof. Under appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be used to mean an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may be used to refer to a natural product. In some cases, again as will be clear from the context, the term may be used to refer to one or more entities that are artificial in that they are designed, engineered, and / or produced through human action and / or are not found in nature. In some embodiments, an agent may be utilized in an isolated or pure form. In some embodiments, the agents may be utilized in crude form. In some embodiments, the agents may be provided as collections or libraries and screened to identify or characterize active agents therein. The agents may bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on a target or target cell. A variety of agents are useful in the compositions and methods described herein.

[0029] For example, the agent can be or include one or more of a chemotherapeutic agent, a hormone therapy, a cell-based therapy, a thermotherapy treatment, a photodynamic therapy, surgery, radiation, or a transplant. In some embodiments, the chemotherapeutic agent includes or is one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., one or more inhibitors of topoisomerase I or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, or a combination thereof. In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, cefotaxime ... In some embodiments, the chemotherapeutic agent comprises or is one or more of: cytabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, rituxan, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, or vinorelbine. In some embodiments, the chemotherapeutic agent comprises or is an antibody-drug conjugate (ADC).In some embodiments, the ADC is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA The compound includes or is an ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, lorvotuzumab mertansine, or a combination thereof.

[0030] In some embodiments, the hormone therapy can be or include tamoxifen, raloxifene, leuprolide, bicalatomid, granisetron, flutamide, or a combination thereof. In some embodiments, the cell-based therapy includes or is chimeric antigen receptor T (CAR-T) cells, TCR-transduced T cells, dendritic cells, tumor-infiltrating lymphocytes (TIL), natural killer (NK) cells, or a combination thereof. In some embodiments, the hyperthermia includes or is local hyperthermia (e.g., external, intraluminal, or interstitial hyperthermia), regional hyperthermia (e.g., deep tissue hyperthermia, regional perfusion, or (continuous hyperthermic peritoneal perifusion), or whole body hyperthermia. In some embodiments, the photodynamic therapy includes or is the administration of a photosensitizer, such as hematoporphyrin and its derivatives, Verteporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxyhypocerin A, 2BA-2-DMHA, or combinations thereof. In some embodiments, the surgery includes or is surgery to remove cancerous or precancerous tissue. In some embodiments, the transplant includes or is a stem cell transplant or an organ transplant.

[0031] Antibody molecule: As used herein, the term "antibody molecule" refers to a polypeptide, e.g., an immunoglobulin chain or fragment thereof, that contains sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target, e.g., an antigen. The term "antibody molecule" encompasses antibodies and antibody fragments. As is known in the art, naturally produced intact antibodies are approximately 150 kD tetrameric agents that contain two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD) that associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains, each about 110 amino acids long: the amino-terminal variable (V H) domain, followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the Y-stalk). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" region connects the CH2 and CH3 domains to the CH1 domain. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody molecule. Each light chain is made up of two domains: the amino-terminal variable (V L The CH2 domain is followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch" region. Naturally produced antibodies are typically glycosylated on the CH2 domain. Each domain in an antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed together in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domain, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to create a single hypervariable antigen binding site located at the tip of a Y structure. The Fc region of a naturally occurring antibody binds to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications.

[0032] In some embodiments, any polypeptide, or complex of polypeptides, that contains sufficient immunoglobulin domain sequences can be referred to and / or used as an "antibody molecule," regardless of whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody molecule is monoclonal. In some embodiments, an antibody molecule is polyclonal. In some embodiments, an antibody molecule has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, one or more antibody molecule sequences are humanized, primatized, or chimeric, as known in the art. Furthermore, the term "antibody molecule" can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies. For example, the antibody molecule may be in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4), multispecific (e.g., bispecific) antibodies, and / or antibody fragments (preferably antigen-binding antibody fragments). The antibody molecule may be an immunoglobulin, a heavy chain antibody, a light chain antibody, a leucine-rich repeat (LRR)-based antibody, or other protein scaffold with antibody-like properties.Antibody molecules include, but are not limited to, scFv, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fd', Fv, Feb, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandab, DVD, BiTe, TandAb, single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals (SMIP™), single chain or tandem diabodies (TandAb®), VHH, Anticalin®, N The antibody may be any immunological binding moiety known in the art, including, but not limited to, an anobody, BiTE, ankyrin repeat protein or DARPIN, Avimer, DART, TCR-like antibody, Adnectin, Affilin, Trans-body, Affibody, TrimerX, microprotein, Fynomer, Centyrin, KALBITOR, or any combination thereof.

[0033] In some embodiments, an "antibody molecule" is a polypeptide that includes or comprises one or more structural elements recognized by those of skill in the art as CDRs. In some embodiments, an antibody molecule includes at least one CDR that is substantially identical to one found in a reference antibody (e.g., at least one V H CDRs and / or at least one V LIn some embodiments, a CDR that is substantially identical to a reference CDR is identical or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, a CDR is substantially identical to a reference CDR with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, a CDR is substantially identical to a reference CDR in that at least one amino acid (e.g., 1-5 amino acids) is deleted, added, or substituted compared to the reference CDR, but the CDR otherwise has an amino acid sequence that is identical to the reference CDR.

[0034] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformation.

[0035] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformation.

[0036] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as the term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, which nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0037] Antigen-binding fragment: As used herein, the term "antigen-binding fragment" refers to a portion of an intact antibody or a recombinant variant thereof that binds to a target, e.g., the antigen to which the intact antibody binds. An antigen-binding fragment refers to an antigen-binding domain, e.g., the antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a particular target, e.g., antigen. Antigen-binding fragments include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Exemplary antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; single chain antibody molecules, e.g., scFv, VHH, or camelid; single domain antibodies, e.g., sdAb(VHH); L or VH or multispecific antibody molecules formed from antibody fragments, such as, but not limited to, fragments comprising two or more Fab fragments linked by disulfide bridges at the hinge region. Antigen-binding fragments can be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, or bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments can contain at least two different variable domains, each capable of specifically binding to a separate antigen or different epitopes of the same antigen. The antigen-binding fragment can be produced by any means. For example, the antigen-binding fragment can be produced enzymatically or chemically by fragmentation of an intact antibody or antibody agent. Alternatively, the antigen-binding fragment can be produced recombinantly. The antigen-binding fragment can be produced completely or partially synthetically. The antigen-binding fragment can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids or more.

[0038] Antibody-dependent cellular cytotoxicity: As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the phenomenon in which target cells bound by antibody molecules are killed by immune effector cells. Without wishing to be bound by theory, ADCC is typically understood to involve effector cells bearing Fc receptors (FcRs) recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing a specific antigen on their surface to which the antibody is bound). Immune effector cells that mediate ADCC include, but are not limited to, NK cells, macrophages, neutrophils, and eosinophils.

[0039] Associated: As used herein, two events or entities are "associated" with one another if the presence, level, extent, type, and / or form of one event or entity correlates with that of the other event or entity. For example, a particular entity (e.g., an antigen, polypeptide, genetic signature, metabolite, or microorganism) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population).

[0040] Binding: As used herein, the term "binding" refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties. Indirect binding involves a physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., while covalently bound or otherwise associated with a carrier entity and / or within a biological system or cell).

[0041] Bispecific antibody molecule: As used herein, the term "bispecific antibody molecule" refers to an antibody molecule having two different binding specificities. Typically, a bispecific binding molecule comprises at least two antigen-binding domains, each of which specifically binds to a different antigen or epitope. A variety of different structures of bispecific antibody molecules are known in the art. In some embodiments, each antigen-binding domain comprises at least one V H Region and / or at least one V L In some embodiments, the V H Area and / or V L The regions are derived from a particular monoclonal antibody. In certain embodiments, the bispecific antibody molecule comprises (i) a first antigen-binding domain that comprises or is an antibody fragment (e.g., an scFv, Fab, Fab', F(ab')2, Fd, Fv, nanobody, or camelid antibody) and (ii) a second antigen-binding domain that comprises or is a full-length antibody molecule.

[0042] Cancer: As used herein, the terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, thereby exhibiting an abnormal growth phenotype characterized by a marked loss of control over cell proliferation. Cancer cells can spread locally or via the bloodstream and lymphatic system to other body sites. In some embodiments, a tumor is or includes precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic cells. In some embodiments, a cancer is or includes a solid tumor. In some embodiments, a cancer is or includes a hematological tumor. Examples of various cancers are described herein, and include, but are not limited to, hematopoietic cell cancers (e.g., leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma, and myeloproliferative disorders), sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the oral cavity, pharynx, larynx, and lung, liver cancer, genitourinary cancer (e.g., prostate, cervical, bladder, uterine, and endometrial cancer, and renal cell carcinoma), bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, thyroid or parathyroid, head and neck cancer, breast cancer, gastrointestinal cancer, nervous system cancer, benign lesions such as papilloma, or any combination thereof, as well as several other types, including cancers described elsewhere herein.

[0043] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, and / or vehicle with which the composition is administered. In some exemplary embodiments, the carrier comprises sterile liquids, such as, for example, water and oils, including, for example, oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid ingredients.

[0044] CDR: As used herein, the term "complementarity determining region" or "CDR" refers to a sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. For example, generally, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. Specific systems for defining CDR boundaries (e.g., Kabat, IMGT, Chothia, or combinations thereof) have been established in the art. Thus, CDRs may be referred to by Kabat, Chothia, IMGT, or any other boundary definition known in the art. For example, in the combined Kabat and Chothia numbering scheme, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both. Despite the different boundaries, each of these systems has some overlap in what constitutes a "hypervariable region" within a variable sequence. CDR definitions according to these systems may differ in length and boundary regions with respect to the adjacent framework regions (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Edition, USDepartment of Health and Human Services, 1992; Chothia et al. (1987) J. Mol. Biol. 196, 901; and MacCallum et al., J. Mol. Biol. (1996) 262, 732). Those skilled in the art will appreciate the differences between these systems and will be able to understand CDR boundaries to the extent necessary to understand and practice the claims and disclosure herein.

[0045] Chimeric antibody: As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence is identical to that found in a first species. H and V LChimeric antibodies refer to an antibody molecule that contains a mouse V constant region sequence linked to a human constant region, as well as a constant region sequence found in a second, different species. In many embodiments, a chimeric antibody is an antibody molecule that contains a mouse V constant region sequence linked to a human constant region. H and V L In some embodiments, the human V region is linked to a non-human constant region (e.g., a mouse constant region). H and V L Antibodies having such regions are called "reverse chimeric antibodies."

[0046] Composition: As used herein, the term "composition" refers to a separate physical entity that contains one or more specified components. Generally, unless otherwise specified, a composition may be in any form, for example, a gas, a gel, a liquid, or a solid.

[0047] Comprise: As used herein, a composition or method described herein as "comprising" one or more recited elements or steps is open-ended, meaning that the recited element(s) or step(s) are essential, but other elements or steps may be added within the scope of the composition or method. Any composition or method described herein as "comprising" one or more recited elements or steps also describes a corresponding, more limited composition or method that "consisting essentially of" the same recited elements or steps, meaning that the composition or method includes the essential recited elements or steps, and may also include additional elements or steps that do not substantially affect the basic and novel characteristic(s) of the composition or method. Any composition or method described herein as "comprising" or "consisting essentially of" one or more recited elements or steps also describes a corresponding, more limited and closed-ended composition or method that "consisting of" the recited elements or steps, to the exclusion of any other unrecited elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any listed essential element or step may be substituted for that element or step.

[0048] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which two or more different therapeutic agents (e.g., a bispecific antibody molecule or antigen-binding fragment thereof and at least one additional agent) are administered in an overlapping regimen such that a subject is exposed to both agents simultaneously. When used in combination therapy, the two or more different therapeutic agents may be administered simultaneously or separately. This combination administration can include simultaneous administration of two or more therapeutic agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more therapeutic agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more therapeutic agents can be administered simultaneously in separate formulations. In another alternative, a first therapeutic agent can be administered, followed by one or more additional therapeutic agents. In separate administration protocols, two or more therapeutic agents may be administered minutes apart, hours apart, days apart, or weeks apart. In some embodiments, two or more therapeutic agents may be administered within a few hours (e.g., less than about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours) apart.

[0049] Effective amount: As used herein, the term "effective amount" refers to a dose adequate to prevent or treat at least one sign and / or symptom of a disease, disorder, or condition (e.g., cancer) in an individual. Amounts effective for therapeutic or prophylactic use may depend, for example, on the stage and severity of the disease, disorder, or condition being treated, the age, weight, and general health of the patient, and the judgment of the prescribing physician. The size of the dose may also be determined by the active ingredient / therapeutic agent selected, the method of administration, the timing and frequency of administration, the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular active ingredient, and the desired physiological effect. Those skilled in the art will appreciate that various diseases, disorders, or conditions may require long-term treatment with multiple administrations. For purposes of this disclosure, the amount or dose of therapeutic agent (e.g., one or more bispecific antibody molecules or antigen-binding fragments thereof described herein) administered should be sufficient to effect a therapeutic or prophylactic response in the subject (e.g., a reduction or other alleviation of the severity or duration of at least one sign or symptom) over a reasonable time frame. For example, the dose should be sufficient to detect, treat, or prevent cancer for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In some embodiments, the period is longer. Dosing is determined by the efficacy of one or more particular therapeutic agents and the condition of the subject (e.g., human), as well as the weight of the subject (e.g., human) to be treated.

[0050] Encode: As used herein, "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand (the nucleotide sequence corresponding to the mRNA sequence, typically provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of that gene or cDNA.

[0051] Epitope: As used herein, the term "epitope" refers to any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts another conformation (e.g., linearized).

[0052] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more bispecific antibody molecules or antigen-binding fragments thereof described herein). In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0053] Fragment: As used herein, the term "fragment" refers to a structure that comprises a distinct portion of an entire structure, but lacks one or more portions found in the entire structure. In some embodiments, a fragment is made up of such distinct portions. In some embodiments, a fragment comprises or is a characteristic structural element or portion found in the entire structure. In some embodiments, a fragment (e.g., an antigen-binding fragment described herein) comprises or is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., amino acids) found in a whole antibody molecule. In some embodiments, a fragment (e.g., an antigen-binding fragment described herein) comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole antibody molecule. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in all nucleotides.

[0054] Framework region: As used herein, the term "framework region" or "FR" refers to the sequence of the variable region minus the CDRs. Since the CDR sequences can be determined by different systems, the framework sequences are also subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 being located between FR1 and FR2, CDR2 being between FR2 and FR3, and CDR3 being between FR3 and FR4. As others refer to, without identifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region represents the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions. For example, FR1 represents the first framework region that is closest to the amino terminus of the variable region and 5' with respect to CDR1. FR represents two or more subregions that make up the framework region.

[0055] HHLA2: As used herein, the term "HHLA2" or "human endogenous retrovirus H long terminal repeat associated protein 2" refers to a member of the B7 family. HHLA2 is also known as HERV-H LTR-associated 2, B7y, B7H7, or B7-H7. HHLA2 protein has limited expression in normal human tissues, but is widely expressed in human cancers. HHLA2 is a membrane protein with three Ig-like domains (IgV-IgC-IgV), whereas other members of the B7 family generally have only two Ig domains (IgV-IgC). HHLA2 in normal human tissues is expressed in the epithelium of kidney, intestine, gallbladder, and breast, as well as placental trophoblast cells. In the immune system, HHLA2 is constitutively expressed on human monocytes and macrophages. HHLA2 regulates the functions of human T cells and HK cells, including, for example, proliferation and cytokine production of T cells and NK cells. HHLA2 is expressed at elevated levels in a wide range of human cancers, including, but not limited to, colon, renal, lung, pancreatic, ovarian, and prostate cancers. HHLA2 is also expressed in human cancers of thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophageal tissues.

[0056] The term "HHLA2" includes fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human HHLA2 cDNA and human HHLA2 protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). Human HHLA2 variants include variant 1 (NM_007072.3 and NP_009003.1 (representing the longest transcript and encoding the longest isoform a)), variant 2 (NM_001282556.1 and NP_001269485.1 (containing an alternative promoter and differing in the 5'UTR compared to variant 1)), variant 3 (NM_001282557.1 and NP_001269486.1 (containing an alternative promoter and differing in the 5'UTR compared to variant 1)), variant 4 (NM_001282558.1 and NP_00126948 These include variant 7.1 (encoding isoform b, containing an alternative promoter, differing in the 5'UTR, and lacking an alternative in-frame exon in the 3' coding region compared to variant 1, resulting in a shorter isoform than isoform a), and variant 5 (NM_001282559.1, and NP_001269488.1 (encoding isoform c, containing an alternative promoter, having an alternative 5'UTR, translation initiation begins at an alternative start codon compared to variant 1, resulting in an alternative N-terminus and a shorter polypeptide than isoform a)).

[0057] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Upon reading this disclosure, one of skill in the art will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., or Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, or P. methanolica), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, or Trichoplusia ni), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadromas). In some embodiments, the host cell comprises or is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the host cell comprises one or more of CHO (e.g., CHO K1, DXB-1 1CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3 A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the foregoing.In some embodiments, the host cell contains one or more viral genes.

[0058] Human antibody: As used herein, the term "human antibody" refers to an antibody having variable and constant regions generated (or assembled) from human immunoglobulin sequences. Antibodies or antigen-binding fragments thereof may be considered "human" even though their amino acid sequences contain residues or elements, e.g., in one or more CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., sequence variations that may be introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0059] Humanized: As used herein, the term "humanized" refers to the modification of a V antibody from a reference antibody produced in a non-human species (e.g., mouse). H and / or V LHumanized antibody molecules or antigen-binding fragments thereof, whose amino acid sequences include domain sequences, but also include modifications of those sequences relative to a reference antibody intended to make them "human-like" or more similar to human germline variable sequences due to human germline variable sequences. In some embodiments, a humanized antibody molecule or antigen-binding fragment thereof is one that immunospecifically binds to an antigen of interest and has FR regions having substantially the amino acid sequence of a human antibody and CDRs having substantially the amino acid sequence of a non-human antibody. A humanized antibody molecule comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to a non-human immunoglobulin (e.g., a donor immunoglobulin) and all or substantially all of the framework regions correspond to human immunoglobulin consensus sequences. In some embodiments, a humanized antibody molecule also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody molecule contains at least the variable domains of both the light and heavy chains. The antibody molecule may also comprise the CH1, hinge, CH2, CH3, and optionally CH4 regions of the heavy chain constant region. In some embodiments, the humanized antibody molecule contains only a humanized VL region. In some embodiments, the humanized antibody molecule contains only a humanized VH region. In some specific embodiments, the humanized antibody molecule comprises humanized VH and VL regions.

[0060] Immunoglobulin: As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibody molecules. Antibody molecules expressed by B cells may be referred to as BCRs (B cell receptors) or antigen receptors. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE, further including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and respiratory and urogenital mucous secretions. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0061] Identity: The term "identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical, e.g., if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., 9 positions in a 10 amino acid long polymer) are identical, then the two amino acid sequences are 90% identical.

[0062] Substantial identity: As used herein, the term "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments the relevant extension is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of CDRs, references to "substantial identity" typically refer to CDRs having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the reference CDR.

[0063] Immune effector cell: As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response, e.g., promoting an immune response. Examples of immune effector cells include, but are not limited to, T cells, natural killer (NK) cells, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans cells, or B lymphocytes.

[0064] Immune response: As used herein, the term "immune response" refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign, induce the formation of antibodies, and / or activate lymphocytes to remove the antigen. In some embodiments, an immune cell response can include proliferation of immune effector cells (e.g., T cells or NK cells), cytokine production by immune effector cells (e.g., T cells or NK cells), and / or release of cytotoxic particles, including perforin and / or granzymes, by immune effector cells (e.g., T cells or NK cells).

[0065] "Improve", "Increase", "Inhibit", or "Decrease": As used herein, the terms "improve", "increase", "inhibit", "decrease", or grammatical equivalents thereof, refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement is or includes a measurement in a particular system (e.g., a single individual) under otherwise equivalent conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement is or includes a measurement in a comparable system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0066] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response and / or a change in the nature of the response in a subject compared to the level and / or nature of the response in an untreated or untreated subject. The term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0067] Monoclonal antibody: As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, in which the individual antibodies are substantially identical and / or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the production of the monoclonal), which variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibody molecules directed against different determinants (epitopes), each monoclonal antibody molecule in such a preparation is directed against a single determinant on the antigen. Thus, "monoclonal" typically indicates that the population of antibodies is not a mixture of separate antibodies.

[0068] Nucleic acid molecule: As used herein, the term "nucleic acid molecule" refers to a polymer of at least three nucleotides. In some embodiments, the nucleic acid molecule comprises DNA. In some embodiments, the nucleic acid molecule comprises RNA. In some embodiments, the nucleic acid molecule is single-stranded. In some embodiments, the nucleic acid molecule is double-stranded. In some embodiments, the nucleic acid molecule comprises both single-stranded and double-stranded portions. In some embodiments, the nucleic acid molecule comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, the nucleic acid molecule comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, the nucleic acid molecule may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, such as, for example, a peptide nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises one or more or all naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, and / or uracil). In some embodiments, the nucleic acid molecule comprises one or more or all non-naturally occurring residues. In some embodiments, the non-naturally occurring residues comprise a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, a methylated base, an intercalating base, or a combination thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose, or combinations thereof) compared to the natural residue. In some embodiments, the nucleic acid molecule has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide.In some embodiments, the nucleic acid molecule has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid molecule can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, (e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid molecule has at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 ... 0, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length.

[0069] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit / risk ratio.

[0070] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, or polyanhydrides; or combinations thereof, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0071] Polypeptide: As used herein, the term "polypeptide" or "protein" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is engineered and / or produced by the action of the human hand. A polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. A polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. A polypeptide may comprise D-amino acids, L-amino acids, or both. A polypeptide may comprise one or more pendant groups or other modifications, e.g., modifications or conjugations of one or more amino acid side chains at the N-terminus, C-terminus, or both. In some embodiments, such pendant groups or modifications are selected from acetylation, amidation, lipidation, methylation, or pegylation, including combinations thereof. A polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, the polypeptide is linear. The polypeptide may be or include a stapled polypeptide. The term "polypeptide" may be added to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to a polypeptide that shares a related activity or structure and can therefore be considered to be a member of the same class or family of polypeptides. For each such class, the disclosure provides exemplary polypeptides within the class whose amino acid sequence and / or function are known and / or the skilled artisan would recognize. In some embodiments, such exemplary polypeptides are the reference polypeptides of the polypeptide class or family.In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity, share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with reference polypeptides of a class. For example, a member polypeptide has an overall degree of sequence homology or identity with a reference polypeptide of at least about 30-40%, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region that may be or may include a characteristic sequence element) that exhibits a very high degree of sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such conserved regions usually encompass at least 3-4, and often up to 20 or more amino acids, and in some embodiments, the conserved region encompasses at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. Useful polypeptides may comprise or consist of fragments of a parent polypeptide. Useful polypeptides may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest or vice versa, and / or the fragments may be present in the polypeptide of interest in a different order than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0072] scFv: As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are linked in sequence via a short flexible polypeptide linker. An scFv can be expressed as a single polypeptide chain and retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv can be expressed as a single polypeptide chain with the V and V chains in any order with respect to the N- and C-termini of the polypeptide. L and V H Thus, the scFv may have a V L -Linker-V H or V H -Linker-V L may include:

[0073] Subject: As used herein, the term "subject" refers to an organism, e.g., a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject is afflicted with a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as set forth herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is predisposed to a disease, disorder, or condition. In some embodiments, a predisposed subject is predisposed to developing a disease, disorder, or condition and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., a clinical symptom of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom one or more of the diagnosis or therapy has been administered.

[0074] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0075] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect can be achieved, for example, by suppression, amelioration, or eradication of a disease state.

[0076] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that induces a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance being delivered, and / or the target cell or tissue. For example, an effective amount of a bispecific antibody molecule or antigen-binding fragment thereof in a formulation for treating a disease, disorder, or condition described herein is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the occurrence of one or more symptoms or features of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is administered in a single dose. In some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0077] TMIGD2: As used herein, the term "TMIGD2" or "transmembrane and immunoglobulin domain-containing 2 (TMIGD2)" refers to a membrane protein with an extracellular IgV-like domain with two tyrosine signaling motifs, a transmembrane region, and a proline-rich cytoplasmic domain. TMIGD2 is constitutively expressed in naive T cells and NK cells, but not in T regulatory cells or B cells. TMIGD2 expression is slowly lost upon repeated stimulation of T cells. Consistent with this, TMIGD2 is expressed in only about half of memory T cells, and TMIGD2-negative T cells have a terminally differentiated senescent phenotype. TMIGD2 is also expressed in endothelial and epithelial cells, where it functions to reduce cell migration and promote capillary tube formation during angiogenesis.

[0078] The term "TMIGD2" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human TMIGD2 cDNA and human TMIGD2 protein sequences are publicly available from NCBI. Human TMIGD2 isoforms include isoform 1 (NM_144615.2 and NP653216.2), isoform 2 (NM_001169126.1 and NP001162597.1 (which uses an alternative in-frame splice site in the 3' coding region compared to variant 1, resulting in a shorter isoform compared to isoform 1)), and isoform 3 (NM_001308232.1 and NP001295161.1 (which lacks an alternative in-frame exon in the 5' coding region compared to variant 1, resulting in a shorter isoform compared to isoform 1)). Nucleic acid and polypeptide sequences of TMIGD2 orthologues in organisms other than humans are also known, including, for example, chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2, and XM_OOl 138228.4 and XP_001138228.3), and bovine TMIGD2 (XM_005208980.3 and XP005209037.1, XM_005208979.3 and XP_005209036.1, and XM_002688933.5 and XP_002688979.1).

[0079] Treat: As used herein, the terms "treat", "treatment", or "treating" refer to partial or complete alleviation, amelioration, delay in onset, inhibition, prevention, reduction, and / or reduction in the incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition described herein. In some embodiments, the treatment is administered to a subject who does not exhibit signs or features of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, the treatment is administered to a subject who exhibits, for example, early or mild signs or features of a disease, disorder, and / or condition described herein, e.g., to reduce the risk of developing pathology associated with a disease, disorder, and / or condition described herein. In some embodiments, the treatment is administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, and / or condition described herein. In some embodiments, the treatment comprises administering to the subject one or more bispecific antibody molecules or antigen-binding fragments thereof described herein.

[0080] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. A tumor may include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic cells. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor is a dispersed or liquid tumor. In some embodiments, a tumor is a solid tumor.

[0081] Throughout this disclosure, various aspects may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, and 3-6, as well as individual numbers within that range, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6. This applies regardless of the breadth of the range. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The present disclosure provides, inter alia, bispecific antibody molecules or antigen-binding fragments thereof that are capable of (i) targeting cancer cells by selectively binding to at least one TAA, and (ii) recruiting and activating immune effector cells (e.g., T cells (e.g., naive T cells and / or effector T cells) and / or NK cells) that contain or express transmembrane and immunoglobulin domain containing 2 (TMIGD2) within the tumor microenvironment (TME), thereby targeting cancer cells for destruction. Thus, the present disclosure provides several examples of such bispecific antibody molecules or antigen-binding fragments thereof that are particularly useful for the treatment of various cancers, including solid or hematological tumors, as well as for modulating immune responses in a subject.

[0083] Bispecific antibody molecules and antigen-binding fragments thereof The present disclosure provides, inter alia, a bispecific antibody molecule or antigen-binding fragment thereof. The bispecific antibody molecule or antigen-binding fragment thereof described herein can have specificity for at least two antigens. The bispecific antibody molecule or antigen-binding fragment thereof can be characterized by a first immunoglobulin variable domain sequence having binding specificity for a first antigen and a second immunoglobulin variable domain sequence having binding specificity for a second antigen.

[0084] A bispecific antibody molecule or antigen-binding fragment thereof described herein can comprise (i) a first antigen-binding domain that selectively binds to transmembrane and immunoglobulin domain-containing 2 (TMIGD2), and (ii) a second antigen-binding domain that selectively binds to at least one tumor-associated antigen (TAA) described herein (e.g., HHLA2). In some embodiments, the first antigen-binding domain activates and / or recruits immune effector cells (e.g., T cells (e.g., naive and / or effector T cells) and / or NK cells) to the tumor microenvironment (TME). In some embodiments, a bispecific antibody molecule or antigen-binding fragment thereof described herein comprises (i) a first antigen-binding domain that selectively binds TMIGD2 on immune effector cells (e.g., T cells and / or NK cells), thereby recruiting and / or activating immune effector cells in the TME, and (ii) a second antigen-binding domain that selectively binds to at least one TAA described herein.

[0085] The first antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein can selectively bind to at least one epitope on TMIGD2. The second antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein can selectively bind to at least one epitope on a TAA described herein. In some embodiments, at least one TAA comprises or is HHLA2, CD123, B7-H3, B7-H4, CD33, CD30, HER2, CD22, CD79b, Nectin-4, Trop-2, BCMA, CD19, folate receptor alpha, EGFR, or a combination thereof. In certain embodiments, at least one TAA comprises or is HHLA2 (also referred to as B7y, B7H7, or B7-H7). In certain embodiments, at least one TAA comprises or is CD123. In certain embodiments, at least one TAA comprises or is B7-H3. In certain embodiments, at least one TAA comprises or is B7-H4. In certain embodiments, at least one TAA comprises or is CD33. In certain embodiments, at least one TAA comprises or is CD30. In certain embodiments, at least one TAA comprises or is HER2. In certain embodiments, at least one TAA comprises or is CD22. In certain embodiments, at least one TAA comprises or is CD79b. In certain embodiments, at least one TAA comprises or is Nectin-4. In certain embodiments, at least one TAA comprises or is Trop-2. In certain embodiments, at least one TAA comprises or is BCMA. In certain embodiments, at least one TAA comprises or is CD19. In certain embodiments, at least one TAA comprises or is folate receptor alpha. In certain embodiments, the at least one TAA comprises or is EGFR.

[0086] In some embodiments, the at least one TAA is selected from the group consisting of HHLA2 (also known as B7y, B7H7, or B7-H7), CD20, CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant I, II (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family members, B cell maturation (BCMA), Tn antigen (TnAg or GalNAcα-Ser / Thr), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor (R OR1), FMS-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); folate receptor alpha; HER2 (also called receptor tyrosine-protein kinase erbB-2 or CD340);Mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase (also called kallikrein 4 (KLK4) or EMSP1), prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrinB2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), LMP2, glycoprotein 100 (gp100), breakpoint cluster region-based oncogene fusion protein (BCR) and and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6);thyroid-stimulating hormone receptor (TSHR);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading frame 61 (CXORF61);CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta-specific 1 (PLAC1);hexasaccharide moiety of globoH glycoceramide (GloboH);mammary differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cellular receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, K9 locus (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17);X antigen family, such as member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma mutant (RAS) corpus;human telomerase reverse transcriptase (hTERT);sarcoma translocation breakpoint;melanoma inhibitor of apoptosis (ML-IAP);ERG;N-acetylglucosaminyltransferase V (NA17);paired box protein Pax-3 (PAX3);androgen receptor;cyclin B1;v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN);Ras homolog family member C (RhoC);tyrosinase-related protein 2 (TRP-2);cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2);CD79a;CD79b;CD72;leukocyte-associated immunoglobulin-like receptor 1 (LAIR1);Fc fragment of IgA receptor (FCAR or CD89);leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2);The antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof may comprise or be one or more of CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican 3 (GPC3), Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1), or a combination thereof. The first antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein may comprise or be an antibody molecule or antigen-binding fragment thereof. The second antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein may comprise or be an antibody molecule or antigen-binding fragment thereof. Antibody molecules or antigen-binding fragments thereof may be any of the following: scFv, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fd', Fv, Feb, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandab, DVD, BiTe, TandAb, single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof), camelid antibodies, full length antibodies, masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals (SMIP™), single chain or tandem diabodies (TandAb®), VHH, Ant, It can be any immunological binding moiety, including, but not limited to, icalin®, Nanobody®, BiTE®, ankyrin repeat protein or DARPIN®, Avimer®, DART, TCR-like antibody, Adnectin®, Affilin®, Trans-body®, Affibody®, TrimerX®, microprotein, Fynomer®, Centyrin®, KALBITOR®, or any combination thereof;

[0087] In some embodiments, the first antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or is an antigen-binding fragment thereof. In some embodiments, the second antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or is an antigen-binding fragment thereof. The antigen-binding fragment may be any V- or V-type antibody derived from any antibody, e.g., an antibody described herein. H Or, V L The antibody may comprise or be any polypeptide or peptide-containing molecule comprising at least a portion of an immunoglobulin comprising at least one complementarity determining region (CDR) of. Antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Such functional antibody fragments can retain the ability to selectively bind to TMIGD2 or at least one TAA as described herein.

[0088] Examples of antigen-binding fragments described herein include (i) Fab fragments, V L , V H (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment containing the CH1 domain, (iv) the V of a single arm of the antibody. L and V H (v) an Fv fragment containing the V H (vi) camelid or camelized variable domains; (vii) scFv, V H and V L (viii) a fusion protein of a domain; (viii) a single domain antibody; or (viii) a heavy chain (and light chain (e.g., half antibody). In certain embodiments, the antigen-binding fragment described herein comprises or is an scFv.

[0089] The first antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein may, for example, comprise or be a full-length antibody comprising an immunoglobulin Fc region. The second antigen-binding domain of the bispecific antibody molecule or antigen-binding fragment thereof described herein may, for example, comprise or be a full-length antibody comprising an Fc region. In some embodiments, the Fc region has reduced effector function compared to a reference antibody or antigen-binding fragment thereof. In some embodiments, the Fc region comprises a modification to reduce glycosylation of the Fc region. In some embodiments, the modification comprises one or more of the following amino acid substitutions: N297A, L234A, L235A, C220, C226S, C229S, P238S, C226S, C229S, E3233P, L234V, L235A, L234F, L235E, P331S, or a combination thereof, numbered according to the EU index. In some embodiments, the modification comprises one or more amino acid substitutions at positions 234, 235, 237 of the CH2 region, numbered according to the EU index, or a combination thereof.

[0090] The antibodies or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein may be H and V L In some embodiments, an antibody or antigen-binding fragment thereof comprises an immunoglobulin molecule of four polypeptide chains, e.g., two heavy chains and two light chains. The heavy chains include the V H The light chain can include a V domain and a heavy chain constant domain. The heavy chain constant domain can include a CH1, hinge, CH2, CH3, and optionally a CH4 region. L The light chain constant domain may comprise a CL domain and a light chain constant domain. The light chain constant domain may comprise a CL domain.

[0091] V H and / or V L can be further subdivided into variable regions, called CDRs, interspersed with more conserved regions, called framework regions (FRs). Hand / or V L Each domain can comprise three CDRs and four framework regions arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (one or more of which can be engineered as described herein). Generally, each V H There are three CDRs (HCDR1, HCDR2, and HCDR3) in each V L There are three CDRs in the region (LCDR1, LCDR2, and LCDR3). Framework regions and CDRs can be defined using a number of well-known schemes (see, for example, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definitions used in Oxford Molecular's AbM antibody modeling software, each of which is incorporated herein by reference in its entirety).

[0092] An antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise: (a) a VH comprising one, two or three VH CDR sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the VH CDRs of Tables 1-5, respectively; and / or (b) a VL comprising one, two or three VL CDR sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the VL CDRs of Tables 1-5, respectively. In some embodiments, an antibody molecule or antigen-binding fragment thereof described herein is, or comprises, (a) a VH comprising one, two, or three VH CDR sequences having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the VH CDRs of Tables 1-5, respectively, and / or (b) a VL comprising one, two, or three VL CDR sequences having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the VL CDRs of Tables 1-5, respectively. In some embodiments, an antibody or antigen-binding fragment thereof described herein is, or comprises, (a) a VH that comprises one, two, or three VH CDR sequences that comprise or consist of the VH CDRs of Tables 1-5, respectively, and / or (b) a VL that comprises one, two, or three VL CDR sequences that comprise or consist of the VL CDRs of Tables 1-5, respectively.

[0093] An antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise: (a) a VH having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a VH of Tables 1-5; and / or (a) a VL having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a VL of Tables 1-5. In some embodiments, the antibody molecule or antigen-binding fragment thereof is or comprises: (a) a VH having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a VH of Tables 1-5, and / or (b) a VL having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a VL of Tables 1-5. In some embodiments, the antibody molecule or antigen-binding fragment thereof is or comprises: (a) a VH comprising or consisting of a VH of Tables 1-5, and / or (b) a VL comprising or consisting of a VL of Tables 1-5.

[0094] An antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise: (a) a heavy chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a heavy chain of Tables 1-5, and / or (a) a light chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a light chain of Tables 1-5. In some embodiments, the antibody molecule or antigen-binding fragment thereof is or comprises: (a) a heavy chain having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a heavy chain of Tables 1-5, and / or (b) a light chain having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a light chain of Tables 1-5. In some embodiments, the antibody molecule or antigen-binding fragment thereof is or comprises: (a) a heavy chain comprising or consisting of a heavy chain of Tables 1-5, and / or (b) a light chain comprising or consisting of a light chain of Tables 1-5.

[0095] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 212, a VH CDR2 amino acid sequence of SEQ ID NO: 213, and a VH CDR3 amino acid sequence of SEQ ID NO: 214, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 223, a VL CDR2 amino acid sequence of SEQ ID NO: 224, and a VL CDR3 amino acid sequence of SEQ ID NO: 225. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 215, a VH CDR2 amino acid sequence of SEQ ID NO: 216, and a VH CDR3 amino acid sequence of SEQ ID NO: 217, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 226, a VL CDR2 amino acid sequence of SEQ ID NO: 227, and a VL CDR3 amino acid sequence of SEQ ID NO: 228. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 218, a VH CDR2 amino acid sequence of SEQ ID NO: 219, and a VH CDR3 amino acid sequence of SEQ ID NO: 220, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 229, a VL CDR2 amino acid sequence of SEQ ID NO: 230, and a VL CDR3 amino acid sequence of SEQ ID NO: 231.

[0096] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO:211, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO:211. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO:222, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO:222. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO:211, and a VL comprising the amino acid sequence of SEQ ID NO:222.

[0097] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 221. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 209. In some embodiments, the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 205.

[0098] In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 207, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 207. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 208, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 208. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 207, and a light chain comprising the amino acid sequence of SEQ ID NO: 208.

[0099] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 241, a VH CDR2 amino acid sequence of SEQ ID NO: 242, and a VH CDR3 amino acid sequence of SEQ ID NO: 243, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 252, a VL CDR2 amino acid sequence of SEQ ID NO: 253, and a VL CDR3 amino acid sequence of SEQ ID NO: 254. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 244, a VH CDR2 amino acid sequence of SEQ ID NO: 245, and a VH CDR3 amino acid sequence of SEQ ID NO: 246, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 255, a VL CDR2 amino acid sequence of SEQ ID NO: 256, and a VL CDR3 amino acid sequence of SEQ ID NO: 257. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH that comprises a VH CDR1 amino acid sequence of SEQ ID NO: 247, a VH CDR2 amino acid sequence of SEQ ID NO: 248, and a VH CDR3 amino acid sequence of SEQ ID NO: 249, and (b) a VL that comprises a VL CDR1 amino acid sequence of SEQ ID NO: 258, a VL CDR2 amino acid sequence of SEQ ID NO: 259, and a VL CDR3 amino acid sequence of SEQ ID NO: 260.

[0100] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 240, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 240. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 251, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 251. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 240, and a VL comprising the amino acid sequence of SEQ ID NO: 251.

[0101] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 250. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 238. In some embodiments, the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 234.

[0102] In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 236, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 236. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 237, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 237. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 236, and a light chain comprising the amino acid sequence of SEQ ID NO: 237.

[0103] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 270, a VH CDR2 amino acid sequence of SEQ ID NO: 271, and a VH CDR3 amino acid sequence of SEQ ID NO: 272, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 281, a VL CDR2 amino acid sequence of SEQ ID NO: 282, and a VL CDR3 amino acid sequence of SEQ ID NO: 283. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 273, a VH CDR2 amino acid sequence of SEQ ID NO: 274, and a VH CDR3 amino acid sequence of SEQ ID NO: 275, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 284, a VL CDR2 amino acid sequence of SEQ ID NO: 285, and a VL CDR3 amino acid sequence of SEQ ID NO: 286. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 276, a VH CDR2 amino acid sequence of SEQ ID NO: 277, and a VH CDR3 amino acid sequence of SEQ ID NO: 278, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 287, a VL CDR2 amino acid sequence of SEQ ID NO: 288, and a VL CDR3 amino acid sequence of SEQ ID NO: 289.

[0104] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 269, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 269. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 280, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 280. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 269, and a VL comprising the amino acid sequence of SEQ ID NO: 280.

[0105] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 279. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 267. In some embodiments, the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 263.

[0106] In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:265, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO:265. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO:266, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO:266. In some embodiments, the anti-CD20 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:265, and a light chain comprising the amino acid sequence of SEQ ID NO:266.

[0107] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 3, a VH CDR2 amino acid sequence of SEQ ID NO: 4, and a VH CDR3 amino acid sequence of SEQ ID NO: 5, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 39, a VL CDR2 amino acid sequence of SEQ ID NO: 40, and a VL CDR3 amino acid sequence of SEQ ID NO: 41. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 6, a VH CDR2 amino acid sequence of SEQ ID NO: 7, and a VH CDR3 amino acid sequence of SEQ ID NO: 8, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 42, a VL CDR2 amino acid sequence of SEQ ID NO: 43, and a VL CDR3 amino acid sequence of SEQ ID NO: 44. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 9, a VH CDR2 amino acid sequence of SEQ ID NO: 10, and a VH CDR3 amino acid sequence of SEQ ID NO: 11, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 45, a VL CDR2 amino acid sequence of SEQ ID NO: 46, and a VL CDR3 amino acid sequence of SEQ ID NO: 47.

[0108] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 12. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 48. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 12, and a VL comprising the amino acid sequence of SEQ ID NO: 48.

[0109] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 14. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 50. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 14, and a light chain comprising the amino acid sequence of SEQ ID NO: 50.

[0110] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 19, the VH CDR2 amino acid sequence of SEQ ID NO: 20, and the VH CDR3 amino acid sequence of SEQ ID NO: 21, and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 30, the VL CDR2 amino acid sequence of SEQ ID NO: 31, and the VL CDR3 amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 22, the VH CDR2 amino acid sequence of SEQ ID NO: 23, and the VH CDR3 amino acid sequence of SEQ ID NO: 24, and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 33, the VL CDR2 amino acid sequence of SEQ ID NO: 34, and the VL CDR3 amino acid sequence of SEQ ID NO: 35. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 25, a VH CDR2 amino acid sequence of SEQ ID NO: 26, and a VH CDR3 amino acid sequence of SEQ ID NO: 27, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 36, a VL CDR2 amino acid sequence of SEQ ID NO: 37, and a VL CDR3 amino acid sequence of SEQ ID NO: 38.

[0111] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 18. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 29. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 29.

[0112] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-TMIGD2 and anti-HHLA2 bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 1.

[0113] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 54, a VH CDR2 amino acid sequence of SEQ ID NO: 55, and a VH CDR3 amino acid sequence of SEQ ID NO: 56, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 90, a VL CDR2 amino acid sequence of SEQ ID NO: 92, and a VL CDR3 amino acid sequence of SEQ ID NO: 93. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 57, a VH CDR2 amino acid sequence of SEQ ID NO: 58, and a VH CDR3 amino acid sequence of SEQ ID NO: 59, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 94, a VL CDR2 amino acid sequence of SEQ ID NO: 95, and a VL CDR3 amino acid sequence of SEQ ID NO: 96. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 60, a VH CDR2 amino acid sequence of SEQ ID NO: 61, and a VH CDR3 amino acid sequence of SEQ ID NO: 62, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 97, a VL CDR2 amino acid sequence of SEQ ID NO: 98, and a VL CDR3 amino acid sequence of SEQ ID NO: 99.

[0114] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 63. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 100. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 63, and a VL comprising the amino acid sequence of SEQ ID NO: 100.

[0115] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 65. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 102. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence of SEQ ID NO: 102.

[0116] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 70, the VH CDR2 amino acid sequence of SEQ ID NO: 71, and the VH CDR3 amino acid sequence of SEQ ID NO: 72, and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 81, the VL CDR2 amino acid sequence of SEQ ID NO: 82, and the VL CDR3 amino acid sequence of SEQ ID NO: 83. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 73, the VH CDR2 amino acid sequence of SEQ ID NO: 74, and the VH CDR3 amino acid sequence of SEQ ID NO: 75, and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 84, the VL CDR2 amino acid sequence of SEQ ID NO: 85, and the VL CDR3 amino acid sequence of SEQ ID NO: 86. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 76, a VH CDR2 amino acid sequence of SEQ ID NO: 77, and a VH CDR3 amino acid sequence of SEQ ID NO: 78, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 87, a VL CDR2 amino acid sequence of SEQ ID NO: 88, and a VL CDR3 amino acid sequence of SEQ ID NO: 89.

[0117] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 69. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 80. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 69, and a VL comprising the amino acid sequence of SEQ ID NO: 80.

[0118] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-TMIGD2 and anti-HHLA2 bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 52.

[0119] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 105, a VH CDR2 amino acid sequence of SEQ ID NO: 106, and a VH CDR3 amino acid sequence of SEQ ID NO: 107, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 141, a VL CDR2 amino acid sequence of SEQ ID NO: 142, and a VL CDR3 amino acid sequence of SEQ ID NO: 143. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 108, a VH CDR2 amino acid sequence of SEQ ID NO: 109, and a VH CDR3 amino acid sequence of SEQ ID NO: 110, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 144, a VL CDR2 amino acid sequence of SEQ ID NO: 145, and a VL CDR3 amino acid sequence of SEQ ID NO: 146. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 111, a VH CDR2 amino acid sequence of SEQ ID NO: 112, and a VH CDR3 amino acid sequence of SEQ ID NO: 113, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 147, a VL CDR2 amino acid sequence of SEQ ID NO: 148, and a VL CDR3 amino acid sequence of SEQ ID NO: 149.

[0120] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 114. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 150. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 114, and a VL comprising the amino acid sequence of SEQ ID NO: 150.

[0121] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 116. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 152. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 116, and a light chain comprising the amino acid sequence of SEQ ID NO: 152.

[0122] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 121, a VH CDR2 amino acid sequence of SEQ ID NO: 122, and a VH CDR3 amino acid sequence of SEQ ID NO: 123, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 132, a VL CDR2 amino acid sequence of SEQ ID NO: 133, and a VL CDR3 amino acid sequence of SEQ ID NO: 134. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 124, a VH CDR2 amino acid sequence of SEQ ID NO: 125, and a VH CDR3 amino acid sequence of SEQ ID NO: 126, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 135, a VL CDR2 amino acid sequence of SEQ ID NO: 136, and a VL CDR3 amino acid sequence of SEQ ID NO: 137. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH that comprises a VH CDR1 amino acid sequence of SEQ ID NO: 127, a VH CDR2 amino acid sequence of SEQ ID NO: 128, and a VH CDR3 amino acid sequence of SEQ ID NO: 129, and (b) a VL that comprises a VL CDR1 amino acid sequence of SEQ ID NO: 138, a VL CDR2 amino acid sequence of SEQ ID NO: 139, and a VL CDR3 amino acid sequence of SEQ ID NO: 140.

[0123] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 120. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 131. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 120, and a VL comprising the amino acid sequence of SEQ ID NO: 131.

[0124] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 130. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 118. In some embodiments, the anti-TMIGD2 and anti-HHLA2 bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 103.

[0125] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 156, a VH CDR2 amino acid sequence of SEQ ID NO: 157, and a VH CDR3 amino acid sequence of SEQ ID NO: 158, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 192, a VL CDR2 amino acid sequence of SEQ ID NO: 193, and a VL CDR3 amino acid sequence of SEQ ID NO: 194. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 159, a VH CDR2 amino acid sequence of SEQ ID NO: 160, and a VH CDR3 amino acid sequence of SEQ ID NO: 161; and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 195, a VL CDR2 amino acid sequence of SEQ ID NO: 196, and a VL CDR3 amino acid sequence of SEQ ID NO: 197. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 162, a VH CDR2 amino acid sequence of SEQ ID NO: 163, and a VH CDR3 amino acid sequence of SEQ ID NO: 164, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 198, a VL CDR2 amino acid sequence of SEQ ID NO: 199, and a VL CDR3 amino acid sequence of SEQ ID NO: 200.

[0126] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 165. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 201, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 201. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 165, and a VL comprising the amino acid sequence of SEQ ID NO: 201.

[0127] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 167. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a light chain comprising the amino acid sequence of SEQ ID NO: 203, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 203. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein may be or comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 167, and a light chain comprising the amino acid sequence of SEQ ID NO: 203.

[0128] In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or can comprise (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 172, a VH CDR2 amino acid sequence of SEQ ID NO: 173, and a VH CDR3 amino acid sequence of SEQ ID NO: 174, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 183, a VL CDR2 amino acid sequence of SEQ ID NO: 184, and a VL CDR3 amino acid sequence of SEQ ID NO: 185. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 175, a VH CDR2 amino acid sequence of SEQ ID NO: 176, and a VH CDR3 amino acid sequence of SEQ ID NO: 177, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 186, a VL CDR2 amino acid sequence of SEQ ID NO: 187, and a VL CDR3 amino acid sequence of SEQ ID NO: 188. In some embodiments, an anti-TMIGD2 antibody or antigen-binding fragment thereof of a bispecific antibody molecule or antigen-binding fragment thereof described herein may be or may comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 178, a VH CDR2 amino acid sequence of SEQ ID NO: 179, and a VH CDR3 amino acid sequence of SEQ ID NO: 180, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 189, a VL CDR2 amino acid sequence of SEQ ID NO: 190, and a VL CDR3 amino acid sequence of SEQ ID NO: 191.

[0129] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 171. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VL comprising the amino acid sequence of SEQ ID NO: 182, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical or more to SEQ ID NO: 182. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or comprise a VH comprising the amino acid sequence of SEQ ID NO: 171, and a VL comprising the amino acid sequence of SEQ ID NO: 182.

[0130] In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises a linker comprising the amino acid sequence of SEQ ID NO: 181. In some embodiments, the anti-TMIGD2 antibody or antigen-binding fragment thereof of the bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 169. In some embodiments, the anti-TMIGD2 and anti-HHLA2 bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or has the amino acid sequence of SEQ ID NO: 154. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

[0131] The antibody molecules or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein can be from any class of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, as well as any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody molecules or antigen-binding fragments thereof described herein can be or include human, humanized, CDR-grafted, or in vitro generated antibodies. The antibody molecules or antigen-binding fragments thereof described herein can have or include a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody molecules or antigen-binding fragments thereof described herein can have or include a light chain selected from, for example, kappa or lambda.

[0132] The antibody molecules or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein can be or include monoclonal antibodies. In some embodiments, monoclonal antibodies directed to a particular epitope are derived from a single cell line (e.g., a B cell line).

[0133] In some embodiments, the antibody molecule or antigen-binding fragment of the bispecific antibody molecule or antigen-binding fragment thereof described herein is or comprises a polyclonal antibody. In contrast to monoclonal antibodies, polyclonal antibodies are typically obtained from a heterogeneous antibody population, such that the antibodies in a particular population contain structural variations, e.g., affinities for different epitopes on a particular target (e.g., TMIGD2 or TAA described herein). Several methods of producing polyclonal antibodies are known, including, for example, the use of multiple subcutaneous and / or intraperitoneal injections of the relevant antigen into animals, optionally including co-administration of one or more adjuvants.

[0134] Many multispecific antibody constructs are known in the art, and the bispecific antibody molecule or antigen-binding fragment thereof provided herein may be provided in the form of any suitable multispecific construct. In some embodiments, the bispecific antibody molecule or antigen-binding fragment thereof provided herein is a multispecific antibody molecule or antigen-binding fragment thereof. In some embodiments, the multispecific antibody molecule or antigen-binding fragment thereof described herein binds to more than one antigen, e.g., at least two antigens, at least three antigens, at least four antigens, or at least five antigens. In some embodiments, the multispecific antibody molecule or antigen-binding fragment thereof described herein binds to more than one epitope on TMIGD2, e.g., at least two or at least three epitopes on TMIGD2. In some embodiments, the multispecific antibody molecule or antigen-binding fragment thereof described herein binds to more than one epitope on a TAA described herein (e.g., HHLA2), e.g., at least two or at least three epitopes on a TAA described herein (e.g., HHLA2). In some embodiments, the multispecific antibody molecule or antigen-binding fragment thereof described herein binds to more than one TAA.

[0135] The bispecific antibody molecules or antigen-binding fragments thereof described herein may comprise or be a first antibody or fragment thereof linked to the N-terminus or C-terminus of a heavy or light chain of a second antibody (see, e.g., Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163, incorporated by reference in its entirety). In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are tetravalent bispecific antibody molecules. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are scFv linked to the C-terminus of the heavy chain of a full-length antibody (e.g., IgG). In some embodiments, a bispecific antibody molecule or antigen-binding fragment thereof described herein comprises or is a hybrid immunoglobulin comprising at least two different heavy chain variable regions and at least two different light chain variable regions (e.g., Milstein and Cuello, Nature, 1983, 305:537-540, and Staerz and Bevan, Proc. Natl. Acad. Sci. USA, 1986, 83:1453-1457, each of which is incorporated by reference in its entirety).

[0136] In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are one or more immunoglobulin chains with modifications to reduce the formation of non-multispecific by-products. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more "knobs-into-holes" modifications (see, e.g., U.S. Pat. No. 5,731,168, incorporated herein by reference in its entirety).

[0137] In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are two half antibodies, comprising a first half antibody having binding specificity for a first epitope and a second half antibody having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are two scFvs, comprising a first scFv having binding specificity for a first epitope and a second scFv having binding specificity for a second epitope. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise or are bispecific single chain molecules (see, e.g., Traunecker et al., EMBO J., 1991, 10:3655-3659, and Gruber et al., J. Immunol., 1994, 152:5368-5374, each of which is incorporated by reference in its entirety).

[0138] The bispecific antibody molecules or antigen-binding fragments thereof described herein may comprise a linker connecting the first and second antigen-binding domains. In some embodiments, the linker comprises or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids in length. Typically, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but rather providing flexibility to the polypeptide (e.g., the bispecific antibody molecules or antigen-binding fragments thereof described herein). Linkers can be used in the bispecific antibody molecules or antigen-binding fragments thereof described herein based on the specific properties they confer to the bispecific antibody molecule or antigen-binding fragment thereof, such as, for example, reduced aggregation and / or increased stability. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more G4S linkers. In some embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more (G4S) n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. In certain embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more (G4S)2 linkers. In certain embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more (G4S)3 linkers. In certain embodiments, the bispecific antibody molecules or antigen-binding fragments thereof described herein comprise one or more (G4S)4 linkers.

[0139] How to create The present disclosure provides, inter alia, methods for making the bispecific antibody molecules or antigen-binding fragments thereof described herein. In some embodiments, the antibodies or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein are identified using a display technology such as yeast display, phage display, or ribosome display. In some embodiments, the antibodies or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein are identified using a hybridoma library (e.g., a mammalian hybridoma library, e.g., a murine hybridoma library), followed by supernatant screening.

[0140] Combinatorial methods for producing antibody molecules are known in the art (e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92 / 18619; Dower et al. International Publication No. WO 91 / 17271; Winter et al. International Publication No. WO 92 / 20791; Markland et al. International Publication No. WO 92 / 15679; Breitling et al. International Publication No. WO 93 / 01288; McCafferty et al. International Publication No. WO 92 / 01047; Garrard et al. International Publication No. WO 92 / 09690; Ladner et al. International Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibody Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffiths et al. (1993) EMBO J 12:725-734, Hawkins et al. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137, and Barbas et al. (1991) PNAS 88:7978-7982, each of which is incorporated herein by reference in its entirety.

[0141] The antibodies or antigen-binding fragments of the bispecific antibody molecules or antigen-binding fragments thereof described herein may be derived from other species. Humanized antibodies are antibodies produced by recombinant DNA technology in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding (e.g., constant and / or framework regions of the variable domain) are used to replace the corresponding amino acids from the light or heavy chain of a cognate non-human antibody. As an example, a humanized version of a mouse antibody for a given antigen has, in both the heavy and light chains, (1) the constant region of a human antibody, (2) FRs from the variable domain of a human antibody, and (3) CDRs from a mouse antibody. The human FRs may be selected based on their highest sequence homology to the mouse FR sequences. If necessary, one or more residues in the human FRs may be changed to the residues at the corresponding positions in the mouse antibody to maintain the binding affinity of the humanized antibody to its target. This change may be referred to as a "back mutation." Similarly, forward mutations may be made to revert to the mouse sequence for desired reasons, e.g., stability or affinity to the target. Because humanized antibodies contain significantly fewer non-human components, humanized antibodies are generally less likely to provoke an immune response in humans than chimeric human antibodies.

[0142] Methods for humanizing non-human antibodies are well known in the art. Suitable methods for producing humanized antibodies according to the present disclosure are described, for example, in Winter EP0239400, Jones et al., Nature 321:522-525(1986), Riechmann et al., Nature 332:323-327(1988), Verhoeyen et al., Science 239:1534-1536(1988), Queen et al., Proc.Nat.Acad.ScL USA 86:10029(1989), U.S. Patent No. 6,180,370, and Orlandi et al., Proc.Natl.Acad.Sd.USA 86:3833(1989), the disclosures of each of which are incorporated herein by reference in their entirety. In general, grafting of non-human (e.g., murine) CDRs into a human antibody is accomplished as follows: cDNAs encoding VH and VL are isolated from hybridomas, and the nucleic acid sequences encoding VH and VL containing CDRs are determined by sequencing. The nucleic acid sequences encoding the CDRs are inserted into the corresponding regions of a human antibody VH or VL coding sequence and linked to human constant region gene segments of the desired isotype (e.g., γl for CH and κ for CL). The humanized heavy and light chain genes are co-expressed in mammalian host cells (e.g., CHO or NSO cells) to produce soluble humanized antibodies. To facilitate large-scale production of antibodies, it is often desirable to select high expressers, for example, using DHFR or GS genes in producer lines.

[0143] The antibody molecule or antigen-binding fragment of the bispecific antibody molecule or antigen-binding fragment described herein can be or include a human antibody molecule or antigen-binding fragment. Fully human antibodies may be particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by a variety of methods known in the art, including the phage display method described above, using antibody libraries derived from human immunoglobulin sequences (see, for example, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98 / 46645, WO98 / 60433, WO98 / 24893, WO98 / 16664, WO96 / 34096, WO96 / 33735, and WO91 / 10741, each of which is incorporated herein by reference in its entirety). Techniques for the preparation of human monoclonal antibodies are also available and can be found, for example, in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985), and Boerner et al., J. Immunol., 147(1):86-95, (1991), each of which is incorporated herein by reference in its entirety.

[0144] The antibody molecule or antigen-binding fragment of the bispecific antibody molecule or antigen-binding fragment thereof described herein can be or include a chimeric antibody molecule or antigen-binding fragment thereof. Exemplary methods for making chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 1984, 81:6851-6855, each of which is incorporated by reference in its entirety. In some embodiments, chimeric antibodies are made by combining non-human variable regions (e.g., variable regions from non-human primates such as mice, rats, hamsters, rabbits, or monkeys) with human constant regions using recombinant techniques.

[0145] Any suitable method can be used to introduce variability into one or more polynucleotide sequences encoding the bispecific antibody molecules or antigen-binding fragments thereof described herein, including error-prone PCR, chain shuffling, and oligonucleotide-directed mutagenesis, such as trinucleotide-directed mutagenesis (TRIM). In some embodiments, several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted for mutations. Introduction of diversity into the variable regions and / or CDRs can be used to generate secondary libraries. The secondary libraries are then screened to identify antibody variants with improved affinity. Affinity maturation by constructing and reselecting secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology, 2001, 178:1-37, incorporated by reference in its entirety.

[0146] nucleic acid The present disclosure provides, inter alia, nucleic acids encoding the bispecific antibody molecules or antigen-binding fragments thereof described herein. The present disclosure includes nucleic acids encoding one or more of the heavy chains, VH domains, heavy chain FRs, heavy chain CDRs, heavy chain constant domains, light chains, VL domains, light chain FRs, light chain CDRs, light chain constant domains, or other immunoglobulin-like sequences, antibodies, or antigen-binding fragments disclosed herein. Such nucleic acids may be present in a vector. Such nucleic acids may be present in the genome of a cell, for example, a cell of a subject in need of treatment, or a cell for the production of an antibody molecule or an antigen-binding fragment thereof, for example, a mammalian cell for the production of at least one bispecific antibody molecule or an antigen-binding fragment thereof described herein.

[0147] Nucleic acids encoding the bispecific antibody molecules or antigen-binding fragments thereof described herein may be modified to contain codons optimized for expression in a particular cell type or organism. Codon-optimized sequences are synthetic sequences, preferably encoding the same polypeptide (or a biologically active fragment of the full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide. In some embodiments, the coding region of a nucleic acid encoding a bispecific antibody molecule or antigen-binding fragment thereof described herein may contain, in whole or in part, modified sequences to optimize codon usage for a particular cell type (e.g., eukaryotic or prokaryotic cells). For example, the coding sequence of the humanized heavy (or light) chain variable region described herein may be optimized for expression in bacterial cells. Alternatively, the coding sequence may be optimized for expression in mammalian cells (e.g., CHO cells). Such sequences may be described as codon-optimized sequences.

[0148] The nucleic acid construct of the present disclosure can be inserted into an expression vector by methods known in the art, and the nucleic acid can be operably linked to an expression control sequence. The term "operably linked" as used herein refers to, for example, a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, in the same reading frame, and, where necessary, to link two protein coding regions.

[0149] Further provided by this disclosure is a vector comprising any of the nucleic acids described herein or fragments thereof. Any of the nucleic acids described herein or fragments thereof can be cloned into any suitable vector and used to transform or transfect any suitable host. The selection of vectors and methods for constructing them are generally known to those skilled in the art (see, for example, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).

[0150] For example, conventional techniques including electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection can be used to introduce foreign nucleic acid (e.g., DNA or RNA) into prokaryotic or eukaryotic host cells. Desirably, depending on whether the vector is DNA or RNA, the vector can optionally contain regulatory sequences, such as transcription and / or translation initiation and / or termination codons, that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced. In some embodiments, the vector contains regulatory sequences that are specific to the genus of the host cell. In some embodiments, the vector contains regulatory sequences that are specific to the species of the host.

[0151] In addition to the replication system and the inserted nucleic acid, the nucleic acid construct can contain one or more marker genes to allow for selection of transformed or transfected hosts. Exemplary marker genes include, for example, biocide resistance (e.g., resistance to antibiotics or heavy metals) or complementation in auxotrophic hosts to provide prototrophy.

[0152] The expression vector may include a native or non-native promoter operably linked to the isolated or purified nucleic acid as described above. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, and / or development-specific) is within the skill of the art. Similarly, combining such a nucleic acid with a promoter is also within the skill of the art.

[0153] Suitable vectors include vectors designed for propagation and expansion, and / or expression. For example, cloning vectors may be selected from the pUC series, pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors that can be used include pBI110, pBI101.2, pBI101.3, pBI121, or pBIN19 (Clontech). Examples of animal expression vectors that can be used include pEUK-C1, pMAM, or pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used following the manufacturer's recommendations.

[0154] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in the isolation of nucleic acids encoding the bispecific antibody molecules or antigen-binding fragments thereof described herein, or improve the introduction of the nucleic acids into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley&Sons, New York, NY (1994), the contents of each of which are incorporated herein by reference in their entirety).

[0155] In some embodiments, the nucleic acids and vectors described herein are isolated and / or purified. Isolated nucleic acids and vectors can be prepared using standard techniques known in the art, including, for example, alkaline / SDS treatment, CsCl binding, column chromatography, and / or agarose gel electrophoresis. The compositions can include other components as further described herein.

[0156] Any method known to those of skill in the art for the insertion of nucleic acids into a vector may be used to construct expression vectors encoding the bispecific antibody molecules or antigen-binding fragments thereof described herein under the control of transcriptional and / or translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (see, for example, Ausubel, supra, or Sambrook, supra).

[0157] Antibodies that bind to the same epitope The bispecific antibody molecules or antigen-binding fragments thereof described herein include antibodies and antibody fragments that bind to one or more of the same epitopes as the antibody molecules or fragments thereof shown in Tables 1-5. As used herein, the term "epitope" refers to any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts another conformation (e.g., when linearized).

[0158] Additional antibody molecules or antigen-binding fragments can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with a bispecific antibody molecule or antigen-binding fragment thereof described herein in a TMIGD2 binding assay and / or a TAA (e.g., HHLA2) binding assay. The ability of a test antibody molecule or antigen-binding fragment to inhibit the binding of a bispecific antibody molecule or antigen-binding fragment thereof described herein to TMIGD2 demonstrates that the test antibody molecule or antigen-binding fragment can compete with the bispecific antibody molecule or antigen-binding fragment for binding to TMIGD2. Thus, such an antibody molecule or antigen-binding fragment can bind to the same or related epitope on TMIGD2 as the bispecific antibody molecule or antigen-binding fragment thereof described herein. The ability of a test antibody molecule or antigen-binding fragment to inhibit the binding of a bispecific antibody molecule or antigen-binding fragment thereof described herein to a TAA (e.g., HHLA2) demonstrates that the test antibody molecule or antigen-binding fragment can compete with the bispecific antibody molecule or antigen-binding fragment for binding to a TAA (e.g., HHLA2). Such antibody molecules or antigen-binding fragments may therefore bind to the same or related epitope on a TAA (e.g., HHLA2) as the bispecific antibody molecules or antigen-binding fragments thereof described herein.

[0159] Additional drugs The present disclosure provides, inter alia, one or more additional agents (e.g., two, three, four, five, or more additional agents) that can be administered with the bispecific antibody molecules or antigen-binding fragments thereof described herein in combination therapy. As used herein, an additional agent can be or include any known treatment for a particular disease, disorder, or condition (e.g., cancer). For example, the additional agent can be or include one or more of a chemotherapeutic agent, hormone therapy, cell-based therapy, hyperthermia treatment, photodynamic therapy, surgery, radiation, or transplantation.

[0160] In some embodiments, the chemotherapeutic agent includes or is one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., one or more inhibitors of topoisomerase I or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, or combinations thereof.

[0161] In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, cefotaxime ... In some embodiments, the chemotherapeutic agent comprises or is one or more of: cytabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, rituxan, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, or vinorelbine. In some embodiments, the chemotherapeutic agent comprises or is an antibody-drug conjugate (ADC).In some embodiments, the ADC is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA The compound includes or is an ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, lorvotuzumab mertansine, or a combination thereof.

[0162] In some embodiments, the hormone therapy can be or include tamoxifen, raloxifene, leuprolide, bicalatomid, granisetron, flutamide, or a combination thereof. In some embodiments, the cell-based therapy includes or is chimeric antigen receptor T (CAR-T) cells, CAR-NK cells, TCR-transduced T cells, dendritic cells, tumor-infiltrating lymphocytes (TIL), natural killer (NK) cells, or a combination thereof. In some embodiments, the hyperthermia includes or is local hyperthermia (e.g., external, intraluminal, or interstitial hyperthermia), regional hyperthermia (e.g., deep tissue hyperthermia, regional perfusion, or (continuous hyperthermic peritoneal perifusion), or whole body hyperthermia. In some embodiments, the photodynamic therapy includes or is the administration of a photosensitizer, such as hematoporphyrin and its derivatives, Verteporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxyhypocerin A, 2BA-2-DMHA, or combinations thereof. In some embodiments, the surgery includes or is surgery to remove cancerous or precancerous tissue. In some embodiments, the transplant includes or is a stem cell transplant or an organ transplant.

[0163] In some embodiments, the additional agent is administered prior to, substantially simultaneously with, or after administration of a bispecific antibody molecule or antigen-binding fragment thereof described herein. In some embodiments, administration of a bispecific antibody molecule or antigen-binding fragment thereof described herein and an additional agent results in an improvement of a disease, disorder, or condition (e.g., cancer) to a degree greater than the improvement provided by either the bispecific antibody molecule or antigen-binding fragment thereof described herein or the additional agent alone. The difference between the effect of the combination and the effect of each agent alone may be a statistically significant difference. In some embodiments, the effect of the combination may be a synergistic effect. In some embodiments, coadministration of a bispecific antibody molecule or antigen-binding fragment thereof described herein with an additional agent allows for the additional agent to be administered at a reduced dose, at a reduced number of doses, and / or at a reduced frequency of administration compared to a standard administration regimen, e.g., an approved administration regimen for the additional agent.

[0164] Conjugates to therapeutic agents The present disclosure provides, inter alia, bispecific antibody molecules or antigen-binding fragments thereof conjugated to one or more therapeutic agents. In some embodiments, the therapeutic agent includes or is a cytotoxic agent, a drug, and / or a radioisotope. When conjugated to a cytotoxic agent, such a conjugate may be referred to as an "immunotoxin." A cytotoxic agent includes any agent that is detrimental to (e.g., capable of killing) cells. Examples of cytotoxic agents include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs of any of the foregoing.

[0165] The bispecific antibody molecules or antigen-binding fragments thereof described herein may be used in combination with other agents, such as antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and / or 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and / or cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and / or or doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and / or anthramycin (AMC), and antimitotic agents (e.g., vincristine and / or vinblastine). The bispecific antibody molecules or antigen-binding fragments thereof described herein can be conjugated to one or more radioisotopes (e.g., radioactive iodine) to generate cytotoxic radiopharmaceuticals for treating a disease, disorder, or condition described herein, such as a cancer described herein.

[0166] Pharmaceutical Compositions The present disclosure provides, inter alia, pharmaceutical compositions comprising one or more bispecific antibody molecules or antigen-binding fragments as described herein in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients.

[0167] When a "therapeutically effective amount" is indicated, the exact amount of a pharmaceutical composition comprising at least one bispecific antibody molecule or antigen-binding fragment thereof as described herein can be determined by the physician, taking into account individual differences in age, weight, immune response, and condition of the subject.

[0168] The pharmaceutical compositions described herein may include, but are not limited to, buffers including neutral buffered saline or phosphate buffered saline (PBS); carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol; proteins, polypeptides, or amino acids (e.g., glycine); antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., free of detectable levels of at least one contaminant (e.g., endotoxin).

[0169] The pharmaceutical compositions described herein may be administered in any manner appropriate for the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will depend on factors such as the condition of the patient, and the type and severity of the patient's disease, disorder, or condition, although appropriate doses may be determined by clinical trials.

[0170] The pharmaceutical compositions described herein may be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0171] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion. In a preferred embodiment, the pharmaceutical compositions described herein are formulated for subcutaneous or intravenous injection or infusion. The pharmaceutical compositions described herein can be formulated for administration by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, incorporated herein by reference in its entirety).

[0172] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0173] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., prior to, substantially simultaneously with, or subsequent to) one or more additional agents. In some embodiments, the additional agents are one or more chemotherapeutic agents (e.g., actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, The treatment may be or may include bone marrow transplantation or lymphodepletion therapy using cyclosporine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, rituxan, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof). In certain embodiments, the subject undergoes standard of care with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives one or more pharmaceutical compositions described herein. In some embodiments, the pharmaceutical compositions described herein are administered before or after surgery.

[0174] The dosage of any of the aforementioned therapeutic agents administered to a subject will vary depending on the disease, disorder, or condition being treated and on the particular subject. Scaling of dosages for administration to humans can be performed according to art-recognized practices.

[0175] The present disclosure provides, inter alia, pharmaceutical compositions comprising one or more bispecific antibody molecules or antigen-binding fragments thereof described herein and immune effector cells (e.g., T cells (e.g., naive T cells and / or effector T cells) and / or NK cells). In some embodiments, the immune effector cells described herein are loaded with one or more bispecific antibody molecules or antigen-binding fragments thereof described herein. In some embodiments, the immune effector cells described herein are pre-loaded with one or more bispecific antibody molecules or antigen-binding fragments thereof described herein prior to cryopreservation. In some embodiments, the immune effector cells described herein are cryopreserved prior to loading with one or more bispecific antibody molecules or antigen-binding fragments thereof described herein. A pharmaceutical composition comprising immune effector cells pre-loaded with one or more bispecific antibody molecules or antigen-binding fragments thereof described herein may be administered to a patient suffering from a disease, disorder, or condition described herein. In some embodiments, the immune effector cells comprise or are T cells (e.g., naive T cells and / or effector T cells). In some embodiments, the immune effector cells comprise or are NK cells. In some embodiments, the immune effector cells described herein are derived from a patient suffering from a disease, disorder, or condition described herein. In some embodiments, the immune effector cells described herein are isolated from umbilical cord tissue, placental tissue, induced pluripotent stem cells (iPSCs), or peripheral blood mononuclear cells (PBMCs).

[0176] Treatment methods The present disclosure provides, inter alia, methods of treating a disease, disorder, or condition described herein in a subject comprising administering a pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein. In some embodiments, a therapeutically effective amount of at least one pharmaceutical composition described herein is administered to a subject having a disease, disorder, or condition.

[0177] In some embodiments, the disclosure provides one or more bispecific antibody molecules or antigen-binding fragments described herein comprising an amino acid sequence found in Table 1 for use as a medicament. In some embodiments, the disclosure provides one or more bispecific antibody molecules or antigen-binding fragments described herein comprising an amino acid sequence found in Table 1 for use in the treatment of a disease, disorder, or condition described herein. In some embodiments, the disclosure provides the use of one or more bispecific antibody molecules or antigen-binding fragments described herein comprising an amino acid sequence found in Table 1 for the manufacture of a medicament for the treatment of a disease, disorder, or condition described herein.

[0178] A pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein (e.g., in Tables 1-5) can be used in the manufacture of a medicament for treating a disease, disorder, or condition in a subject. A pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein can be used in the manufacture of a medicament for modulating an immune response in a subject. A pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein can be administered to a subject according to the dosing regimens described herein alone or in combination with (e.g., before, substantially simultaneously with, or after) one or more additional agents that may be or may include one or more of a therapeutic agent, procedure, or modality.

[0179] The subject treated with the methods described herein may be a mammal (e.g., a primate, a mouse, a humanized mouse, a rat, a non-human mammal, a domestic animal such as a dog, a cat, a cow, or a horse), and is preferably a human (e.g., a patient having or at risk of having a disease, disorder, or condition described herein). The subject may be an animal model of cancer, such as a xenograft animal model of a human-derived cancer. In some embodiments, the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immune checkpoint therapy. In another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immune checkpoint therapy. In certain embodiments, the subject has undergone surgery to remove cancerous or pre-cancerous tissue. In other embodiments, the cancerous tissue has not been removed, for example, the cancerous tissue may be located in an inoperable area of ​​the body, such as tissue vital to survival, or in an area where surgical procedures would be at high risk of causing damage to the subject.

[0180] Methods are provided for treating cancer or tumor in a subject (e.g., reducing, inhibiting, or delaying the progression of cancer or tumor) using pharmaceutical compositions comprising one or more bispecific antibody molecules or antigen-binding fragments described herein. The subject may have an adult or childhood form of cancer. The cancer may be in early, middle, or late stage, or may be a metastatic cancer.

[0181] Methods are provided for treating (e.g., reducing, inhibiting, or slowing the progression of) at least one sign or symptom of cancer in a subject using a pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein. In some embodiments, the pharmaceutical compositions described herein are useful for delaying the onset, slowing the progression, or ameliorating one or more signs or symptoms of cancer. In some embodiments, the physiological signs or symptoms of cancer include or are one or more of an increase in tumor volume, an increase in the number of cancer cells, an increase in the number of metastases, a decrease in life expectancy, an increase in cancer cell proliferation, or an increase in cancer cell survival. In some embodiments, the physical signs or symptoms of cancer include or are one or more of skin lesions (e.g., lumps or moles), weight loss, digestive problems, discomfort, fatigue, pain, difficulty swallowing, coughing, abnormal bleeding and / or discharge, changes in bowel and / or bladder habits, or mental confusion.

[0182] Cancer can include, but is not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, or myeloma, e.g., multiple myeloma), metastatic lesions, or combinations thereof. Examples of solid tumors include, but are not limited to, malignant tumors, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of various organ systems, such as those affecting the lung, breast, ovaries, lymph nodes, reproductive organs, gastrointestinal tract (e.g., colon), and genitourinary tract (e.g., kidney, urinary tract, bladder cells, or prostate), pharynx, CNS (e.g., brain cells, neuronal cells, or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0183] In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC (e.g., non-small cell lung cancer (NSCLC) or NSCLC adenocarcinoma of squamous and / or non-squamous tissue) or small cell lung cancer (SCLC)), skin cancer (e.g., Merkel cell carcinoma or melanoma (e.g., advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (e.g., hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal carcinoma (e.g., renal cell carcinoma)), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptors, progesterone receptors, or Her2 / neu, e.g., triple negative breast cancer (TNBC)), colon cancer (e.g., recurrent large intestinal cancer, The cancer may include or be a colon cancer or metastatic colon cancer, e.g., microsatellite unstable colon cancer, microsatellite stable colon cancer, mismatch repair competent colon cancer, or mismatch repair deficient colon cancer), nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., anaplastic thyroid carcinoma), cervical cancer (e.g., squamous cell carcinoma of the cervix), neuroendocrine tumors (e.g., atypical pulmonary carcinoid tumors), lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders), lymphomas (e.g., T-cell lymphoma, B-cell lymphoma, or non-Hodgkin's lymphoma), myelomas (e.g., multiple myeloma), leukemias (e.g., myeloid leukemia or lymphocytic leukemia), or a combination thereof.

[0184] In some embodiments, the cancer comprises or is a brain tumor, e.g., glioblastoma, gliosarcoma, or recurrent brain tumor. In some embodiments, the cancer comprises or is a pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer comprises or is a skin cancer, e.g., melanoma (e.g., stage II-IV melanoma, HLA-A2 positive melanoma, unresectable melanoma, or metastatic melanoma), or Merkel cell carcinoma. In some embodiments, the cancer comprises or is a kidney cancer, e.g., renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer comprises or is a breast cancer, e.g., metastatic breast cancer or stage IV breast cancer, e.g., TNBC. In some embodiments, the cancer comprises or is a virus-associated cancer. In some embodiments, the cancer comprises or is anal canal cancer (e.g., squamous cell carcinoma of the anal canal). In some embodiments, the cancer comprises or is cervical cancer (e.g., squamous cell carcinoma of the cervix). In some embodiments, the cancer comprises or is gastric cancer (e.g., Epstein-Barr virus (EBV) positive gastric cancer, or gastric or gastroesophageal junction cancer). In some embodiments, the cancer comprises or is head and neck cancer (e.g., HPV positive or negative squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the cancer comprises or is nasopharyngeal carcinoma (NPC). In some embodiments, the cancer comprises or is colorectal cancer, e.g., recurrent colorectal cancer, metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair competent colorectal cancer, or mismatch repair deficient colorectal cancer.

[0185] In some embodiments, the cancer comprises or is a hematological cancer. In some embodiments, the cancer comprises or is a leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the cancer comprises or is a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or DLBCL (e.g., relapsed or refractory HL or DLBCL). In some embodiments, the cancer comprises or is a myeloma, such as multiple myeloma.

[0186] Administration of a pharmaceutical composition comprising one or more bispecific antibody molecules or antigen-binding fragments described herein can be by any medically appropriate method (e.g., injection, ingestion, infusion, inhalation, implantation, or transplantation). In some embodiments, the pharmaceutical compositions described herein are administered by injection or infusion. The pharmaceutical compositions described herein can be administered to a patient intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous, intravenous, intramuscular, or intrasternal infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be directly injected into the subject at the site of inflammation, the site of local disease, lymph nodes, organs, tumors, or the site of infection.

[0187] In some embodiments, one or more bispecific antibody molecules or antigen-binding fragments described herein are administered in combination with one or more other therapeutic agents or modalities. In some embodiments, the one or more additional agents (e.g., therapeutic agents or modalities) include or are anti-cancer agents or modalities. In some embodiments, such combination therapy exhibits synergistic effects in the treatment of cancer. Known additional agents (e.g., compounds or therapeutic agents that exhibit therapeutic efficacy in the treatment of cancer) may include, for example, one or more chemotherapeutic agents, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics, angiogenesis inhibitors, immunomodulatory agents, vaccines, cell-based therapies (e.g., allogeneic or autologous stem cell transplants), organ transplants, radiation therapy, or surgery.

[0188] In some embodiments, the chemotherapeutic agent may be or include one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., one or more inhibitors of topoisomerase I or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, or combinations thereof. In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, cefotaxime ... The chemotherapeutic agent may be or include one or more of: tabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, rituxan, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, or vinorelbine. In some embodiments, the chemotherapeutic agent may be an antibody-drug conjugate (ADC).In some embodiments, the ADC is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA The compound includes or is an ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, lorvotuzumab mertansine, or a combination thereof.

[0189] kit The present disclosure provides, inter alia, kits comprising one or more bispecific antibody molecules or antigen-binding fragments described herein and instructions for use and / or administration. In some embodiments, the kit comprises one or more bispecific antibody molecules or antigen-binding fragments described herein and a pharma- ceutically acceptable carrier, and instructions for use and / or administration.

[0190] In some embodiments, the kit includes instructions for use in any of the methods described herein. The instructions can include instructions for administering the first pharmaceutical composition and the second composition (e.g., additional agents) to a subject to achieve an intended activity in the subject. The kit can further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions include instructions for administering the first pharmaceutical composition and the second pharmaceutical composition to a subject in need of treatment.

[0191] The instructions for the first and second pharmaceutical compositions (e.g., additional agents) described herein generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. The instructions provided with the kits described herein are typically written instructions on a label or package insert. The label or package insert indicates that one or more pharmaceutical compositions are used to treat, delay the onset of, and / or alleviate a disease, disorder, or condition in a subject.

[0192] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and / or flexible packaging. Packages for use in combination with specific devices, such as injection devices, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierce that can be inserted by a hypodermic injection needle). The container may also have a sterile access port.

[0193] The kit may optionally provide additional components such as buffers and / or interpretive information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0194] Incorporation by Reference All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described herein.

[0195] The present disclosure is further illustrated by the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present disclosure in any way. EXAMPLES

[0196] The following examples are provided to illustrate to one of ordinary skill in the art how to make and use the methods and compositions described herein, and are not intended to limit the scope of the disclosure.

[0197] Example 1: Characterization of specific TMIGD2-TAA bispecific antibody molecules This example demonstrates methods for characterizing bispecific antibody molecules or antigen-binding fragments thereof, specifically anti-TMIGD2 and anti-tumor associated antigen (TAA) bispecific antibody molecules or antigen-binding fragments thereof described herein. This example further provides various methods for determining and / or characterizing the relevant functional activities of the bispecific antibody molecules or antigen-binding fragments thereof described herein.

[0198] A schematic diagram of an exemplary bispecific antibody molecule comprising a scFv targeting TMIGD2 linked to the C-terminus of the heavy chain of an IgG antibody targeting TAA (tetravalent Morrison bispecific format) is shown in Figure 1. To reduce effector function and prevent destruction of circulating TMIGD2-expressing immune cells, L234A / L235A mutations were introduced in the Fc region. (V H Domain and V L The internal linker within the scFv domain (between the Fc tail and the scFv domain) may comprise or be a (G4S)4 repeat. The linker between the Fc tail and the scFv domain may comprise or be a (G4S)2, (G4S)3, or (G4S)4 repeat.

[0199] An exemplary bispecific antibody molecule comprising a TMIGD2-targeting scFv (Amplimmune4.5 (Amp4.5) described in WO2014100823A1 (herein incorporated by reference in its entirety)) linked to the C-terminus of the heavy chain of an IgG antibody (rituximab) targeting CD20 was constructed and used in the assays described herein. Additional exemplary anti-TMIGD2 / anti-CD20 bispecific antibodies (TK20-17C7-01, TK20-29F8-16, and TK20-39C9-2134) were also constructed and used in the assays herein.

[0200] Jurkat reporter assay To evaluate the enhancement of TCR stimulation by the exemplary anti-TMIGD2 / anti-CD20 antibody molecules, TMIGD2 was overexpressed in Jurkat cells expressing an NFAT-driven luciferase reporter. Binding of TMIGD2 on these cells with the anti-TMIGD2 / anti-CD20 antibody molecules provided herein increased T cell activation more than anti-CD3 stimulation alone when the bispecific antibody molecules were immobilized on plastic (Figure 2A) or bound to CD20+Raji cells (Figure 3). These results were corroborated by additional anti-TMIGD2 / anti-CD20 antibody molecules provided herein (TK20-17C7-01, TK20-29F8-16, and TK20-39C9-2134), which confirmed that such anti-TMIGD2 / anti-CD20 antibody molecules increased T cell activation when bound to CD20+Raji cells (Figure 4).

[0201] The enhancement of T cell activation induced by the anti-TMIGD2 / anti-CD20 bispecific antibody molecule was superior to that observed with the human IgG4 version of Amp4.5 (Figure 2A). When the anti-TMIGD2 / anti-CD20 bispecific antibody molecule was added in solution (Figure 2B), no enhancement of T cell signaling was observed, and plate-bound anti-TMIGD2 / anti-CD20 bispecific antibody molecule did not induce T cell activation in the absence of anti-CD3 antibody. These data indicate that the TMIGD2 / anti-CD20 bispecific antibody molecule exhibited agonistic activity and enhanced T cell activation in the TMIGD2-jurkat reporter assay, for example, in the presence of TAA (CD20)-expressing Raji target cells.

[0202] Plate binding assay Anti-TMIGD2 / anti-CD20 bispecific antibody molecules, anti-TMIGD2 IgG4 (Amp4.5), or human IgG4 isotype control were coated onto wells of a 96-well white flat-bottom tissue culture plate at 10 μg / mL with anti-CD3 antibody (OKT3) at 0.5 μg / mL in 50 μL of PBS overnight at 4° C. Plates were washed three times with PBS and 4×10 4TMIGD2-Jurkat NFAT reporter cells were added to each well in 100 μl complete RPMI containing 10% FBS. In some wells, anti-TMIGD2 / anti-CD20 bispecific antibody molecules were added at 10 μg / mL in the medium. Cells were incubated at 37° C. for 6 hours and 100 μl of Bio-Glo™ Luciferase Assay Reagent (Promega®, G7940) was added to each well. Luciferase activity was measured in a plate reader.

[0203] Raji co-culture assay Raji cells (2×10 4 ) were seeded into 96-well white flat-bottom tissue culture plates containing 10 μg / mL of anti-TMIGD2 / anti-CD20 bispecific antibody molecules in 50 μl of complete RPMI containing 10% FBS and incubated for 30 min at 37° C. TMIGD2-Jurkat NFAT reporter cells (4×10 4 ) was added to each well along with 1 μg / mL of anti-CD3 antibody (OKT3) or moue IgG2a isotype control in 50 μl of complete RPMI. Cells were incubated at 37° C. for 6 hours and 100 ul of Bio-Glo™ Luciferase Assay Reagent (Promega®, G7940) was added to each well. Luciferase activity was measured on a plate reader.

[0204] Raji cells (2×10 4 ) were seeded in 96-well white flat-bottom tissue culture plates with anti-TMIGD2 / anti-CD20 bispecific antibodies (TK20-17C7-01, TK20-29F8-16, and TK20-39C9-2134) in 50 μl of R10 medium (RPMI1640 + 10% fetal bovine serum) at 3-fold serial dilutions ranging from 10 to 0.5 μg / mL. Anti-CD20 (IgG1 LALA) was included as a negative control. Cells were incubated for 30 min at 37 °C. TMIGD2-Jurkat NFAT reporter cells (4 × 10 4) was added to each well along with 1 μg / mL anti-CD3 antibody (OKT3) in 50 μl of R10 medium. Cells were incubated at 37° C. for 6 hours and 100 ul of Bio-Glo™ Luciferase Assay Reagent (Promega®, G7940) was added to each well. Luciferase activity was measured on a plate reader.

[0205] Example 2: Characterization of certain TMIGD2-TAA bispecific antibody molecules This example demonstrates methods for characterizing the bispecific antibody molecules or antigen-binding fragments thereof described herein, specifically anti-TMIGD2 and anti-TAA (e.g., HHLA2) bispecific antibody molecules or antigen-binding fragments thereof. This example further provides various methods for determining and / or characterizing the relevant functional activities of the bispecific antibody molecules or antigen-binding fragments thereof described herein. An exemplary bispecific antibody molecule comprising a TMIGD2-targeting scFv linked to the C-terminus of the heavy chain of an HHLA2-targeting IgG antibody is constructed and used in the assays described herein (see, e.g., Tables 1-4).

[0206] Jurkat reporter assay To evaluate the enhancement of TCR stimulation by the exemplary anti-TMIGD2 / anti-HHLA2 antibody molecule, TMIGD2 is overexpressed in Jurkat cells expressing NFAT-driven luciferase reporter.The binding of TMIGD2 to anti-TMIGD2 / anti-HHLA2 antibody molecule in these cells is evaluated in comparison with anti-CD3 antibody stimulation alone with TMIGD2 / anti-HHLA2 bispecific antibody molecule immobilized on plastic, in solution, or in the presence of HHLA2-expressing Raji cells.

[0207] Plate binding assay Anti-TMIGD2 / anti-HHLA2 antibody molecules with Fc silencing mutation isotype control, anti-TMIGD2 IgG, or human IgG1 are coated onto wells of a 96-well white flat-bottom tissue culture plate at approximately 10 μg / mL with approximately 0.5 μg / mL of anti-CD3 antibody (OKT3) in approximately 50 μL of PBS overnight at 4° C. The plates are washed three times with PBS and approximately 4×10 4 TMIGD2-Jurkat NFAT reporter cells are added to each well in about 100 μl of complete RPMI containing about 10% FBS. In some wells, anti-TMIGD2 / anti-HHLA2 bispecific antibody molecules are added at about 10 μg / mL in the medium. The cells are incubated at about 37° C. for about 6 hours and about 100 μl of Bio-Glo™ Luciferase Assay Reagent (Promega®, G7940) is added to each well. Luciferase activity is measured on a plate reader.

[0208] Raji co-culture assay Raji cells (2×10 4 ) are seeded into a 96-well white flat-bottom tissue culture plate containing about 10 μg / mL of anti-TMIGD2 / anti-HHLA2 bispecific antibody molecules in about 50 μl of complete RPMI containing about 10% FBS and incubated at about 37° C. for about 30 minutes. TMIGD2-Jurkat NFAT reporter cells (about 4×10 4 ) is added to each well with about 1 μg / mL of anti-CD3 (OKT3) or moue IgG isotype control in about 50 μl of complete RPMI. Cells are incubated at about 37° C. for about 6 hours and about 100 ul of Bio-Glo™ Luciferase Assay Reagent (Promega®, G7940) is added to each well. Luciferase activity is measured on a plate reader.

[0209] equivalent It will be understood by those skilled in the art that various changes, modifications, and improvements of the present disclosure will be easily thought of by those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the present disclosure. Therefore, the foregoing description and drawings are by way of example only, and any inventions described in this disclosure are further described in detail by the following claims.

[0210] Those of ordinary skill in the art will understand the typical standards of deviation or error attributable to values ​​obtained in the assays or other processes described herein. Publications, websites, and other reference materials referenced herein to describe the background of this disclosure and to provide additional details regarding its practice are hereby incorporated by reference in their entireties.

Claims

1. (i) a first antigen-binding domain that selectively binds to the transmembrane and immunoglobulin domain containing 2 (TMIGD2); and (ii) a second antigen-binding domain that selectively binds to at least one tumor-associated antigen (TAA), or an antigen-binding fragment thereof.

2. The bispecific antibody molecule or antigen-binding fragment thereof of claim 1 , wherein the first antigen-binding domain activates an immune effector cell.

3. 3. The bispecific antibody molecule or antigen-binding fragment thereof of claim 1 or 2, wherein the first antigen-binding domain recruits immune effector cells to the tumor microenvironment.

4. 4. The bispecific antibody molecule or antigen-binding fragment thereof of claim 2 or 3, wherein the immune effector cells comprise or are T cells and / or NK cells.

5. 5. The bispecific antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the at least one TAA comprises or is HHLA2, CD123, B7-H3, B7-H4, CD33, CD30, HER2, CD22, CD79b, Nectin-4, Trop-2, BCMA, CD19, folate receptor alpha, EGFR, or a combination thereof.

6. The bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the first antigen-binding domain comprises or is an antibody or antigen-binding fragment thereof.

7. The bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the second antigen-binding domain comprises or is an antibody or antigen-binding fragment thereof.

8. The antigen-binding fragment may be an scFv, Fab, Fab', F(ab') ２ 8. A bispecific antibody molecule or an antigen-binding fragment thereof as described in claim 6 or 7, which comprises or is a nanobody or a camelid antibody.

9. the antibody or antigen-binding fragment thereof (i) a chimeric, human, or humanized antibody molecule, or an antigen-binding fragment thereof, and / or (ii) A bispecific antibody molecule or an antigen-binding fragment thereof according to any one of claims 6 to 8, which is or comprises a monoclonal antibody molecule or an antigen-binding fragment thereof.

10. the antibody or antigen-binding fragment thereof (i) a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, and / or (ii) a light chain constant region selected from a kappa or lambda light chain constant region, or an antigen-binding fragment thereof, according to any one of claims 6 to 9.

11. The bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 6 to 9, wherein the antibody or antigen-binding fragment comprises an Fc region with reduced effector function compared to a reference antibody or antigen-binding fragment thereof.

12. 12. The bispecific antibody molecule or antigen-binding fragment thereof of claim 11 , wherein the Fc region comprises one or more amino acid substitutions L234A and / or L235A, numbered according to the EU index.

13. 2. The bispecific antibody molecule or antigen-binding fragment thereof according to any one of the preceding claims, wherein the first antigen-binding domain comprises or is an scFv and the second antigen-binding domain comprises or is an antibody molecule.

14. The bispecific antibody molecule or antigen-binding fragment thereof of claim 13, wherein the scFv and the antibody molecule are covalently linked by a peptide linker.

15. A pharmaceutical composition comprising at least one bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

16. 16. A method of treating a subject having a disease, disorder, or condition and / or modulating an immune response in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 15.

17. 17. The method of claim 15 or 16, wherein the subject has or is at risk of developing cancer.

18. 18. The method of claim 17, wherein the subject has a solid tumor or a hematological cancer.

19. 20. The method of claim 18, wherein the solid tumor is or comprises one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer.

20. 19. The method of claim 18, wherein the hematological cancer comprises or is leukemia or lymphoma.

21. 21. The method of claim 20, wherein the leukemia comprises or is acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia.

22. 21. The method of claim 20, wherein the lymphoma comprises or is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

23. The method of any one of claims 16 to 22, wherein the bispecific antibody molecule or antigen-binding fragment thereof is administered in combination with at least one additional agent.

24. A nucleic acid molecule encoding a bispecific antibody molecule or an antigen-binding fragment thereof according to any one of claims 1 to 14.

25. 25. An expression vector comprising the nucleic acid molecule of claim 24.

26. A host cell comprising or expressing a bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 14, a nucleic acid molecule according to claim 24, or an expression vector according to claim 25.

27. 1. A method for making a bispecific antibody molecule or antigen-binding fragment thereof, comprising the steps of: (i) culturing a host cell comprising the nucleic acid molecule of claim 24 or the expression vector of claim 25 under conditions suitable for expression of the bispecific antibody molecule or antigen-binding fragment thereof; (ii) recovering the bispecific antibody molecule or antigen-binding fragment thereof.

28. A method for detecting the presence or level of a TMIGD2 polypeptide and / or a TAA in a sample, the method comprising detecting the TMIGD2 polypeptide and / or the TAA in the sample using a bispecific antibody molecule or an antigen-binding fragment thereof described in any one of claims 1 to 14.

29. A kit comprising at least one bispecific antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 14 and instructions for use.

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