HHLA2 binding agents with novel activity

Abstract

The present disclosure relates to novel active HHLA2 binding agents and uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 142,832, filed January 28, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Immunotherapy has been investigated for many diseases and disorders, including cancer, but faces functional limitations that still need to be addressed. The immune system is tightly controlled by a network of costimulatory and co-inhibitory ligands and receptors. Immune checkpoints negatively regulate the progression of immune responses based on complex interactions. Currently available immune checkpoint inhibitors can modulate immune responses in some patients, but immune checkpoint expression and interactions with natural binding partners can vary from patient to patient.

[0003] Thus, there is a need for the development of new therapeutics optimized to target immune checkpoint pathways. Summary of the Invention [Means for solving the problem]

[0004] HERV-H LTR-associated 2 (HHLA2), a member of the B7 gene family, is widely expressed in various tumors and antigen-presenting cells. HHLA2 is known to interact with both inhibitory and stimulatory receptors to regulate T cell function. Killer cell immunoglobulin-like receptor (KIR) proteins contain two (KIR2D) or three (KIR3D) immunoglobulin-like extracellular domains, and KIR3DL3 is the inhibitory HHLA2 receptor found on T cells and NK cells. Binding of HHLA2 to KIR3DL3 has been shown to inhibit the immune response of activated T cells and the cytotoxic activity of NK cells.

[0005] In contrast, transmembrane and immunoglobulin domain-containing 2 (TMIGD2) is an activating receptor for HHLA2. Upon T cell receptor (TCR) signaling, TMIGD2 on naive T cells interacts with HHLA2 and costimulates T cell activation through a pathway involving AKT phosphorylation. Upon repeated T cell activation, the expression of the stimulatory receptor TMIGD2 is gradually lost, and the expression of the inhibitory receptor KIR3DL3 becomes predominant.

[0006] While HHLA2 binding agents have been investigated as immunotherapeutics, the present disclosure encompasses, inter alia, the discovery of HHLA2 binding agents described herein with novel activities that result in (i) inhibition of HHLA2 binding to KIR3DL3; and / or (ii) enhancement of HHLA2 binding to TMIGD2. In some embodiments, the HHLA2 binding agents can: (i) inhibit HHLA2 binding to KIR3DL3; and (ii) enhance HHLA2 binding to TMIGD2. In some embodiments, the HHLA2 binding agents described herein cause an allosteric change in HHLA2, thereby resulting in a conformational change in HHLA2 that enhances the binding of HHLA2 to TMIGD2. In some embodiments, the HHLA2 binding agents described herein directly compete with at least one binding site for TMIGD2 on HHLA2. In some embodiments, the HHLA2 binding agents described herein enhance early stage immune responses and / or late stage immune responses. In some embodiments, the HHLA2 binding agents described herein enhance HHLA2 binding to TMIGD2 on naive immune effector cells. In some embodiments, the HHLA2 binding agents described herein block HHLA2 binding to KIR3DL3 on exhausted immune effector cells. In some embodiments, exhausted immune effector cells contain or express certain cell surface markers, such as, for example, PD-1, CTLA-4, LAG-3, TIM-3, 2B4 / CD244 / SLAMF4, CD160, and / or TIGIT.

[0007] Thus, the present disclosure provides several examples of such HHLA2 binding agents that are particularly useful for treating a variety of cancers, including solid cancers such as non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), cholangiocarcinoma, or breast cancer, and hematological tumors, and for modulating an immune response in a subject.

[0008] In some embodiments, the HHLA2 binding agents described herein are used for tumor targeting of at least one cytotoxic agent. In some embodiments, the HHLA2 binding agents described herein are administered or co-formulated with a cytotoxic agent. In some embodiments, the HHLA2 binding agents described herein are used for delivering at least one radionuclide to a tumor (e.g., a tumor described herein). In some embodiments, the HHLA2 binding agents described herein are administered or co-formulated with a radionuclide. In some embodiments, the HHLA2 binding agents described herein are used for targeting a tumor (e.g., a tumor described herein) in combination with a monoclonal antibody that binds to an Fc receptor (FcR) and thereby mediates antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the HHLA2 binding agents described herein are administered or co-formulated with a monoclonal antibody that binds to an FcR.

[0009] In one aspect, the disclosure provides HHLA2 binding agents that are capable of (i) inhibiting binding of HHLA2 to KIR3DL3 and / or (ii) enhancing binding of HHLA2 to TMIGD2.

[0010] In some embodiments, the HHLA2 binding agent is or comprises an antibody or antigen-binding fragment thereof, a small molecule, a polypeptide, or an aptamer.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (i) a chimeric, human or humanized antibody, or an antigen-binding fragment thereof; (ii) a monospecific or bispecific antibody, or an antigen-binding fragment thereof; and / or (iii) a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment comprises an scFv, Fab, Fab', F(ab')2, Fc, nanobody, or camelid antibody. 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.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is, or comprises: (a) a heavy chain variable region (VH) comprising one, two, or three VH CDR sequences, each of which is at least about 90% identical to a VH CDR of Table 1; and / or (b) a light chain variable region (VL) comprising one, two, or three VL CDR sequences, each of which is at least about 90% identical to a VL CDR of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is, or comprises, (a) a VH comprising one, two, or three VH CDR sequences that are at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical, respectively, to the VH CDRs of Table 1; and / or (b) a VL comprising one, two, or three VL CDR sequences that are at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical, respectively, to the VL CDRs of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is, or comprises, (a) a VH comprising one, two, or three VH CDR sequences each comprising or consisting of a VH CDR of Table 1, and / or (b) a VL comprising one, two, or three VL CDR sequences each comprising or consisting of a VL CDR of Table 1.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a VH that is at least about 90% or more identical to a VH of Table 1; and / or (b) a VL that is at least about 90% or more identical to a VL of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a VH that is at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a VH of Table 1; and / or (b) a VL that is at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a VL of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a VH that comprises or consists of a VH of Table 1, and / or (b) a VL that comprises or consists of a VL of Table 1.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain that is at least about 90% or more identical to a heavy chain of Table 1; and / or (b) a light chain that is at least about 90% or more identical to a light chain of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain that is at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a heavy chain of Table 1; and / or (b) a light chain that is at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a light chain of Table 1. In some embodiments, the antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain that comprises or consists of a heavy chain of Table 1, and / or (b) a light chain that comprises or consists of a light chain of Table 1.

[0015] In another aspect, the disclosure provides an agent that binds to and / or competes for binding to the same epitope on HHLA2 as an HHLA2 binding agent of any aspect or embodiment described herein.

[0016] In some embodiments, the HHLA2 binding agent enhances binding of HHLA2 to TMIGD2 on naive immune effector cells. In some embodiments, the HHLA2 binding agent blocks binding of HHLA2 to KIR3DL3 on exhausted immune effector cells. In some embodiments, the immune effector cells include or are T cells and / or NK cells. In some embodiments, the T cells include or are CD4+ T cells and / or CD8+ T cells.

[0017] In some embodiments, the HHLA2 binding agent binds HHLA2 with a KD of about 5 nM or less. In some embodiments, the HHLA2 binding agent binds HHLA2 with a KD of about 15 nM or less. In some embodiments, the HHLA2 binding agent binds human HHLA2 with an affinity that is at least about 50-fold to about 800-fold over background. In some embodiments, the HHLA2 binding agent enhances binding of HHLA2 to TMIGD2 by a ratio of greater than about 2.

[0018] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one HHLA2 binding agent of any aspect or embodiment described herein and a pharma- ceutically acceptable carrier.

[0019] In another aspect, the present disclosure provides a method of treating a subject having a disease, disorder, or condition, comprising administering a therapeutically effective amount of at least one HHLA2 binding agent of any aspect or embodiment described herein, or a pharmaceutical composition of any aspect or embodiment described herein.

[0020] In another aspect, the present disclosure provides a method of regulating an immune response in a subject, comprising administering a therapeutically effective amount of at least one HHLA2 binding agent of any aspect or embodiment described herein, or a pharmaceutical composition of any aspect or embodiment described herein.

[0021] In some embodiments, the subject has cancer 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, colorectal 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).

[0022] In some embodiments, the disease, disorder, or condition is associated with aberrant HHLA2 expression.

[0023] In some embodiments, the HHLA2 binding agent is administered parenterally. In some embodiments, parenteral administration is or includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion.

[0024] In some embodiments, the HHLA2 binding agent is administered in combination with a second agent.

[0025] In another aspect, the disclosure provides a nucleic acid encoding at least one HHLA2 binding agent, or an antigen-binding fragment thereof, of any aspect or embodiment described herein.

[0026] In another aspect, the disclosure provides an expression vector comprising at least one nucleic acid of any aspect or embodiment described herein.

[0027] In another aspect, the disclosure provides a host cell comprising or expressing at least one HHLA2 binding agent of any aspect or embodiment described herein, a host cell comprising at least one nucleic acid of an aspect or embodiment described herein, or a host cell comprising at least one expression vector of an aspect or embodiment described herein.

[0028] In another aspect, the disclosure provides a method of making an HHLA2 binding agent, comprising: (i) culturing a host cell comprising at least one nucleic acid of any aspect or embodiment described herein, or at least one expression vector of any aspect or embodiment described herein, under conditions suitable for expression of the HHLA2 binding agent; and (ii) recovering the HHLA2 binding agent.

[0029] In another aspect, the present disclosure provides a method for detecting the presence or level of an HHLA2 polypeptide in a sample, comprising detecting an HHLA2 polypeptide in the sample using at least one HHLA2 binding agent of any aspect or embodiment described herein.

[0030] In another aspect, the present disclosure provides a kit comprising at least one HHLA2 binding agent of any aspect or embodiment described herein and instructions for use and / or administration.

[0031] In some embodiments, the HHLA2 binding agent forms a complex with the HHLA2 polypeptide. In some embodiments, the complex is detected by an assay including an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and / or a Western blot. In some embodiments, the HHLA2 binding agent is directly labeled.

[0032] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of example only, and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0033] The figures described below, together constituting a drawing, are for illustrative purposes only and not for limiting purposes. [Brief description of the drawings]

[0034] [Figure 1A] FIG. 1 is a schematic diagram showing the alignment of the heavy chain variable domains of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665. [Figure 1B] FIG. 1 is a schematic diagram showing the alignment of the light chain variable domains of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665.

[0035] [Figure 2A] 1 is a graph showing the binding affinity of an exemplary anti-HHLA2 antibody Ab-60638 to recombinant HHLA2-Fc using ForteBio's Octet system. [Figure 2B] 1 is a graph showing the binding affinity of an exemplary anti-HHLA2 antibody Ab-60665 to recombinant HHLA2-Fc using ForteBio's Octet system.

[0036] [Figure 3A] 1 is a graph of a flow cytometry histogram showing binding of an exemplary anti-HHLA2 antibody Ab-60638 to 300.19 cells overexpressing human HHLA2 compared to an isotype control. [Figure 3B] 1 is a graph of a flow cytometry histogram showing binding of an exemplary anti-HHLA2 antibody Ab-60665 to 300.19 cells overexpressing human HHLA2 compared to an isotope control. [Figure 3C]1 is a flow cytometry histogram graph showing binding of exemplary anti-HHLA2 antibodies Ab-65885 / Ab-65886 / Ab-65887 / Ab-65889 / Ab-65890 to 300.19 cells overexpressing human HHLA2 compared to an isotope control.

[0037] [Figure 4A] 1 is a graph of flow cytometry data showing the ability of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665 to block binding of human HHLA2-Fc to 300.19 cells overexpressing human KIR3DL3, compared to an isotype control. [Figure 4B] 1 is a graph of flow cytometry data showing the ability of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665 to enhance binding of human TMIGD2-Fc to 300.19 cells overexpressing human HHLA2 compared to an isotype control.

[0038] [Figure 5A] 1 is a graph of flow cytometry data showing the ability of exemplary anti-HHLA2 antibodies Ab-65885, Ab-65886, Ab-65887, Ab-65889 and Ab-65890 to block binding of human HHLA2-Fc to 300.19 cells overexpressing human KIR3DL3 compared to an isotype control. [Figure 5B] 1 is a graph of flow cytometry data showing the ability of exemplary anti-HHLA2 antibodies Ab-65885, Ab-65886, Ab-65887, Ab-65889 and Ab-65890 to enhance binding of human TMIGD2-Fc to 300.19 cells overexpressing human HHLA2 compared to an isotype control.

[0039] [Figure 6]1 is a bar graph showing that exemplary anti-HHLA2 antibodies Ab-65885, Ab-65886, Ab-65887, Ab-65889 and Ab-65890 modulate the activity of HHLA2-expressing CHO cells over adjacent TMIGD2-expressing Jurkat cells using a luciferase reporter in Jurkat cells; this assay shows that these five exemplary antibodies not only do not block HHLA2-mediated signaling via TMIGD2, but also enhance HHLA2-mediated signaling via TMIGD2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, and other terms, can be found throughout the specification. Publications and other reference materials referred to herein are incorporated by reference in order to explain the background of the invention and to provide further details regarding its practice.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an agent" means one agent or multiple agents.

[0042] About: As used herein, the term "about" as applied to one or more values ​​of interest refers to a value similar to the reference value described. 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 (above or below) 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).

[0043] Affinity maturation: The term "affinity maturation" as used herein refers to an antibody that has one or more changes in one or more of its CDRs that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have these one or more changes. In some embodiments, an affinity matured antibody will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies may be produced by a variety of procedures known in the art. Affinity maturation by VH and VL domain shuffling is described in Marks et al., BioTechnology, 10:779-783 (1992). Random mutagenesis of CDR and / or framework residues has been 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).

[0044] Agent: As used herein, the term "agent" refers to a biological entity and / or compound, such as, for example, an antibody 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 saccharide, a lipid, a growth factor, an enzyme, a synthetic molecule, a carbohydrate, a lipid, a hormone, a polymer, or a derivative, variant, conjugate, or any combination thereof. In appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be utilized to mean an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively, or in addition, as will be clear from the context, the term may be used to refer to a natural product. In some cases, also as will be clear from the context, the term may be used to refer to one or more entities that are man-made in that they are designed, engineered, and / or produced by human action and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or pure form. In some embodiments, an agent may be utilized in crude form. In some embodiments, the agents may be provided as a collection or library and screened to identify or characterize active agents therein. An agent may bind 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.

[0045] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, an intact antibody, as produced in nature, is a tetrameric agent of about 150 kD composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains, each about 110 amino acids long: an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the bottom of the base of the Y). A short region known as a "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, which are separated from each other by another "switch". An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by one disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, thus connecting the dimers to form a tetramer. In addition, naturally produced antibodies are usually glycosylated in the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two beta sheets (e.g., three-, four-, or five-stranded sheets) packed together into an antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2 and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3 and FR4).When a natural antibody folds, the FR regions form beta sheets to provide a structural framework for the domains, with the CDR loop regions from both the heavy and light chains joining 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 elements of the complement system and also to receptors on effector cells, such as effector cells mediating cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, an antibody produced and / or utilized in accordance with the present invention comprises a glycosylated Fc domain, e.g., an Fc domain with modified or engineered glycosylation. In some embodiments, any polypeptide, or complex of polypeptides, that includes a sufficient immunoglobulin domain sequence as found in a natural antibody, can be referred to and / or used as an "antibody," whether such a 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, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, or chimeric, as known in the art. Furthermore, the term "antibody" as used herein can refer, in appropriate embodiments (unless otherwise stated or clear from the context), to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations.For example, in some embodiments, the antibodies utilized in accordance with the present invention are of a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); and / or antibody fragments (preferably antibody fragments exhibiting the desired antigen-binding activity). The antibodies described herein can be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as any other immunological binding moieties known in the art, e.g., Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIP, antibodies, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandb, DVD, BiTe, TandAb, or any combination thereof. The subunit structures and three-dimensional configurations of different classes of antibodies are known in the art. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0046] Antibody Agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, or chimeric, as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies or fragments thereof, such as IgNAR), camelid antibodies; masked antibodies (e.g., Probodies®); small molecule immunopharmaceutical agents ("SMIPs™"); single chain or tandem diabodies. Formats include, but are not limited to, (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTEs®, Anrikin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; or KALBITOR®.In some embodiments, the antibody lacks a covalent modification (e.g., glycan attachment) that it has when produced in nature. In some embodiments, the antibody contains a covalent modification (e.g., glycan attachment, a payload, e.g., a detectable, therapeutic, or catalytic moiety), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are identical in sequence or contain 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs by at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that one to five amino acids within the included CDRs are deleted, added, or substituted relative to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDR is substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids in the included CDR are deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0047] Antibody heavy chain: As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibodies in their naturally occurring conformations.

[0048] Antibody light chain: As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibodies in their naturally occurring conformation.

[0049] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include either antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule, including 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 that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these 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 is 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.

[0050] Antigen-binding fragment: As used herein, the term "antigen-binding fragment" refers to a portion of an intact antibody that binds the antigen to which the intact antibody binds. Antigen-binding fragments of antibodies include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Exemplary antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, VHH, camelid antibodies, or only VH or VL domains); or multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragments of the antibodies described herein are scFvs. In some embodiments, the antigen-binding fragments of the antibodies described herein are only VHH domains. As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies may include at least two different variable domains, each of which can specifically bind to a separate antigen or a different epitope of the same antigen. The antigen-binding fragment may be produced by any means. For example, in some embodiments, the antigen-binding fragment is enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, the antigen-binding fragment is recombinantly produced. In some embodiments, the antigen-binding fragment is wholly or partially synthetically produced. In some embodiments, the antigen-binding fragment has 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.

[0051] Antibody-dependent cellular cytotoxicity: As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to the phenomenon in which antibody-bound target cells are killed by immune effector cells. Without wishing to be bound by theory, ADCC is generally understood to involve effector cells bearing Fc receptors (FcRs) that recognize and subsequently kill antibody-coated target cells (e.g., cells expressing on their surface a specific antigen to which the antibody binds). Effector cells that mediate ADCC include immune cells, including, but not limited to, natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0052] Aptamer: As used herein, the term "aptamer" refers to a polymer composed of nucleic acids (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., umbrella topology glycans). A particular aptamer may be described by a linear nucleotide sequence, typically about 15-60 nucleotides in length. While not wishing to be bound by theory, it is contemplated that the chain of nucleotides within an aptamer forms intramolecular interactions that fold the molecule into a complex three-dimensional shape that allows the aptamer to bind tightly to the surface of its target molecule. Given the incredible diversity of molecular shapes that exist within the realm of all possible nucleotide sequences, aptamers may be obtained for a wide range of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers typically have very high affinity for their targets (e.g., picomolar to low nanomolar range affinity for proteins). In many embodiments, aptamers are chemically stable and can be boiled or frozen without loss of activity. Because aptamers are synthetic molecules, they can be variously modified to optimize their function for specific applications. For example, aptamers can be modified for in vivo applications to dramatically reduce their susceptibility to degradation by enzymes in the blood, and in addition, aptamers can be modified to alter their biodistribution and plasma residence time.

[0053] Associated: Two events or entities, as that term is used herein, are "associated" with one another if the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic trait, 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). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly to be in physical proximity and / or remain in close proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0054] Binding: As used herein, the term "binding" refers to a non-covalent association between two or more entities. "Direct" binding includes physical contact between the entities or moieties. Indirect binding involves a physical interaction through physical contact with one or more intermediate entities. In general, binding between two or more entities can 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 or otherwise bound to a carrier entity and / or within a biological system or cell).

[0055] Cancer: As used herein, the terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth and thus exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cancer cells can spread locally or via the bloodstream and lymphatic system to other parts of the body. In some embodiments, a tumor is or includes pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. In some embodiments, the cancer is or includes a solid tumor. In another embodiment, the cancer is or includes a hematological tumor. Examples of various cancers are described herein, including, but are not limited to, cancers of the hematopoietic system, including leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma, and myeloproliferative disorders; sarcomas, melanomas, adenomas, and carcinomas of solid tissues; squamous cell carcinoma of the mouth, throat, larynx, and lung; liver cancer; genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, endometrial cancer, and renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; cancers of the endocrine system; cancer of the thyroid gland; cancer of the parathyroid gland; head and neck cancer; breast cancer; gastrointestinal cancer; cancer of the nervous system; as well as benign lesions such as papilloma, as well as several other types, including those described elsewhere herein.

[0056] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in administering a composition. In some exemplary embodiments, a carrier includes sterile liquids such as, for example, water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, a carrier is or includes one or more solid ingredients.

[0057] CDR: As used herein, "CDR" refers to the complementarity determining region in the variable region of an antibody. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each of the variable regions. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs present in either a single variable region capable of binding an antigen, or the CDRs of cognate heavy and light chain variable regions capable of binding an antigen. The exact definitional CDR boundaries and lengths are subject to various classification and numbering systems. Specific systems for defining CDR boundaries have been established in the art (e.g., Kabat, IMGT, Chothia, or combinations thereof). Thus, CDRs may be referred to by Kabat, Chothia, IMGT, or other boundary definitions known in the art. Despite the different boundaries, each of these systems has some overlap in what constitutes a "hypervariable region" within the variable sequence. Thus, the definition of a CDR 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, each of which is incorporated by reference in its entirety). Those of skill in the art will understand the differences between these systems and will be able to understand the boundaries of CDRs to the extent necessary to understand and practice the claims and disclosures herein.

[0058] Chemotherapeutic Agent: As used herein, the term "chemotherapeutic agent" has its art-understood meaning referring to one or more proapoptotic, cytostatic and / or cytotoxic agents, and specifically includes, for example, agents that are available and / or recommended for use in the treatment of one or more diseases, disorders or conditions associated with undesired cell proliferation. In many embodiments, the chemotherapeutic agent is useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be or may include one or more alkylating agents, 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., inhibitors of topoisomerase I and / 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, and / or one or more analogs of one or more of the following (i.e., sharing related antiproliferative activity): 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, fluorouracil, gemcitabine, hydroxyurea, The chemotherapeutic agent may be or may include one or more of idarubicin, imatinib, irinotecan, maytansine and / or analogs thereof (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in conjunction with an antibody drug conjugate.In some embodiments, the chemotherapeutic agent 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 and antibody drug conjugates including 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 ADCs, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and / or lorvotuzumab mertansine.

[0059] Chimeric antibody: as used herein refers to an antibody whose amino acid sequence comprises VH and VL region sequences found in a first species and constant region sequences found in a second species different from the first species. In many embodiments, a chimeric antibody has mouse VH and VL regions linked to human constant regions. In some embodiments, an antibody with human VH and VL regions linked to non-human constant regions (e.g., mouse constant regions) is referred to as a "reverse chimeric antibody."

[0060] Composition: One of ordinary skill in the art will understand that the term "composition" may be used to refer to a physically separate entity that includes one or more specific components. In general, unless otherwise specified, a composition may be in any form, such as, for example, a gas, a gel, a liquid, or a solid.

[0061] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but that other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is understood that any composition or method described as "comprising" (or "comprises") one or more named elements or steps also describes a corresponding, more limited composition or method that "consistes essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps, and may include additional elements or steps that do not materially affect the basic and novel property(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes a corresponding, more limited, closed-end composition or method that "consists of" (or "consists of") the named elements or steps, excluding any other elements or steps not named. In any composition or method disclosed herein, known or disclosed equivalents of any of the required named elements or steps may be substituted for that element or step.

[0062] Conservative sequence modifications: As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody or antigen-binding fragment thereof containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into antibodies conforming to various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in an antibody CDR region can be replaced with another amino acid residue from the same side chain family and the altered antibodies can be tested for their ability to bind antigen using the functional assays described herein.

[0063] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which two or more different therapeutic agents are administered in overlapping dosing schedules such that the subject is exposed to both agents at the same time. When used in combination therapy, two or more different therapeutic agents may be administered simultaneously or separately. This administration in combination can include simultaneous administration of two or more 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, where the therapeutic agents are in separate formulations. In another alternative, the first therapeutic agent can be administered followed by one or more additional therapeutic agents. In separate administration protocols, the two or more therapeutic agents can be administered minutes apart, or hours, days, or weeks apart. In some embodiments, the two or more therapeutic agents can be administered within an interval of a few hours (e.g., less than about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours).

[0064] Effective amount: As used herein, "effective amount" refers to a dose that is sufficient 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 will 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 will also be determined by the selected activity, the method of administration, the timing and frequency of administration, the presence, nature, and extent of adverse side effects that may accompany the administration of a particular active agent, and the desired physiological effect. Those skilled in the art will understand that various diseases or disorders may require long-term treatment, including multiple administrations. For purposes of this disclosure, the amount or dose of the therapeutic agent (e.g., at least one HHLA2 binding agent described herein) administered should be sufficient to effect a therapeutic or prophylactic response in the subject over a reasonable time frame (e.g., a reduction or other alleviation of the severity or duration of at least one sign or symptom). For example, the dose should be sufficient to detect, treat, or prevent cancer within a period of about 2 hours or more, such as about 12 hours to about 24 hours or more, from the time of administration. In some embodiments, the period is longer. The dose will be 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.

[0065] Encode: As used herein, the term "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, either having a defined sequence of nucleotides (i.e., rRNA, tRNA, or mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes 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, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding a protein or other product of that gene or cDNA.

[0066] Engineered: As used herein, the term "engineered" refers to aspects that have been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not linked in that order in nature are manipulated by the hand of man to be directly linked to each other in an engineered polynucleotide. For example, in some embodiments, an engineered polynucleotide comprises or is a regulatory sequence found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, which is linked by the hand of man to be operably associated with the second coding sequence. Equivalently, a cell or organism is considered to be "engineered" when it has been engineered to change its genetic information (e.g., new genetic material not previously present is introduced, for example, by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, for example, by substitution or deletion mutations, or mating protocols). In some embodiments, an engineered antibody or antigen-binding fragment thereof (e.g., an engineered monoclonal antibody or antigen-binding fragment thereof) comprises sequences of the VH and / or VL regions derived from a reference antibody produced in a non-human species (e.g., a mouse) and includes modifications of those sequences relative to the reference antibody intended to make them more "human" or more similar to human germline variable sequences.

[0067] As is common practice and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell will usually still be referred to as "engineered," even if the actual manipulation was performed on an earlier entity.

[0068] 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 a proper three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such structure. In some embodiments, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative structure (e.g., linearized).

[0069] 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 an anti-HHLA2 antibody or antigen-binding fragment 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) generation 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.

[0070] Fragment: As used herein, the term "fragment" refers to a structure that comprises a distinct portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, a fragment consists of such a distinct portion. In some embodiments, a fragment consists of or includes a characteristic structural element or portion found in the whole. In some embodiments, an antigen-binding fragment comprises or consists of 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. In some embodiments, an antigen-binding 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 the whole antibody. 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 the whole antibody.

[0071] Framework region: As used herein, the term "framework region" 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 likewise 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 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without identifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region refers to the combined FRs in the variable region of a single, naturally occurring immunoglobulin chain, as referred to elsewhere. As used herein, FR refers to one of the four subregions, with FR1 representing, for example, the first framework region closest to the amino terminus and 5' of the variable region relative to CDR1, and multiple FRs representing two or more of the subregions that make up the framework region.

[0072] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that code for a particular product). In some embodiments, a gene includes non-coding sequences. In some embodiments, a gene may include both coding (e.g., exon) and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory elements that may, for example, control or affect one or more aspects of gene expression (e.g., cell type specific expression and / or inducible expression).

[0073] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acids (e.g., DNA and / or RNA) and / or between polypeptides. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, various algorithms are available that allow for the comparison of sequences to determine the degree of homology, including, for example, by allowing gaps of a specified length in one sequence to another sequence when considering which residues in different sequences "correspond" to each other. Calculation of the percentage of homology between two nucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-corresponding sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. The molecules are identical at a position in the first sequence if that position is occupied by the same nucleotide as the corresponding position in the second sequence, and similar at that position if that position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence. The percent homology between two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0074] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art will understand upon reading this disclosure that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may exist in subsequent generations, either due to mutations or environmental influences, such progeny may not actually 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., Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, 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 cells comprise or are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell selected from CHO (e.g., CHO K1, DXB-1 1CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, 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, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes.

[0075] 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. An antibody or antigen-binding fragment thereof may be considered "human" in one or more CDRs, particularly CDR3, even though its amino acid sequence contains residues or elements not encoded in human germline immunoglobulin sequences (e.g., sequence alterations that may have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0076] Humanized: As used herein, the term "humanized" refers to an antibody or antigen-binding fragment thereof that contains VH and / or VL region sequences derived from a reference antibody whose amino acid sequence was generated in a non-human species (e.g., mouse), but also includes modifications of those sequences relative to the reference antibody intended to make the sequences more "human-like" or similar to human germline variable sequences. In some embodiments, a humanized antibody or antigen-binding fragment thereof is one that immunospecifically binds to an antigen of interest and has FR regions that have substantially the amino acid sequences of a human antibody and CDRs that have substantially the amino acid sequences of a non-human antibody. A humanized antibody comprises substantially all of at least one, and usually 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 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 comprises both a light chain and at least the variable domains of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and optionally CH4 regions of the heavy chain constant region. In some embodiments, a humanized antibody contains only a humanized VL region. In some embodiments, a humanized antibody contains only a humanized VH region. In some specific embodiments, a humanized antibody contains humanized VH and VL regions.

[0077] Identity: As used herein, the term "identity" 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.

[0078] Immune cell: As used herein, the term "immune cell" refers to a cell that is involved in an immune response, such as promoting an immune response. Examples of immune 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.

[0079] Immune checkpoint: As used herein, the term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells as well as NK cells that fine-tune the immune response by downregulating or inhibiting anti-tumor immune responses. Immune checkpoint proteins are well known in the art and include, but are not limited to, HHLA2, KIR family receptors, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPα, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, CD226, CD155, CD112. butyrophilin, and A2aR. In some embodiments, the NK cells comprise TIGIT, CD226, and / or CD96. The term further encompasses nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof, as well as biologically active protein fragments, in some embodiments, the term further encompasses any fragments according to the homology descriptions provided herein.

[0080] 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 an antigen molecule as foreign, induce the formation of antibodies, and / or activate lymphocytes to eliminate the antigen. In some embodiments, an immune cell response can include proliferation of immune effector cells (e.g., T cells), cytokine production by immune effector cells (e.g., T cells), and / or release of cytotoxic granules including perforin and / or granzymes by immune effector cells (e.g., T cells).

[0081] Immunoglobulin: As used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of 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 with no known antibody function, but may serve 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.

[0082] "Improve", "Increase", "Inhibit" or "Reduce": As used herein, the terms "improve", "increase", "inhibit", "reduce", or their grammatical equivalents 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 comparable conditions in the absence of a particular agent or treatment (e.g., before and / or after a particular agent or treatment) or in the presence of a suitable 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 manner in the presence of a relevant agent or treatment.

[0083] Isolated: As used herein, the term "isolated" refers to something that has been altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0084] K D As used herein, "K D The term "K" refers to the dissociation constant of a binding agent (e.g., an antibody or antigen-binding fragment thereof) from a complex with its partner (e.g., the epitope to which the antibody or antigen-binding fragment thereof binds). D The term "K" off K on It is equal to divided by.

[0085] K off As used herein, "K off The term "off rate constant" refers to the off rate constant for dissociation of a binding agent (e.g., an antibody or antigen-binding fragment thereof) from a complex with its partner (e.g., the epitope to which the antibody or antigen-binding fragment thereof binds).

[0086] K on As used herein, "K on The term "on rate constant" refers to the on rate constant for the association of a binding agent (e.g., an antibody or antigen-binding fragment thereof) with its partner (e.g., the epitope to which the antibody or antigen-binding fragment thereof binds).

[0087] Modulate: As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level and / or change in the nature of a response in a subject compared to the level and / or nature of the response in a non-treated 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.

[0088] Monoclonal antibody: "Monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies such that the individual antibodies of the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., mutations that occur naturally or that arise during production of the monoclonal), although such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0089] Nucleic Acid: As used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single stranded and double stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester bonds. For example, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, e.g., as in peptide nucleic acids. In some embodiments, a nucleic acid 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, a nucleic acid comprises one or more or all non-naturally occurring residues. In some embodiments, the non-natural residue comprises 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, O(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, and / or hexose) relative to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns.In some embodiments, nucleic acids may be prepared by isolation from natural sources, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., by replication in recombinant cells or systems, in vivo or in vitro, or by chemical synthesis). In some embodiments the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 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, 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.

[0090] Operably linked: As used herein, the term "operably linked" refers to a functional link between, for example, 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 and, where necessary to link two protein coding regions, in the same reading frame.

[0091] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0092] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in the transport or transfer of a subject compound from one organ or body part 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 examples of materials which can function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., 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, and / or polyanhydrides; and other non-toxic, compatible substances employed in pharmaceutical formulations.

[0093] Polynucleotide: As used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0094] Polypeptide: As used herein, the terms "polypeptide" or "protein," as used interchangeably herein, refer to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through the action of the human hand. A polypeptide may include or consist of natural amino acids, unnatural amino acids, or both. A polypeptide may include or consist of only natural amino acids or only unnatural amino acids. A polypeptide may include D-amino acids, L-amino acids, or both. A polypeptide may include one or more pendant groups or other modifications at the N-terminus, C-terminus, or both, e.g., that modify or link one or more amino acid side chains. In some embodiments, such pendant groups or modifications are selected from acetylation, amidation, lipidation, methylation, pegylation, including combinations thereof. A polypeptide may be cyclic and / or include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not include a cyclic portion. 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; 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 present specification provides exemplary polypeptides within the class whose amino acid sequence and / or function are known and / or the skilled artisan will be aware of; in some embodiments, such exemplary polypeptides are the reference polypeptides of the class or family of polypeptides.In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with reference polypeptides of that class, share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a comparable level or within a specified range). For example, a member polypeptide may have an overall degree of sequence homology or identity with the reference polypeptide that is at least about 30-40%, and often is about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region that exhibits very high sequence identity (e.g., a conserved region that may be or may include a characteristic sequence element), often exceeding 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; in some embodiments, the conserved regions encompass at least one 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 are found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest, such that the polypeptide of interest is a derivative of that parent polypeptide (e.g., fragments that are directly linked in 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).

[0095] Single chain antibody: As used herein, the term "single chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered stretch of amino acids. Various methods for producing single chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988), Science, 242:423-442; Huston et al. (1988), Proc. Natl. Acad. Sci. USA, 85:5879-5883; Ward et al. (1989), Nature, 334:54454; and Skerra et al. (1988), Science, 242:1038-1041.

[0096] Recombinant: As used herein, refers to a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library (Hoogenboom, TIB Tech, 15:62, 1997; Azzazy Clin. Biochem. 35:425, 2002; Gavilondo BioTechniques, 29:128, 2002; Hoogenboom, Immunology Today, 21:371, 2000), an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor Nuc. Acids Res. 20:6287, 1992; Little, Immunology Today, 12:364, 2000; Kellermann, Curr. Opin. Biotechnol, 13:593, 2002; Murphy, Proc. Natl. Acad. Sci. USA, 111:5153, 2104), or any other means involving splicing selected sequence elements together. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements result, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources. For example, in some embodiments, the recombinant antibody polypeptide is comprised of sequences found in the germline of a biological source of interest (e.g., human, mouse, etc.). In some embodiments, recombinant antibodies have amino acid sequences that have been generated by mutagenesis (e.g., in vitro or in vivo, e.g., in transgenic animals) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to germline VH and VL sequences, are sequences that do not naturally occur within the germline antibody repertoire in vivo.

[0097] Small molecule: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. In general, a "small molecule" is a molecule less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 Daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mole, less than about 1500 g / mole, less than about 1000 g / mole, less than about 800 g / mole, or less than about 500 g / mole. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymer moiety. In some embodiments, a small molecule is not a protein or polypeptide and / or does not include a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, the small molecule is not a polynucleotide and / or does not include a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, the small molecule is not a polysaccharide and / or does not include a polysaccharide. For example, in some embodiments, the small molecule is not a glycoprotein, proteoglycan, or glycolipid. In some embodiments, the small molecule is not a lipid. In some embodiments, the small molecule is a modulator (e.g., an inhibitor or activator). In some embodiments, the small molecule is biologically active. In some embodiments, the small molecule is detectable (e.g., includes at least one detectable moiety). In some embodiments, the small molecule is a therapeutic agent. One of skill in the art will understand, upon reading this disclosure, that a particular small molecule compound may be provided and / or utilized in any of a variety of forms, such as, for example, a crystalline form, a salt form, a protected form, a prodrug form, an ester form, an isomeric form (e.g., an optical isomer and / or structural isomer), or an isotopic form. One of skill in the art will understand that a particular small molecule compound has a structure that can exist in one or more stereoisomeric forms.In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers; in some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of a racemic mixture. Those of skill in the art will appreciate that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual tautomers, or in forms that interconvert between tautomers. Those of skill in the art will appreciate that certain small molecule compounds may be utilized in accordance with the present disclosure in the form of isotopic substitutions (e.g., for H. 2 H or 3 Regarding H;12C 11 C. 13 C or 14 Regarding C and 14N 13 N or 15 N)16O 17 O or 18 Regarding O and XXC 36 For Cl and XXF 18It will be understood that the small molecule has a structure that allows for the isotopically modified form (e.g., 131I for XXXI, 132F for XXXI, etc.). In some embodiments, such small molecules may be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof. In some embodiments, reference to a particular small molecule compound may refer to a particular form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in the form of a salt (e.g., in the form of an acid addition salt or a base addition salt, depending on the compound); in some such embodiments, the salt form may be a pharma- ceutically acceptable salt form. In some embodiments, when a small molecule compound is a compound that exists or is found in nature, the compound may be provided and / or utilized in accordance with the present disclosure in a form that is different from the form that exists or is found in nature. Those skilled in the art will understand that in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of a compound or a particular form thereof that differs from the absolute or relative amount (e.g., with respect to other components of the preparation that contain another form of the compound) of the compound or form present in a reference preparation of interest (e.g., in a primary sample from a source of interest, such as a biological or environmental source) will be different from the compound present in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound is considered to be a different form of the compound from a racemic mixture of the compound; a particular salt of a small molecule compound is considered to be a different form of the compound from another salt form of the compound; a preparation that contains only a form of the compound that contains one conformational isomer of the double bond ((Z) or (E)) is considered to be different from a form of the compound that contains the other conformational isomer of the double bond ((E) or (Z)); or a preparation in which one or more atoms are isotopically different from those present in the reference preparation is considered to be a different form.

[0098] 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 provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, a susceptible subject has a predisposition to a disease, disorder, or condition and / or exhibits an increased risk of developing a disease, disorder, or condition (relative 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 symptom or characteristic of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is being administered.

[0099] 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 may be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN for nucleotide sequences, BLASTP for amino acid sequences, gapped BLAST, and PSI-BLAST. 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 the 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 section 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.

[0100] Substantially: As used herein, the term "substantially" refers to a qualitative state exhibiting the entire or nearly entire 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.

[0101] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of the disease, disorder, and / or condition.

[0102] Target: As used herein, the term "target" refers to a cell, tissue, organ, or site in the body that is the subject of the methods, systems, and / or compositions provided, e.g., a cell, tissue, organ, or site in the body that is in need of treatment or that is preferentially bound by, e.g., an HHLA2 binding agent described herein.

[0103] Therapeutic: As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is achieved, for example, by suppression, amelioration, or eradication of a disease state.

[0104] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent can be an agent that can prevent undesirable side effects when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Therapeutic agents include, but are not limited to, at least one HHLA2 binding agent as described herein.

[0105] 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 delivered, and / or the target cell or tissue. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / 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.

[0106] 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 incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, the treatment is administered to a subject who does not exhibit signs or characteristics of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, the treatment is administered to a subject who exhibits only early or mild signs or characteristics of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition. In some embodiments, the treatment is administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, the treatment comprises administering to the subject at least one HHLA2 binding agent described herein.

[0107] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. A tumor may contain cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic. In some embodiments, a tumor is associated with or is an indication of cancer. In some embodiments, a tumor is a dispersed or liquid tumor. In some embodiments, a tumor is a solid tumor.

[0108] Throughout this disclosure, various aspects of the invention can 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 invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, as well as the individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0109] Detailed Description The present disclosure provides, inter alia, HHLA2 binding agents that result in (i) inhibition of HHLA2 binding to KIR3DL3; and / or (ii) enhancement of HHLA2 binding to TMIGD2. In some embodiments, the HHLA2 binding agent can: (i) inhibit HHLA2 binding to KIR3DL3, and (ii) enhance HHLA2 binding to TMIGD2. HHLA2 is a member of the B7 family that regulates NK cell and T cell function. HHLA2 is widely expressed in various tumors and antigen-presenting cells, and has been implicated as both an activating and inhibitory ligand for NK cells and T cells. HHLA2 is a specific ligand for TMIGD2, and the interaction of HHLA2 and TMIGD2 selectively stimulates T cell proliferation and cytokine production. HHLA2 also binds to KIR3DL3, a receptor on T cells and NK cells, and inhibits T cell and NK cell activation. The present disclosure provides HHLA2 binding agents for treating various cancers, including solid tumors and hematological tumors, and / or for modulating the immune response in a subject.

[0110] HHLA2 binding agents The present disclosure provides, inter alia, HHLA2 binding agents. In some embodiments, the HHLA2 binding agents described herein exhibit the ability to (i) inhibit the binding of HHLA2 to one or more receptors (e.g., TMIGD2) that inhibit immune response, and / or (ii) enhance the binding of HHLA2 to one or more receptors (e.g., TMIGD2) that promote immune response. Thus, the HHLA2 binding agents described herein are particularly useful for treating various cancers, including solid tumors and hematological tumors, as well as for regulating immune response in a subject.

[0111] 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). In normal human tissues, HHLA2 is expressed in placental trophoblast cells, as well as in kidney, intestine, gallbladder, and breast epithelium. In the immune system, HHLA2 is constitutively expressed in human monocytes and macrophages. HHLA2 regulates human T cell functions, such as T cell proliferation and cytokine production. HHLA2 is expressed at high levels in a wide range of human cancers of colorectal, renal, lung, pancreatic, ovarian, and prostate origin. HHLA2 is also expressed in human cancers of the thyroid, melanoma, liver, bladder, colon, kidney, breast, and esophagus.

[0112] 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, showing the longest transcript and encoding the longest isoform a), variant 2 (NM_001282556.1 and NP_001269485.1, showing the use of an alternative promoter and differing in the 5'UTR compared to variant 1), variant 3 (NM_001282557.1 and NP_001269486.1, showing the use of an alternative promoter and differing in the 5'UTR compared to variant 1), and variant 4 (NM_001282558.1 and NP_001269487.1, encoding isoform b). These include variant 1 (NM_001282559.1 and NP_001269488.1, which encodes isoform c, shows alternative promoter usage, has multiple differences compared to variant 2 resulting in a distinct 5'UTR, and causes translation initiation at an alternative start codon compared to variant 1, resulting in a distinct N-terminus and a shorter isoform than isoform a).

[0113] In some embodiments, binding of the HHLA2 binding agents described herein to HHLA2 is assessed using assays such as, for example, biolayer interferometry (BLI), immunohistochemistry (IHC), Western blot, intracellular flow, ELISA, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other methods known in the art. In some embodiments, the HHLA2 binding agents described herein have a K of about 20 nM to about 0.1 nM, e.g., about 10 nM to about 0.1 nM, e.g., about 5 nM to about 0.5 nM. DIn some embodiments, the HHLA2 binding agents described herein bind to HHLA2 with a K of about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less. D It binds to HHLA2.

[0114] In some embodiments, the HHLA2 binding agents described herein inhibit the binding of HHLA2 to KIR3DL3. As used herein, the term "KIR3DL3" or "killer cell immunoglobulin-like receptor 3DL3" refers to a member of the killer cell immunoglobulin-like receptor transmembrane glycoprotein family expressed by NK cells and T cells. KIR3DL3 is also known as KIRC1, CD158Z, KIR3DL7, and KIR44. Killer cell immunoglobulin-like receptor (KIR) genes are polymorphic and highly homologous genes found in a cluster on chromosome 19q13.4 within the 1 Mb leukocyte receptor complex (LRC). Several "framework" genes are found in all haplotypes (KIR3DL3, KIR3DL1, KIR3DL4, and KIR3DL2), but the genetic content of the KIR gene cluster differs between haplotypes. KIR proteins are classified according to the number of extracellular immunoglobulin domains (2D or 3D) and whether they have a long (L) or short (S) cytoplasmic domain. KIR proteins with long cytoplasmic domains transmit inhibitory signals upon ligand binding via immunotyrosine-based inhibitory motifs (ITIMs), whereas KIR proteins with short cytoplasmic domains lack ITIM motifs and instead transmit activating signals by associating with TYRO protein tyrosine kinase-binding proteins. The ligands for some KIR proteins are a subset of HLA class I molecules; therefore, KIR proteins are thought to play an important role in regulating immune responses. KIR3DL3 proteins have an N-terminal signal sequence, three Ig domains, a transmembrane region lacking positively charged residues, and a long cytoplasmic tail containing ITIMs. KIR3DL3 lacks the stalk region found in other KIRs.

[0115] The term "KIR3DL3" includes fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human KIR3DL3 cDNA and human KIR3DL3 polypeptide sequences are publicly available from NCBI. For example, at least one human KIR3DL3 isoform is known: human KIR3DL3 (NM_153443.4) encoded by transcript (NP_703144.3). Nucleic acid and polypeptide sequences of KIR3DL3 orthologs in organisms other than humans are also known, including, but not limited to, chimpanzee KIR3DL3 (XM_003316679.3 and XP_003316727.3), rhesus macaque KIR3DL3 (NM_001104552.2 and NP_001098022.1), mouse KIR3DL3 (NM_001310690.1 and NP_001297619.1, NM_177749.4 and NP_808417.2, NM_177748.2 and NP_808416.1), and rat KIR3DL3 (NM_181479.2 and NP_852144.1).

[0116] In some embodiments, inhibition of HHLA2 binding to KIR3DL3 by the HHLA2 binding agents described herein is assessed using assays such as cell binding competition assays (e.g., assays of soluble HHLA2 binding to KIR3DL3-expressing cells (e.g., 300.19 mouse precursor B leukemia cells expressing KIR3DL3), surface plasmon resonance (SPR), or any other method known in the art. In some embodiments, the HHLA2 binding agents described herein inhibit HHLA2 binding to KIR3DL3 at a ratio of about 0.8 to about 0.0, for example, compared to an isotopic control. In some embodiments, the HHLA2 binding agents described herein inhibit HHLA2 binding to KIR3DL3 at a ratio of about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, or about 0.0, for example, compared to an isotopic control. In some embodiments, the HHLA2 binding agents described herein abolish binding of HHLA2 to KIR3DL3.

[0117] In some embodiments, the HHLA2 binding agents described herein enhance HHLA2 binding to 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, a transmembrane region, and a proline-rich cytoplasmic domain with two tyrosine signaling motifs. TMIGD2 is constitutively expressed in naive T cells and natural killer (NK) cells, but not in regulatory T 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.

[0118] The term "TMIGD2" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human MIGD2 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 use 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 lack 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 orthologs in organisms other than humans are also known, including, for example, chimpanzee TMIGD2 (XM_009434393.2 and XP_009432668.2, and XM_OO1138228.4 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).

[0119] In some embodiments, the enhancement of HHLA2 binding to TMIGD2 by the HHLA2 binding agents described herein is assessed using an assay such as a cell binding competition assay (e.g., an assay of soluble TMIGD2 binding to HHLA2-expressing cells (e.g., 300.19 mouse precursor B leukemia cells expressing HHLA2) or any other method known in the art. In some embodiments, the HHLA2 binding agents described herein enhance the binding of HHLA2 to TMIGD2, for example, by a ratio of about 2.0 to about 8.0 compared to an isotopic control. In some embodiments, the HHLA2 binding agents described herein enhance the binding of HHLA2 to TMIGD2, for example, by a ratio of about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or more compared to an isotopic control.

[0120] In some embodiments, the HHLA2 binding agent is or comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the HHLA2 binding agent is or comprises an organic molecule (e.g., a small molecule). In some embodiments, the HHLA2 binding agent is or comprises a polypeptide (e.g., a fusion polypeptide). In some embodiments, the HHLA2 binding agent is or comprises an aptamer. In some embodiments, the HHLA2 binding agent is or comprises a nucleic acid. In some embodiments, the HHLA2 binding agent is or comprises a chimeric antigen receptor (e.g., a CAR comprising an anti-HHLA2 antigen-binding fragment described herein, such as an scFv).

[0121] Anti-HHLA2 antibodies and their fragments The present disclosure provides, inter alia, anti-HHLA2 antibodies or antigen-binding fragments thereof. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein specifically bind to an epitope on HHLA2. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein can be or include other immunological binding moieties known in the art, including immunoglobulins, heavy chain antibodies, light chain antibodies, or other protein scaffolds with antibody-like properties, as well as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments, scFv fragments, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandavs, BiTe, or any combination thereof. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein inhibit binding of HHLA2 to KIR3DL3. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein enhance binding of HHLA2 to TIMGD2.

[0122] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises or is a monoclonal antibody. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises or is a full-length antibody, e.g., comprising an immunoglobulin Fc region. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises or is a multispecific antibody, e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises or is a bispecific antibody molecule. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein is or has been affinity matured.

[0123] The anti-HHLA2 antibody or antigen-binding fragment thereof can comprise a heavy chain variable domain sequence (VH) and a light chain variable domain sequence (VL). In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof comprises an immunoglobulin molecule with four polypeptide chains, e.g., two heavy chains and two light chains. The heavy chain can comprise a VH and a heavy chain constant domain. The heavy chain constant domain can also comprise a CH1, hinge, CH2, CH3, and optionally a CH4 region. The light chain can comprise a VL and a light chain constant domain. The light chain constant domain can comprise a CL domain.

[0124] VH and / or VL can be further subdivided into regions of variability, termed complementarity determining regions (CDRs), and interspersed, more conserved regions, termed framework regions (FRs). Each such VH and / or VL 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 VH has three CDRs (HCDR1, HCDR2, and HCDR3) and each VL has three CDRs (LCDR1, LCDR2, and LCDR3). The extent of framework regions and CDRs can be defined using a number of well-known schemes (see, e.g., 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).

[0125] The anti-HHLA2 antibodies or antigen-binding fragments thereof described herein can be derived from any class of antibodies, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The anti-HHLA2 antibodies or antigen-binding fragments thereof described herein can be or include a human antibody, a humanized antibody, a CDR-grafted antibody, or an antibody generated in vitro. The anti-HHLA2 antibodies 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 anti-HHLA2 antibodies or fragments can have or include a light chain selected from, for example, kappa or lambda.

[0126] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein is or comprises a monoclonal antibody. Usually, monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, so that the individual antibodies that make up the population are substantially identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" as used herein indicates the characteristic of the antibody that it is not a mixture of separate antibodies. In some embodiments, the monoclonal antibody against a particular epitope is derived from a single cell line (e.g., a B cell line).

[0127] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein is or comprises a polyclonal antibody. In contrast to monoclonal antibodies, polyclonal antibodies are usually obtained from a heterogeneous antibody population, so that the antibodies within a particular population contain structural variations, such as affinity for various epitopes on a particular target (e.g., HHLA2). Several methods of producing polyclonal antibodies are known in the art, including the use of multiple subcutaneous and / or intraperitoneal injections of the relevant antigen into animals, optionally with the co-administration of one or more adjuvants.

[0128] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising one, two, or three VH CDR sequences that are at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the VH CDRs of Table 1, respectively; and / or (b) a VL comprising one, two, or three VL CDR sequences that are at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the VL CDRs of Table 1, respectively. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a VH of Table 1; and / or (a) a VL that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to a VL of Table 1. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a heavy chain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a heavy chain of Table 1; and / or (a) a light chain that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a light chain of Table 1.

[0129] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 1, the VH CDR2 amino acid sequence of SEQ ID NO: 2, and the VH CDR3 amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 14, the VL CDR2 amino acid sequence of SEQ ID NO: 15, and the VL CDR3 amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 4, the VH CDR2 amino acid sequence of SEQ ID NO: 5, and the VH CDR3 amino acid sequence of SEQ ID NO: 6; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 17, the VL CDR2 amino acid sequence of SEQ ID NO: 18, and the VL CDR3 amino acid sequence of SEQ ID NO: 19, each of which is disclosed in Table 1. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 7, the VH CDR2 amino acid sequence of SEQ ID NO: 8, and the VH CDR3 amino acid sequence of SEQ ID NO: 9; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 20, the VL CDR2 amino acid sequence of SEQ ID NO: 21, and the VL CDR3 amino acid sequence of SEQ ID NO: 22, each of which is disclosed in Table 1.

[0130] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 10. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 23. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 23.

[0131] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 12. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 25. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 25.

[0132] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 27, the VH CDR2 amino acid sequence of SEQ ID NO: 28, and the VH CDR3 amino acid sequence of SEQ ID NO: 29; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 40, the VL CDR2 amino acid sequence of SEQ ID NO: 41, and the VL CDR3 amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 30, the VH CDR2 amino acid sequence of SEQ ID NO: 31, and the VH CDR3 amino acid sequence of SEQ ID NO: 32; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 43, the VL CDR2 amino acid sequence of SEQ ID NO: 44, and the VL CDR3 amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 33, a VH CDR2 amino acid sequence of SEQ ID NO: 34, and a VH CDR3 amino acid sequence of SEQ ID NO: 35; and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 46, a VL CDR2 amino acid sequence of SEQ ID NO: 47, and a VL CDR3 amino acid sequence of SEQ ID NO: 48.

[0133] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 36. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 49. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 49.

[0134] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 38. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 51, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 51. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 51.

[0135] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 53, the VH CDR2 amino acid sequence of SEQ ID NO: 54, and the VH CDR3 amino acid sequence of SEQ ID NO: 55; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 66, the VL CDR2 amino acid sequence of SEQ ID NO: 67, and the VL CDR3 amino acid sequence of SEQ ID NO: 68. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 56, the VH CDR2 amino acid sequence of SEQ ID NO: 57, and the VH CDR3 amino acid sequence of SEQ ID NO: 58; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 69, the VL CDR2 amino acid sequence of SEQ ID NO: 70, and the VL CDR3 amino acid sequence of SEQ ID NO: 71. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 59, a VH CDR2 amino acid sequence of SEQ ID NO: 60, and a VH CDR3 amino acid sequence of SEQ ID NO: 61; and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 72, a VL CDR2 amino acid sequence of SEQ ID NO: 73, and a VL CDR3 amino acid sequence of SEQ ID NO: 74.

[0136] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 62. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 75. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0137] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 64. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 77. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 77.

[0138] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 79, the VH CDR2 amino acid sequence of SEQ ID NO: 80, and the VH CDR3 amino acid sequence of SEQ ID NO: 81; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 92, the VL CDR2 amino acid sequence of SEQ ID NO: 93, and the VL CDR3 amino acid sequence of SEQ ID NO: 94. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 82, the VH CDR2 amino acid sequence of SEQ ID NO: 83, and the VH CDR3 amino acid sequence of SEQ ID NO: 84; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 95, the VL CDR2 amino acid sequence of SEQ ID NO: 96, and the VL CDR3 amino acid sequence of SEQ ID NO: 97. In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 85, a VH CDR2 amino acid sequence of SEQ ID NO: 86, and a VH CDR3 amino acid sequence of SEQ ID NO: 87; and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 98, a VL CDR2 amino acid sequence of SEQ ID NO: 99, and a VL CDR3 amino acid sequence of SEQ ID NO: 100.

[0139] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 88. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 101. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 88 and a VL comprising the amino acid sequence of SEQ ID NO: 101.

[0140] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 90. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 103. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising the amino acid sequence of SEQ ID NO: 103.

[0141] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 105, the VH CDR2 amino acid sequence of SEQ ID NO: 106, and the VH CDR3 amino acid sequence of SEQ ID NO: 107; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 118, the VL CDR2 amino acid sequence of SEQ ID NO: 119, and the VL CDR3 amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 108, the VH CDR2 amino acid sequence of SEQ ID NO: 109, and the VH CDR3 amino acid sequence of SEQ ID NO: 110; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 121, the VL CDR2 amino acid sequence of SEQ ID NO: 122, and the VL CDR3 amino acid sequence of SEQ ID NO: 123. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 111, the VH CDR2 amino acid sequence of SEQ ID NO: 112, and the VH CDR3 amino acid sequence of SEQ ID NO: 113; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 124, the VL CDR2 amino acid sequence of SEQ ID NO: 125, and the VL CDR3 amino acid sequence of SEQ ID NO: 126.

[0142] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 114. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 127. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein 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: 127.

[0143] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 116. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 129. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein 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: 129.

[0144] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 131, the VH CDR2 amino acid sequence of SEQ ID NO: 132, and the VH CDR3 amino acid sequence of SEQ ID NO: 133; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 144, the VL CDR2 amino acid sequence of SEQ ID NO: 145, and the VL CDR3 amino acid sequence of SEQ ID NO: 146. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 134, the VH CDR2 amino acid sequence of SEQ ID NO: 135, and the VH CDR3 amino acid sequence of SEQ ID NO: 136; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 147, the VL CDR2 amino acid sequence of SEQ ID NO: 148, and the VL CDR3 amino acid sequence of SEQ ID NO: 149. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 137, the VH CDR2 amino acid sequence of SEQ ID NO: 138, and the VH CDR3 amino acid sequence of SEQ ID NO: 139; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 150, the VL CDR2 amino acid sequence of SEQ ID NO: 151, and the VL CDR3 amino acid sequence of SEQ ID NO: 152.

[0145] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 140. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 153. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 140 and a VL comprising the amino acid sequence of SEQ ID NO: 153.

[0146] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 142. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 155. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 142 and a light chain comprising the amino acid sequence of SEQ ID NO: 155.

[0147] In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 157, the VH CDR2 amino acid sequence of SEQ ID NO: 158, and the VH CDR3 amino acid sequence of SEQ ID NO: 159; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 170, the VL CDR2 amino acid sequence of SEQ ID NO: 171, and the VL CDR3 amino acid sequence of SEQ ID NO: 172. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 160, the VH CDR2 amino acid sequence of SEQ ID NO: 161, and the VH CDR3 amino acid sequence of SEQ ID NO: 162; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 173, the VL CDR2 amino acid sequence of SEQ ID NO: 174, and the VL CDR3 amino acid sequence of SEQ ID NO: 175. In some embodiments, an anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises: (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 163, the VH CDR2 amino acid sequence of SEQ ID NO: 164, and the VH CDR3 amino acid sequence of SEQ ID NO: 165; and (b) a VL comprising the VL CDR1 amino acid sequence of SEQ ID NO: 176, the VL CDR2 amino acid sequence of SEQ ID NO: 177, and the VL CDR3 amino acid sequence of SEQ ID NO: 178.

[0148] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 166. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VL comprising the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 179. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a VH comprising the amino acid sequence of SEQ ID NO: 166 and a VL comprising the amino acid sequence of SEQ ID NO: 179.

[0149] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 168. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence that is at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 181. In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 181. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

[0150] antigen binding fragment The present disclosure provides, inter alia, anti-HHLA2 antigen-binding fragments. As used herein, "anti-HHLA2 antigen-binding fragments" includes or is any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that contains at least one complementarity determining region (CDR) of VH or VL, or an HHLA2 binding portion derived from any of the antibodies described herein. Antibody 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 HHLA2.

[0151] Examples of anti-HHLA2 antigen-binding fragments described herein include (i) a Fab fragment, which is a monovalent fragment containing the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which contains the VH and CH1 domains; (iv) an Fv fragment, which contains the VL and VH domains of a single arm of an antibody; (v) a diabody (dAb) fragment, which contains the VH domain; (vi) a camel or camelized variable domain; (vii) an scFv, which is a fusion protein of the VH and VL regions, or (viii) a single domain antibody. In some embodiments, the anti-HHLA2 antigen-binding fragment described herein contains or is a heavy chain and a light chain (e.g., a half antibody).

[0152] Method of preparation The present disclosure provides, inter alia, a method for producing the anti-HHLA2 antibody or antigen-binding fragment thereof described herein.In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein is identified using display technology such as yeast display, phage display, or ribosome display.In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein is identified using a hybridoma library (e.g., a mammalian hybridoma library, e.g., a mouse hybridoma library) followed by supernatant screening.

[0153] Combinatorial methods for generating antibodies are known in the art (see, 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. al.(1992),Hum.Antibody Hybridomas,3:81-85;Huse et al.(1989),Science,246:1275-1281;Griffths et al.(1993),EMBO.J.12:725-734;Hawkins et al. 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. al. (1991), Bio / Technology, 9:1373-1377; Hoogenboom et al. (1991), Nuc. Acid Res. 19:4133-4137; and Barbas et al. al. (1991), PNAS, 88:7978-7982).

[0154] In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein may be derived from other species. Humanized antibodies are antibodies created by recombinant DNA techniques 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., framework regions of the constant region and / or 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 would have (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 in both the heavy and light chains. The human FRs may be selected based on the highest sequence homology to the mouse FR sequences. If necessary, to maintain the binding affinity of the humanized antibody to the target, one or more residues of the human FRs can be changed to the residues at the corresponding positions in the mouse antibody. This change is sometimes referred to as a "back mutation." Similarly, forward mutations can be made to revert to the mouse sequences for desired reasons, such as stability or affinity to the target. Humanized antibodies are generally less likely to elicit an immune response in humans than chimeric human antibodies, since the former contain significantly fewer non-human components.

[0155] 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, EP 0 239 400; 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 nucleic acid sequences encoding VH and VL containing CDRs are determined by sequencing. The nucleic acid sequences encoding CDRs are inserted into the corresponding regions of the coding sequence of human antibody VH or VL and linked to human constant region gene segments of the desired isotype (e.g., γ1 for CH, κ for CL). 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-expressing strains, for example, using DHFR or GS genes in the production strains.

[0156] In some embodiments, the anti-HHLA2 antibody or antigen-binding fragment thereof described herein comprises or is a human antibody. Fully human antibodies may be particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by various methods known in the art, including the above-mentioned phage display method 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). For example, the techniques 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, can also be used to prepare human monoclonal antibodies.

[0157] nucleic acid The present disclosure provides, inter alia, nucleic acids encoding the HHLA2 binding agents (e.g., anti-HHLA2 antibodies 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 thereof disclosed herein. Such nucleic acids may be present in a vector. Such nucleic acids may be present in the genome of a cell, e.g., a cell of a subject in need of treatment, or a cell for antibody production, e.g., a mammalian cell for producing an anti-HHLA2 antibody or antigen-binding fragment thereof described herein.

[0158] Nucleic acids encoding HHLA2 binding agents (e.g., anti-HHLA2 antibodies or antigen-binding fragments thereof) described herein may be modified to include codons that are 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 a full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by a parent polynucleotide that is not codon-optimized. In some embodiments, all or a portion of the coding region of a nucleic acid encoding an HHLA2 binding agent described herein may include a sequence that has been modified to optimize codon usage for a particular cell type (e.g., eukaryotic or prokaryotic cells). For example, the coding sequence of a 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.

[0159] The nucleic acid constructs of the present disclosure may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid may be operably linked to an expression control sequence. Further provided by the present 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, may 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 of skill in the art (see, for example, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).

[0160] For example, exogenous nucleic acid (e.g., DNA or RNA) may be introduced into prokaryotic or eukaryotic host cells using conventional techniques including electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Desirably, the vector may contain regulatory sequences, such as transcription and / or translation initiation and / or termination codons, specific for the type of host into which the vector is to be introduced (e.g., bacterial, fungal, plant, or animal), taking into account whether the vector is DNA or RNA, as appropriate. In some embodiments, the vector contains regulatory sequences specific to the genus of the host cell. In some embodiments, the vector contains regulatory sequences specific to the species of the host.

[0161] 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.

[0162] The expression vector may include a native or non-native promoter operably linked to the nucleic acid isolated or purified as described above. The selection of a promoter, e.g., a strong, weak, inducible, tissue-specific, and / or development-specific promoter, is within the skill of the art. Similarly, the combination of the above nucleic acid and a promoter is also within the skill of the art.

[0163] Suitable vectors include those designed for propagation and propagation 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.

[0164] 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 the nucleic acid encoding the HHLA2 binding agent described herein, or improve the introduction of the nucleic acid into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, for example, 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), each of which is incorporated herein by reference in its entirety).

[0165] In some embodiments, the nucleic acids and vectors of the disclosure are isolated and / or purified. The disclosure also provides compositions comprising isolated or purified nucleic acids, optionally in the form of vectors. Isolated nucleic acids and vectors may be prepared using standard techniques known in the art, including, for example, alkaline / SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis, and / or other techniques known in the art. The compositions may include other components as further described herein.

[0166] Any method known to one of skill in the art for inserting a nucleic acid into a vector may be used to construct expression vectors encoding the anti-human HHLA2 antibodies 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, e.g., Ausubel, supra; or Sambrook, supra).

[0167] Antibodies that bind to the same epitope In some embodiments, the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein include antibodies and antibody fragments that bind to the same epitope as the HHLA2-binding antibodies shown in Table 1. Thus, additional antibodies and antibody fragments can be identified based on their ability to cross-compete (e.g., compete and inhibit the binding in a statistically significant manner) with other antibodies described herein in an HHLA2 binding assay. The ability of a test antibody to inhibit the binding of the antibodies and antibody fragments described herein to an HHLA2 protein (e.g., human HHLA2) demonstrates that the test antibody can compete with that antibody or antibody fragment for binding to HHLA2; such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximate) epitope on the HHLA2 protein as the competing antibody or antibody fragment. In some embodiments, an antibody that binds to the same epitope on HHLA2 as the anti-HHLA2 antibodies or antigen-binding fragments thereof described herein is a human monoclonal antibody or a humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0168] Treatment method The present disclosure provides, inter alia, a method for treating a disease, disorder, or condition in a subject (e.g., a disease, disorder, or condition described herein) comprising administering a pharmaceutical composition comprising at least one HHLA2 binding agent 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.

[0169] A pharmaceutical composition comprising at least one HHLA2 binding agent as described herein can be for use in the manufacture of a medicament for treating a disease, disorder or condition in a subject, or for stimulating an immune response in a subject.A pharmaceutical composition comprising at least one HHLA2 binding agent as described herein can be administered to a subject alone or in combination with one or more therapeutic agents, procedures, or treatments according to the dosage regimen as described herein.

[0170] The subject treated by the methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having or at risk of having a disease, disorder, or condition described herein). Methods are provided for treating (e.g., alleviating, inhibiting, or delaying the progression of) cancer or tumor in a subject using pharmaceutical compositions comprising at least one HHLA2 binding agent described herein. The subject can have adult or pediatric forms of cancer. The cancer can be in early, middle, or late stages, or can be metastatic cancer.

[0171] Methods are provided for treating (e.g., alleviating, inhibiting, or delaying the progression of) signs or symptoms of cancer in a subject using a pharmaceutical composition comprising at least one HHLA2 binding agent described herein. In some embodiments, the pharmaceutical compositions described herein are useful for delaying the onset of cancer, slowing its progression, or ameliorating one or more signs or symptoms thereof. In some embodiments, the physiological signs or symptoms of cancer include or are 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, and / or an increase in the survival of cancer cells. In some embodiments, the physical signs or symptoms of cancer include or are skin lesions (e.g., lumps or moles), weight loss, digestive disorders, discomfort, fatigue, pain, difficulty swallowing, coughing, abnormal bleeding and / or discharge, changes in bowel and / or bladder habits, and / or mental confusion.

[0172] Cancers can include, but are not limited to, solid tumors, hematological cancers (e.g., leukemia, lymphoma, or myeloma such as multiple myeloma), or metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas, e.g., adenocarcinomas, including those affecting the lung, breast, ovary, lymphatic system, gastrointestinal (e.g., colon), anus, reproductive and genitourinary tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0173] In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., non-small cell lung cancer (NSCLC) of squamous and / or non-squamous tissue, or NSCLC adenocarcinoma), 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 cell carcinoma (e.g., renal cell carcinoma), cystic 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 the estrogen receptor, progesterone receptor, or Her2 / neu, e.g., triple-negative breast cancer), colorectal cancer (e.g., recurrent colorectal cancer or metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair proficient, repair proficient 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., cervical squamous cell carcinoma), neuroendocrine tumors (NETs) (e.g., atypical lung carcinoid tumors)), lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders), lymphomas (e.g., T-cell lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma), myelomas (e.g., multiple myeloma), or leukemias (e.g., myeloid leukemia or lymphocytic leukemia). In some embodiments, the subject has renal cell carcinoma.

[0174] In some embodiments, the cancer is a brain tumor, e.g., a glioblastoma, a gliosarcoma, or a recurrent brain tumor. In some embodiments, the cancer is a pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer is a skin cancer, e.g., a melanoma (e.g., stage II-IV melanoma, HLA-A2 positive melanoma, unresectable melanoma, or metastatic melanoma), or a Merkel cell carcinoma. In some embodiments, the cancer is a kidney cancer, e.g., a renal cell carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is a breast cancer, e.g., metastatic breast cancer, or stage IV breast cancer, e.g., triple negative breast cancer (TNBC). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is anal canal cancer (e.g., squamous cell carcinoma of the anal canal). In some embodiments, the cancer is cervical cancer (e.g., squamous cell carcinoma of the cervix). In some embodiments, the cancer is gastric cancer (e.g., Epstein-Barr virus (EBV) positive gastric cancer, or gastric or gastroesophageal junction cancer). In some embodiments, the cancer is head and neck cancer (e.g., HPV positive and negative squamous cell carcinoma of the head and neck (SCCHN)). In some embodiments, the cancer is nasopharyngeal carcinoma (NPC). In some embodiments, the cancer is colorectal cancer, e.g., recurrent colorectal cancer, metastatic colorectal cancer, e.g., microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair proficient colorectal cancer, or mismatch repair deficient colorectal cancer.

[0175] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer 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 is a lymphoma, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory HL or DLBCL). In some embodiments, the cancer is a myeloma, such as multiple myeloma.

[0176] Administration of the pharmaceutical composition of at least one HHLA2 binding agent described herein may be by any convenient method (e.g., injection, ingestion, infusion, inhalation, implantation, or implantation). In some embodiments, the pharmaceutical composition described herein is administered by injection or infusion. The pharmaceutical composition described herein may be administered to a patient intraarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous, intravenous, intramuscular, or substernal infusion or injection. In some embodiments, the pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection. The pharmaceutical composition described herein may 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.

[0177] In some embodiments, at least one HHLA2 binding agent described herein is utilized in combination with one or more other therapeutic agents or modalities. In some embodiments, the one or more other therapeutic agents or modalities are also anti-cancer agents or anti-cancer modalities. In some embodiments, the combination exhibits synergistic effects in the treatment of cancer. Known compounds or therapies that exhibit therapeutic effects 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, and / or surgery.

[0178] Pharmaceutical Compositions The present disclosure provides, inter alia, pharmaceutical compositions comprising at least one HHLA2 binding agent in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients.

[0179] Where a "therapeutically effective amount," "immunologically effective amount," "anti-immune response effective amount," or "immune response inhibiting effective amount" is indicated, the exact amount of a pharmaceutical composition comprising at least one HHLA2 binding agent described herein can be determined by a physician taking into account individual differences in the age, weight, immune response, and condition of the patient (subject).

[0180] The pharmaceutical compositions described herein may include a buffer, including neutral buffered saline or phosphate buffered saline (PBS); a carbohydrate, such as glucose, mannose, sucrose, dextran, or mannitol; a protein, polypeptide, or amino acid (e.g., glycine); an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., free of detectable levels of contaminants (e.g., endotoxins).

[0181] The pharmaceutical compositions described herein may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. The amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, disorder, or condition, although appropriate dosages may be determined by clinical trials.

[0182] 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. A preferred composition may be an injectable or infusible solution. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0183] 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 substernal injection or infusion. In a preferred embodiment, the pharmaceutical compositions described herein are formulated for injection of subcutaneous or intravenous infusion. The pharmaceutical compositions described herein can be formulated for administration using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, which is incorporated herein by reference in its entirety).

[0184] As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0185] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, concurrently with, or after) bone marrow transplantation or lymphodepletion therapy using chemotherapeutic agents (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or Rituxan). 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 may be administered before or after surgery.

[0186] The dosage of any of the aforementioned therapies administered to a subject will vary depending on the disease, disorder, or condition being treated, and based on the particular subject. Scaling of dosages for administration to humans can be performed according to art-recognized practices.

[0187] kit The present disclosure provides, inter alia, a kit comprising at least one HHLA2 binding agent described herein and instructions for use and / or administration. In some embodiments, the kit comprises at least one HHLA2 binding agent described herein, a pharma- ceutically acceptable carrier, and instructions for use and / or administration.

[0188] 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 and second pharmaceutical compositions 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 and second pharmaceutical compositions to a subject in need of treatment.

[0189] The instructions for the first and second pharmaceutical compositions described herein typically include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit doses. The instructions provided with the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease, disorder, or condition in a subject.

[0190] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Also contemplated are packaging for use in combination with a specific device, such as an infusion device. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container may also have a sterile access port.

[0191] The kit may optionally provide additional components such as buffers and 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.

[0192] Incorporation by Reference All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. In addition, 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0193] 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

[0194] 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.

[0195] Example 1: Characterization of specific HHLA2 binding agents This example demonstrates methods for characterizing the HHLA2 binding agents described herein, particularly anti-HHLA2 antibodies and / or antigen-binding fragments thereof. This example further provides various methods for determining and / or characterizing the relevant functional activities of the HHLA2 binding agents described herein. Alignment of the heavy and light chain variable domains of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665 described herein is shown in Figures 1A-1B.

[0196] The strong affinity of anti-HHLA2 antibodies to recombinant HHLA2-Fc was examined. The strong affinity of anti-HHLA2 antibodies to recombinant HHLA2-Fc was measured using ForteBio's Octet system. Strong affinity was determined by capturing each antibody on an AHC sensor and then transferring it to a well containing 100 nM HHLA2-Fc antigen in solution. Strong affinity for exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665 showed strong affinity of single-digit nanomolar or higher to recombinant HHLA2 (Table 2 and Figures 2A-2B). [Table 2]

[0197] The monovalent affinity of exemplary anti-HHLA2 antibodies Ab-65885, Ab-65886, Ab-65887, Ab-65889, and Ab-65890 for recombinant HHLA2-His was measured using Cytiva's Biacore system. Monovalent affinity was determined by capturing each antibody on a CM5 chip followed by capture of various concentrations of HHLA2-His analyte. The monovalent affinity of exemplary anti-HHLA2 antibodies Ab-65885, Ab-65886, Ab-65887, Ab-65889, and Ab-65890 for recombinant HHLA2 ranged from about 750 pM to about 15 nM (Table 3). [Table 3]

[0198] The ability of anti-HHLA2 antibodies to bind 300.19 murine precursor B leukemia cells overexpressing human HHLA2 was examined. 300.19-human HHLA2 cells were incubated with 15 μg / ml of each antibody for 30 minutes on ice, followed by incubation with a 1:10 dilution of PE-conjugated anti-human secondary antibody. Flow cytometry histograms demonstrated the ability of exemplary anti-HHLA2 antibodies Ab-60638 and Ab-60665 to bind to 300.19 cells overexpressing human HHLA2 (Table 4 and Figures 3A-3B). Data are presented as median fluorescence intensity (MFI) and fold over background (FOB). [Table 4]

[0199] The ability of anti-HHLA2 antibodies to block HHLA2 binding to KIR3DL3 and enhance HHLA2 binding to TMIGD2 was examined. A total of 10 μg / mL of IgG was incubated with 4 μg / mL of biotinylated human HHLA2-Fc or TMIGD2-Fc for 30 minutes on ice, and then added to 300.19 mouse pre-B leukemia cells expressing KIR3DL3 or HHLA2, respectively, and incubation continued on ice for 30 minutes. Alexa Fluor 633-conjugated streptavidin was added as a secondary detection reagent. Flow cytometry data demonstrated the ability of exemplary anti-HHLA2 antibodies to completely block HHLA2 binding to KIR3DL3 while increasing HHLA2 binding to TMIGD2 (Table 5, Figures 4A-4B and 5A-5B). These data demonstrate that the anti-HHLA2 antibodies described herein (i) can inhibit the binding of HHLA2 to KIR3DL3; and (ii) can enhance the binding of HHLA2 to TMIGD2. [Table 5] Equivalent

[0200] It should be understood that various changes, modifications, and improvements to the present disclosure will be readily apparent to 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 invention. Accordingly, the foregoing description and drawings are by way of example only, and the inventions described in this disclosure are more particularly described by the following claims.

[0201] Those of 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 the invention and to provide further details regarding its practice are hereby incorporated by reference in their entireties.

Claims

1. 1. An HHLA2 binding agent comprising: (i) capable of inhibiting binding of HHLA2 to KIR3DL3; (ii) The HHLA2 binding agent, which is capable of enhancing the binding of HHLA2 to TMIGD2.

2. The HHLA2 binding agent is or comprises an antibody or antigen-binding fragment thereof, a small molecule, a polypeptide or an aptamer, and the antibody or antigen-binding fragment thereof is (i) a chimeric, human, or humanized antibody, or an antigen-binding fragment thereof; (ii) a monospecific or bispecific antibody, or an antigen-binding fragment thereof; (iii) a monoclonal antibody or an antigen-binding fragment thereof; (iv) an scFv, Fab, Fab', F(ab')2, Fc, nanobody, or camelid antibody; (v) a heavy chain constant region chosen from IgG1, IgG2, IgG3, or IgG4; and / or (vi) the HHLA2 binding agent of claim 1, which is or comprises a light chain constant region selected from a kappa or lambda light chain constant region.

3. the antibody or antigen-binding fragment thereof (I) (a) a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO: 105, the VH CDR2 amino acid sequence of SEQ ID NO: 106, and the VH CDR3 amino acid sequence of SEQ ID NO: 107; and (b) a light chain variable region (VL) comprising the VL CDR1 amino acid sequence of SEQ ID NO: 118, the VL CDR2 amino acid sequence of SEQ ID NO: 119, and the VL CDR3 amino acid sequence of SEQ ID NO: 120; (II) (a) a VH comprising the VH CDR1 amino acid sequence of SEQ ID NO: 108, the VH CDR2 amino acid sequence of SEQ ID NO: 109, and the 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: 121, a VL CDR2 amino acid sequence of SEQ ID NO: 122, and a VL CDR3 amino acid sequence of SEQ ID NO: 123; (III) (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: 124, a VL CDR2 amino acid sequence of SEQ ID NO: 125, and a VL CDR3 amino acid sequence of SEQ ID NO: 126; (IV) (a) a VH comprising an amino acid sequence having at least 90% or more identity to SEQ ID NO: 114; and a VL comprising an amino acid sequence having at least 90% or more identity to SEQ ID NO: 127; or (b) a VH comprising the amino acid sequence of SEQ ID NO: 114; and a VL comprising the amino acid sequence of SEQ ID NO: 127; and / or (V) (a) a heavy chain comprising an amino acid sequence that has at least 90% or more identity to SEQ ID NO: 116; and a light chain comprising an amino acid sequence that has at least 90% or more identity to SEQ ID NO: 129; or (b) 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:

129.

3. The HHLA2 binding agent of claim 2, which is or comprises:

4. An agent which binds to and / or competes for binding to the same epitope on HHLA2 as the HHLA2 binding agent of any one of claims 1 to 3. (i) enhancing binding of HHLA2 to TMIGD2 in naive immune effector cells; (ii) blocking binding of HHLA2 to KIR3DL3 on exhausted immune effector cells, said exhausted immune effector cells comprising or being T cells and / or NK cells, said T cells comprising or being CD4+ T cells and / or CD8+ T cells; (iii) binds HHLA2 with a K D of 15 nM or less; (iv) binds human HHLA2 with an affinity that is at least 50-800-fold greater than background; and / or (v) enhancing the binding of HHLA2 to TMIGD2 by a ratio of greater than 2; The HHLA2 binding agent according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising at least one HHLA2 binding agent according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier. (a) for treating a subject having a disease, disorder, or condition; and / or (b) modulating an immune response in said subject; The pharmaceutical composition according to claim 6. (i) the subject has cancer or is at risk of developing cancer; (ii) the disease, disorder, or condition is associated with aberrant HHLA2 expression; (iii) the subject has a solid tumor or a hematological cancer; (a) the solid tumor is or comprises one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer; or (b) the hematological cancer comprises or is leukemia or lymphoma; (b1) 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; or (b2) the lymphoma comprises or is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL); and / or (iv) the HHLA2 binding agent is (a) administered parenterally, said parenteral administration being or including subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion; and / or (b) administered in combination with a second agent; The pharmaceutical composition according to claim 7.

9. A nucleic acid encoding at least one HHLA2 binding agent, or an antigen-binding fragment thereof, according to any one of claims 1 to 5.

10. An expression vector comprising at least one nucleic acid according to claim 9.

11. A host cell comprising or expressing at least one HHLA2 binding agent according to any one of claims 1 to 5, comprising at least one nucleic acid according to claim 9, or comprising at least one expression vector according to claim 10.

12. 1. A method of making an HHLA2 binding agent, comprising: (i) culturing a host cell comprising at least one nucleic acid according to claim 9 or at least one expression vector according to claim 10 under conditions suitable for the expression of said HHLA2 binding agent; (ii) recovering the HHLA2 binding agent.

13. A method for detecting the presence or level of an HHLA2 polypeptide in a sample, the method comprising detecting said HHLA2 polypeptide in said sample using at least one HHLA2 binding agent described in any one of claims 1 to 5.

14. A kit comprising at least one HHLA2 binding agent according to any one of claims 1 to 5 and instructions for use and / or administration, (i) the at least one HHLA2 binding agent forms a complex with an HHLA2 polypeptide, and the complex is detected by an assay including an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and / or a Western blot; and / or (ii) A kit, wherein said HHLA2 binding agent is directly labeled.

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