KIR3DL3 inhibitors and immune cell activators

Abstract

The present disclosure relates to KIR3DL3 inhibitors (e.g., anti-KIR3DL3 antibodies or antigen-binding fragments thereof) and immune cell activators, and their uses, such as for modifying immune effector cells. Killer cell immunoglobulin-like receptor (KIR) proteins contain either two (KIR2D) or three (KIR3D) immunoglobulin-like extracellular domains. KIR3DL3 is a member of the KIR family and is a receptor that has been demonstrated to be found on both T cells and NK cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 312,507, filed February 22, 2022, and U.S. Provisional Patent Application No. 63 / 418,105, filed October 21, 2022, the contents of each of which are incorporated by reference in their entirety herein. [Background technology]

[0002] Immunotherapy has been explored for many diseases and disorders, including cancer, but has encountered functional limitations that still need to be addressed. The immune system is tightly controlled by a network of co-stimulatory 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 regulate immune responses in some patients, but immune checkpoint expression and interactions with natural binding partners can vary between patients.

[0003] Thus, there is a need for the development of new therapeutic modalities optimized to target immune checkpoint pathways. Summary of the Invention

[0004] Killer cell immunoglobulin-like receptor (KIR) proteins contain either two (KIR2D) or three (KIR3D) immunoglobulin-like extracellular domains. KIR3DL3 is a member of the KIR family and is a receptor that has been demonstrated to be found on both T cells and NK cells. HHLA2, a member of the B7 gene family, is widely expressed in a variety of tumors and antigen-presenting cells. HHLA2 binding to KIR3DL3 has been shown to inhibit the immune response of activated T cells.

[0005] The present disclosure encompasses, inter alia, the discovery of anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein. Importantly, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein may have one or more of the following properties: (1) specifically bind to KIR3DL3 (e.g., human KIR3DL3) with high affinity, (2) specifically bind to KIR3DL3 expressed on NK cells and / or T cells, (3) block KIR3DL3 binding to HHLA2, (4) block HHLA2-mediated suppressive activity in T cells, (5) enhance NK cell killing of HHLA2-expressing tumor cells, and (6) enhance anti-tumor activity. Thus, the present disclosure provides anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein that are useful in methods of treating diseases, disorders, and conditions, such as, for example, cancer, as described herein, and methods of modulating immune responses.

[0006] While inhibitors of KIR3DL3 are being investigated as immunotherapies, the present disclosure encompasses, inter alia, the recognition that immune cell activators described herein can (i) increase KIR3DL3 expression and / or (ii) increase the strength of the KIR3DL3 promoter (e.g., epigenetically) to enhance functional KIR3DL3 inhibition. Accordingly, the present disclosure provides several examples of such immune cell activators that are particularly useful in combination with KIR3DL3 inhibitors to treat various cancers, including solid tumors such as renal cell carcinoma (RCC).

[0007] In one aspect, the disclosure provides a method of treating a subject having a disease, disorder, or condition, comprising administering a population of modified immune effector cells, where prior to administration, the population of immune effector cells is contacted with at least one immune cell activator and at least one KIR3DL3 inhibitor, thereby forming the population of modified immune effector cells.

[0008] In another aspect, the disclosure provides a method of treating a subject having a disease, disorder, or condition, comprising: (i) administering to the subject a population of modified immune effector cells, where the population of immune effector cells is contacted with at least one immune cell activator prior to administration, thereby forming a population of modified immune effector cells; and (ii) administering to the subject at least one KIR3DL3 inhibitor.

[0009] In some embodiments, the at least one KIR3DL3 inhibitor is or comprises an anti-KIR3DL3 antibody or antigen-binding fragment thereof, miRNA, shRNA, siRNA, CRISPR / Cas guide system, TALEN, ZFN, and / or a demethylating agent. In some embodiments, the antigen-binding fragment comprises an scFv, Fab, Fab', F(ab')2, Fc, or nanobody.

[0010] In some embodiments, the demethylating agent comprises or is 5-aza-2-deoxycytidine (Aza), 5-azacytidine, 1-β-D-arabinofuranosyl-5-azacytosine, or dihydro-5-azacytidine.

[0011] In some embodiments, the immune cell activator results in increased T cell proliferation and / or endogenous expression of at least one cytokine.

[0012] In some embodiments, the immune cell activator comprises or is a cytokine agent. In some embodiments, the cytokine agent is or comprises IL-2, IL-15, IL-12, IL-17, IL-18, IL-21, IFNγ, and / or TNFα. In some embodiments, IL-2 binds to IL-2Rα, IL-2Rβ, or IL-2Rγ. In some embodiments, IL-2 expands only T cells and does not substantially expand Tregs. In some embodiments, the cytokine agent is or comprises an inhibitor of suppressors of cytokine signaling (SOCS) proteins.

[0013] In some embodiments, the immune cell activator comprises or is a costimulatory antibody or antigen-binding fragment thereof, a small molecule, a polypeptide, a glycoprotein, or an exogenous cell. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof binds to 4-1BB, CD3, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TGF-β, LAG-3, CD39, or CD73. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is OKT3. In some embodiments, the costimulatory small molecule binds to 4-1BB, CD3, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TGF-β, LAG-3, CD39, or CD73. In some embodiments, the costimulatory polypeptide comprises or is a soluble HHLA2 polypeptide (e.g., an HHLA2 fusion polypeptide, e.g., an HHLA2 Fc fusion polypeptide) or a fragment thereof. In some embodiments, the costimulatory glycoprotein comprises or is a fibronectin protein or a fragment thereof. In some embodiments, the costimulatory exogenous cell comprises or is an artificial antigen-presenting cell.

[0014] In some embodiments, the immune effector cells are isolated from peripheral blood mononuclear cells (PBMCs) or tumor cells. In some embodiments, the modified immune effector cells include or are NK cells and / or T cells. In some embodiments, the T cells include or are CD4+ T cells and / or CD8+ T cells. In some embodiments, the modified immune effector cells include at least one CAR.

[0015] In some embodiments, the modified immune effector cells are administered to the subject within less than about 3 hours of contact with at least one KIR3DL3 inhibitor. In some embodiments, the modified immune effector cells are administered to the subject within less than about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 30 minutes, or about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours of contact with at least one KIR3DL3 inhibitor.

[0016] In some embodiments, the modified immune effector cell population and / or at least one KIR3DL3 inhibitor is administered parenterally, hi some embodiments, parenteral administration is or includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion.

[0017] In some embodiments, the methods comprise sequential administration of a population of modified immune effector cells and at least one KIR3DL3 inhibitor, in some embodiments (i) the population of modified immune effector cells is administered prior to administration of the at least one KIR3DL3 inhibitor, or (ii) the population of modified immune effector cells is administered after administration of the at least one KIR3DL3 inhibitor.

[0018] In some embodiments, the method comprises co-administration of the population of modified immune effector cells with at least one KIR3DL3 inhibitor, hi some embodiments, the method comprises co-administration by injection.

[0019] In some embodiments, the subject has cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the solid tumor is or includes one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer. In some embodiments, the subject has a blood 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). In some embodiments, the subject has a cancer that is resistant to a treatment that includes a cytokine agent.

[0020] In some embodiments, the methods described herein further comprise determining expression of TMIGD2 and / or KIR3DL3 by the modified immune effector cells. In some embodiments, the methods described herein further comprise determining activation of the immune effector cells. In some embodiments, the methods described herein further comprise determining expression of CD25, CD69, CD137, CD16, CD56, CD96, CD226, KIR2DL5, and / or NKG2D.

[0021] In some embodiments, the methods described herein further comprise formulating the population of modified immune cells into a composition for administration to a subject.

[0022] In another aspect, the disclosure provides a method of producing a population of modified immune effector cells, the method comprising: (i) contacting the population of immune effector cells with at least one immune cell activator; and (ii) contacting the population of immune effector cells with at least one KIR3DL3 inhibitor, thereby producing the population of modified immune effector cells.

[0023] In another aspect, the present disclosure provides a composition comprising a population of modified immune effector cells, at least one immune cell activator, and at least one KIR3DL3 inhibitor.

[0024] In another aspect, the disclosure provides a composition comprising a population of modified immune effector cells and at least one KIR3DL3 inhibitor, wherein the immune effector cells are contacted with at least one immune cell activator.

[0025] In another aspect, the present disclosure provides a kit comprising at least one immune cell activator, at least one KIR3DL3 inhibitor, and instructions for use and / or administration.

[0026] In another aspect, the present disclosure provides a kit comprising a population of modified immune effector cells, at least one KIR3DL3 inhibitor, and instructions for use and / or administration, wherein the immune effector cells are contacted with at least one immune cell activator.

[0027] In another aspect, the disclosure provides an anti-KIR3DL3 antibody or antigen-binding fragment thereof that is, or comprises, (a) a heavy chain variable region (VH) comprising one, two, or three VH CDR sequences, each having at least about 90% identity to the VH CDRs of Table 1, and / or (b) a light chain variable region (VL) comprising one, two, or three VL CDR sequences, each having at least about 90% identity to the VL CDRs of Table 1. Such an anti-KIR3DL3 antibody or antigen-binding fragment thereof may be used in any aspect or embodiment described herein.

[0028] In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises: (a) a VH comprising one, two, or three VH CDR sequences having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VH CDRs, respectively, of Table 1, and / or (b) a VL comprising one, two, or three VL CDR sequences having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VL CDRs, respectively, of Table 1. In some embodiments, an anti-KIR3DL3 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.

[0029] In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises (a) a VH having at least about 90% or more identity to the VH of Table 1, and / or (b) a VL having at least about 90% or more identity to the VL of Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises (a) a VH having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VH of Table 1, and / or (b) a VL having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VL of Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises (a) a VH comprising or consisting of a VH of Table 1, and / or (b) a VL comprising or consisting of a VL of Table 1.

[0030] In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain having at least about 90% or greater identity to a heavy chain of Table 1, and / or (b) a light chain having at least about 90% or greater identity to a light chain of Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to a heavy chain of Table 1, and / or (b) a light chain having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to a light chain of Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof is or comprises: (a) a heavy chain comprising or consisting of a heavy chain of Table 1, and / or (b) a light chain comprising or consisting of a light chain of Table 1.

[0031] In another aspect, the disclosure provides a nucleic acid encoding an anti-KIR3DL3 antibody, or antigen-binding fragment thereof, of any aspect or embodiment described herein.

[0032] In another aspect, the disclosure provides an expression vector comprising a nucleic acid of any aspect or embodiment described herein.

[0033] In another aspect, the disclosure provides a host cell comprising or expressing an anti-KIR3DL3 antibody, or antigen-binding fragment thereof, of any aspect or embodiment described herein, comprising a nucleic acid of any aspect or embodiment described herein, or comprising an expression vector of any aspect or embodiment described herein.

[0034] In another aspect, the disclosure provides a pharmaceutical composition comprising at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof of any aspect or embodiment described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0035] In another aspect, the disclosure provides a method of treating a subject having a disease, disorder, or condition comprising administering a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0036] In another aspect, the present disclosure provides a method of modulating an immune response in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of any aspect or embodiment described herein.

[0037] In some embodiments, the subject has or is at risk of developing cancer. In some embodiments, the subject has a solid tumor or a blood cancer. In some embodiments, the solid tumor is or includes one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer. In some embodiments, the blood cancer includes or is a leukemia or lymphoma. In some embodiments, the leukemia includes or is acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the lymphoma includes or is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

[0038] The figures described below, which together constitute a drawing, are for illustration purposes only and not for limitation. [Brief description of the drawings]

[0039] [Figure 1A] FIG. 1 is a schematic diagram of the HHLA2 checkpoint axis showing that HHLA2 expressed on tumor cells regulates NK and T cell activity through interaction with KIR3DL3 or TMIGD2. [Figure 1B]FIG. 1 is a schematic diagram of the HHLA2 checkpoint axis showing that HHLA2 expressed on tumor cells regulates NK and T cell activity through interaction with KIR3DL3 or TMIGD2.

[0040] [Diagram 2] 1 is a graph showing the monovalent binding affinity of an anti-KIR3DL3 antibody described herein (NPX267) to recombinant KIR3DL3 protein as determined by SPR using a Biacore instrument.

[0041] [Figure 3A] FIG. 13 is a graph showing binding of NPX267 to KIR3DL3 expressed on 300.19-KIR3DL3 cells by flow cytometry using an anti-human phycoerythrin (PE) secondary antibody. [Figure 3B] FIG. 13 is a graph showing binding of NPX267 to KIR3DL3 expressed on NK92MI cells by flow cytometry using an anti-human phycoerythrin (PE) secondary antibody. [Figure 3C] FIG. 1 is a graph showing binding of NPX267 to KIR3DL3 expressed on primary human NK cells by flow cytometry using an anti-human phycoerythrin (PE) secondary antibody.

[0042] [Figure 4] 1 is a series of graphs showing binding of NPX267 to KIR3DL3 expressed on tumor-infiltrating CD56+ NK cells.

[0043] [Diagram 5] FIG. 13 is a graph showing the percentage of KIR3DL3 binding to HHLA2 following treatment of 300.19-KIR3DL3 cells with NPX267 or an IgG4 isotype control antibody at concentrations ranging from 10 mg / mL to 0.0005 mg / mL.

[0044] [Figure 6]FIG. 13 is a graph showing the fold induction of luminescence in a T cell reporter assay of HHLA2 / TCR / CHO cells and Jurkat / IL-2 / KIR3DL3 cells precomplexed with NPX267 and an anti-CD28 agonist antibody as determined using a luminometer (BioTek Synergy™ 2 microplate reader).

[0045] [Figure 7A] Graph showing NK92MI effector cell killing of K562 cells following treatment with NPX267 or IgG4 isotype control as assessed using flow cytometry. [Figure 7B] 13 is a graph showing KIR3DL3+ human NK cell killing of HCC827 cells following treatment with NPX267 or an IgG4 isotype control as assessed using flow cytometry.

[0046] [Figure 8] Graph showing tumor growth, as assessed by imaging, in NSG mice injected intraperitoneally with luciferase-tagged HCC827 cells, and after tumor establishment, injected with KIR3DL3+ primary human NK cells, followed by either NPX267 parental Ab (26E10) or mIgG1 every other day for a total of five injections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] 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, are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.

[0048] 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 element" means one element or more than one element.

[0049] Approximately or about: As used herein, the term "approximately" or "about" refers to a value similar to a stated reference value when applied to one or more subject values. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is 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).

[0050] Agent: As used herein, the term "agent" refers to a molecule that may be delivered to a target by an immune effector cell described herein or that may be expressed, released, or secreted from a target. Agents include, but are not limited to, cytokine agents (e.g., cytokines (IL-2, IL-15, IL-12, IL-12, IL-17, and / or IL-18)), nucleic acids, antibiotics, anti-inflammatory agents, antibodies or fragments thereof, chimeric antigen receptors, antibody agents or fragments thereof, glycoproteins, artificial antigen presenting cells, growth factors, enzymes, proteins (e.g., RNAse inhibitors), peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. An agent can bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a cell and act within the cell.

[0051] Immunotherapeutic Agent: The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent capable of stimulating the host's immune system to generate an immune response against a tumor or cancer in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0052] Affinity maturation (or "affinity matured antibody"): as used herein refers to an antibody with one or more mutations in one or more CDRs that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not have the one or more mutation(s) in one or more CDRs. In some embodiments, an affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies may be produced by any of a variety of procedures known in the art. Marks et al., BioTechnology 10:779-783 (1992) describe affinity matured antibodies as a method for the development of affinity matured antibodies. H and V L Affinity maturation by domain shuffling has been described. 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).

[0053] Antibody: As used herein, the term "antibody" refers to a polypeptide that contains sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As known in the art, naturally produced intact antibodies are approximately 150 kD tetrameric agents that contain two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain contains at least four domains, each about 110 amino acids long, 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 base of the stem of the Y). A short region, known as the "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 and two other disulfide bonds connect the heavy chain hinge regions to each other to form a tetramer. Naturally produced antibodies are also typically 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 "complement 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 domain, and the CDR loop regions of both the heavy and light chains join in three-dimensional space to create one 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, including, for example, effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure include a glycosylated Fc domain, including Fc domains 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," regardless of whether such polypeptide is produced naturally (e.g., produced by an animal in response to an antigen) or produced by recombinant genetic 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, chimeric, etc., as known in the art. Furthermore, the term "antibody" as used herein, in appropriate embodiments (unless otherwise stated or clear from the context), can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations.For example, in some embodiments, the antibodies according to the invention are in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments as used herein in the broadest sense, encompassing a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments thereof that exhibit the desired antigen-binding activity). The antibodies described herein may be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds with antibody-like properties, as well as other immunological binding moieties known in the art, including, for example, Fab, Fab', Fab'2, Fab2, Fab3, F(ab')2, Fd, Fv, Feb, scFv, SMIPs, antibodies, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandab, DVD, BiTe, TandAb, etc., 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 covalent modifications (e.g., glycan attachments) that it would have if it were produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachments, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0054] Antigen-binding fragment: "Antigen-binding fragment" refers to a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds 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 or VHH or VH or VL domains only), and 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 VHH domains only. 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 capable of specifically binding to a separate antigen or a different epitope of the same antigen.

[0055] Antigen-presenting cells: As used herein, the term "antigen-presenting cells" or APCs includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), in addition to other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0056] 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 antibody molecules in their naturally occurring conformation.

[0057] 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 antibody molecules in their naturally occurring conformation.

[0058] Activated: As used herein, the term "activated" refers to the state of a cell, e.g., an immune effector cell as described herein, that has been stimulated sufficiently to induce detectable cell proliferation or to exert its effector function. Activation can also be associated with induced proliferation, cytokine production, cytokine secretion, cell signaling (e.g., gene expression changes), target cell killing, metabolic changes, production of inflammatory mediators, and / or antigen processing and presentation.

[0059] Synthetic antibody: As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0060] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" by that term. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, which nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids.

[0061] Autologous: As used herein, the term "autologous" refers to any material derived from an individual that is later reintroduced into the same individual.

[0062] Allogeneic: As used herein, the term "allogeneic" refers to any material (eg, cell population) derived from a different animal of the same species.

[0063] Xenogeneic: As used herein, the term "xenogeneic" refers to any material (e.g., cell population) derived from an animal of a different species.

[0064] Chimeric Antigen Receptor: As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial cell surface receptor engineered to be expressed on immune effector cells as described herein and specifically target the cells and / or bind antigens. CARs can be used as therapeutics, for example, by adoptive cell transfer. CARs can include at least one extracellular domain, at least one transmembrane domain, and at least one intracellular domain.

[0065] Cancer: As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Cancer can include both solid tumors and blood cancers. Examples of various cancers are described herein, including, but not limited to, renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, thyroid cancer, leukemia, or lymphoma, as well as several other types, including those described elsewhere herein. In certain embodiments, the cancer is renal cell carcinoma (RCC).

[0066] CDR: as used herein, refers to a complementarity determining region within an antibody variable region. 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. Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.), and one of skill in the art will appreciate the differences between these systems and will be able to understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0067] Chemotherapeutic Agent: As used herein, the term "chemotherapeutic agent" has its art-understood meaning to refer to one or more pro-apoptotic, cytostatic, and / or cytotoxic agents, specifically including, for example, agents used and / or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with unwanted cell proliferation. In many embodiments, chemotherapeutic agents are beneficial in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be 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., those that share 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 The chemotherapeutic agent may be or include one or more of: hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, barbicine, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. In some embodiments, the 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 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, antibody drug conjugates including 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.

[0068] Conservative sequence modification: As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of an antibody 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 the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for the ability to bind antigen using the functional assays described herein.

[0069] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which two or more different therapeutic agents (e.g., a population of modified immune effector cells described herein and at least one KIR3DL3 inhibitor described herein) are administered in an overlapping regimen such that the subject is exposed to both agents simultaneously. When used in combination therapy, the two or more different therapeutic agents may be administered simultaneously or separately. This combination administration can include simultaneous administration of two or more therapeutic agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more therapeutic agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more therapeutic agents can be administered simultaneously, where the agents are in separate formulations. In another alternative, a first therapeutic agent can be administered, followed by one or more additional therapeutic agents. In separate administration protocols, the two or more therapeutic agents may be administered minutes apart, or hours apart, days apart, or weeks apart. In some embodiments, the two or more therapeutic agents can be administered within a few hours (e.g., less than about 3 hours) apart.

[0070] Composition: One of skill in the art will understand that the term "composition" may be used to refer to a separate physical entity that includes one or more specified components. In general, unless otherwise specified, a composition may be in any form, e.g., a gas, a gel, a liquid, or a solid.

[0071] Comprising: A composition or method described herein as "comprising" one or more recited elements or steps is open-ended, meaning that the recited 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, any composition or method described as "comprising" (or "comprises") one or more recited elements or steps is also understood to describe a corresponding, more limited composition or method that "consisting essentially of" (or "consists essentially of") the same recited elements or steps, meaning that the composition or method includes the recited essential elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristic(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 recited elements or steps also describes the corresponding more limited and closed-ended composition or method that "consisting of" (or "consists of") the recited elements or steps, to the exclusion of any other unrecited elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any recited required element or step may be substituted for that element or step.

[0072] Co-administration: As used herein, the term "co-administration" with respect to two or more therapeutic agents described herein (e.g., a population of immune effector cells described herein and at least one KIR3DL3 inhibitor described herein) is administration that is performed using doses and time intervals such that the therapeutic agents administered are present together in the body, e.g., at one or more sites of action in the body, in non-negligible amounts and over a period of time. The time interval can be minutes (e.g., at least 1 minute, 1-30 minutes, 30-60 minutes), hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12 hours, 12-24 hours), days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7 days, etc.), or weeks (e.g., at least 1 week, 2 weeks, or 3 weeks, etc.). Thus, the therapeutic agents may, but need not, be administered together, e.g., as part of a single composition. Additionally, the therapeutic agents may, but need not, be administered essentially simultaneously (e.g., within less than 5 minutes, or within less than 1 minute) or a short time apart from each other (e.g., less than 1 hour, less than 30 minutes, less than 10 minutes, approximately 5 minutes apart). According to various embodiments of the present disclosure, therapeutic agents administered within such time intervals may be considered to be administered substantially simultaneously. In certain embodiments of the present disclosure, the therapeutic agents administered simultaneously are present in the body at effective concentrations over the time interval. When administered simultaneously, the effective concentration of each therapeutic agent required to elicit a particular biological response may be lower than the effective concentration of each therapeutic agent when administered alone, thereby allowing for a reduced dose of one or more therapeutic agents compared to the dose required when the agent is administered as a single agent. The effect of multiple therapeutic agents may, but is not necessarily, additive or synergistic. The therapeutic agents may be administered multiple times.

[0073] Conservative sequence modification: As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties 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 the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for the ability to bind antigen using the functional assays described herein.

[0074] Cytotoxicity: As used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or damaging a cell. In one embodiment, the cytotoxicity of a metabolically enhanced cell is improved, e.g., the cytolytic activity of an immune effector cell (e.g., a T cell or a NK cell) described herein is increased.

[0075] Effective amount: As used herein, the "effective amount" described herein refers to a dose sufficient to prevent or treat at least one sign and / or symptom of 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 active agent, the method of administration, the timing and frequency of administration, the presence, nature and extent of any adverse side effects that may accompany the administration of a particular active agent, and the desired physiological effect. It will be understood by those skilled in the art that various diseases or disorders may require chronic treatment with multiple administrations. For purposes of this disclosure, the amount or dose of the therapeutic agent (e.g., modified immune effector cells described herein and / or at least one KIR3DL3 inhibitor 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 for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the particular therapeutic agent or agents and the condition of the subject (e.g., human), as well as the body weight of the subject (e.g., human) to be treated.

[0076] Effector function: As used herein, "effector function" or "effector activity" refers to a specific activity carried out by an immune cell in response to stimulation of the immune cell. For example, the effector function of a T lymphocyte includes recognizing an antigen and killing a cell expressing the antigen.

[0077] Epitope: As used herein, the term "epitope" refers to any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts another conformation (e.g., linearized).

[0078] "Framework" or "framework region": as used herein, refers to the sequence of the variable region minus the CDRs. Since the CDR sequences can be determined by different systems, the framework sequences are also subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 being located between FR1 and FR2, CDR2 being between FR2 and FR3, and CDR3 being between FR3 and FR4. Without identifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region, as referred to elsewhere, represents the combined FRs in the variable region of a single, naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, e.g., FR1 represents the first framework region closest to the amino terminus of the variable region and 5' with respect to CDR1, and FR represents two or more of the subregions that make up the framework region.

[0079] Immune effector function: As used herein, "immune effector function" or "immune effector response," as that term is used herein, refers to a function or response of an immune effector cell (e.g., a T cell or an NK cell), e.g., as described herein, that enhances or promotes immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell and / or an NK cell that promotes inhibition of growth or proliferation of a target cell. For example, in the case of a T cell, primary stimulation and costimulation are examples of immune effector functions or responses.

[0080] Encode: As used herein, "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and 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, which is the nucleotide sequence that is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0081] Endogenous: As used herein, "endogenous" refers to any material that is produced from or within a particular organism, cell, tissue, or system.

[0082] Exogenous: As used herein, the term "exogenous" refers to any material that is introduced from or produced outside a particular organism, cell, tissue, or system.

[0083] Expansion: As used herein, the term "expansion" refers to an increase in number, as in an increase in the number of cells, e.g., immune effector cells described herein, e.g., T cells or NK cells. In one embodiment, ex vivo expanded immune cells are increased in number compared to the number initially present in the culture. In another embodiment, the number of ex vivo expanded immune cells is increased compared to other cell types in the culture. In some embodiments, expansion can occur in vivo. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0084] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. 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: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0085] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that includes 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, a nucleotide 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., nucleic acid) found throughout the nucleotide sequence. 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 entire nucleotide.

[0086] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. 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 sequence homology, including allowing for gaps of a specified length in one sequence relative to another sequence when considering which residues in the different sequences "correspond" to each other. Calculation of the percentage 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 reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position, and if a position in the first sequence is occupied by a nucleotide similar to the corresponding position in the second sequence, the molecules are similar at that position.The percentage of homology between the 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.

[0087] 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 occur in subsequent generations, either due to mutations or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term host cell as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., or Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, or P. methanolica), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, or Trichoplusia ni), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadromas). In some embodiments, the cells comprise or are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell selected from the following: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3 A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes.

[0088] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, antibodies (or antibody components) may be considered to be "human" notwithstanding that their amino acid sequences contain residues or elements (e.g., including sequence mutations that may be (initially) introduced by, for example, random or site-specific mutagenesis in vitro or by somatic mutation in vivo) that are not encoded by human germline immunoglobulin sequences, e.g., in one or more CDRs, particularly CDR3.

[0089] Humanized: As known in the art, the term "humanized" refers to a method in which the amino acid sequence is modified from a V antibody derived from a reference antibody produced in a non-human species (e.g., mouse). H and V L The term "humanized" is generally used to refer to antibodies (or antibody components) that contain variable domain sequences, but also contain modifications of those sequences relative to a reference antibody intended to make them more "human-like", i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and has framework (FR) regions that have substantially the amino acid sequence of a human antibody and complementarity determining regions (CDRs) that have substantially the amino acid sequence of a non-human antibody. A humanized antibody contains substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin) and all or substantially all of the framework regions are of human immunoglobulin consensus sequences. In some embodiments, a humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains at least the variable domains of both the light and heavy chains. H1, hinge, C H 2. C H 3, and optionally, a C of the heavy chain constant region. H In some embodiments, the humanized antibody may comprise a humanized V L In some embodiments, the humanized antibody comprises only a humanized V H In some particular embodiments, the humanized antibody comprises only a humanized V H and V L Includes the area.

[0090] Identity: The term "identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that are matched or 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 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0091] Substantially identical: As used herein, the term "substantially identical" refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, 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 of the two sequences are identical over the relevant stretch of residues. In some embodiments, the relevant stretch is the complete sequence. In some embodiments the relevant extension is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of CDRs, references to "substantial identity" typically refer to CDRs having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the reference CDR.

[0092] Immune cell: As used herein, the term "immune cell" refers to a cell that is involved in immune response, for example, promoting 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. The source of immune cells (e.g., T cells or NK cells) can be obtained from a subject.

[0093] 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 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, KIR family receptors, HHLA2, 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, butyrophilin, and A2aR. The term further encompasses biologically active protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof, in some embodiments, the term further encompasses any fragment according to the homology descriptions provided herein.

[0094] Immune response: As used herein, the term "immune response" refers to a cellular and / or systemic response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0095] 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 called BCRs (B cell receptors) or antigen receptors. The five members of this protein class 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 respiratory and urogenital mucous secretions. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0096] Isolated: As used herein, the term "isolated" refers to something that is changed 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.

[0097] "Improve", "Increase", "Inhibit", "Decrease": As used herein, the terms "improve", "increase", "inhibit", "decrease", or grammatical equivalents thereof, refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement is or includes a measurement in a particular system (e.g., a single individual) under otherwise equivalent conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, a suitable reference measurement is or includes a measurement in a comparable system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0098] K D: As used herein, "K D The term "dissociation constant" 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). As used herein 、 "K D The term "K off K on It is equal to divided by.

[0099] K off As used herein, "K off The term "off rate constant" refers to the off rate constant for dissociation of a binder (e.g., an antibody or antigen-binding fragment thereof) from the complex and its partner (e.g., the epitope to which an antibody or antigen-binding fragment thereof binds).

[0100] K on As used herein, "K on The term "on rate constant" refers to the on rate constant of 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).

[0101] 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 the subject in the absence of a treatment or compound and / or compared to the level and / or nature of the response in an otherwise identical but untreated subject. The term encompasses mediating a beneficial therapeutic response in a subject, preferably a human, by perturbing and / or affecting a native signal or response. Monoclonal antibody: "Monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during production of the monoclonal antibody preparation), such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0102] 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, in some embodiments, 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., "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, 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, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars compared to the natural residue (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). 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, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template, e.g., in vivo or in vitro, regeneration in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid has 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, 200, 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.

[0103] 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, in the same reading frame, and, where necessary, to link two protein coding regions.

[0104] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0105] 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, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some 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, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as 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 used in pharmaceutical formulations.

[0106] 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 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.

[0107] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it is artificially designed and / or created. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., modifications of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, and the like, e.g., combinations thereof. In some embodiments, the 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 moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be added to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and thus can be considered members of the same class or family of polypeptides.For each such class, the present specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known. In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (in some embodiments, with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (in some embodiments, with all polypeptides in the class). For example, in some embodiments, a member polypeptide exhibits an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or contains at least one region (e.g., a conserved region which, in some embodiments, may be or contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically encompasses at least 3-4, and often up to 20 or more amino acids, and in some embodiments the conserved region encompasses 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. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide, hi some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of its parent polypeptide.

[0108] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a sequence of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., it may be a glycoprotein, a proteoglycan, etc.) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may include, for example, two or more polypeptide chains linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0109] Recombinant: As used herein, refers to designed, engineered by recombinant means, prepared, expressed, produced, manufactured, and / or isolated, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant human polypeptide library (see, e.g., 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 (see, e.g., Taylor Nuc. Acids Res. 20:6287, 1992; Little Immunology Today 12:364, 2000; Kellermann Curr. Opin. Biotechnol. 20:131, 2000; 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 arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources. For example, in some embodiments, the recombinant antibody polypeptide consists of sequences found in the germline of the source organism of interest (e.g., human, mouse, etc.).In some embodiments, a recombinant antibody has amino acid sequences that result from mutagenesis (e.g., in vitro or in vivo, e.g., in a transgenic animal) such that the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to germline VH and VL sequences, but are sequences that do not naturally occur in vivo within the germline antibody repertoire.

[0110] Signal transduction pathway: As used herein, the term "signal transduction pathway" refers to the biochemical relationship between multiple signaling molecules that are responsible for transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0111] 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 span 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, Skerra et al. (1988) Science 242:1038-1041.

[0112] 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 that is 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 / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. 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 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, e.g., 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., is 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 reading this disclosure will understand that a particular small molecule compound may be provided and / or utilized in any of a variety of forms, such as, for example, crystalline forms, salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), or isotopic forms. 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, and in some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of a racemic mixture. Those skilled 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 skilled in the art will appreciate that certain small molecule compounds have structures that allow for isotopic substitution (e.g., for H. 2 H or 3 H 、 Regarding 12C 11 C. 13 C, or 14 Regarding C and 14N 13 N or 15 For N and 16O 17 O or 18 Regarding O and XXC 36 For Cl and XXF 18It will be understood that such small molecules may have one or more isotopically modified forms, or mixtures thereof, according to the present disclosure. In some embodiments, reference to a particular small molecule compound may relate to a particular form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., in the form of an acid addition or base addition salt, depending on the compound), and 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 naturally occurring or found compound, the compound may be provided and / or utilized in accordance with the present disclosure in a form that is different from that in which it is naturally occurring or found in nature. One of skill in the art will understand that in some embodiments, an absolute or relative amount of a compound, or a preparation of a particular small molecule compound containing a particular form thereof, that differs from the absolute or relative amount (e.g., another component of the preparation, including another form of the compound) of a compound or form present in a reference preparation of interest (e.g., a primary sample from a source of interest, such as a biological or environmental source) will differ 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 distinct form from a racemic mixture of the compound, a particular salt of a small molecule compound is considered to be a distinct form from another salt form of the compound, a preparation that contains only a form of the compound that contains one stereoisomer of a double bond ((Z) or (E)) is considered to be a distinct form from a form of the compound that contains the other stereoisomer of the double bond (E) or (Z), or a preparation in which one or more atoms are a different isotope than present in a reference preparation is considered to be a distinct form.

[0113] 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 predisposed to a disease, disorder, or condition, and in some embodiments, a predisposed subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or a reference population) of developing a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., a clinical symptom of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, a subject is an individual to whom and / or has been administered a diagnosis and / or therapy.

[0114] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0115] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0116] 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., a population of modified immune effector cells described herein, or a KIR3DL3 inhibitor described herein.

[0117] Treatment: As used herein, the term "treatment" refers to treatment and / or prophylaxis. The therapeutic effect is obtained, for example, by suppression, amelioration, or eradication of a disease state.

[0118] Therapeutic Agent: As used herein, the term "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 the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Therapeutic agents include, but are not limited to, a population of modified immune effector cells as described herein and / or at least one KIR3DL3 inhibitor as described herein.

[0119] 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.

[0120] Treatment: As used herein, the terms "treat", "treatment", or "treating" refer to partial or complete alleviation, amelioration, delay in onset, inhibition, prevention, mitigation, and / or reduction of the incidence and / or severity of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who does not exhibit signs or features of a disease, disorder, and / or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of a disease, disorder, and / or condition, e.g., for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject who exhibits established, severe, and / or late signs of a disease, disorder, or condition. In some embodiments, treatment can include administering to the subject a population of modified immune effector cells (e.g., T cells or NK cells) as described herein and / or at least one KIR3DL3 inhibitor as described herein.

[0121] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may include precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic cells. In some embodiments, a tumor is associated with or is a sign of cancer. In some embodiments, a tumor may be a dispersed or liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0122] 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 individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0123] In particular, provided herein are anti-KIR3DL3 antibodies and antigen-binding fragments thereof. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein specifically bind to KIR3DL3 (e.g., human KIR3DL3) with high affinity. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein block KIR3DL3 binding to HHLA2. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein specifically bind to KIR3DL3 expressed on NK cells. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein block HHLA2-mediated suppressive activity in T cells. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein enhance NK cell killing of HHLA2-expressing tumor cells. In some embodiments, the anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein enhance anti-tumor activity. Thus, the present disclosure provides anti-KIR3DL3 antibodies and antigen-binding fragments thereof described herein that may be useful in methods of treating various diseases, disorders and conditions, such as cancer, as described herein, and in methods of modulating immune responses.

[0124] Immune effector cells The present disclosure provides, inter alia, immune effector cells (e.g., NK cells or T cells) as described herein modified with at least one immune cell activator and / or at least one KIR3DL3 inhibitor to produce a population of modified immune effector cells. Thus, in some embodiments, the population of modified immune effector cells exhibits enhanced proliferation, e.g., compared to proliferation prior to modification. In some embodiments, the population of modified immune effector cells exhibits increased endogenous expression of at least one cytokine, e.g., compared to endogenous expression of at least one cytokine prior to modification. A method of making a population of modified immune effector cells as described herein can include (i) contacting the population of immune effector cells with at least one immune cell activator as described herein, and / or (ii) contacting the population of immune effector cells with at least one KIR3DL3 inhibitor as described herein.

[0125] As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response, e.g., promoting an immune response. Examples of immune effector cells include, but are not limited to, natural killer (NK) cells, T cells (e.g., alpha / beta T cells or gamma / delta T cells), natural killer T (NKT) cells, B cells, mast cells, and bone marrow-derived phagocytes.

[0126] As used herein, the term "modified" or "modification" refers to an altered state or structure of a cell (e.g., an immune effector cell as described herein) or a molecule as described herein. The cell may be modified by the introduction of one or more agents as described herein (e.g., at least one immune cell activator as described herein or at least one KIR3DL3 inhibitor as described herein). The molecule may be modified in many ways, including chemically, structurally, and functionally. In some embodiments, the modified immune effector cell has improved effector function as a result of the modification, e.g., contacting the immune effector cell with at least one immune cell activator as described herein (e.g., a cytokine agent as described herein) and / or at least one KIR3DL3 inhibitor as described herein. In some embodiments, the at least one KIR3DL3 inhibitor is one or more anti-KIR3DL3 antibodies or antigen-binding fragments thereof as described herein.

[0127] T cells In some embodiments, the immune effector cells include or are T cells. T cells can have effector functions (Teff) that increase immune responses by expression of one or more T cell receptors (TCRs). In some embodiments, the effector functions include or are one or more of cytokine secretion, cytotoxic activity, and / or anti-self recognition. Conventional T cells (Tconv or Teff) can be any T cell population that is not a T regulatory cell (Treg), including, but not limited to, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated, for example, into Thl or Th2 lineages. In some embodiments, the Teff includes or is a CD4+ Teff, such as a CD4+ helper T cell (e.g., ThO, Thl, Tfh, or Thl7). In some embodiments, the Teff includes or is a CD8+ cytotoxic T cell. In some embodiments, the Teff includes or is a subset of non-Treg T cells. In some embodiments, the cytotoxic T cells are CD8+ T lymphocytes.

[0128] Prior to the modification of T cells as described herein, a source of T cells can be obtained from a subject. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumor. In some embodiments, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation or magnetic bead separation. Any number of T cell lines available in the art can also be used.

[0129] T cells may be expanded by any method known in the art, such as contacting T cells with a surface bound to an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. T cell populations may be stimulated using a variety of methods known in the art, such as contacting with an anti-CD3 antibody, or an antigen-binding fragment thereof. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule may be used. For example, an anti-CD28 antibody may mimic the activity of B7-1 or B7-2 by activating naive T cells via CD28 prior to translocation of CTLA4 to the cell surface and subsequent T cell suppression. In some embodiments, a population of T cells (e.g., CD4+ T cells or CD8+ T cells) may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. In some embodiments, a population of T cells (e.g., CD4+ T cells or CD8+ T cells) can be contacted with an effective amount of a cytokine (e.g., IL-2 or IL-15) under conditions suitable to stimulate proliferation of the T cells.

[0130] NK cells In some embodiments, the immune effector cells include or are natural killer (NK cells). NK cells can exhibit cytolytic activity against various targets through exocytosis of cytoplasmic granules containing various proteins, including perforin and granzyme proteases. Killing by NK cells can be triggered in a contact-dependent, non-phagocytic process that does not require prior sensitization to antigen. Human NK cells can be characterized by the presence of cell surface markers CD16 and CD56, and the absence of T cell receptor (CD3).

[0131] Mature NK cells, NK progenitor cells, or a mixed population of NK progenitor cells and mature NK cells can be used in the methods and compositions described herein. Mature NK cells include or are committed NK cells that have characteristic surface markers (e.g., CD16 and CD56) and NK cell functions and lack the potential for further differentiation. NK progenitor cells can be derived from common lymphoid progenitors (CLPs).

[0132] Prior to the modification of NK cells as described herein, a source of NK cells can be obtained from a subject. NK cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, NK cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation or magnetic bead isolation. Any number of NK cell lines available in the art (e.g., NK-92 cell line) can be used.

[0133] NK cells may be stimulated using a variety of methods known in the art, such as by incubating with anti-CD20 antibodies or by co-culturing with CD20-expressing cells. In some embodiments, the activation state of NK cells is determined by assessing the expression of markers (e.g., CD25, CD69, CD137, CD16, CD56, CD96, CD226, TIGIT, KIR2DL5, and / or NKG2D). Cell surface expression of markers may be determined, for example, by FACS analysis or immunohistological staining techniques.

[0134] Antibody-dependent cell-mediated cytotoxicity (ADCC) can also be determined for the NK cells described herein. ADCC refers to a form of cytotoxicity in which secreted antibodies bound to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells. To assess the ADCC activity of NK cells, an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 can be performed.

[0135] The immune effector cells described herein can generally be produced using any of the methods described in, e.g., U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,86 9, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005, each of which is incorporated by reference in its entirety.

[0136] Chimeric Antigen Receptor In some embodiments, an immune effector cell (e.g., a T cell or an NK cell) described herein may comprise at least one CAR. Thus, in some embodiments, an immune effector cell comprising at least one CAR comprises (a) an extracellular domain (e.g., an extracellular domain described herein), (b) a transmembrane domain (e.g., a transmembrane domain described herein), and (c) an intracellular domain (e.g., an intracellular domain described herein).

[0137] In some embodiments, the CAR comprises an antigen binding domain that binds, for example, to an antigen on a target cell. In some embodiments, the tumor antigen comprises CD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), TNF receptor family member B-cell maturation (BCMA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, or CD24.

[0138] In some embodiments, the CAR comprises one or more extracellular leader domains, one or more extracellular hinge domains, and / or one or more intracellular costimulatory domains. In some embodiments, the CAR comprises a leader sequence at the N-terminus. In some embodiments, the CAR comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0139] In some embodiments, a CAR comprises a transmembrane domain, e.g., connecting the extracellular domain to the intracellular domain. In some embodiments, the transmembrane domain is naturally associated with one or more other domains of the CAR. In some embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, ROR1, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4) , CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C.

[0140] In some embodiments, the CAR comprises one or more intracellular domains. In some embodiments, the intracellular domain of the CAR comprises at least one domain involved in signal activation and / or transduction. In some embodiments, the intracellular domain is or comprises at least one signaling domain. In some embodiments, the intracellular signaling domain comprises or is a functional signaling domain from one or more of TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain can further comprise a costimulatory signaling domain. A costimulatory signaling domain refers to a portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Examples of such costimulatory molecules include, but are not limited to, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1 (also known as PD1), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0141] KIR3DL3 inhibitors The present disclosure provides, inter alia, KIR3DL3 inhibitors. In some embodiments, a population of immune effector cells described herein is contacted with at least one KIR3DL3 inhibitor and at least one immune cell activator (e.g., prior to or substantially simultaneously with at least one KIR3DL3 inhibitor) to form a population of modified immune effector cells. In some embodiments, at least one KIR3DL3 inhibitor is administered to a subject in combination with a population of modified immune effector cells (e.g., immune effector cells that are contacted with at least one immune cell activator to form a population of modified immune effector cells prior to administration to the subject).

[0142] HHLA2 is a B7 family member 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 with TMIGD2 selectively stimulates cell proliferation and cytokine production. HHLA2 also binds to KIR3DL3, a receptor on T cells and NK cells, resulting in the inhibition of T cell and NK cell activation. The present disclosure provides KIR3DL3 inhibitors for use in combination with immune cell activators to treat various cancers, including solid tumors and hematological tumors.

[0143] 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, KIR3DP1, KIR3DL4, and KIR3DL2), but the gene content of the KIR gene cluster differs between haplotypes. KIR proteins are classified by 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 transduce inhibitory signals upon ligand binding via immune tyrosine-based inhibitory motifs (ITIMs), whereas KIR proteins with short cytoplasmic domains lack ITIM motifs and instead associate with TYRO protein tyrosine kinase binding proteins to transduce activating signals. The ligands of some KIR proteins are a subset of HLA class I molecules, and thus 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.

[0144] 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 the National Center for Biotechnology Information (NCBI). For example, at least one human KIR3DL3 isoform is known, namely human KIR3DL3 (NM_153443.4) encoded by transcript (NP_703144.3). Nucleic acid and polypeptide sequences of KIR3DL3 orthologues in organisms other than humans are known and include, but are 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).

[0145] In some embodiments, the KIR3DL3 inhibitor exhibits binding affinity to KIR3DL3 or a fragment thereof (e.g., as assessed by a diagnostic assay such as immunohistochemistry (IHC), Western blot, intracellular flow, or ELISA). In some embodiments, the KIR3DL3 inhibitor exhibits the ability to inhibit KIR3DL3 binding to HHLA2.

[0146] Anti-KIR3DL3 antibodies and antigen-binding fragments Disclosed herein are methods, compositions, and formulations that include inhibitors of KIR3DL3, such as anti-KIR3DL3 antibodies or antigen-binding fragments thereof. For example, the anti-KIR3DL3 antibodies or fragments thereof may specifically bind to an epitope on KIR3DL3.

[0147] In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is a monoclonal antibody. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is a full-length antibody, for example, comprising an immunoglobulin Fc region. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is a multispecific antibody, for example, comprising a plurality of immunoglobulin variable domain sequences, where a plurality of first immunoglobulin variable domain sequences have binding specificity for a first epitope and a plurality of second immunoglobulin variable domain sequences have binding specificity for a second epitope. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is a bispecific antibody molecule. In some embodiments, the antibody or antigen-binding fragment thereof is affinity matured or has been affinity matured.

[0148] An anti-KIR3DL3 antibody or antigen-binding fragment thereof can comprise a heavy (H) chain variable domain sequence (VH) and a light (L) chain variable domain sequence (VL). In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is a heavy chain and a light chain (half antibody). In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises or is two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modification of whole antibodies or de novo synthesized antibodies using recombinant DNA technology. Such functional antibody fragments can retain the ability to selectively bind to KIR3DL3.

[0149] Examples of antigen-binding fragments of anti-KIR3DL3 antibodies include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) Fd fragments consisting of the VH and CH1 domains, (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody, (v) diabody (dAb) fragments consisting of the VH domain, (vi) camelid or camelized variable domains, (vii) scFv, and (viii) single domain antibodies. Antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Anti-KIR3DL3 antibodies and antibody fragments can be derived from any class of antibody, including but not limited to IgG, IgA, IgM, IgD, and IgE, as well as any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of anti-KIR3DL3 antibodies or antigen-binding fragments thereof can be monoclonal or polyclonal. Anti-KIR3DL3 antibodies or antigen-binding fragments thereof can also be human, humanized, CDR-grafted, or in vitro generated. Anti-KIR3DL3 antibodies or fragments can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Anti-KIR3DL3 antibodies or antigen-binding fragments can also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody."

[0150] The VH and VL regions can be subdivided into regions of hypervariability called "complementarity determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FR or FW). As used herein, the terms "complementarity determining region" and "CDR" refer to sequences of amino acids within an antibody variable region that confer antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The extent of the framework regions and CDRs can be precisely 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).

[0151] In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (VH) comprising one, two, or three VH CDR sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VH CDRs, respectively, of Table 1, and / or (b) a light chain variable region (VL) comprising one, two, or three VL CDR sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to the VL CDRs, respectively, of Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises (a) a VH having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VH of Table 1, and / or (a) a VL having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL of Table 1. In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a heavy chain of Table 1, and / or (a) a light chain having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a light chain of Table 1.

[0152] In some embodiments, the anti-KIR3DL3 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-KIR3DL3 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, respectively, as disclosed in Table 1. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (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.

[0153] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 10. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 23. In some embodiments, the anti-KIR3DL3 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.

[0154] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 12. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 25. In some embodiments, the anti-KIR3DL3 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.

[0155] In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 27, a VH CDR2 amino acid sequence of SEQ ID NO: 28, and a VH CDR3 amino acid sequence of SEQ ID NO: 29, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 40, a VL CDR2 amino acid sequence of SEQ ID NO: 41, and a VL CDR3 amino acid sequence of SEQ ID NO: 42. In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 30, a VH CDR2 amino acid sequence of SEQ ID NO: 31, and a VH CDR3 amino acid sequence of SEQ ID NO: 32, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 43, a VL CDR2 amino acid sequence of SEQ ID NO: 44, and a VL CDR3 amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (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.

[0156] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 36. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 49. In some embodiments, the anti-KIR3DL3 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.

[0157] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 38. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 51. In some embodiments, the anti-KIR3DL3 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.

[0158] In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 53, a VH CDR2 amino acid sequence of SEQ ID NO: 54, and a VH CDR3 amino acid sequence of SEQ ID NO: 55, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 66, a VL CDR2 amino acid sequence of SEQ ID NO: 67, and a VL CDR3 amino acid sequence of SEQ ID NO: 68. In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 56, a VH CDR2 amino acid sequence of SEQ ID NO: 57, and a VH CDR3 amino acid sequence of SEQ ID NO: 58, and (b) a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 69, a VL CDR2 amino acid sequence of SEQ ID NO: 70, and a VL CDR3 amino acid sequence of SEQ ID NO: 71. In some embodiments, an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprises (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.

[0159] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 62. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 75. In some embodiments, the anti-KIR3DL3 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.

[0160] In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 64. In some embodiments, the anti-KIR3DL3 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% identical or more identical to SEQ ID NO: 77. In some embodiments, the anti-KIR3DL3 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. [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

[0161] Phage display and 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. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137, and Barbas et al. al.(1991)PNAS 88:7978-7982, each of which is incorporated herein by reference in its entirety).

[0162] By way of example, anti-KIR3DL3 antibodies suitable for detecting KIR3DL3 protein are known in the art and include, for example, antibody Cat#s: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U, and FAB8919V (R&D systems); antibody AP52374PU-N (Origene); antibody PA5-26178 (ThermoFisher Scientific); antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988; and OASG01190 (Aviva Systems Biology).

[0163] antigen binding fragment The present disclosure provides, inter alia, anti-KIR3DL3 antigen-binding fragments. As used herein, "anti-KIR3DL3 antigen-binding fragments" includes or is any protein- or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule that contains at least one complementarity determining region (CDR) of VH or VL or KIR3DL3-binding portion 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 KIR3DL3.

[0164] Examples of anti-KIR3DL3 antigen-binding fragments described herein may include (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL, and CH1 domains, (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment comprising the VH and CH1 domains, (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment comprising a VH domain, (vi) a camelid or camelized variable domain, (vii) a scFv, a fusion protein of the VH and VL domains, or (viii) a single domain antibody. In some embodiments, the anti-KIR3DL3 antigen-binding fragment described herein comprises or is a heavy chain and a light chain (e.g., a half antibody).

[0165] How to create The present disclosure provides, inter alia, a method for producing the anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein.In some embodiments, the anti-KIR3DL3 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-KIR3DL3 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.

[0166] Combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92 / 18619; Dower et al. International Publication No. WO 91 / 17271; Winter et al. International Publication No. WO 92 / 20791; Markland et al. International Publication No. WO 92 / 15679; Breitling et al. International Publication No. WO 93 / 01288; McCafferty et al. International Publication No. WO 92 / 01047; Garrard et al. International Publication No. WO 92 / 09690; Ladner et al. International Publication No. WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibody Hybridomas 3:81-85, Huse et al. (1989) Science 246:1275-1281, Griffiths et al. (1993) EMBO J 12:725-734, Hawkins et al. (1992) J Mol Biol 226:889-896, Clackson et al. (1991) Nature 352:624-628, Gram et al. (1992) PNAS 89:3576-3580, Garrad et al. (1991) Bio / Technology 9:1373-1377, Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137, and Barbas et al. (1991) PNAS 88:7978-7982, each of which is incorporated herein by reference in its entirety).

[0167] In some embodiments, the anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein may be derived from other species. Humanized antibodies are antibodies produced by recombinant DNA technology in which some or all amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding (e.g., constant and / or framework regions of the variable domain) are used to replace the corresponding amino acids from the light or heavy chain of a cognate non-human antibody. As an example, a humanized version of a mouse antibody for a given antigen would have, in both the heavy and light chains, (1) the constant region of a human antibody, (2) FRs from the variable domain of a human antibody, and (3) CDRs from a mouse antibody. The human FRs may be selected based on their highest sequence homology to the mouse FR sequences. If necessary, one or more residues in the human FRs may be changed to the residues at the corresponding positions in the mouse antibody to maintain the binding affinity of the humanized antibody to its target. This change is sometimes referred to as a "back mutation." Similarly, forward mutations may be made back to the mouse sequence for desired reasons, e.g., stability or affinity to the target. Because the former contain significantly fewer non-human components, humanized antibodies are generally less likely to provoke an immune response in humans than chimeric human antibodies.

[0168] Methods for humanizing non-human antibodies are well known in the art. Suitable methods for producing humanized antibodies according to the present disclosure are described, for example, in Winter EP0239400, Jones et al., Nature 321:522-525(1986), Riechmann et al., Nature 332:323-327(1988), Verhoeyen et al., Science 239:1534-1536(1988), Queen et al., Proc.Nat.Acad.ScL USA 86:10029(1989), U.S. Patent No. 6,180,370, and Orlandi et al., Proc.Natl.Acad.Sd.USA 86:3833(1989), the disclosures of each of which are incorporated herein by reference in their entirety. In general, grafting of non-human (e.g., murine) CDRs into a human antibody is accomplished as follows: cDNAs encoding VH and VL are isolated from hybridomas, and the nucleic acid sequences encoding VH and VL containing CDRs are determined by sequencing. The nucleic acid sequences encoding the CDRs are inserted into the corresponding regions of a human antibody VH or VL coding sequence and linked to human constant region gene segments of the desired isotype (e.g., γl for CH and κ for CL). The humanized heavy and light chain genes are co-expressed in mammalian host cells (e.g., CHO or NSO cells) to produce soluble humanized antibodies. To facilitate large-scale production of antibodies, it is often desirable to select high expressers, for example, using DHFR or GS genes in producer lines.

[0169] In some embodiments, the anti-KIR3DL3 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 a variety of 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). Techniques for the preparation of human monoclonal antibodies are also available, e.g., 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.

[0170] nucleic acid The present disclosure provides, inter alia, nucleic acids encoding KIR3DL3 binding agents (e.g., anti-KIR3DL3 antibodies or antigen-binding fragments thereof) described herein. The present disclosure includes nucleic acids encoding one or more heavy chains, VH domains, heavy chain FRs, heavy chain CDRs, heavy chain constant domains, light chains, VL domains, light chain FRs, light chain CDRs, light chain constant domains, or other immunoglobulin-like sequences, antibodies, or antigen-binding fragments disclosed herein. Such nucleic acids may be present in a vector. Such nucleic acids may be present in the genome of a cell, e.g., a cell of a subject in need of treatment, or a cell for the production of an antibody, e.g., a mammalian cell for the production of an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein.

[0171] Nucleic acids encoding KIR3DL3 binding agents (e.g., anti-KIR3DL3 antibodies or antigen-binding fragments thereof) described herein may be modified to include codons optimized for expression in a particular cell type or organism. Codon-optimized sequences are synthetic sequences, preferably encoding the same polypeptide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by a non-codon-optimized parent polynucleotide. In some embodiments, the coding region of a nucleic acid encoding a KIR3DL3 binding agent described herein may, in whole or in part, include modified sequences 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.

[0172] The nucleic acid constructs of the present disclosure can be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid can be operably linked to an expression control sequence. Vectors comprising any of the nucleic acids described herein or fragments thereof are further provided by the present disclosure. Any of the nucleic acids described herein or fragments thereof can be cloned into any suitable vector and used to transform or transfect any suitable host. The selection of vectors and methods for constructing them are generally known to those of skill in the art (see, for example, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)).

[0173] For example, conventional techniques including electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection can be used to introduce foreign nucleic acid (e.g., DNA or RNA) into prokaryotic or eukaryotic host cells. Desirably, the vector can optionally include regulatory sequences, such as transcription and / or translation initiation and / or termination codons, that are specific to the type of host into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA or RNA. In some embodiments, the vector includes regulatory sequences that are specific to the genus of the host cell. In some embodiments, the vector includes regulatory sequences that are specific to the species of the host.

[0174] In addition to the replication system and the inserted nucleic acid, the nucleic acid construct can include one or more marker genes that allow for the 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 nutrient-deficient hosts to provide protrophy.

[0175] The expression vector may comprise a native or non-native promoter operably linked to the isolated or purified nucleic acid as described above. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific, and / or development-specific, is within the skill of the artisan. Similarly, combining such a nucleic acid with a promoter is also within the skill of the artisan.

[0176] Suitable vectors include vectors designed for propagation and expansion, and / or expression. For example, cloning vectors may be selected from the pUC series, pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors that can be used include pBI110, pBI101.2, pBI101.3, pBI121, or pBIN19 (Clontech). Examples of animal expression vectors that can be used include pEUK-C1, pMAM, or pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used following the manufacturer's recommendations.

[0177] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, aid in the isolation of nucleic acids encoding KIR3DL3-binding agents described herein, or improve the introduction of nucleic acids 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), the contents of each of which are incorporated herein by reference in their entirety).

[0178] 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.

[0179] Any method known to those of skill in the art for the insertion of a nucleic acid into a vector may be used to construct expression vectors encoding the anti-human KIR3DL3 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).

[0180] Antibodies that bind to the same epitope In some embodiments, the anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein include antibodies and antibody fragments that bind to the same epitope as the KIR3DL3-binding antibodies shown in Table 1 described herein. Thus, additional antibodies and antibody fragments can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies described herein in KIR3DL3 binding assays. The ability of a test antibody to inhibit binding of the antibodies and antibody fragments described herein to a KIR3DL3 protein (e.g., human KIR3DL3) demonstrates that the test antibody can compete with that antibody or antibody fragment for binding to KIR3DL3. Such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximal) epitope on the KIR3DL3 protein as the antibody or antibody fragment it competes with. In some embodiments, an antibody that binds to the same epitope on KIR3DL3 as the anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0181] KIR3DL3 gene expression regulator Disclosed herein are methods, compositions, and formulations that include KIR3DL3 inhibitors that modulate gene expression, such as miRNA, shRNA, siRNA, CRISPR / Cas guide systems, TALENs, or ZFNs.

[0182] In some embodiments, the KIR3DL3 inhibitor comprises or is a gene expression regulator. The gene expression regulator can comprise RNAi molecules (e.g., double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA)) and triplex-forming oligonucleotides (TFOs). The gene expression regulator can also comprise any modified version of the aforementioned RNA molecules, thus including synthetic chemically modified RNA.

[0183] In some embodiments, the RNAi can be an miRNA that reduces the level of KIR3DL3 in a cell (e.g., an NK cell or a T cell). In some embodiments, the RNAi can be an shRNA that reduces the level of KIR3DL3 in a cell (e.g., an NK cell or a T cell). In some embodiments, the RNAi can be an siRNA that reduces the level of KIR3DL3 in a cell (e.g., an NK cell or a T cell). Modulation of KIR3DL3 expression using miRNAs and siRNAs is described, for example, in Nutalai et al., Genes (Basel). 2019; 10(8):603, which is incorporated by reference herein in its entirety.

[0184] Commercially available siRNAs and shRNAs for reducing KIR3DL3 expression include shRNA products #TF303684, TR303684, TG303684, TL303684, and TL303684V (HexaBiogen Groupe CliniSciences); shRNA products #sc-60892-SH and sc-60892-V (Santa Cruz Biotechnology, Inc.); siRNA product #SR314516 (HexaBiogen Groupe CliniSciences); and siRNA product #sc-60892 (Santa Cruz Biotechnology, Inc.).

[0185] In some embodiments, the KIR3DL3 inhibitor comprises or is an endonuclease. The endonuclease generates a double-stranded DNA break at a desired location in the genome and can repair the break using host cell mechanisms, for example, using homologous recombination or non-homologous end joining. Classes of endonucleases that can be used for gene editing include clustered regularly interspaced short palindromic repeats(s) (CRISPR), transcription activator-like effector nucleases (TALENs) (see, for example, U.S. Patent No. 8,697,853, and U.S. Publication Nos. 20150118216, 20150079064, and 20140087426), zinc finger nucleases (ZFNs) (see, for example, U.S. Patent Nos. 8,956,828, 8,921,112, and 8,922,113). Nos. 8,846,578, 8,569,253), meganucleases (see, e.g., U.S. Patent Nos. 8,802,437, 8,445,251, and 8,338,157, and U.S. Publication Nos. 20130224863, 20110113509, and 20110033935), and homing endonucleases (see, e.g., U.S. Publication No. 20150166969, and U.S. Patent No. 9,005,973).

[0186] In some embodiments, the KIR3DL3 inhibitor comprises or is a CRISPR / Cas system. As used herein, the term "CRISPR" or "CRISPR for KIR3DL3" or "CRISPR inhibiting KIR3DL3" refers to a set of clustered regularly interspaced short palindromic repeats or a system that comprises such a set of repeats. "Cas" as used herein refers to CRISPR-associated proteins. "CRISPR / Cas system" refers to a system derived from CRISPR and Cas, which can be used to silence or mutate the KIR3DL3 gene in immune effector cells, such as NK cells or T cells, as described herein.

[0187] Commercially available CRISPR constructs for regulating KIR3DL3 expression include CRISPR product #KN224383, KN224383BN, KN224383RB, and KN224383LP (Origene Technologies), CRISPR product #K1151421, K1151401, K1151402, K1151403, K1151404, K1151405, K1151406, K1151407, K1151408, and K1151411 (Applied Biological Materials), and CRISPR product #sc-406227, sc-406227-KO-2, sc-406227-HDR-2, sc-406227-NIC, and sc-406227-NIC-2 (Santa Cruz). Biotechnology).

[0188] In some embodiments, the KIR3DL3 inhibitor comprises or is TALEN.As used herein, the term "TALEN" or "TALEN to KIR3DL3" or "TALEN inhibiting KIR3DL3" refers to the transcription activator-like effector nuclease as an artificial nuclease that can be used to edit the KIR3DL3 gene in immune effector cells, such as NK cells or T cells, as described.

[0189] In some embodiments, the KIR3DL3 inhibitor comprises or is a ZFN. As used herein, the term "ZFN" or "zinc finger nuclease" or "ZFN to KIR3DL3" or "ZFN inhibiting KIR3DL3" refers to zinc finger nuclease as an artificial nuclease that can be used to edit the KIR3DL3 gene in immune effector cells, such as NK cells or T cells, as described herein.

[0190] Demethylating Agents In some embodiments, the KIR3DL3 inhibitor comprises or is a demethylating agent. As used herein, "demethylating agent" refers to a chemical that can inhibit methylation. In some embodiments, the demethylating agent comprises or is 5-aza-2-deoxycytidine (Aza), as described, for example, in Trundley et al, Immunogenetics (2006) 57:904-916, the entirety of which is incorporated herein by reference. In some embodiments, the demethylating agent comprises or is 5-azacytidine. In some embodiments, the demethylating agent comprises or is 1-β-D-arabinofuranosyl-5-azacytosine. In some embodiments, the demethylating agent comprises or is dihydro-5-azacytidine.

[0191] Immune cell activator Disclosed herein are methods, compositions, and formulations comprising at least one immune cell activator, such as a cytokine agent, a costimulatory antibody or antigen-binding fragment thereof, a polypeptide, a glycoprotein, or an exogenous cell.

[0192] As used herein, the term "immune cell activator" refers to an agent that activates immune effector cells (e.g., NK cells or T cells) described herein, thereby modifying the immune effector cells (e.g., by increasing proliferation and / or endogenous expression of interleukins). Immune cell activators can include, but are not limited to, cytokine agents (e.g., interleukins, e.g., cytokines (e.g., IL-2, IL-15, IL-12, IL-17, and / or IL-18)), antibodies or fragments thereof (e.g., costimulatory antibodies or fragments thereof), polypeptides, glycoproteins, exogenous cells (e.g., artificial antigen presenting cells), nucleic acids, antibiotics, anti-inflammatory agents, chimeric antigen receptors, growth factors, enzymes, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. Immune cell activators can be endogenously expressed or exogenous to the immune effector cells described herein. The immune cell activator may bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on an immune effector cell as described herein. The immune cell activator may diffuse into, be transported into, or act within the cell. The immune activator may be an antibody or antigen-binding fragment thereof, or a small molecule that reduces or blocks the inhibitory activity of one or more of a checkpoint protein (e.g., 4-1BB, CD40, CD28, OX40, GITR, PD-1, PD-L1, PD-L2, TIM-3, TGF-β, or LAG-3), an enzyme (e.g., CD39 or CD73), and / or a receptor (e.g., CTLA-4 or CD3).

[0193] In some embodiments, contacting an immune effector cell described herein with an immune cell activator described herein increases proliferation and / or endogenous expression of at least one interleukin (e.g., a cytokine) by the immune effector cell. In some embodiments, contacting an immune effector cell described herein with at least one immune cell activator increases proliferation, e.g., compared to immune effector cells not contacted with at least one immune cell activator. In some embodiments, contacting an immune effector cell described herein with at least one immune cell activator increases endogenous expression of at least one interleukin described herein, e.g., compared to immune effector cells not contacted with at least one immune cell activator.

[0194] In some embodiments, the immune cell activator comprises or is a cytokine agent. In some embodiments, the immune cell activator comprises or is an interleukin. In some embodiments, the cytokine agent is or comprises IL-2, IL-15, IL-12, IL-17, IL-18, IL-21, IFNγ, or TNFα. In some embodiments, the cytokine agent is or comprises IL-2. In some embodiments, the cytokine agent is or comprises IL-15. In some embodiments, the cytokine agent is or comprises IL-12. In some embodiments, the cytokine agent is or comprises IL-17. In some embodiments, the cytokine agent is or comprises IL-18. In some embodiments, the cytokine agent is or comprises IL-21.

[0195] IL-2 is a member of a cytokine family that includes IL-4, IL-7, IL-9, IL-15, and IL-21, each member of the family having four alpha-helical bundles. IL-2 can be a T cell growth factor and is endogenously secreted in vivo by both CD4+ helper T cells and CD8+ T cells. IL-2 signals through the IL-2 receptor complex, which consists of three chains: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2R (CD132). In some embodiments, IL-2 binds to IL-2Rα (CD25), IL-2Rβ (CD122), and / or IL-2R (CD132) to activate immune effector cells, or proliferation of immune effector cells, as described herein. In some embodiments, IL-2 expands T cells (e.g., CD4+ helper T cells and / or CD8+ T cells). In some embodiments, IL-2 does not substantially expand Tregs, hi some embodiments, IL-2 enhances cytotoxicity and / or expands NK cells.

[0196] In some embodiments, the cytokine agent comprises or is an inhibitor of a suppressor of cytokine signaling (e.g., an endogenous suppressor of cytokine signaling). In some embodiments, the cytokine agent comprises or is a suppressor of cytokine signaling (SOCS) protein.

[0197] In some embodiments, the immune cell activator comprises or is a costimulatory antibody or antigen-binding fragment thereof, or a costimulatory small molecule. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof binds to CD3, 4-1BB, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TGF-β, or LAG-3. In some embodiments, the costimulatory small molecule binds to CD3, 4-1BB, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TGF-β, LAG-3, CD39, or CD73.

[0198] In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-CD3 antibody or fragment thereof. In some embodiments, the anti-CD3 antibody comprises or is OKT3 (also known as muromonab-CD3 or orthoclon OKT3). In some embodiments, the anti-CD3 antibody or fragment thereof binds to T3, a T cell molecule that associates with the T cell antigen receptor resulting in activation of the T cell. In some embodiments, contacting an immune effector cell described herein with an anti-CD3 antibody or fragment thereof increases cytokine production by the immune effector cell, for example, compared to an immune effector cell not contacted with an anti-CD3 antibody or fragment thereof. In some embodiments, contacting an immune effector cell described herein with an anti-CD3 antibody or fragment thereof enhances proliferation of the immune effector cell, for example, relative to an immune effector cell not contacted with an anti-CD3 antibody or fragment thereof.

[0199] In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-4-1BB antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-CD40 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-CD28 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-OX40 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-GITR antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-CTLA-4 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-PD-1 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-PD-L1 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-PD-L2 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-TIM-3 antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-TGF-β antibody or fragment thereof. In some embodiments, the costimulatory antibody or antigen-binding fragment thereof comprises or is an anti-LAG-3 antibody or fragment thereof.

[0200] In some embodiments, the immune cell activator comprises or is a costimulatory polypeptide. In some embodiments, the costimulatory polypeptide comprises or is a soluble HHLA2 Fc fusion polypeptide. In some embodiments, the soluble HHLA2 Fc fusion polypeptide comprises the extracellular domain of human HHLA2. In some embodiments, the soluble HHLA2 Fc fusion polypeptide comprises a human IgG Fc region. In some embodiments, the soluble HHLA2 Fc fusion polypeptide comprises a human IgM Fc region. In some embodiments, the IgG is IgG1. In some embodiments, the soluble HHLA2 Fc fusion polypeptide binds to KIR3DL3 in immune effector cells described herein. In some embodiments, the soluble HHLA2 Fc fusion polypeptide blocks the interaction of KIR3DL3 and HHLA2 in immune effector cells described herein. Soluble HHLA2 Fc fusion polypeptides are described in WO2014 / 133728, which is incorporated herein by reference in its entirety.

[0201] In some embodiments, the immune cell activator comprises or is a glycoprotein. In some embodiments, the costimulatory glycoprotein comprises or is a fibronectin protein or a fragment thereof. Fibronectin is an endogenously expressed high molecular weight (about 440 kDa) glycoprotein of the extracellular matrix known to bind to integrins spanning membranes. In some embodiments, the immune cell activator comprises or is a recombinant human fibronectin protein or a fragment thereof. In some embodiments, the recombinant human fibronectin fragment comprises a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. In some embodiments, the fibronectin protein or a fragment thereof comprises or is RetroNectin® (Takara Bio Inc.). In some embodiments, contacting the immune effector cells described herein with a fibronectin protein or a fragment thereof (e.g., RetroNectin®) increases the proliferation of the immune effector cells described herein, for example, compared to immune effector cells not contacted with a fibronectin protein or a fragment thereof.

[0202] In some embodiments, the immune cell activator comprises or is a costimulatory exogenous cell. In some embodiments, the costimulatory exogenous cell comprises or is an artificial antigen presenting cell (aAPC). In some embodiments, the aAPC comprises or is a K562-based aAPC. In some embodiments, contacting the immune effector cells described herein with aAPC enhances immune effector cell cytokine production (e.g., IL-2 production) to enhance immune effector cell stimulation, for example, compared to immune effector cells not contacted with aAPC. In some embodiments, contacting the immune effector cells described herein with aAPC enhances immune effector cell proliferation, for example, compared to immune effector cells not contacted with aAPC.

[0203] Treatment methods The present disclosure provides, inter alia, methods of 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 KIR3DL3 inhibitor described herein. In some embodiments, the at least one KIR3DL3 inhibitor is or comprises one or more anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein.

[0204] In some embodiments, the disclosure provides at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprising an amino acid sequence found in Table 1 for use as a medicament. In some embodiments, the disclosure provides at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprising an amino acid sequence found in Table 1 for use in treating a disease, disorder, or condition described herein. In some embodiments, the disclosure provides the use of at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein comprising an amino acid sequence found in Table 1 for the manufacture of a medicament for the treatment of a disease, disorder, or condition described herein.

[0205] The present disclosure provides, inter alia, a method of treating a disease, disorder, or condition in a subject (e.g., a disease, disorder, or condition described herein) comprising administering a pharmaceutical composition comprising a population of modified immune effector cells described herein. In some embodiments, prior to administration, the population of immune effector cells described herein is contacted with at least one immune cell activator described herein and at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof), thereby forming a population of modified immune effector cells.

[0206] The present disclosure also provides, inter alia, a method of treating a disease, disorder, or condition in a subject (e.g., a disease, disorder, or condition described herein) comprising delivering to the subject a pharmaceutical composition comprising a population of modified immune effector cells described herein and administering to the subject a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, prior to administration, the population of immune effector cells is contacted with at least one immune cell activator, thereby forming a population of modified immune effector cells.

[0207] 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. The pharmaceutical compositions described herein can be used to manufacture a medicament for treating a disease, disorder, or condition in a subject, or for stimulating an immune response in a subject.

[0208] In some embodiments, a pharmaceutical composition comprising an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein is administered to a subject. In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein is administered to a subject prior to administering to the subject a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein is administered to a subject after administering to the subject a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein is administered to a subject substantially simultaneously (e.g., co-administered by injection) with a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein and at least one KIR3DL3 inhibitor (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein) is administered to a subject (e.g., by injection).

[0209] In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein is administered to a subject within less than about 3 hours of contacting the immune effector cells with at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a population of modified immune effector cells described herein is administered to a subject within less than about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 30 minutes, or about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours of contacting the immune effector cells with at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein).

[0210] The subject treated with the methods described herein may 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). In some embodiments, the immune effector cells (e.g., NK cells or T cells) may be autologous, allogeneic, or xenogeneic with respect to the subject. The pharmaceutical compositions described herein may be administered to a subject according to the administration regimens described herein, alone or in combination with one or more therapeutic agents, procedures, or modalities.

[0211] Methods are provided for treating (e.g., reducing, inhibiting, or delaying one or more of the progression of) a cancer or tumor in a subject with a pharmaceutical composition comprising an immune cell (e.g., an NK cell or a T cell) described herein and / or a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). The subject may have an adult or pediatric form of cancer. The cancer may be in early, mid, or late stage, or may be a metastatic cancer. In some embodiments, the subject has a cancer that is resistant to a therapeutic agent, including, for example, a cytokine agent described herein.

[0212] Methods are provided for treating (e.g., one or more of reducing, inhibiting, or delaying progression of) a sign or symptom of cancer in a subject using a pharmaceutical composition comprising an immune cell (e.g., an NK cell or a T cell) described herein and / or a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, the pharmaceutical compositions described herein are useful for delaying the onset, slowing the progression, or ameliorating one or more signs or symptoms of cancer. In some embodiments, the physiological sign or symptom of cancer includes or is 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 cancer cell survival. In some embodiments, the physical signs or symptoms of cancer include or are skin lesions (e.g., a lump or mole), weight loss, digestive problems, discomfort, fatigue, pain, difficulty swallowing, coughing, abnormal bleeding and / or discharge, changes in bowel and / or bladder habits, and / or mental confusion.

[0213] 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, such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as those affecting the lung, breast, ovaries, lymphatic system, gastrointestinal tract (e.g., colon), anus, reproductive and genitourinary tract (e.g., kidney, urinary tract, bladder cells, prostate), pharynx, CNS (e.g., brain cells, neuronal cells or glial cells), head and neck, skin (e.g., melanoma, e.g., cutaneous melanoma), pancreas, and bone (e.g., chordoma).

[0214] 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 cancer (e.g., renal cell carcinoma), cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two or all of 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 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.

[0215] 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., an 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., a metastatic renal cell carcinoma). In some embodiments, the cancer is a breast cancer, e.g., a metastatic breast carcinoma or a stage IV breast carcinoma, e.g., a triple negative breast cancer (TNBC). In some embodiments, the cancer is a virus-associated cancer. In some embodiments, the cancer is an anal canal cancer (e.g., a squamous cell carcinoma of the anal canal). In some embodiments, the cancer is a cervical cancer (e.g., a 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.

[0216] 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.

[0217] Administration of the pharmaceutical compositions described herein can be by any convenient method (e.g., injection, ingestion, infusion, inhalation, implantation, or implantation). In some embodiments, the pharmaceutical compositions disclosed herein are administered by injection or infusion. The pharmaceutical compositions disclosed herein can be administered to a patient intra-arterially, subcutaneously, intravenously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous, intravenous, intramuscular, or intrasternal infusion or injection. In some embodiments, the pharmaceutical compositions described herein are administered by intramuscular or subcutaneous injection. The pharmaceutical compositions described herein can be directly injected into the subject at the site of inflammation, the site of local disease, lymph nodes, organs, tumors, or the site of infection.

[0218] The subject treated with the methods described herein may be a mammal (e.g., a primate, a mouse, a humanized mouse, a rat, a non-human mammal, a domestic animal such as a dog, a cat, a cow, or a horse), and preferably a human (e.g., a patient having or at risk of having a disease, disorder, or condition described herein). The subject may be an animal model of cancer, such as a xenograft animal model of a human-derived cancer. In some embodiments, the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immune checkpoint therapy. In another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and / or immune checkpoint therapy. In certain embodiments, the subject has undergone surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, for example, the cancerous tissue may be located in an inoperable area of ​​the body, such as tissue essential to survival, or in an area where surgical procedures may cause substantial harm to the subject.

[0219] Additional drugs The present disclosure provides, inter alia, one or more additional agents (e.g., two, three, four, five, or more additional agents) that can be administered with an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein in a combination therapy. As used herein, an additional agent may be or include any known treatment for a particular disease, disorder, or condition (e.g., cancer). For example, the additional agent may be or include one or more of a chemotherapeutic agent, an immune checkpoint inhibitor, a gene expression regulator, an immunomodulatory interleukin, an immunomodulatory chemokine, a hormone therapy, a cell-based therapy, a cancer vaccine, an epigenetic modifier (e.g., a histone deacetylase (HDAC) modifier, an immunomodulatory drug, an immunomodulatory antibody, a nutritional supplement, hyperthermia, photodynamic therapy, surgery, radiation, or a transplant.

[0220] In some embodiments, the chemotherapeutic agent includes or is one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., one or more inhibitors of topoisomerase I or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, or combinations thereof.

[0221] In some embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, cefotaxime ... In some embodiments, the chemotherapeutic agent comprises or is one or more of: cytabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, rituxan, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, or vinorelbine. In some embodiments, the chemotherapeutic agent comprises or is an antibody-drug conjugate (ADC).In some embodiments, the ADC is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA The compound includes or is an ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, lorvotuzumab mertansine, or a combination thereof.

[0222] In some embodiments, the immune checkpoint inhibitor comprises or is an agent that targets one or more of the following: CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR.

[0223] In some embodiments, the gene expression regulator comprises or is one or more of an inhibitory nucleic acid, a CRISPR / Cas guide system, a TALEN, or a ZFN. The inhibitory nucleic acid can comprise an RNAi molecule (e.g., double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA)), and a triplex forming oligonucleotide (TFO)). The gene expression regulator can also comprise a modified version of any of the aforementioned RNA molecules, thus including synthetic chemically modified RNA. In some embodiments, the CRISPR / Cas guide system can comprise Cas9, Cas12 (e.g., Cas12a), or Cas13.

[0224] In some embodiments, the immunomodulatory interleukin comprises or is one or more of IL-2, IL-6, IL-7, IL-12, IL-17, or IL-23. In some embodiments, the immunomodulatory chemokine comprises or is one or more of CCL3, CCL26, and CXCL7.

[0225] In some embodiments, the immunomodulatory agent is an immune cell suppressant, a glucocorticoid, a cell suppressant, an immunophilin and its modulators (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, cyclosporin (cyclosporin), pimecrolimus, avetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, or zotarolimus), hydrocortisone (cortisol), cortisone acetate, , prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, leflunomide, teriflunomide, folic acid analogues, methotrexate, antithymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin Min, catechin, opioids, IMPDH inhibitors, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitors, raloxifene, drotrecogin alfa, denosumab, NF-kB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopirin The therapeutic agent may include or be: dol, a nonsteroidal anti-inflammatory drug (NSAID), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), I3C (indole-3-carbinol) / DIM (di-indolemethane) (13C / DIM), Bay11-7082, luteolin, the cell penetrating peptide SN-50, IKBa.-super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any of the foregoing.

[0226] In some embodiments, the immunomodulatory antibody is an antibody that binds to CD40, a toll-like receptor (TLR), OX40, GITR, CD27, or 4-1BB, a T cell bispecific antibody, an anti-IL-2 receptor antibody, an anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CD11 antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, Afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, anti-CD23 antibody, rumiliximab, anti-CD40 antibody, teneliximab, toralizumab, anti-CD40L antibody, ruplizumab, anti-CD62L antibody, acelizumab, anti-CD80 antibody, galiximab, anti-CD147 antibody, gavilimomab, B-lymphocyte stimulator (BLyS) inhibitor antibody, belimumab, CTLA4-Ig fusion protein, abatacept, belatacept, anti-CTLA4 antibody, ipilimumab Mab, tremelimumab, anti-eotaxin 1 antibody, bertilimumab, anti-a4-integrin antibody, natalizumab, anti-IL-6R antibody, tocilizumab, anti-LFA-1 antibody, ozlimomab, anti-CD25 antibody, basiliximab, daclizumab, inolimomab, anti-CD5 antibody, zolimomab, anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atolimumab, cedelizumab, dorlimomab alitox, drixizumab, fontolizumab, gante including or being one or more of nelumab, gomiliximab, levrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, terimomab alitox, bapaliximab, beparimomab, aflibercept, alefacept, rilonacept, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, mepolizumab, IgE inhibitors, omalizumab, IL12 inhibitors, IL23 inhibitors, or ustekinumab.

[0227] In some embodiments, the hormone therapy can be or include tamoxifen, raloxifene, leuprolide, bicalatomid, granisetron, flutamide, or a combination thereof. In some embodiments, the cell-based therapy includes or is chimeric antigen receptor T (CAR-T) cells, CAR-NK cells, TCR-transduced T cells, dendritic cells, tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, irradiated autologous or allogeneic tumor cells, or a combination thereof. In some embodiments, the hyperthermia includes or is local hyperthermia (e.g., external, intraluminal, or interstitial hyperthermia), regional hyperthermia (e.g., deep tissue hyperthermia, local perfusion, or (continuous hyperthermic peritoneal perifusion), or whole body hyperthermia. In some embodiments, the photodynamic therapy includes or is administration of a photosensitizer, such as hematoporphyrin and its derivatives, Verteporfin (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxyhypocerin A, 2BA-2-DMHA, or combinations thereof. In some embodiments, the surgery includes or is surgery to remove cancerous or precancerous tissue. In some embodiments, the transplant includes or is stem cell transplant or organ transplant.

[0228] In some embodiments, the dietary supplement includes or is one or more of vitamin A, vitamin E, vitamin C, and the like (see, e.g., U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publication No. WO 2004 / 004483).

[0229] In some embodiments, the additional agent is administered prior to, substantially simultaneously with, or after administration of an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein. In some embodiments, administration of an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein and an additional agent results in an improvement of the disease, disorder, or condition (e.g., cancer) to a degree greater than the improvement produced by either the bispecific antibody molecule or antigen-binding fragment thereof described herein or the additional agent alone. The difference between the effect of the combination and the effect of each agent alone may be a statistically significant difference. In some embodiments, the effect of the combination may be a synergistic effect. In some embodiments, administration of an anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein in combination with an additional agent allows for the additional agent to be administered at a reduced dose, a reduced number of doses, and / or a reduced frequency of administration compared to a standard administration regimen, e.g., an approved administration regimen for the additional agent.

[0230] Conjugates to therapeutic agents The present disclosure provides, inter alia, anti-KIR3DL3 antibodies or antigen-binding fragments thereof conjugated to one or more therapeutic agents. In some embodiments, the therapeutic agents include or are cytotoxic agents, drugs, and / or radioisotopes. When conjugated to a cytotoxic agent, such conjugates may be referred to as "immunotoxins." Cytotoxic agents include any agent that is detrimental to (e.g., capable of killing) cells. Examples of cytotoxic agents include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs of any of the foregoing.

[0231] The anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein may be administered in combination with other agents, including antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and / or 5-fluorouracil decarbazine), topoisomerase I inhibitors (e.g., deruxtecan), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomas, The anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein can be conjugated to one or more therapeutic agents (e.g., one or more drugs), including, but not limited to, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and / or doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and / or anthramycin (AMC)), and antimitotic agents (e.g., vincristine and / or vinblastine). The anti-KIR3DL3 antibodies or antigen-binding fragments thereof described herein can be conjugated to one or more radioisotopes (e.g., radioactive iodine) to generate cytotoxic radiopharmaceuticals for treating the diseases, disorders, or conditions described herein, such as the cancers described herein.

[0232] Pharmaceutical Compositions The present disclosure provides, inter alia, pharmaceutical compositions comprising a population of immune effector cells (e.g., NK cells or T cells) described herein in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients.

[0233] The present disclosure also provides, inter alia, pharmaceutical compositions comprising at least one KIR3DL3 inhibitor in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical compositions comprise an anti-KIR3DL3 antibody or antigen-binding fragment thereof in combination with one or more pharma-ceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical compositions comprise a population of modified immune effector cells as described herein and at least one KIR3DL3 inhibitor as described herein.

[0234] 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 a population of modified immune effector cells (e.g., NK cells or T cells) described herein and / or at least one KIR3DL3 inhibitor (e.g., an anti-KIR3DL3 antibody or antigen-binding fragment thereof) described herein can be determined by a physician, taking into consideration individual differences in the patient's (subject's) age, weight, immune response, and condition.

[0235] The pharmaceutical compositions described herein may include buffers, including neutral buffered saline or phosphate buffered saline (PBS), carbohydrates such as glucose, mannose, sucrose, dextran, or mannitol, proteins, polypeptides, or amino acids (e.g., glycine), antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. In some embodiments, the pharmaceutical compositions are substantially free of contaminants, e.g., no detectable levels of contaminants (e.g., endotoxins).

[0236] The pharmaceutical compositions disclosed herein can be administered in a manner appropriate to the disease, disorder, or condition being 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 doses can be determined by clinical trials.

[0237] The pharmaceutical compositions disclosed herein may be in various forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

[0238] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion. In a preferred embodiment, the pharmaceutical compositions described herein are formulated for subcutaneous or intravenous injection of infusion. The pharmaceutical compositions described herein can be formulated for administration by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988, incorporated herein by reference in its entirety).

[0239] As used herein, the terms "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumor, and intrasternal injection and infusion.

[0240] A pharmaceutical composition comprising the modified immune effector cells (e.g., NK cells or T cells) described herein may be administered in an amount of about 10 4 ~about 10 9 Cells / kg body weight (e.g., about 10 5 ~about 10 6In some embodiments, the dose of the modified immune effector cells (e.g., NK cells or T cells) described herein is at least about 1×10 6 , about 1.1×10 6 , about 2×10 6 , about 3.6×10 6 , about 5×10 6 , about 1×10 7 , about 1.8×10 7 , about 2×10 7 , about 5×10 7 , about 1×10 8 , about 2×10 8 , about 5×10 8 , about 1×10 9 , about 2×10 9 , or about 5 × 10 9 The pharmaceutical compositions described herein may also be administered multiple times at a particular dosage. The optimal dosage and treatment regime for a particular patient can be readily determined by one of skill in the art by monitoring the patient for symptoms of a disease, disorder, or condition and adjusting the treatment accordingly.

[0241] In some embodiments, the pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously 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 an infusion of one or more pharmaceutical compositions described herein. In some embodiments, the pharmaceutical compositions described herein may be administered before or after surgery.

[0242] The dosage of any of the aforementioned therapies administered to a subject will vary depending on the disease, disorder, or condition being treated and on the particular subject. Scaling of dosages for administration to humans can be performed according to art-recognized practices.

[0243] kit The present disclosure provides, inter alia, kits comprising at least one KIR3DL3 inhibitor described herein and instructions for use and / or administration. In some embodiments, the kits may comprise one or more containers comprising a pharmaceutical composition comprising at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof described herein, and instructions for use and / or administration.

[0244] The present disclosure provides, inter alia, a kit comprising at least one immune cell activator as described herein, at least one KIR3DL3 inhibitor as described herein, and instructions for use and / or administration. Such a kit may comprise one or more containers comprising a first pharmaceutical composition comprising at least one immune cell activator as described herein and a pharma- ceutically acceptable carrier, and a second pharmaceutical composition comprising at least one KIR3DL3 inhibitor as described herein and a pharma- ceutically acceptable carrier. In some embodiments, the kits described herein comprise one or more anti-KIR3DL3 antibodies or antigen-binding fragments thereof as described herein, and instructions for use and / or administration.

[0245] The present disclosure provides, inter alia, kits comprising a population of modified immune effector cells, at least one KIR3DL3 inhibitor, and instructions for use and / or administration. Such kits may comprise one or more containers comprising a first pharmaceutical composition comprising a population of modified immune effector cells as described herein and a pharma- ceutically acceptable carrier, and a second pharmaceutical composition comprising at least one KIR3DL3 inhibitor as described herein and a pharma- ceutically acceptable carrier. In some embodiments, such kits comprise at least one KIR3DL3 antibody or antigen-binding fragment thereof as described herein and a pharma- ceutically acceptable carrier.

[0246] 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.

[0247] The instructions for the first and second pharmaceutical compositions described herein generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package) or sub-unit dose. The instructions provided in the kit 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 of the subject.

[0248] The kit provided herein is suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packages for use in combination with specific devices, such as injection devices, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierce that can be inserted by a hypodermic needle). The container may also have a sterile access port.

[0249] 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 that includes the contents of the kit described above.

[0250] Incorporation by Reference All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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. EXAMPLES

[0251] 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.

[0252] Example 1: First-in-class monoclonal antibody that binds to human KIR3DL3 To explore the therapeutic potential of HHLA2-KIR3DL3 blockade, we generated a first-in-class monoclonal antibody called NPX267 (Hu26E10z7p7 in Table 1) that binds with high affinity to human killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 3 (KIR3DL3). KIR3DL3 is a member of the killer cell Ig-like (KIR) receptor family and is expressed by both NK and T cells. KIR3DL3 has recently been shown to be a co-inhibitory receptor for the B7 ligand, human endogenous retroviral H long terminal repeat-associated protein 2 (HHLA2) (see Bhatt et al, Cancer Immunol Res 2021;9:156-169 and Wei et al. Science Immunol. 2021;6:eab9792, each of which is incorporated herein by reference in its entirety).

[0253] KIR3DL3 expressed on T cells and NK cells in the tumor microenvironment suppresses immune responses after engagement with HHLA2 (Figures 1A and 1B). Upon HHLA2-induced KIR3DL3 activation, it recruits SHP-1 and SHP-2 to the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) of KIR3DL3, blunting downstream activation signals. As a result, T cell and NK cell activity is suppressed. HHLA2 has limited expression in normal tissues, but is highly expressed in many cancers and is often associated with poor patient outcomes. In renal cell carcinoma (RCC), HHLA2 expression is often not co-expressed with PD-L1. However, co-expression of HHLA2 and PD-L1 in tumors from RCC patients is associated with worse progression-free survival than patients with tumors that exclusively express PD-L1. Thus, the KIR3DL3-HHLA2 axis represents a novel immune checkpoint pathway, and blockade of KIR3DL3 signaling may be a promising strategy to promote antitumor immunity.

[0254] NPX267 is a humanized IgG4 monoclonal antibody that was shown to bind specifically to KIR3DL3 (Figure 2). The monovalent binding affinity of NPX267 to recombinant KIR3DL3 protein was determined by SPR using a Biacore instrument, where KIR3DL3 protein was immobilized on a CM5 chip and NPX267 was run as the analyte. The affinity of NPX267 for human KIR3DL3 was 679 pM. No off-target binding was found in a screen of over 5800 plasma membrane proteins.

[0255] NPX267 was shown to specifically bind to KIR3DL3 expressed on 300.19-KIR3DL3 cells, NK92MI cells, and primary human NK cells (Figures 3A-3C). 300.19-KIR3DL3 cells were treated with NPX267 or an IgG4 isotype control antibody at concentrations ranging from 10 mg / mL to 0.0005 mg / mL for 30 minutes (Figure 3A). Flow cytometric analysis of primary antibody binding was detected using an anti-human PE secondary antibody. The mean and standard deviation of duplicate data points are shown. Data run in duplicate were entered into GraphPad Prism software and EC 50 It was decided.

[0256] NK92MI cells were treated with NPX267 at concentrations ranging from 10 mg / mL to 0.0005 mg / mL for 30 min (Figure 3B). Flow cytometry analysis of primary antibody binding was detected using an anti-human PE secondary antibody. The mean and standard deviation of triplicate data points are shown.

[0257] For primary human NK cells, human whole blood cells were treated with NPX267 at concentrations ranging from 10 mg / mL to 0.00016351 mg / mL for 30 min (Figure 3C). Red blood cells were lysed and cells were stained with a cocktail of anti-human CD3, anti-human CD56, and anti-human IgG4 PE. Flow cytometry analysis of primary antibody binding was detected using an anti-human PE secondary antibody.

[0258] NPX267 inhibits tumor-infiltrating CD56 + It was shown to bind KIR3DL3 on NK cells (Figure 4). Cryopreserved human dissociated tumor cells were thawed and split into NPX276 and fluorescence minus one (FMO) conditions. Cells were stained with an antibody cocktail of viability dyes, CD45-BV421, EpCAM-FITC, and CD56-PE + / - NPX267-APC for 30 minutes. Samples were immediately acquired on a flow cytometer. NPX267+ gates were determined from the FMO samples for each donor. KIR3DL3 expression ranged from 6-15% of infiltrating CD56+ NK cells.

[0259] NPX267 was shown to block the binding of KIR3DL3 to HHLA2 (Figure 5). 300.19-KIR3DL3 cells were treated with NPX267 or IgG4 isotype control antibody at concentrations ranging from 10 mg / mL to 0.0005 mg / mL for 30 min. Recombinant biotinylated HHLA2 at 5 mg / mL was added to the cells and incubated for 30 min. Cells were treated with 1:250 APC-streptavidin for 30 min to detect recombinant biotinylated HHLA2 bound to the cells. The gMFI of APC-streptavidin in 300.19-KIR3DL3 cells was measured by flow cytometry and the inhibition percentage was calculated. The maximum binding signal (0% inhibition) was established using samples treated with FACS buffer alone, recombinant biotinylated HHLA2 protein, and APC-streptavidin, whereas the minimum binding signal (100% inhibition) was set using samples treated with biotinylated HHLA2 without the addition of APC-streptavidin. NPX267 blocked recombinant HHLA2-Fc from binding to its inhibitory receptor KIR3DL3 in a dose-dependent manner.

[0260] NPX267 was shown to block HHLA2-mediated suppressive activity in a T cell reporter assay (Figure 6). HHLA2 / TCR / CHO cells were seeded in white clear-bottom 96-well plates and allowed to adhere overnight. The following day, Jurkat / IL-2 / KIR3DL3 cells were incubated with NPX267 for 1 h. Tissue culture medium was removed from HHLA2 / TCR / CHO cells and Jurkat / IL-2 / KIR3DL3 cells precomplexed with experimental antibodies and anti-CD28 agonist antibodies were added for 5-6 h. Luciferase assays were performed using the One-Step Luciferase Assay System. Luminescence was measured using a luminometer (BioTek Synergy™ 2 microplate reader). All treatment conditions were performed in triplicate. Luminescence intensity data was analyzed using GraphPad Prism software. Luminescence intensity (L t) was defined as 1. The fold induction of luminescence in the presence of each compound was calculated according to the following formula: Fold induction = (L-Lb) / (Lt-Lb), where L = luminescence intensity in the presence of compound, Lb = luminescence intensity in the absence of cells, and Lt = luminescence intensity in the absence of compound. The fold induction values ​​were plotted with GraphPad Prism software and the IC was calculated using a four-parameter nonlinear regression equation. 50 It was decided.

[0261] NPX267 was shown to enhance NK cell killing of HHLA2-expressing tumor cells (Figures 7A and 7B). NK92MI effector cells were plated in round-bottom 96-well plates and treated with 10 mg / mL NPX267 for 30 minutes. After treatment, NK92MI cells were mixed with K562 cells engineered to express HHLA2 at a 1:1 effector-to-target ratio and incubated at 37°C and 5% CO2 for 3 hours. Cells were then stained with the apoptosis marker Annexin V and analyzed via flow cytometry to assess target cell death. NPX267 increased target cell death by up to 2-fold in a dose-dependent manner compared to the IgG4 isotype control (Figure 7A).

[0262] KIR3DL3+ human NK cells were incubated with 10 mg / mL NPX267 or IgG4 isotype control for 30 min and then added to CellTrace Violet-labeled HCC827 cells for 6 h at a ratio of 5 NK cells to 1 HCC827 cell. Cells were stained with 7-AAD. Flow cytometry was used to detect non-viable HCC827 cells (CellTrace Violet+7-AAD+). Specific lysis was calculated by: specific lysis = (CellTrace Violet+7-AAD+ cells) / (CellTrace Violet+ cells)*100. The mean and standard deviation of triplicate data points for each condition are shown. P values ​​were calculated using a two-tailed Student's T-test. NPX267 increased target cell death in two different donors with various levels of KIR3DL3 (Figure 7B).

[0263] NPX267 is a humanized version of 26E10, which was shown to enhance NK cell-mediated antitumor activity in the HCC827 model in vivo (Figure 8). Six to eight week old NSG mice (n=6 / group) were inoculated with luciferase-labeled HCC827 cells (4 × 10 6 ) was injected intraperitoneally (i.p.) into the mice. When tumors were established, mice were injected with KIR3DL3+ NK cells (1 × 10 7 ), along with 1 μg rhIL-2, 1 μg rhIL-15, and 200 μg NPX267 (or mIgG1) were injected ip every other day for a total of five times. Tumor growth was assessed by imaging.

[0264] In summary, the data presented herein demonstrate that NPX267 blocked HHLA2 engagement with KIR3DL3 in primary human NK and T cells in a dose-dependent manner. The ability of NPX267 to block KIR3DL3-mediated inhibition of T cell activation was demonstrated using a T cell reporter system and primary CD8 + T cell function assays were used to assess this. KIR3DL3 blockade with NPX267 inhibited HHLA2-mediated suppression of T cell activation in a dose-dependent manner. The antitumor activity of KIR3DL3 blockade with NPX267 was also demonstrated in an NK cell-mediated cytotoxicity assay. NPX267 treatment enhanced the HHLA2 suppression of human NK cell lines and primary NK cells in vitro. + Finally, NPX267 blockade of HHLA2-mediated KIR3DL3 signaling enhanced HHLA2’s ability to kill tumor cells. + Enhanced antitumor immunity in a humanized mouse model bearing human tumors.

[0265] Taken together, these data demonstrate that the KIR3DL3-HHLA2 pathway is a novel immune checkpoint axis that promotes tumor escape by attenuating both innate and adaptive antitumor immune responses. NPX267, a first-in-class KIR3DL3-blocking antibody, inhibits HHLA2 + It represents a promising approach for enhancing anti-tumor immunity against tumors and for treating certain diseases, disorders or conditions, particularly cancer.

[0266] equivalent It will be understood by those skilled in the art that various changes, modifications, and improvements of the present disclosure will be readily suggested 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 any inventions described in this disclosure are further described in detail by the following claims.

[0267] One of ordinary skill in the art will understand the typical standards of deviation or error attributable to values ​​obtained in the assays or other processes described herein. Publications, websites, and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference in their entirety.

Claims

1. (a) a VH comprising a VH CDR1 amino acid sequence of SEQ ID NO: 1, a VH CDR2 amino acid sequence of SEQ ID NO: 2, and a VH CDR3 amino acid sequence of SEQ ID NO: 3; and a VL comprising a VL CDR1 amino acid sequence of SEQ ID NO: 14, a VL CDR2 amino acid sequence of SEQ ID NO: 15, and a VL CDR3 amino acid sequence of SEQ ID NO: 16; (b) 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 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; or (c) an anti-KIR3DL3 antibody or antigen-binding fragment thereof, which is or comprises: 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 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. (a) a VH comprising an amino acid sequence having at least about 85% identity to SEQ ID NO: 10; and / or (b) the anti-KIR3DL3 antibody or antigen-binding fragment thereof according to claim 1, which is or comprises a VL comprising an amino acid sequence having at least about 85% or more identity to SEQ ID NO:

23. (a) a heavy chain comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 12; and / or (b) the anti-KIR3DL3 antibody or antigen-binding fragment thereof according to claim 1, which is or comprises a light chain comprising an amino acid sequence having at least about 80% or more identity to SEQ ID NO:

25.

4. A nucleic acid encoding the anti-KIR3DL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell containing or expressing the expression vector described in claim 5.

7. A pharmaceutical composition comprising at least one anti-KIR3DL3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier, diluent, or excipient.

8. 10. The pharmaceutical composition of claim 7, for use in treating a subject having a disease, disorder, or condition, said use comprising administering a therapeutically effective amount of said pharmaceutical composition to said subject.

9. 8. The pharmaceutical composition of claim 7 for use in modulating an immune response in a subject, said use comprising administering a therapeutically effective amount of said pharmaceutical composition to said subject.

10. 9. The pharmaceutical composition of claim 8, wherein the subject has or is at risk of developing cancer.

11. The pharmaceutical composition of claim 10, wherein the subject has a solid tumor or a hematological cancer.

12. The pharmaceutical composition of claim 11, wherein (a) the solid tumor is or comprises one or more of renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer, or (b) the blood cancer comprises or is leukemia or lymphoma.

13. (a) 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; or 13. The pharmaceutical composition of claim 12, wherein (b) the lymphoma comprises or is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

14. A composition comprising a population of modified immune effector cells for use in a method of treating a subject having a disease, disorder, or condition, said method comprising: administering the population of modified immune effector cells to the subject, wherein prior to administering, the population of immune effector cells is contacted with at least one immune cell activator, thereby forming a population of modified immune effector cells; The composition, wherein said population of modified immune effector cells is administered to said subject in combination with at least one KIR3DL3 inhibitor.

15. A composition comprising at least one KIR3DL3 inhibitor for use in a method of treating a subject having a disease, disorder, or condition, said method comprising: administering said at least one KIR3DL3 inhibitor to said subject; The composition, wherein the at least one KIR3DL3 inhibitor is administered to the subject in combination with a population of modified immune effector cells.

16. A composition comprising a population of modified immune effector cells for use in a method of treating a subject having a disease, disorder, or condition, said method comprising administering said population of modified immune effector cells; The composition, wherein prior to administration, the population of immune effector cells is contacted with at least one immune cell activator and at least one KIR3DL3 inhibitor, thereby forming a population of modified immune effector cells.

17. A combination comprising a population of modified immune effector cells and at least one KIR3DL3 inhibitor for use in a method of treating a subject having a disease, disorder, or condition, said method comprising: (i) administering the population of modified immune effector cells to the subject, wherein prior to administration, the population of immune effector cells is contacted with at least one immune cell activator, thereby forming a population of modified immune effector cells; (ii) administering to said subject at least one KIR3DL3 inhibitor; A combination comprising:

18. A method for producing a population of modified immune effector cells, comprising: (i) contacting a population of immune effector cells with at least one immune cell activator; (ii) contacting said population of immune effector cells with at least one KIR3DL3 inhibitor, thereby producing a population of modified immune effector cells; A method comprising:

19. A composition comprising a population of modified immune effector cells, at least one immune cell activator, and at least one KIR3DL3 inhibitor.

20. A composition comprising a population of modified immune effector cells and at least one KIR3DL3 inhibitor, wherein the immune effector cells are contacted with at least one immune cell activator.

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