CN118742323A

CN118742323A - KIR3DL3 inhibitors and immune cell activators

Info

Publication number: CN118742323A
Application number: CN202380022655.6A
Authority: CN
Inventors: S·查佩尔; B·埃特马德-吉尔博特逊; D·比尼斯凯维奇
Original assignee: Nextpoint Treatment Co
Current assignee: Nextpoint Treatment Co
Priority date: 2022-02-22
Filing date: 2023-02-21
Publication date: 2024-10-01

Abstract

The present disclosure relates to KIR3DL3 inhibitors (e.g., anti-KIR 3DL3 antibodies or antigen-binding fragments thereof) and immune cell activators and uses thereof, such as to modify immune effector cells.

Description

KIR3DL3 inhibitors and immune cell activators

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/312,507, filed on 22 nd 2, 2022, and U.S. provisional patent application No. 63/418,105, filed on 21 th 10, 2022, the contents of each of these provisional patent applications being incorporated herein by reference in their entirety.

Background

Although immunotherapy has been studied for many diseases and conditions, including cancer, functional limitations are encountered 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 immune response progression based on complex interactions. Currently available immune checkpoint inhibitors may modulate the immune response of some patients, but immune checkpoint expression and interaction with natural binding partners may vary from patient to patient.

Thus, there is a need to develop new therapeutic approaches optimized to target immune checkpoint pathways.

Disclosure of Invention

Killer cell immunoglobulin-like receptor (KIR) proteins comprise two (KIR 2D) or three (KIR 3D) immunoglobulin-like extracellular domains. KIR3DL3 is a member of the KIR family and is a receptor that has been demonstrated to be present on both T cells and NK cells. HHLA2 is a B7 gene family member that is widely expressed in a variety of tumor and antigen presenting cells. HHLA2 binding to KIR3DL3 has been shown to suppress the immune response of activated T cells.

The present disclosure encompasses, inter alia, the discovery of anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein. Importantly, the anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein may have one or more of the following properties: (1) specifically binds KIR3DL3 (e.g., human KIR3DL 3) with high affinity, (2) specifically binds KIR3DL3 expressed on NK cells and/or T cells, (3) blocks KIR3DL3 from binding to HHLA, (4) blocks HHLA 2-mediated inhibitory activity in T cells, (5) enhances NK cell killing of tumor cells expressing HHLA2, and (6) enhances antitumor activity. Accordingly, the present disclosure provides anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein, which are useful in methods of treating diseases, disorders, and conditions such as cancers described herein, and methods for modulating immune responses.

Although inhibitors of KIR3DL3 have been studied as immunotherapy, the present disclosure covers, inter alia, the following insights: the immune cell activators described herein can (i) increase KIR3DL3 expression to enhance functional KIR3DL3 inhibition, and/or (ii) increase (e.g., by epigenetic) the strength of KIR3DL3 promoters. Thus, the present disclosure provides several examples of such immune cell activators, which may be used in combination with KIR3DL3 inhibitors, inter alia, to treat a variety of cancers, including solid tumors, such as Renal Cell Carcinoma (RCC).

In one aspect, the present disclosure provides a method of treating a subject having a disease, disorder, or condition, the method comprising administering a modified population of immune effector cells, 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 modified population of immune effector cells.

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

In some embodiments, the at least one KIR3DL3 inhibitor is or comprises an anti-KIR 3DL3 antibody or antigen-binding fragment thereof, miRNA, shRNA, siRNA, CRISPR/Cas guide system, TALENs, ZFNs, and/or demethylators. In some embodiments, the antigen binding fragment comprises scFv, fab, fab ', F (ab') 2, fc, or nanobody.

In some embodiments, the demethylating agent includes or is 5-Aza-2-deoxycytidine (Aza), 5-azacytidine, 1- β -D-arabinofuranosyl-5-azacytidine, or dihydro-5-azacytidine.

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

In some embodiments, the immune cell activator comprises or is a cytokine agent. In some embodiments, the cytokine agent is or includes IL-2, IL-15, IL-12, IL-17, IL-18, IL-21, IFN gamma and/or TNF alpha. In some embodiments, IL-2 binds 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 includes an inhibitor of a cytokine signaling inhibitor (SOCS) protein.

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

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 comprise or are cd4+ T cells and/or cd8+ T cells. In some embodiments, the modified immune effector cell comprises at least one CAR.

In some embodiments, the modified immune effector cells are administered to the subject in less than about 3 hours of contact with the 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 the at least one KIR3DL3 inhibitor.

In some embodiments, the modified population of immune effector cells and/or the at least one KIR3DL3 inhibitor are administered parenterally. In some embodiments, parenteral administration is or includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion.

In some embodiments, the method comprises sequentially administering a modified population of immune effector cells and at least one KIR3DL3 inhibitor. In some embodiments, (i) administering a modified population of immune effector cells prior to administering at least one KIR3DL3 inhibitor; or (ii) administering the modified population of immune effector cells after administration of the at least one KIR3DL3 inhibitor.

In some embodiments, the modified population of immune effector cells and the at least one KIR3DL3 inhibitor are co-administered. In some embodiments, the method comprises co-administration by injection.

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, colorectal cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer. In some embodiments, the subject has hematological cancer. In some embodiments, the hematological cancer comprises or is leukemia or lymphoma. In some embodiments, the leukemia comprises or is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the lymphoma comprises 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 treatment including a cytokine agent.

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 immune effector cells. In some embodiments, the methods described herein further comprise determining the expression of CD25, CD69, CD137, CD16, CD56, CD96, CD226, KIR2DL5, and/or NKG 2D.

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

In another aspect, the present disclosure provides a method of preparing a modified population of immune effector cells, the method comprising: (i) Contacting a 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 a modified population of immune effector cells.

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

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

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.

In another aspect, the present disclosure provides a kit comprising a modified population of immune effector cells and 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.

In another aspect, the present disclosure provides an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, which 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 a VH CDR 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 a VL CDR of table 1. Such anti-KIR 3DL3 antibodies, or antigen-binding fragments thereof, may be used in any aspect or embodiment described herein.

In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) VH comprising one, two or three VH CDR sequences each having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VH CDR of table 1; and/or (b) a VL comprising one, two, or three VL CDR sequences each having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL CDR of table 1. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (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.

In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) VH, which has at least about 90% or greater identity to VH of table 1; and/or (b) a VL that has at least about 90% or greater identity to a VL of table 1. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) VH having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to VH of table 1; and/or (b) a VL that has at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL of table 1. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) VH comprising or consisting of VH of table 1; and/or (b) a VL comprising or consisting of the VL of table 1.

In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) A heavy chain having at least about 90% or greater identity to the 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-KIR 3DL3 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 more identity to the heavy chain of table 1; and/or (b) a light chain having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a light chain of table 1. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, is or comprises: (a) a heavy chain comprising or consisting of the heavy chain of table 1; and/or (b) a light chain comprising or consisting of the light chain of table 1.

In another aspect, the present disclosure provides nucleic acids encoding an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of any aspect or embodiment described herein.

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

In another aspect, the present disclosure provides a host cell comprising or expressing an anti-KIR 3DL3 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.

In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of any aspect or embodiment described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

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

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

In some embodiments, the subject has or is at risk of developing cancer. In some embodiments, the subject has a solid tumor or hematological cancer. In some embodiments, the solid tumor is or includes one or more of the following: renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer. In some embodiments, the hematological cancer comprises or is leukemia or lymphoma. In some embodiments, the leukemia comprises or is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, the lymphoma comprises or is Hodgkin's Lymphoma (HL), non-hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

Drawings

The drawings described below (which together constitute the figures) are for illustration purposes only and are not intended to be limiting.

Fig. 1A-1B are schematic diagrams of HHLA checkpoint axes showing that HHLA2 expressed on tumor cells modulates NK and T cell activity via interaction with KIR3DL3 or TMIGD 2.

FIG. 2 is a graph showing monovalent binding affinities of the anti-KIR 3DL3 antibody described herein (NPX 267) for recombinant KIR3DL3 proteins as determined by SPR using a Biacore instrument.

Fig. 3A-3C are graphs showing binding of NPX267 to KIR3DL3 expressed on 300.19-KIR3DL3 cells (fig. 3A), NK92MI cells (fig. 3B), and primary human NK cells (fig. 3C) as measured by flow cytometry using an anti-human Phycoerythrin (PE) secondary antibody.

Fig. 4 is a series of graphs showing binding of NPX267 to KIR3DL3 expressed on tumor-infiltrating CD56 ⁺ NK cells.

FIG. 5 is a graph showing the percent (%) of KIR3DL3 binding to HHLA2 after treatment of 300.19-KIR3DL3 cells with NPX267 or IgG4 isotype control antibodies at concentrations ranging from 10mg/mL to 0.0005 mg/mL.

FIG. 6 is a graph showing luminescence induction fold in T cell reporter assays of HHLA/TCR/CHO cells and Jurkat/IL-2/KIR3DL3 cells pre-complexed with NPX267 and anti-CD 28 agonist antibodies, as determined using a photometer (BioTek Synergy ^TM 2 microplate reader).

Fig. 7A-7B are graphs showing NK92MI effector cell killing of K562 cells (fig. 7A) or kir3dl3+ human NK cell killing of HCC827 cells (fig. 7B) after treatment with NPX267 or IgG4 isotype control, as assessed using flow cytometry.

Fig. 8 is a graph showing tumor growth in NSG mice assessed by imaging, which were intraperitoneally injected with luciferase-labeled HCC827 cells, and after tumor establishment, KIR3DL3 ⁺ primary human NK cells were injected, followed by NPX267 parent Ab (26E 10) or mIgG1 every other day for a total of 5 times.

Definition of the definition

For easier understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification. Publications and other references cited herein are hereby incorporated by reference to describe the background of the invention and provide additional details regarding its practice.

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. For example, "an element" refers to one element or more than one element.

About or about: as used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value that is similar to the stated reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the stated reference value unless otherwise indicated or clearly seen by the context (except where such numbers would exceed 100% of the possible values).

The preparation method comprises the following steps: as used herein, the term "agent" refers to a molecule that can be delivered to or expressed, released or secreted from a target by an immune effector cell as described herein. Agents include, but are not limited to, cytokine agents (e.g., cytokines (IL-2, IL-15, 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. The agent may bind to any cellular moiety, such as a receptor, an epitope, or other binding site present on the target or target cell. The agent may diffuse or be transported into the cell where it may act within the cell.

Immunotherapeutic agent: the term "immunotherapeutic" may include any molecule, peptide, antibody, or other agent that may stimulate the host immune system to generate an immune response against a tumor or cancer in a subject. Various immunotherapeutic agents can be used in the compositions and methods described herein.

Affinity matured (or "affinity matured antibody"): as used herein, refers to an antibody having one or more alterations in one or more CDRs thereof that result in improved affinity of the antibody for the antigen as compared to the parent antibody without those alterations. In some embodiments, affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be produced by any of a variety of procedures known in the art. Marks et al, biotechnology 10:779-783 (1992) describe affinity maturation by shuffling of the V _H and V _L domains. The following documents describe random mutagenesis of CDRs and/or framework residues: barbas et al, proc.Nat.Acad.Sci.U.S. A91:3809-3813 (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).

Antibody: as used herein, the term "antibody" refers to a polypeptide that includes typical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As known in the art, an intact antibody as produced in nature is an approximately 150kD tetrameric agent comprising two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD) that associate with each other to form what is commonly referred to as a "Y-shaped" structure. Each heavy chain comprises at least four domains (each of about 110 amino acids in length) -an amino terminal Variable (VH) domain (located at the top of the Y structure), followed by three constant domains: CH1, CH2, and CH3 at the carboxy terminus (at the base of the stem of Y). The short region called the "switch" connects the heavy chain variable and constant regions. The "hinge" connects the CH2 domain and the CH3 domain to the rest of the antibody. Two disulfide bonds in the hinge region link the two heavy chain polypeptides in the intact antibody to each other. Each light chain comprises two domains, an amino-terminal Variable (VL) domain followed by a carboxy-terminal Constant (CL) domain, separated from each other by another "switch". The intact antibody tetramer comprises two heavy chain-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to each other, such that the dimers are connected to each other and form a tetramer. naturally occurring antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., 3,4, or 5 folds) stacked on top of each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops called "complementarity determining regions" (CDR 1, CDR2, and CDR 3) and four somewhat invariant "framework" regions (FR 1, FR2, FR3, and FR 4). When the natural antibody is folded, the FR regions form a β -sheet that provides the structural framework for the domain, and the CDR loop regions from both the heavy and light chains are clustered together in three dimensions such that they create a single hypervariable antigen binding site at the top of the Y structure. The Fc region of naturally occurring antibodies 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 Fc receptors may be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and/or utilized according to the present disclosure comprise glycosylated Fc domains, including Fc domains having modified or engineered glycosylation. In some embodiments, any polypeptide or polypeptide complex that comprises sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and/or used as an "antibody," whether such polypeptide is naturally-occurring (e.g., produced by the reaction of an organism with an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the antibody is polyclonal. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody has a constant region sequence specific for a mouse, rabbit, primate, or human antibody. 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 indicated or clear from context), may refer to any construct or form known or developed in the art that utilizes the structural and functional characteristics of an antibody in alternative presentations. For example, in some embodiments, the antibodies utilized in accordance with the present invention are in a form selected from, but not limited to: intact IgA, igG, igE or IgM antibodies; bispecific or multispecific antibodies (e.g.,Etc.); antibody fragments, such as those used herein in the broadest sense and encompass 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, those fragments that exhibit the desired antigen-binding activity). The antibodies described herein can be immunoglobulins, heavy chain antibodies, light chain antibodies, LRR-based antibodies, or other protein scaffolds having antibody-like properties, as well as other immune binding moieties known in the art, including, for example, fab '2, fab3, F (ab') 2, fd, fv, feb, scFv, SMIP, antibodies, diabodies, triabodies, tetrabodies, minibodies, tandab, DVD, biTe, tandAb, and the like, or any combination thereof. 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., linkages of glycans) that would be had if naturally occurring. In some embodiments, the antibodies can contain covalent modifications (e.g., linkages to glycans, payloads [ e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc. ] or other pendant groups [ e.g., polyethylene glycol, etc. ].

Antigen binding fragment: an "antigen binding fragment" refers to a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Antigen binding fragments of an antibody include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Exemplary antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv or VHH or VH or VL domain only); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding fragment of an antibody described herein is an scFv. In some embodiments, the antigen binding fragment of an antibody described herein is only a VHH domain. As with whole antibody molecules, antigen binding fragments may be monospecific or multispecific (e.g., bispecific). The multispecific antigen-binding fragment of an antibody may comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or a different epitope of the same antigen.

Antigen presenting cells: as used herein, the term "antigen presenting cell" or APC includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, langerhans cells) and other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

Antibody heavy chain: as used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains that are present in all antibody molecules in their naturally occurring conformation.

Antibody light chain: as used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains that are present in all antibody molecules in their naturally occurring conformation.

Activating: as used herein, the term "activate" 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 has been stimulated to exert its effector function. Activation may also be associated with induced proliferation, cytokine production, cytokine secretion, cell signaling (e.g., changes in gene expression), target cell killing, metabolic changes, production of inflammatory mediators, and/or antigen processing and presentation.

Synthesis of antibodies: as used herein, the term "synthetic antibody" refers to an antibody that is generated using recombinant DNA techniques, such as an antibody expressed by a phage as described herein. The term should also be construed to mean an antibody produced by synthesizing a DNA molecule encoding the antibody (which expresses an antibody protein) or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

Antigen: as used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. The immune response may involve antibody production, activation of specific immunocompetent cells, or both. The skilled artisan will appreciate that any macromolecule, including almost any protein or peptide, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full length nucleotide sequence of a gene. It will be apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, the skilled artisan will appreciate that antigens need not be encoded by a "gene" at all. It is apparent that the antigen may be synthetically produced or may be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

And (3) autologous: as used herein, the term "autologous" refers to any material derived from an individual that is later reintroduced into the same individual.

Allograft: as used herein, the term "allogeneic" refers to any material (e.g., a population of cells) derived from different animals of the same species.

Different species: as used herein, the term "xenogenic" refers to any material (e.g., a population of cells) derived from animals of different species.

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 described herein and specifically target cells and/or bind antigen. CARs may be used as therapies, for example, with adoptive cell transfer. The CAR may comprise at least one extracellular domain, at least one transmembrane domain, and at least one intracellular domain.

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 blood stream and lymphatic system to other parts of the body. Cancers may include solid tumors and hematological cancers. Examples of various cancers are described herein and include, but are not limited to, kidney, bone, skin, breast, cervical, colorectal, endometrial, lung, ovarian, liver, bile duct, thyroid, leukemia or lymphoma, as well as several other types, including those as described elsewhere herein. In certain embodiments, the cancer is Renal Cell Carcinoma (RCC).

CDR: as used herein, refers to complementarity determining regions within antibody variable regions. There are three CDRs in each of the variable regions of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3 for each variable region. "set of CDRs" or "set of CDRs" refers to a set of three or six CDRs that occur in a single variable region capable of binding an antigen or in CDRs of homologous heavy and light chain variable regions capable of binding an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., kabat, chothia, etc.); those skilled in the art understand the differences between these systems and are able to understand the CDR boundaries to the extent necessary to understand and practice the claimed invention.

Chemotherapeutic agents: the term "chemotherapeutic agent" as used herein has its art-understood meaning one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents for and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesired cell proliferation. In many embodiments, the chemotherapeutic agent may be used to treat cancer. In some embodiments, the chemotherapeutic agent may be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule-targeting agents such as taxanes, maytansinoids, and the like), one or more epothilones, one or more histone deacetylase inhibitors HDAC), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogues or nucleotide precursor analogues one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, One or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., sharing related antiproliferative activity). In some embodiments, the chemotherapeutic agent may be or include one or more of the following: actinomycin, all-trans retinoic acid, auristatin, azacytidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunomycin, docetaxel, deoxyfluorouridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and/or analogs thereof (e.g., DM 1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. In some embodiments, a chemotherapeutic agent may be used in the context of an antibody-drug conjugate. In some embodiments, the chemotherapeutic agent is an antibody-drug conjugate comprising: hLL 1-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 (gemtuzumab ozogamicin), rituximab Shan Kangwei statin (brentuximab vedotin), trastuzumab maytansine (trastuzumab emtansine), infliximab (inotuzumab ozogamicin), amitraz, GmbH Mo Shankang Vidolatine (glembatumomab 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、 is resistant to PSMAADCs, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vomitozumab Ma Foduo (vorsetuzumab mafodotin) and Luo Fozhu monoclonal antibody mistatin (lorvotuzumab mertansine).

Modification of a conserved sequence: as used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into antibodies compatible with the various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following: 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 within the CDR regions of an antibody may be replaced with other amino acid residues from the same side chain family, and altered antibodies may be tested for their ability to bind antigen using the functional assays described herein.

Combination therapy: the term "combination therapy" as used herein refers to those instances in which two or more different therapeutic agents (e.g., a modified population of 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, two or more different therapeutic agents may be administered simultaneously or separately. Such combined administration may 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 may be formulated together and administered simultaneously in the same dosage form. Alternatively, two or more therapeutic agents may be administered simultaneously, wherein the therapeutic agents are present in separate formulations. In another alternative, a first therapeutic agent may be administered followed by one or more additional therapeutic agents. In a single administration regimen, two or more therapeutic agents may be administered minutes apart, or hours apart, days apart, or weeks apart. In some embodiments, two or more therapeutic agents may be administered within a few hours (e.g., less than about 3 hours) of each other.

Composition: those skilled in the art will appreciate that the term "composition" may be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise indicated, the composition may have any form-e.g., gas, gel, liquid, or solid.

Comprising: a composition or method described herein as "comprising" one or more specified elements or steps is open ended, meaning that the specified elements or steps are essential, but that other elements or steps can be added within the scope of the composition or method. To avoid complications, it should also be understood that any composition or method described as "comprising" (or "including") one or more specified elements or steps also describes a corresponding, more limited composition or method "consisting essentially of (or" consisting essentially of ") the same specified elements or steps, meaning that the composition or method includes the specified essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method. It will be further understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more specified elements or steps also describes a corresponding, more limited, and closed composition or method "consisting of (or" consisting of ") the specified elements or steps to exclude any other unspecified elements or steps. Any known or disclosed equivalents of the specified elements or steps may be substituted for those elements or steps in any of the compositions or methods disclosed herein.

Simultaneous application: as used herein, the term "simultaneous administration" with respect to two or more therapeutic agents described herein (e.g., an immune effector cell population described herein and at least one KIR3DL3 inhibitor described herein) is administration using a dose and time interval such that the administered therapeutic agents are present together in the body, e.g., at one or more sites of action, in the body in non-negligible amounts over the time interval. The time interval may be a few minutes (e.g., at least 1 minute, 1-30 minutes, 30-60 minutes), a few hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12 hours, 12-24 hours), a few days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7 days, etc.), or a few weeks (e.g., at least 1,2, or 3 weeks, etc.). Thus, the therapeutic agents may, but need not, be administered together, e.g., as part of a single composition. Furthermore, the therapeutic agents may, but need not, be administered substantially simultaneously (e.g., within less than 5 minutes, or less than 1 minute apart) or within a short time of each other (e.g., less than 1 hour apart, less than 30 minutes apart, less than 10 minutes apart, about 5 minutes apart). According to various embodiments of the present disclosure, therapeutic agents administered during such time intervals may be considered to be administered at substantially the same time. In certain embodiments of the present disclosure, the concurrently administered therapeutic agent is present in the body at an effective concentration during the time interval. When administered simultaneously, the effective concentration of each therapeutic agent required to elicit a particular biological response may be less than the effective concentration of each therapeutic agent when administered alone, allowing for a reduction in the dosage of one or more therapeutic agents relative to the dosage required if the agents were administered as a single agent. The effects of the multiple therapeutic agents may be, but are not necessarily, additive or synergistic. The therapeutic agent may be administered multiple times.

Modification of a conserved sequence: as used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antigen-binding fragment thereof that contains the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into antibodies compatible with the various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following: 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 within the C DR region of an antibody can be replaced with other amino acid residues from the same side chain family, and altered antibodies can be tested for their ability to bind antigen using the functional assays described herein.

Cytotoxicity: as used herein, the term "cytotoxic" or "cytotoxicity" refers to killing or destroying cells. In one embodiment, the cytotoxicity of the metabolically enhanced cells is improved, e.g., the cytolytic activity of immune effector cells (e.g., T cells or NK cells) described herein is increased.

Effective amount of: as used herein, an "effective amount" as described herein refers to a dose sufficient to prevent or treat at least one sign and/or symptom of cancer in an individual. The effective amount 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 discretion of the prescribing physician. The size of the dose will also be determined by the active substance selected, the method of administration, the time and frequency of administration, the presence, nature and extent of any adverse side effects that may accompany the administration of a particular active substance, and the desired physiological effect. Those skilled in the art will appreciate that a variety of diseases or conditions may require long-term treatment involving multiple administrations. For the purposes of this disclosure, the amount or dose of the therapeutic agent administered (e.g., modified immune effector cells described herein and/or at least one KIR3DL3 inhibitor described herein) should be sufficient to achieve a therapeutic or prophylactic response (e.g., a reduction in the severity or duration of at least one sign or symptom or other reduction) in the subject over a reasonable time frame. For example, the dose should be sufficient to detect, treat, or prevent cancer over a period of time from about 2 hours or more, such as from about 12 hours to about 24 hours or more, from the time of administration. In certain embodiments, the period of time may be even longer. The dosage will be determined by the efficacy of the particular therapeutic agent or agents and the condition of the subject (e.g., human) and the weight of the subject (e.g., human) to be treated.

Effect function: as used herein, "effector function" or "effector activity" refers to a particular activity that an immune cell performs in response to stimulation of the immune cell. For example, effector functions of T lymphocytes include recognizing antigens and killing cells expressing antigens.

Epitope: as used herein, the term "epitope" refers to any moiety specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts a related three-dimensional conformation. In some embodiments, when the antigen adopts such a conformation, such chemical atoms or groups are in spatial physical proximity to each other. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearization), at least some of such chemical atoms or groups are physically separated from each other.

"Frame" or "frame region": as used herein, refers to the sequence of the variable region minus the CDRs. Since CDR sequences can be determined by different systems, the same framework sequences are affected by correspondingly different interpretations. Six CDRs divide the framework regions on the heavy and light chains into four sub-regions on each chain (FRl, FR2, FR3 and FR 4), with CDRl located between FRl and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. In the case where a specific sub-region is not designated as FR1, FR2, FR3 or FR4, the framework regions as otherwise mentioned represent the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR means one of four sub-regions, for example FR1 means the first framework region closest to the amino terminus of the variable region and 5' relative to CDR1, and FR means two or more sub-regions constituting the framework region.

Immune effector function: as used herein, "immune effector function" or "immune effector response" (terms as used herein) refers to a function or response such as that described herein that enhances or promotes immune attack by a target cell by an immune effector cell (e.g., a T cell or NK cell). For example, immune effector function or response refers to the property of T cells and/or NK cells that promote inhibition of growth or proliferation of target cells. For example, in the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

Encoding: as used herein, "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to be used in a biological process as a template for the synthesis of other polymers and macromolecules, the template having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand, which has a nucleotide sequence identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a transcription template for a gene or cDNA, may both be referred to as a protein or other product encoding the gene or cDNA.

Endogenous: as used herein, "endogenous" refers to any material from or produced within a particular organism, cell, tissue, or system.

Exogenous: as used herein, the term "exogenous" refers to any material introduced from or produced outside a particular organism, cell, tissue or system.

Amplification: as used herein, the term "expansion" refers to an increase in a number of, e.g., cells (e.g., immune effector cells, e.g., T cells or NK cells, as described herein). In one embodiment, the number of immune cells expanded ex vivo is increased relative to the number originally present in the culture. In another embodiment, the ex vivo expanded immune cells are increased in number relative to other cell types in culture. In some embodiments, amplification may occur in vivo. The term "ex vivo" as used herein refers to cells that have been removed from a living organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

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 may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of the nucleic acid sequence involves one or more of the following: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by splicing, editing, 5 'cap formation, and/or 3' end formation); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

Fragments: as used herein, the term "fragment" or "portion" refers to a structure that includes discrete portions that are integral but lacks one or more portions found in the entire structure. In some embodiments, the fragments consist of such discrete portions. In some embodiments, a fragment consists of or comprises a feature element or portion found in 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 monomer units (e.g., nucleic acids) as found throughout the nucleotide. 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 monomer units (e.g., residues) found in the entire nucleotide.

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 "homologous" to each other 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 "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., contain residues with related chemical properties at the corresponding positions). As will be appreciated by those skilled in the art, sequences can be compared using a variety of algorithms to determine their degree of homology, including allowing gaps of a specified length in one sequence relative to another when considering which residues in different sequences "correspond" to each other. For example, the calculation of the percent homology between two nucleic acid sequences may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first nucleic acid sequence and the second nucleic acid sequence to achieve optimal alignment, and non-corresponding sequences may be omitted for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a nucleotide that is similar to the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

Host cell: as used herein, the term "host cell" refers to a cell into which exogenous DNA has been introduced (recombinantly or otherwise). Those skilled in the art will appreciate 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 succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term host cell as used herein. In some embodiments, the host cell includes prokaryotic and eukaryotic cells selected from any life kingdom suitable for expression of exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic cells (single or multiple cells), bacterial cells (e.g., E.coli), strains of Bacillus species (Bacillus spp.) or Streptomyces species (Streptomyces spp.), mycobacterial cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae, schizosaccharomyces pombe (S.pombe), pichia pastoris (P.pastoris), or Pichia methanolica (P.methanol)), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells or Trichoplusia ni (Trichoplusia ni)), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadromas). In some embodiments, the cell comprises or is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell selected from the group consisting of: CHO (e.g., CHO Kl, DXB-1 CHO, veggie-CHO), COS (e.g., COS-7), retinal cells, vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, haK, BHK), heLa, hepG2, WI38, MRC 5, colo205, HB 8065, HL-60 (e.g., BHK 21), jurkat, daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, saltoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the above. In some embodiments, the cell comprises one or more viral genes.

Human antibodies: as used herein, the term "human antibody" is intended to include antibodies having variable and constant regions that are generated (or assembled) from human immunoglobulin sequences. In some embodiments, antibodies (or antibody components) may be considered "human" even though their amino acid sequences include residues or elements not encoded by human germline immunoglobulin sequences (e.g., including sequence variations, such as may have been introduced (initially) by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in one or more CDRs, particularly CDR 3.

Humanization: as known in the art, the term "humanized" is generally used to refer to antibodies (or antibody components) whose amino acid sequences comprise the V _H and V _L region sequences of a reference antibody produced in a non-human species (e.g., mouse), but also include modifications in those sequences relative to the reference antibody that are 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 an antibody (or antibody component) that immunospecifically binds to an antigen of interest and has a Framework Region (FR) having substantially the same amino acid sequence as that of a human antibody and a Complementarity Determining Region (CDR) having substantially the same amino acid sequence as that of a non-human antibody. Humanized antibodies comprise 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 (e.g., a donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises a light chain and at least a variable domain of a heavy chain. Antibodies may also comprise the C _H, hinge, C _H2、C_H 3 and optionally C _H regions of the heavy chain constant region. In some embodiments, the humanized antibody only comprises a humanized V _L region. In some embodiments, the humanized antibody only comprises a humanized V _H region. In some certain embodiments, the humanized antibody comprises a humanized V _H region and a V _L region.

Identity: as used herein, the term "identity" refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules. When two amino acid sequences have identical residues at identical positions; for example, if a position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree of identity of two amino acid sequences with identical residues at identical positions in an alignment is typically expressed as a percentage. Identity between two amino acid sequences is a direct function of the number of matches or identical positions; for example, if half of the positions in two sequences (e.g., five positions in a polymer ten amino acids in length) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are identical, then the two amino acid sequences are 90% identical.

Basic identity: as used herein, the term "substantial identity" refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially identical" if they contain identical residues in the 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 commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, notch BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant residue segment. In some embodiments, the relevant segment is a complete sequence. In some embodiments, the relevant stretch 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 residues or more. In the context of CDRs, reference to "substantial identity" generally refers 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.

Immune cells: as used herein, the term "immune cell" refers to a cell that is involved in an immune response (e.g., that promotes an immune response). Examples of immune cells include, but are not limited to, T cells, natural Killer (NK) cells, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, langerhans cells, or B lymphocytes. The source of immune cells (e.g., T cells or NK cells) can be obtained from a subject.

Immune checkpoints: as used herein, the term "immune checkpoint" refers to a set of molecules on the cell surface of cd4+ and/or cd8+ T cells that fine-tune an immune response by down-regulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well known in the art and include, but are not limited to, KIR family receptors 、HHLA2、CTLA-4、PD-l、VISTA、B7-H2、B7-H3、PD-L1、B7-H4、B7-H6、ICOS、HVEM、PD-L2、CD160、gp49B、PIR-B、TIM-l、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、 milk fat proteins and A2aR. The term also 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 also encompasses any fragment described in terms of homology provided herein.

Immune response: as used herein, the term "immune response" refers to a cellular and/or systemic response to an antigen that occurs when lymphocytes identify an antigen molecule as a foreign and induce the formation of antibodies and/or activate lymphocytes to remove antigen.

Immunoglobulin: as used herein, the term "immunoglobulin" or "Ig" refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptor. Five members included in this class of proteins are IgA, igG, igM, igD and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced in the primary immune response of most subjects. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody responses, and is important for protection against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergens.

Separating: as used herein, the term "isolated" refers to a substance that is altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from its naturally occurring coexisting materials, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment such as a host cell.

"Improve", "increase", "inhibit" or "decrease": as used herein, the terms "improve," "increase," "inhibit," "decrease," or grammatical equivalents thereof denote 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., in a single individual) in the absence (e.g., before and/or after) of a particular agent or treatment or in other comparable conditions in the presence of a suitable comparable reference agent. In some embodiments, the appropriate reference measurement is or includes a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of the relevant agent or treatment.

K _D： as used herein, the term "K _D" 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., an epitope to which the antibody or antigen binding fragment thereof binds). The term "K _D" as used herein is equal to K _off divided by K _on.

K _off: as used herein, the term "K _off" refers to the dissociation rate constant of a binding agent (e.g., an antibody or antigen binding fragment thereof) from a complex with its partner (e.g., an epitope to which the antibody or antigen binding fragment thereof binds).

K _on： as used herein, the term "K _on" refers to the association rate constant of a binding agent (e.g., an antibody or antigen binding fragment thereof) associated with its partner (e.g., an epitope to which the antibody or antigen binding fragment thereof binds).

And (3) adjusting: as used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response and/or a change in the nature of a response in a subject as compared to the level and/or nature of a response in a subject in the absence of a treatment or compound and/or as compared to the level and/or nature of a response in an otherwise identical but untreated subject. The term encompasses disruption and/or influencing of a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human. Monoclonal antibodies: "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homologous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during monoclonal antibody production), such variants typically being present in minor amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.

Nucleic acid: as used herein, the term "nucleic acid" refers to a polymer of at least three nucleotides. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the nucleic acid is single stranded. In some embodiments, the nucleic acid is double stranded. In some embodiments, the nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a backbone comprising phosphodiester linkages and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5' -N-phosphoramidite linkages and/or one or more peptide linkages, e.g., as in a "peptide nucleic acid". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all non-natural residues. In some embodiments, the unnatural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-adenosine, 0 (6) -methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2 '-fluoro ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by complementary template-based polymerization, e.g., in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、20、225、250、275、300、325、350、375、400、425、450、475、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000 or more residues in length.

Operatively connected to: as used herein, the term "operably linked" refers to a functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence such that the latter is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Pharmaceutically acceptable: as used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable 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.

Pharmaceutically acceptable carrier: as used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. 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 that can be used as pharmaceutically 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

Polynucleotide (c): as used herein, the term "polynucleotide" refers to a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the following general knowledge: a nucleic acid is a polynucleotide that can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means (i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR ^TM, etc.), as well as by synthetic means.

Polypeptide: as used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) that are typically joined by peptide bonds. In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that is not found in nature. In some embodiments, the polypeptide has an engineered amino acid sequence in that it is designed and/or produced by artificial action. In some embodiments, the polypeptide may comprise, consist of, or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, the polypeptide may comprise only natural amino acids or only unnatural amino acids, or consist of only such amino acids. In some embodiments, the polypeptide may comprise a D-amino acid, an L-amino acid, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may comprise one or more pendant groups or other modifications, such as modification at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or attachment to one or more amino acid side chains, or any combination thereof. In some embodiments, such pendent groups or modifications may be selected from the group consisting of: acetylation, amidation, lipidation, methylation, pegylation, and the like, including combinations thereof. In some embodiments, the polypeptide may be cyclic, and/or may comprise a cyclic moiety. In some embodiments, the polypeptide is not cyclic and/or does not comprise any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or include a stapled polypeptide (stapled polypeptide). In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity or structure; in this case, 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 specification provides and/or one of skill in the art will know exemplary polypeptides within that class, whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides of a polypeptide class or family. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity to a reference polypeptide of the class (in some embodiments, to all polypeptides within the class), share a common sequence motif (e.g., a characteristic sequence element) with a reference polypeptide of the class (in some embodiments, to all polypeptides within the class), and/or share a common activity (in some embodiments, at a comparable level or within a specified range). for example, in some embodiments, one member polypeptide exhibits at least about 30-40% and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of overall sequence homology or degree of identity to a reference polypeptide, and/or includes at least one region exhibiting very high sequence identity (typically greater than 90% or even 95%, 96%, 97%, 98% or 99%) such as may be or include a conserved region of a characteristic sequence element in some embodiments. Such conserved regions typically cover at least 3-4 and typically up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least a stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In 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 found in the polypeptide of interest (e.g., fragments directly linked in the parent may be spatially separated in the polypeptide of interest and vice versa, and/or fragments may be present in the polypeptide of interest in a different order than in the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.

Protein: as used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked to each other by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. One of ordinary skill in the art will appreciate that a "protein" may be an intact polypeptide chain (with or without a signal sequence) as produced by a cell, or may be a characteristic portion thereof. It will be appreciated by those of ordinary skill that a protein may sometimes include more than one polypeptide chain linked, for example, by one or more disulfide bonds or otherwise associated. The polypeptide 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, and the like. In some embodiments, the protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides that are less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

Recombinant: as used herein, it is intended to refer to polypeptides designed, engineered, prepared, expressed, produced, manufactured, and/or isolated by recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells; polypeptides isolated from a recombinant combinatorial 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); antibodies isolated from animals (e.g., mice) that are 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 13:593,2002;Murphy Proc.Natl Acad Sci USA 111:5153,2104); or polypeptides prepared, expressed, produced, or isolated by any other means, in some embodiments, one or more of such selected sequence elements are present in nature, one or more of such selected sequence elements are computer-designed, in some embodiments, for example, in some embodiments, the recombinant antibody polypeptide consists of sequences that are present in the germline of an organism of interest (e.g., human, mouse, etc.) in some embodiments, the recombinant antibody has amino acid sequences that are generated by 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 sequences that, while derived from and associated with germline VH and VL sequences, are not naturally present in the germline antibody repertoire in vivo.

Signal transduction pathways: as used herein, the term "signal transduction pathway" refers to a biochemical relationship between a plurality of signal transduction molecules that function in the transfer of a signal from one portion of a cell to another portion of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes capable of receiving signals and transmitting signals across the plasma membrane of a cell.

Single chain antibody: as used herein, the term "single chain antibody" refers to an antibody formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to Fv regions via engineered amino acid spans. Various methods of generating single chain antibodies are known, including those described in the following documents: U.S. patent No. 4,694,778; bird (1988) Science242:423-442; 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.

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

The subject: as used herein, the term "subject" refers to an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, experimental animal, mouse, rat, hamster, gerbil, cat, or dog). In some embodiments, the human subject is an adult, adolescent, or pediatric subject. In some embodiments, the subject has a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or tumor listed herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or exhibits an increased risk of developing a disease, disorder, or condition (as compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit a disease, disorder, or particular symptom (e.g., clinical manifestation of the disease) or feature of the disease. In some embodiments, the subject does not exhibit any symptoms or features of the disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is administered and/or has been administered diagnosis and/or therapy.

Basically: as used herein, the term "substantially" refers to a qualitative condition that exhibits a feature or characteristic of interest in an overall or near-overall range or degree. It will be appreciated by those of ordinary skill in the biological arts that little, if any, biological and chemical phenomena may be accomplished and/or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to achieve inherent completeness that is potentially lacking in many biological and chemical phenomena.

Is provided with: an individual "suffering from" a disease, disorder, and/or condition has been diagnosed as suffering from and/or exhibiting one or more symptoms of the disease, disorder, and/or condition.

And (3) target: as used herein, the term "target" refers to a cell, tissue, organ or site in the body of a subject that is a provided method, system, and/or composition, 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 modified population of immune effector cells as described herein or a KIR3DL3 inhibitor as described herein.

Therapeutic properties: as used herein, the term "therapeutic" refers to treatment and/or prevention. Therapeutic effects are obtained, for example, by inhibiting, alleviating or eradicating a disease state.

Therapeutic agent: as used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and/or causes a desired biological and/or pharmacological effect when administered to a subject. In some embodiments, the therapeutic agent may be an agent that prevents undesirable side effects when administered to a subject. In some embodiments, a therapeutic agent is any substance that is useful for alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. Therapeutic agents include, but are not limited to, a modified population of immune effector cells described herein and/or at least one KIR3DL3 inhibitor described herein.

Therapeutically effective amount of: 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 elicits a desired biological response when administered as part of a therapeutic 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 one of ordinary skill in the art, the effective amount of the substance may vary depending on factors such as the desired biological endpoint, the substance to be 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 reduces, ameliorates, alleviates, inhibits, prevents, delays onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, the therapeutically effective amount is administered in a single dose. In some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

Treatment: as used herein, the term "treatment" refers to the partial or complete alleviation, amelioration, onset delay, inhibition, prevention, alleviation 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, the treatment may be administered to a subject that does not exhibit signs or characteristics of the disease, disorder, and/or condition (e.g., may be prophylactic). In some embodiments, the treatment may be administered to subjects that exhibit only early or mild signs or features of the disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject exhibiting a defined, severe, and/or advanced sign of the disease, disorder, or condition. In some embodiments, treatment can include administering to a subject a population of modified immune effector cells (e.g., T cells or NK cells) described herein and/or at least one KIR3DL3 inhibitor described herein.

Tumor: as used herein, the term "tumor" refers to abnormal growth of cells or tissue. In some embodiments, the tumor may comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, and/or non-metastatic cells. In some embodiments, the tumor is associated with or is a manifestation of cancer. In some embodiments, the tumor may be a dispersed tumor or a liquid tumor. In some embodiments, the tumor may be a solid tumor.

Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as limiting the scope of the invention. Accordingly, the description of a range should be considered to have all possible subranges as specifically disclosed, as well as individual values within the range. For example, descriptions of ranges such as 1 to 6 should be considered to have specifically disclosed sub-ranges (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) as well as individual values within the range (e.g., 1,2, 2.7, 3,4, 5, 5.3, and 6). This applies regardless of the width of the range.

Detailed Description

Provided herein, inter alia, are anti-KIR 3DL3 antibodies and antigen-binding fragments thereof. In some embodiments, the anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein specifically bind KIR3DL3 (e.g., human KIR3DL 3) with high affinity. In some embodiments, an anti-KIR 3DL3 antibody described herein, and antigen-binding fragments thereof, blocks the binding of KIR3DL3 to HHLA. In some embodiments, the anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein specifically bind KIR3DL3 expressed on NK cells. In some embodiments, an anti-KIR 3DL3 antibody described herein, and antigen-binding fragments thereof, blocks HHLA 2-mediated inhibitory activity in T cells. In some embodiments, the anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein enhance NK cell killing of HHLA-expressing tumor cells. In some embodiments, the anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein enhance anti-tumor activity. Accordingly, the present disclosure provides anti-KIR 3DL3 antibodies and antigen-binding fragments thereof described herein, which are useful in methods of treating diseases, disorders, and conditions such as the various cancers described herein, as well as methods for modulating immune responses.

Immune effector cells

The present disclosure provides, inter alia, immune effector cells (e.g., NK cells or T cells) described herein that are modified with at least one immune cell activator and/or at least one KIR3DL3 inhibitor to produce a modified immune effector cell population. Thus, in some embodiments, for example, the modified population of immune effector cells exhibits enhanced proliferation relative to proliferation prior to modification. In some embodiments, for example, the modified population of immune effector cells exhibits an increase in endogenous expression of at least one cytokine relative to the endogenous expression of the at least one cytokine prior to modification. The method of preparing a modified population of immune effector cells described herein may comprise: (i) Contacting a population of immune effector cells with at least one immune cell activator described herein, and/or (ii) contacting a population of immune effector cells with at least one KIR3DL3 inhibitor described herein.

As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response (e.g., that promotes an immune response). Examples of immune cell 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 myelogenous phagocytes.

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 molecule as described herein. The cells can be modified by introducing one or more agents described herein (e.g., at least one immune cell activator described herein or at least one KIR3DL3 inhibitor described herein). Molecules can be modified in a number of ways, including chemical, structural and functional. In some embodiments, the modified immune effector cell has improved effector function due to the modification, e.g., an immune effector cell contacted with at least one immune cell activator (e.g., cytokine agents described herein) and/or at least one KIR3DL3 inhibitor described herein. In some embodiments, the at least one KIR3DL3 inhibitor is one or more anti-KIR 3DL3 antibodies described herein, or antigen-binding fragments thereof.

T cell

In some embodiments, the immune effector cell comprises or is a T cell. T cells may have effector functions (Teff) to increase immune responses by expressing one or more T Cell Receptors (TCRs). In some embodiments, effector functions include or are one or more of cytokine secretion, cytotoxic activity, and/or anti-self recognition. Conventional T cells (Tconv or Teff) may be any T cell population that is not a T regulatory cell (Treg) and includes, but is not limited to, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon that has differentiated into, for example, the Thl or Th2 lineages. In some embodiments, teff comprises or is a cd4+ Teff, such as a cd4+ helper T cell (e.g., thO, thl, tfh or Thl 7). In some embodiments, teff comprises or is a cd8+ cytotoxic T cell. In some embodiments, teff comprises non-Treg T cells or a subset of non-Treg T cells. In some embodiments, the cytotoxic T cell is a cd8+ T lymphocyte.

Prior to modification of the T cells described herein, a source of T cells may be obtained from the subject. T cells can be obtained from a number of sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells may be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll ^TM isolation or magnetic bead isolation. Any number of T cell lines available in the art may also be used.

T cells can be expanded by any method known in the art, such as by contacting the T cells with a surface to which are attached an agent that stimulates a CD3/TCR complex-associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. The T cell population may be stimulated using a variety of methods known in the art, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof. To co-stimulate the accessory molecules on the surface of the T cells, ligands that bind the accessory molecules may be used. For example, an anti-CD 28 antibody can 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 inhibition. In some embodiments, a population of T cells (e.g., cd4+ T cells or cd8+ T cells) may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. In some embodiments, a population of T cells (e.g., cd4+ T cells or cd8+ T cells) may be contacted with an effective amount of a cytokine (e.g., IL-2 or IL-15) under conditions suitable to stimulate T cell proliferation.

NK cells

In some embodiments, the immune effector cells comprise or are natural killer (NK cells). NK cells can exhibit cytolytic activity against a variety of targets via exocytosis of cytoplasmic granules containing a variety of proteins, including perforin and granzyme proteases. NK cell killing may 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 the cell surface markers CD16 and CD56 and the absence of the T cell receptor (CD 3).

Mature NK cells, NK progenitor cells, or mixed populations of NK progenitor cells and mature NK cells can be used in the methods and compositions described herein. Mature NK cells include or target NK cells that have characteristic surface markers (e.g., CD16 and CD 56) and NK cell function and lack the potential for further differentiation. NK progenitor cells can be derived from common lymphoid progenitor Cells (CLPs).

Prior to modification of NK cells described herein, a source of NK cells may be obtained from the subject. NK cells can be obtained from a number of sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, NK cells may be obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll ^TM isolation or magnetic bead isolation. Any number of NK cell lines available in the art (e.g., NK-92 cell line) may also be used.

NK cells may be stimulated using a variety of methods known in the art, such as by incubation with anti-CD 20 antibodies or co-culture with cells expressing CD 20. In some embodiments, the activation state of NK cells is determined by assessing expression of a marker (e.g., CD25, CD69, CD137, CD16, CD56, CD96, CD226, TIGIT, KIR2DL5, and/or NKG 2D). Cell surface expression of the markers may be determined, for example, via FACS analysis or immunohistological staining techniques.

Antibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells as described herein can also be determined. ADCC refers to a form of cytotoxicity in which secreted antibodies that bind to Fc receptors (FcR) present on cytotoxic cells (e.g., natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind and subsequently kill antigen-bearing target cells. To assess ADCC activity of NK cells, an in vitro ADCC assay may be performed, such as described in U.S. Pat. nos. 5,500,362 or 5,821,337.

The immune effector cells described herein can generally be activated and expanded using methods as described, for example, in U.S. patent 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、7,172,869、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 hereby incorporated by reference in its entirety.

Chimeric antigen receptor

In some embodiments, an immune effector cell (e.g., a T cell or NK cell) described herein can 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).

In some embodiments, the CAR comprises an antigen binding domain that binds to an antigen on, for example, 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 CLECL 1); 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 (ROR 1); fms-like tyrosine kinase 3 (FLT 3); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD 276); KIT (CD 117); interleukin-13 receptor subunit alpha-2 (IL-13 Ra2 or CD213 A2); mesothelin; vascular endothelial growth factor receptor 2 (VEGFR 2); lewis (Y) antigen; or CD24.

In some embodiments, the CAR comprises one or more extracellular lead domains, one or more extracellular hinge domains, and/or one or more intracellular co-stimulatory 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, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

In some embodiments, the CAR comprises a transmembrane domain that connects an extracellular domain to an intracellular domain, for example. In some embodiments, the transmembrane domain is naturally associated with one or more other domains of the CAR. In some embodiments, the transmembrane domain comprises one or more of the following: the α, β or ζ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., ,CD8α、CD8β)、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、IL2Rβ、IL2Rγ、IL7Rα、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.

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 responsible for signal activation and/or transduction. In some embodiments, the intracellular domain is or includes at least one signaling domain. In some embodiments, the intracellular signaling domain comprises or is derived from one or more of TCR zeta, fcrgamma, fcrbeta, cd3zeta, cd3gamma, cd3delta, cd3epsilon, CD5, CD22, CD79a, CD79b, and CD66 d. In some embodiments, the intracellular signaling domain may further comprise a costimulatory signaling domain. The co-stimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of the co-stimulatory molecule. Examples of such co-stimulatory molecules include, but are not limited to, CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1 (also known as PD 1), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3.

KIR3DL3 inhibitors

The present disclosure provides, inter alia, KIR3DL3 inhibitors. In some embodiments, the 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 simultaneous with the at least one KIR3DL3 inhibitor) to form a modified population of immune effector cells. In some embodiments, at least one KIR3DL3 inhibitor is administered to a subject in combination with a modified population of immune effector cells (e.g., immune effector cells are contacted with at least one immune cell activator to form a modified population of immune effector cells prior to administration to the subject).

HHLA2 are members of the B7 family that regulate NK cell and T cell functions. HHLA2 is widely expressed in a variety of tumor and antigen presenting cells and has been considered as an activating and inhibitory ligand for NK cells and T cells. HHLA2 are specific ligands for TMIGD2, and interaction of HHLA2 and TMIGD2 selectively stimulates cell proliferation and cytokine production. HHLA2 also binds to the receptor KIR3DL3 on T cells and NK cells, resulting in inhibition of T cell and NK cell activation. The present disclosure provides KIR3DL3 inhibitors for use in combination with immune cell activators for the treatment of a variety of cancers including solid and hematological tumors.

The term "KIR3DL3" or "killer cell immunoglobulin-like receptor 3DL3" as used herein refers to members of the killer cell immunoglobulin-like receptor transmembrane glycoprotein family expressed by NK cells and T cells. KIR3DL3 is also known as KIRC1, CD158Z, KIR DL7, and KIR44. The killer cell immunoglobulin-like receptor (KIR) gene is a polymorphic and highly homologous gene on chromosome 19q13.4 that resides within the 1Mb Leukocyte Receptor Complex (LRC). The genetic content of the KIR gene cluster varies between haplotypes, but several "framework" genes are found in all haplotypes (KIR 3DL3, KIR3DP1, KIR3DL4 and KIR3DL 2). KIR proteins are classified according to the number of extracellular immunoglobulin domains (2D or 3D) and whether they have long (L) or short (S) cytoplasmic domains. KIR proteins with long cytoplasmic domains transduce inhibitory signals via an immune tyrosine-based inhibitory motif (ITIM) upon ligand binding, whereas KIR proteins with short cytoplasmic domains lack ITIM motifs and instead associate with TYRO protein tyrosine kinase binding proteins to transduce activation signals. The ligands of several KIR proteins are a subset of HLA class I molecules; thus KIR proteins are believed to play an important role in the regulation of immune responses. KIR3DL3 proteins have an N-terminal signal sequence, 3 Ig domains, a transmembrane region lacking positively charged residues, and a long cytoplasmic tail containing ITIM. KIR3DL3 lacks stem regions present in other KIRs.

The term "KIR3DL3" includes fragments, variants (e.g., allelic variants) and derivatives thereof. Representative human KIR3DL3 cdnas 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: human KIR3DL3 (nm_ 153443.4) encoded by transcript (np_ 703144.3). Nucleic acid and polypeptide sequences of KIR3DL3 orthologs in organisms other than humans are also known, including but not limited to chimpanzee KIR3DL3 (xm_ 003316679.3 and xp_ 003316727.3), rhesus 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).

In some embodiments, the KIR3DL3 inhibitor exhibits binding affinity for KIR3DL3 or a fragment thereof (e.g., as assessed in a diagnostic assay such as Immunohistochemistry (IHC), western blot, intercellular flow, or ELISA). In some embodiments, KIR3DL3 inhibitors exhibit the ability to inhibit KIR3DL3 binding to HHLA.

Anti-KIR 3DL3 antibodies and antigen-binding fragments

Disclosed herein are methods, compositions, and formulations comprising inhibitors of KIR3DL3 (e.g., anti-KIR 3DL3 antibodies or antigen-binding fragments thereof). For example, an anti-KIR 3DL3 antibody or fragment thereof may specifically bind to an epitope on KIR3DL 3.

In some embodiments, the anti-KIR 3DL3 antibody or antigen-binding fragment thereof comprises or is a monoclonal antibody. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises or is a full-length antibody, e.g., comprising an immunoglobulin Fc region. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises or is a multispecific antibody, e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences has binding specificity for a second epitope. In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises or is a bispecific antibody molecule. In some embodiments, the antibody or antigen binding fragment thereof is or has been affinity matured.

An anti-KIR 3DL3 antibody or antigen-binding fragment thereof may comprise a heavy (H) chain variable domain sequence (VH) and a light (L) chain variable domain sequence (VL). In some embodiments, the anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises or is a heavy chain and a light chain (half antibody). In some embodiments, the anti-KIR 3DL3 antibody or antigen-binding fragment thereof comprises or is a two heavy (H) chain variable domain sequence and two light (L) chain variable domain sequences, thereby forming two antigen binding sites, such as Fab, fab ', F (ab ') 2, fc, 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 those antibodies synthesized de novo using recombinant DNA techniques. Such functional antibody fragments may retain the ability to selectively bind KIR3DL 3.

Examples of antigen binding fragments of anti-KIR 3DL3 antibodies may include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody single arm, (v) diabody (dAb) fragments consisting of VH domains; (vi) a camelidae antibody or a camelidae variable domain; (vii) scFv; and (viii) single domain antibodies. Antibody fragments can be obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as whole antibodies. anti-KIR 3DL3 antibodies and antibody fragments may be from any class of antibodies, including but not limited to IgG, igA, igM, igD and IgE, as well as from any subclass of antibodies (e.g., igG1, igG2, igG3, and IgG 4). The preparation of the anti-KIR 3DL3 antibody or antigen-binding fragment thereof may be monoclonal or polyclonal. The anti-KIR 3DL3 antibodies or antigen-binding fragments thereof may also be human, humanized, CDR-grafted, or generated in vitro. The anti-KIR 3DL3 antibody or fragment may have a heavy chain constant region selected from, for example, igG1, igG2, igG3, or IgG 4. The anti-KIR 3DL3 antibodies or antigen-binding fragments may also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody".

VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FR or FW). The terms "complementarity determining region" and "CDR" as used herein refer to amino acid sequences within the variable regions of an antibody that confer antigen specificity and binding affinity. Generally, there are three CDRs (HCDR 1, HCDR2 and HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2 and LCDR 3) in each light chain variable region. The framework regions and CDR ranges can be precisely defined using a variety of well known protocols (see, e.g., kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Pat. No. HEALTH AND Human Services, NIH publication No. 91-3242; chothia, C. Et al, (1987) J.mol. Biol.196:901-917; and AbM definitions used by Oxford Molecular's AbM antibody modeling software, each of which is hereby incorporated by reference in its entirety).

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises: (a) A heavy chain variable region (VH) comprising one, two or three VH CDR sequences each having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VH CDR in table 1; and/or (b) a light chain variable region (VL) comprising one, two, or three VL CDR sequences each having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL CDR in table 1. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprises: (a) VH having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to VH in table 1; and/or (a) a VL that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL in table 1. In some embodiments, an anti-KIR 3DL3 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 the heavy chain in 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 the light chain in table 1.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (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 (b) 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. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (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 a VL CDR1 amino acid sequence of SEQ ID NO:17, a VL CDR2 amino acid sequence of SEQ ID NO:18 and a VL CDR3 amino acid sequence of SEQ ID NO:19, each of which are disclosed in Table 1. In some embodiments, an anti-KIR 3DL3 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 a VL CDR1 amino acid sequence of SEQ ID NO:20, a VL CDR2 amino acid sequence of SEQ ID NO:21 and a VL CDR3 amino acid sequence of SEQ ID NO:22, each of which are disclosed in Table 1.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VH comprising the amino acid sequence of SEQ ID No. 10 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 10. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VL comprising the amino acid sequence of SEQ ID No. 23 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 23. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: VH comprising the amino acid sequence of SEQ ID No. 10; and VL comprising the amino acid sequence of SEQ ID NO. 23.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 12 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 12. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a light chain comprising the amino acid sequence of SEQ ID No. 25 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 25. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: 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.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (a) A VH comprising the VH CDR1 amino acid sequence of SEQ ID No. 27, the VH CDR2 amino acid sequence of SEQ ID No. 28, and the VH CDR3 amino acid sequence of SEQ ID No. 29; and (b) a VL comprising 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-KIR 3DL3 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, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (a) A VH comprising the VH CDR1 amino acid sequence of SEQ ID No. 33, the VH CDR2 amino acid sequence of SEQ ID No. 34, and the 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.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VH comprising the amino acid sequence of SEQ ID No. 36 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 36. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VL comprising the amino acid sequence of SEQ ID No. 49 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 49. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: a VH comprising the amino acid sequence of SEQ ID NO. 36; and VL comprising the amino acid sequence of SEQ ID NO. 49.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 38 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 38. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a light chain comprising the amino acid sequence of SEQ ID No. 51 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 51. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: 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.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (a) A VH comprising the VH CDR1 amino acid sequence of SEQ ID No. 53, the VH CDR2 amino acid sequence of SEQ ID No. 54, and the VH CDR3 amino acid sequence of SEQ ID No. 55; and (b) a VL comprising 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-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (a) A VH comprising the VH CDR1 amino acid sequence of SEQ ID No. 56, the VH CDR2 amino acid sequence of SEQ ID No. 57, and the VH CDR3 amino acid sequence of SEQ ID No. 58; and (b) a VL comprising 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-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: (a) A VH comprising the VH CDR1 amino acid sequence of SEQ ID No. 59, the VH CDR2 amino acid sequence of SEQ ID No. 60, and the 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.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VH comprising the amino acid sequence of SEQ ID No. 62 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 62. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a VL comprising the amino acid sequence of SEQ ID No. 75 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 75. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: VH comprising the amino acid sequence of SEQ ID No. 62; and VL comprising the amino acid sequence of SEQ ID NO. 75.

In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 64 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 64. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises a light chain comprising the amino acid sequence of SEQ ID No. 77 or an amino acid sequence having at least 85%, 90%, 95% or 99% or more identity to SEQ ID No. 77. In some embodiments, an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprises: 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. Amino acid and nucleotide sequences of exemplary anti-KIR 3DL3 antibodies.

Phage display and combinatorial methods for antibody production are known in the art (as described, for example, in 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 93/0188; mcCafferty et al, international publication No. WO 92/01047; gargard et al, international publication No. WO 92/09690; ladner et al, international publication No. WO 90/02809; fuchs et al, (1991) Bio/Technology9:1370-1372; hay et al, (1992) Hum Antibody Hybridomas:81-85; huse et al, (1989) Science 246:5-1281; grifms et al, (1993) J12-0120:3535-226; mcAb-353; biotechnology et al, (1993) scintin-35:3535-37; biosupport, 1996:3537-37; biosupport, 1996:1371; biosupport, 1996:3537-37:1371; biosupport, 1999:37:37:37:1999); and Barbas et al, (1991) PNAS 88:7978-7982, each of which is hereby incorporated by reference in its entirety.

For example, anti-KIR 3DL3 antibodies suitable for detecting KIR3DL3 proteins are known in the art and include, for example, antibody accession numbers: FAB8919R, MAB8919, FAB8919G, FAB8919N, FAB8919S, FAB8919T, FAB8919U and FAB8919V (R & D systems); antibody AP52374PU-N (origin); antibody PA5-26178 (ThermoFisher Scientific); antibodies OAAB05761, OAAF08125, OAAN04122, OACA09134, OACA09135, OACD04988; and OASG01190 (AVIVA SYSTEMS Biology).

Antigen binding fragments

The present disclosure provides, inter alia, anti-KIR 3DL3 antigen-binding fragments. As used herein, an "anti-KIR 3DL3 antigen-binding fragment" includes or is any protein or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule that contains at least one Complementarity Determining Region (CDR) derived from a VH or VL or KIR3DL3 binding portion of any of the antibodies described herein. Antibody fragments can be obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as whole antibodies. Such functional antibody fragments may retain the ability to selectively bind KIR3DL 3.

Examples of anti-KIR 3DL3 antigen-binding fragments described herein may include: (i) Fab fragments, which are monovalent fragments comprising VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment comprising VH and CH1 domains; (iv) Fv fragments comprising the VL and VH domains of a single arm of an antibody, (v) diabody (dAb) fragments comprising the VH domain; (vi) a camelidae antibody or a camelidae variable domain; (vii) fusion proteins of scFv, VH and VL regions; or (viii) a single domain antibody. In some embodiments, an anti-KIR 3DL3 antigen-binding fragment described herein comprises or is a heavy chain and a light chain (e.g., a half antibody).

Preparation method

The present disclosure provides, inter alia, methods of making an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein. In some embodiments, the anti-KIR 3DL3 antibodies or antigen-binding fragments thereof described herein are identified using a display technique such as yeast display, phage display, or ribosome display. In some embodiments, KIR3DL3 antibodies or antigen-binding fragments thereof described herein are identified using a hybridoma library (e.g., a mammalian hybridoma library, such as a mouse hybridoma library), followed by supernatant screening.

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 93/01188; mcCafferty et al, international publication No. WO 92/01047; gargard 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:81-85; huse et al, (1989) Science 246:5-1281; griffs et al, (1993) J12:725-226; mcCaffertl et al, (1993) P3) J12:226-226-McPhe-1275-1271; mcAb et al, (1996) Nad3:35:35:35:35:35-35; bio35-35:359; bio35, 1996:35, 1996; and Barbas et al, (1991) PNAS 88:7978-7982, each of which is incorporated by reference in its entirety.

In some embodiments, an anti-KIR 3DL3 antibody described herein, or antigen-binding fragment thereof, may be derived from other species. Humanized antibodies are antibodies produced by recombinant DNA techniques in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding (e.g., the constant and/or framework regions of a variable domain) are used to replace the corresponding amino acids of a light or heavy chain from a homologous non-human antibody. For example, humanized versions of murine antibodies to a given antigen have on the heavy and light chains: (1) a constant region of a human antibody; (2) FR from the variable domain of a human antibody; and (3) CDRs from a murine antibody. Human FR can be selected based on its highest sequence homology with the mouse FR sequence. If desired, one or more residues in the human FR may be changed to residues at corresponding positions in the murine antibody in order to maintain the binding affinity of the humanized antibody to the target. Such changes are sometimes referred to as "back mutations". Similarly, for desired reasons, such as stability or affinity to the target, a positive mutation can be made to revert back to the murine sequence. Humanized antibodies are generally less likely to elicit an immune response in humans than chimeric human antibodies because the former contain relatively few non-human components.

Methods of humanizing non-human antibodies are well known in the art. Methods suitable for preparing humanized antibodies according to the present disclosure are described, for example, in Winter EP 0 239 400; jones et al, nature 321:522-525 (1986); riechmann et al Nature 332:323-327 (1988); verhoeyen et al, science 239:1534-1536 (1988); queen et al, proc.Nat.Acad.ScL USA86:10029 (1989); U.S. Pat. nos. 6,180,370; and Orlandi et al, proc.Natl. Acad.Sd.USA86:3833 (1989); the disclosures of each of these documents are incorporated herein by reference in their entirety. Typically, the grafting of non-human (e.g., murine) CDRs onto a human antibody is accomplished as follows. The cdnas encoding VH and VL were isolated from hybridomas, and the nucleic acid sequences encoding VH and VL comprising CDRs were determined by sequencing. Nucleic acid sequences encoding CDRs are inserted into the corresponding regions of the human antibody VH or VL coding sequences and ligated to human constant region gene segments of the desired isotype (e.g., γl for CH and k for CL). Humanized heavy and light chain genes are co-expressed in mammalian host cells (e.g., CHO or NSO cells) to produce soluble humanized antibodies. To facilitate large-scale production of antibodies, it is often desirable to select for high expressors using, for example, the DHFR gene or the GS gene in the production line.

In some embodiments, an anti-KIR 3DL3 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 prepared by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences (see, e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/60433, WO 98/24893, WO 98/16664, WO 96/34096, WO 96/33735 and WO 91/10741; each of which is incorporated herein by reference in its entirety). Techniques such as those described 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, may be used to prepare human monoclonal antibodies.

Nucleic acid

The present disclosure provides, inter alia, nucleic acids encoding KIR3DL3 binding agents described herein (e.g., anti-KIR 3DL3 antibodies or antigen-binding fragments thereof). The 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 thereof disclosed herein. Such nucleic acids may be present in vectors. Such nucleic acid may be present in the genome of a cell, e.g., a cell of a subject in need of treatment or a cell for producing an antibody, e.g., a mammalian cell for producing an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein.

Nucleic acids encoding KIR3DL3 binding agents described herein (e.g., anti-KIR 3DL3 antibodies or antigen-binding fragments thereof) can be modified to include codons optimized for expression in a particular cell type or organism. The codon-optimized sequence is a synthetic sequence and preferably encodes 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 the non-codon-optimized parent polynucleotide. In some embodiments, the coding region of a nucleic acid encoding a KIR3DL3 binding agent described herein, in whole or in part, can comprise an altered sequence to optimize codon usage for a particular cell type (e.g., eukaryotic or prokaryotic cells). For example, the coding sequences for the humanized heavy (or light) chain variable regions described herein can 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.

The nucleic acid constructs of the present disclosure may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid may be operably linked to expression control sequences. The present disclosure further provides vectors comprising any of the nucleic acids described herein or fragments thereof. Any of the nucleic acids or fragments thereof described herein 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 ordinary skill in the art (see, e.g., "Recombinant DNA Part D," Methods in Enzymology, vol.153, wu and Grossman, eds., ACADEMIC PRESS (1987)).

Conventional techniques include, for example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection, which may be used to introduce exogenous nucleic acids (e.g., DNA or RNA) into prokaryotic or eukaryotic host cells. Advantageously, the vector may comprise regulatory sequences, such as transcription and/or translation initiation and/or termination codons, which are specific for the type of host (e.g. bacterial, fungal, plant or animal) into which the vector is to be introduced, as appropriate and considering whether the vector is DNA or RNA. In some embodiments, the vector comprises regulatory sequences specific for an genus of host cells. In some embodiments, the vector comprises regulatory sequences specific for the species of host.

In addition to replication systems and inserted nucleic acids, the nucleic acid construct may comprise one or more marker genes that allow 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 an auxotrophic host to provide prototrophy.

The expression vector may comprise a native or non-native promoter operably linked to the isolated or purified nucleic acid described above. The choice of promoters, such as strong promoters, weak promoters, inducible promoters, tissue-specific promoters and/or development-specific promoters, is within the skill of the person skilled in the art. Similarly, combining a nucleic acid as described above with a promoter is also within the skill of the art.

Suitable vectors include those designed for proliferation and amplification and/or expression. For example, the cloning vector may be selected from the pUC series, the pBluescript series (Stratagene, laJolla, calif.), the pET series (Novagen, madison, wis.), the pGEX series (PHARMACIA BIOTECH, uppsala, sweden) or the pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, λ ZapII (Stratagene), λEMBL4, and λNM1149 can be used. Examples of plant expression vectors that may be used include pBI110, pBI101.2, pBI101.3, pBI121, or pBIN19 (Clontech). Examples of animal expression vectors that may be used include pEUK-C1, pMAM, or pMAMneo (Clontech). The TOPO cloning system (Invitrogen, carlsbad, calif.) can also be used according to manufacturer's recommendations.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression to aid in isolating nucleic acids encoding KIR3DL3 binding agents described herein, or to improve the introduction of nucleic acids into cells. The use of cloning vectors, expression vectors, adaptors, and linkers are well known in the art (see, e.g., sambrook et al, molecular Cloning, a Laboratory Manual, 2 nd edition, cold Spring Harbor Press, cold Spring Harbor, n.y. (1989); and Ausubel et al, current Protocols in Molecular Biology, greene Publishing Associates and John Wiley & Sons, new York, n.y. (1994), each of which is hereby incorporated by reference in its entirety).

In some embodiments, the nucleic acids and vectors of the present disclosure are isolated and/or purified. The present disclosure also provides compositions comprising isolated or purified nucleic acids, optionally in the form of vectors. The isolated nucleic acids and vectors can be prepared using standard techniques known in the art, including, for example, alkali/SDS treatment, csCl binding, column chromatography, agarose gel electrophoresis, and/or other techniques well known in the art. The composition may comprise other components as further described herein.

Any method known to those of skill in the art for inserting nucleic acids into vectors that encode an anti-human KIR3DL3 antibody or antigen-binding fragment thereof described herein under the control of transcriptional and/or translational control signals may be used to construct expression vectors. These methods may include recombinant DNA in vitro and synthetic techniques and recombinant in vivo (see, e.g., ausubel, supra; or Sambrook, supra).

Antibodies binding to the same epitope

In some embodiments, the anti-KIR 3DL3 antibodies or antigen-binding fragments thereof described herein comprise antibodies and antibody fragments that bind the same epitope as KIR3DL 3-binding antibodies shown in table 1 described herein. Thus, other 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 an antibody and antibody fragment described herein to KIR3DL3 protein (e.g., human KIR3DL 3) demonstrates that the test antibody competes with the antibody or antibody fragment for binding to KIR3DL3; such antibodies may bind to epitopes on KIR3DL3 proteins that are identical or related (e.g., structurally similar or spatially close) to antibodies or antibody fragments that compete with them, according to non-limiting theory. In some embodiments, the antibody that binds to the same epitope on KIR3DL3 as the anti-KIR 3DL3 antibodies described herein, or antigen-binding fragments thereof, is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

KIR3DL3 gene expression modulators

Disclosed herein are methods, compositions, and formulations comprising KIR3DL3 inhibitors that modulate gene expression (e.g., miRNA, shRNA, siRNA, CRISPR/Cas guide system, TALEN, or ZFN).

In some embodiments, the KIR3DL3 inhibitor comprises or is a gene expression modulator. Gene expression modulators may comprise RNAi molecules (e.g., double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micrornas (miRNA), short interfering RNAs (siRNA), short hairpin RNAs (shRNA)) and triplex-forming oligonucleotides (TFO)). Modulators of gene expression may also comprise modified forms of any of the foregoing RNA molecules, and thus comprise synthetic chemically modified RNAs.

In some embodiments, the RNAi can be a miRNA that reduces KIR3DL3 levels in a cell (e.g., NK cell or T cell). In some embodiments, the RNAi can be a shRNA that reduces KIR3DL3 levels in cells (e.g., NK cells or T cells). In some embodiments, the RNAi can be an siRNA that reduces KIR3DL3 levels in cells (e.g., NK cells or T cells). Use of mirnas and sirnas to modulate KIR3DL3 expression is described, for example, in Nutalai et al, genes (Basel). 2019;10 603, which is hereby incorporated by reference in its entirety.

Commercially available sirnas and shrnas for reducing KIR3DL3 expression include shRNA product numbers TF303684, TR303684, TG303684, TL303684, and TL303684V (HexaBiogen Groupe CliniSciences); shRNA product numbers sc-60892-SH and sc-60892-V (Santa Cruz Biotechnology, inc.); siRNA product number SR314516 (HexaBiogen Groupe CliniSciences); and siRNA product No. sc-60892 (Santa Cruz Biotechnology, inc.).

In some embodiments, the KIR3DL3 inhibitor comprises or is an endonuclease. Endonucleases can form breaks in double stranded DNA at desired locations in the genome and repair the break using host cell mechanisms using, for example, homologous recombination or nonhomologous end joining. Classes of endonucleases that can be used for gene editing include, but are not limited to, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), transcription activator-like effector nucleases (TALENs) (see, e.g., U.S. patent No. 8,697,853; and U.S. publications nos. 20150118216, 20150079064, and 20140087426), zinc Finger Nucleases (ZFNs) (see, e.g., U.S. patent nos. 8,956,828;8,921,112;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. publications nos. 20130224863, 20110113509, and 20110033935), and homing endonucleases (see, e.g., U.S. publication No. 20150166969; and U.S. patent No. 9,005,973).

In some embodiments, the KIR3DL3 inhibitor comprises or is a CRISPR/Cas system. The term "CRISPR" or "CRISPR of KIR3DL 3" or "CRISPR that inhibits KIR3DL 3" as used herein refers to a set of clustered regularly interspaced short palindromic repeats or a system comprising such a set of repeats. As used herein, "Cas" refers to a CRISPR-associated protein. "CRISPR/Cas system" refers to a system derived from CRISPR and Cas that can be used to silence or mutate KIR3DL3 genes in immune effector cells (e.g., NK cells or T cells) as described herein.

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

In some embodiments, the KIR3DL3 inhibitor comprises or is a TALEN. The term "TALEN" or "TALEN of KIR3DL 3" or "TALEN that inhibits KIR3DL 3" as used herein refers to a transcriptional activator-like effector nuclease that is an artificial nuclease that can be used to edit KIR3DL3 genes in the immune effector cells (e.g., NK cells or T cells).

In some embodiments, the KIR3DL3 inhibitor comprises or is a ZFN. The term "ZFN" or "zinc finger nuclease" or "ZFN that inhibits KIR3DL 3" as used herein refers to a zinc finger nuclease that is an artificial nuclease that can be used to edit KIR3DL3 genes in immune effector cells (e.g., NK cells or T cells) described herein.

Demethylating agents

In some embodiments, the KIR3DL3 inhibitor comprises or is a demethylating agent. "demethylating agent" as used herein refers to a chemical that inhibits methylation. In some embodiments, the demethylating agent includes or is 5-Aza-2-deoxycytidine (Aza), e.g., as described in Trundley et al, immunogenetics (2006) 57:904-916, which is hereby incorporated by reference in its entirety. In some embodiments, the demethylating agent comprises or is 5-azacytidine. In some embodiments, the demethylating agent includes or is 1- β -D-arabinofuranosyl-5-azacytosine. In some embodiments, the demethylating agent includes or is dihydro-5-azacytidine.

Immune cell activator

Disclosed herein are methods, compositions, and formulations comprising at least one immune cell activator (e.g., a cytokine agent, a co-stimulatory antibody or antigen binding fragment thereof, a polypeptide, a glycoprotein, or an exogenous cell).

As used herein, the term "immune cell activator" refers to an agent that activates an immune effector cell (e.g., NK cell or T cell) described herein, thereby modifying the immune effector cell (e.g., by increasing proliferation and/or endogenous expression of interleukin). Immune cell activators may include, but are not limited to, cytokine agents (e.g., interleukins, such as cytokines (e.g., IL-2, IL-15, IL-12, IL-17, and/or IL-18)), antibodies or fragments thereof (e.g., co-stimulatory 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. The immune cell activator may be endogenously expressed or exogenously expressed with respect to the immune effector cells described herein. Immune cell activators may bind to any cellular moiety, such as a receptor, epitope, or other binding site present on immune effector cells described herein. Immune cell activators may diffuse or transport into cells where it may act within the cell. The immune activator can 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-L2, TIM-3, TGF-beta, or LAG-3), an enzyme (e.g., CD39 or CD 73), and/or a receptor (e.g., CTLA-4 or CD 3).

In some embodiments, contacting an immune effector cell described herein with an immune cell activator described herein increases proliferation of the immune effector cell and/or increases endogenous expression of at least one interleukin (e.g., cytokine) of the immune effector cell. In some embodiments, for example, contacting an immune effector cell described herein with at least one immune cell activator increases proliferation relative to an immune effector cell not contacted with the at least one immune cell activator. In some embodiments, for example, contacting an immune effector cell described herein with at least one immune cell activator increases endogenous expression of at least one interleukin described herein relative to an immune effector cell not contacted with the at least one immune cell activator.

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 includes IL-2, IL-15, IL-12, IL-17, IL-18, IL-21, IFN gamma or TNF alpha. In some embodiments, the cytokine agent is or includes IL-2. In some embodiments, the cytokine agent is or includes IL-15. In some embodiments, the cytokine agent is or includes IL-12. In some embodiments, the cytokine agent is or includes IL-17. In some embodiments, the cytokine agent is or includes IL-18. In some embodiments, the cytokine agent is or includes IL-21.

IL-2 is a member of a cytokine family comprising IL-4, IL-7, IL-9, IL-15 and IL-21, wherein each member of the family has a bundle of four alpha helices. IL-2 may be a T cell growth factor and is secreted endogenously in vivo by both CD4+ helper T cells and CD8+ T cells. IL-2 signals through an IL-2 receptor complex consisting of three chains, namely IL-2Rα (CD 25), IL-2Rβ (CD 122), and IL-2R (CD 132). In some embodiments, IL-2 binds IL-2Rα (CD 25), IL-2Rβ (CD 122), and/or IL-2R (CD 132) to activate immune effector cells or proliferation of immune effector cells 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 amplify tregs. In some embodiments, IL-2 enhances cytotoxicity and/or expands NK cells.

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

In some embodiments, the immune cell activator comprises or is a co-stimulatory antibody or antigen binding fragment thereof, or a co-stimulatory 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-L2, TIM-3, TGF-beta, or LAG-3. In some embodiments, the co-stimulatory small molecule binds CD3, 4-1BB, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L2, TIM-3, TGF-beta, LAG-3, CD39, or CD73.

In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-CD 3 antibody or fragment thereof. In some embodiments, the anti-CD 3 antibody comprises or is OKT3 (also known as molsingle-CD 3 or Orthoclone OKT 3). In some embodiments, the anti-CD 3 antibody or fragment thereof binds to T cell molecule T3 associated with a T cell antigen receptor, resulting in T cell activation. In some embodiments, for example, contacting an immune effector cell described herein with an anti-CD 3 antibody or fragment thereof increases cytokine production by the immune effector cell relative to an immune effector cell not contacted with the anti-CD 3 antibody or fragment thereof. In some embodiments, for example, contacting an immune effector cell described herein with an anti-CD 3 antibody or fragment thereof enhances proliferation of the immune effector cell relative to an immune effector cell not contacted with the anti-CD 3 antibody or fragment thereof.

In some embodiments, the costimulatory antibody, or antigen-binding fragment thereof, comprises or is an anti-4-1 BB antibody, or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-CD 40 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-CD 28 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-OX 40 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-GITR antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-CTLA-4 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-PD-1 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-PD-L1 antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-PD-L2 antibody or fragment thereof. For 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 co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-TGF- β antibody or fragment thereof. In some embodiments, the co-stimulatory antibody or antigen binding fragment thereof comprises or is an anti-LAG-3 antibody or fragment thereof.

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

In some embodiments, the immune cell activator comprises or is a glycoprotein. In some embodiments, the co-stimulatory glycoprotein comprises or is fibronectin or a fragment thereof. Fibronectin is an endogenously expressed high molecular weight (about 440 kDa) extracellular matrix glycoprotein known to bind transmembrane integrins. In some embodiments, the immune cell activator comprises or is recombinant human fibronectin 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 or fragment thereof comprises or is(Takara Bio Inc.). In some embodiments, for example, an immune effector cell described herein is contacted with fibronectin or a fragment thereof (e.g.,) The contacting increases proliferation of the immune effector cells described herein.

In some embodiments, the immune cell activator comprises or is a co-stimulatory exogenous cell. In some embodiments, the co-stimulatory exogenous cells include or are artificial antigen presenting cells (aapcs). In some embodiments, aapcs comprise or are K562-based aapcs. In some embodiments, for example, contacting an immune effector cell described herein with an aAPC increases cytokine production (e.g., IL-2 production) of the immune effector cell relative to an immune effector cell not contacted with the aAPC to increase immune effector cell stimulation. In some embodiments, for example, contacting an immune effector cell described herein with an aAPC enhances proliferation of the immune effector cell relative to an immune effector cell not contacted with the aAPC.

Therapeutic method

The present disclosure provides, inter alia, methods of treating a disease, disorder, or condition (e.g., a disease, disorder, or condition described herein) in a subject 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-KIR 3DL3 antibodies described herein, or antigen-binding fragments thereof.

In some embodiments, the present disclosure provides at least one anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, comprising an amino acid sequence set forth in table 1, as described herein for use as a medicament. In some embodiments, the present disclosure provides at least one anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprising an amino acid sequence set forth in table 1, for use in treating a disease, disorder, or condition described herein. In some embodiments, the present disclosure provides the use of at least one anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein comprising an amino acid sequence set forth in table 1, in the manufacture of a medicament for the treatment of a disease, disorder, or condition described herein.

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 a modified population of 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-KIR 3DL3 antibody or antigen-binding fragment thereof), thereby forming a modified population of immune effector cells.

The present disclosure also provides, inter alia, methods of treating a disease, disorder, or condition in a subject (e.g., a disease, disorder, or condition described herein) comprising delivering a pharmaceutical composition comprising a modified population of immune effector cells described herein, and administering to the subject a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR 3DL3 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 modified population of immune effector cells.

In some embodiments, a therapeutically effective amount of at least one pharmaceutical composition described herein is administered to a subject suffering from a disease, disorder, or condition. The pharmaceutical compositions described herein are useful for the manufacture of a medicament for treating a disease, disorder or condition in a subject or stimulating an immune response in a subject.

In some embodiments, a pharmaceutical composition comprising an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein is administered to a subject. In some embodiments, a pharmaceutical composition comprising a modified population of immune effector cells described herein is administered to a subject prior to administration of a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein) to a subject. In some embodiments, after administering a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein) to a subject, a pharmaceutical composition comprising a modified population of immune effector cells described herein is administered to the subject. In some embodiments, a pharmaceutical composition comprising a modified population of immune effector cells described herein is administered (e.g., co-administered by injection) to a subject substantially simultaneously with a pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a modified population of immune effector cells and at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein) is administered (e.g., by injection) to a subject.

In some embodiments, a pharmaceutical composition comprising a modified population of immune effector cells described herein is administered to a subject in less than about 3 hours of contacting the immune effector cells with at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, a pharmaceutical composition comprising a modified population of 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 an immune effector cell with at least one KIR3DL3 inhibitor described herein (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein).

The subject to be treated with the methods described herein can be a mammal, e.g., a primate, e.g., a human (e.g., a patient having or at risk of having a disease, disorder, or condition described herein). In some embodiments, immune effector cells (e.g., NK cells or T cells) may be autologous, allogenic, or xenogenic to the subject. The pharmaceutical compositions described herein may be administered to a subject according to the dosing regimen described herein, alone or in combination with one or more therapeutic agents, procedures or forms.

Provided is a method of treating a cancer or tumor (e.g., one or more of reducing, inhibiting, or delaying progression thereof) in a subject with a pharmaceutical composition comprising an immune cell (e.g., NK cell or T cell) described herein and/or a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein). The subject may have cancer in the form of an adult or a child. The cancer may be in early, mid or late stages, or may be metastatic cancer. In some embodiments, the subject has a cancer that is resistant to a therapeutic agent (e.g., comprising a cytokine agent described herein).

Provided is a method of treating a sign or symptom (e.g., one or more of reducing, inhibiting, or delaying progression of) of cancer in a subject with a pharmaceutical composition comprising an immune cell (e.g., NK cell or T cell) described herein and/or a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein). In some embodiments, the pharmaceutical compositions described herein can be used to delay the onset of cancer, slow the progression of cancer, or ameliorate 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 sign or symptom of the cancer includes or is a skin lesion (e.g., a lump or mole), weight loss, digestive problems, discomfort, fatigue, pain, dysphagia, cough, unusual bleeding and/or secretion, changes in bowel movement and/or urination habits, and/or confusion.

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

In some embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer (NSCLC) or NSCLC adenocarcinoma with squamous and/or non-squamous histology) 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., angioblastoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal cell carcinoma)), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two or all of the estrogen receptor, progesterone receptor, or Her 2/neu), such as triple negative breast cancer), colorectal cancer (such as recurrent or metastatic colorectal cancer, such as microsatellite unstable colorectal cancer, microsatellite stable colorectal cancer, mismatch repair proficient colorectal cancer or mismatch repair deficient colorectal cancer), nasopharyngeal cancer, duodenum cancer, endometrial cancer, pancreatic cancer, head and neck cancer (such as Head and Neck Squamous Cell Carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (such as undifferentiated thyroid cancer), cervical cancer (such as cervical squamous cell carcinoma), neuroendocrine tumor (NET) (such as atypical lung carcinoid tumor), pancreatic cancer, head and neck cancer (such as head and neck squamous cell carcinoma), lymphoproliferative disease (e.g., post-transplant lymphoproliferative disease), lymphoma (e.g., T-cell lymphoma, B-cell lymphoma, or non-hodgkin's lymphoma), myeloma (e.g., multiple myeloma), or leukemia (e.g., myelogenous leukemia or lymphoblastic leukemia). In some embodiments, the subject has renal cell carcinoma.

In some embodiments, the cancer is a brain tumor, such as glioblastoma, gliosarcoma, or recurrent brain tumor. In some embodiments, the cancer is pancreatic cancer, e.g., advanced pancreatic cancer. In some embodiments, the cancer is a skin cancer, such as melanoma (e.g., stage II-IV melanoma, HLA-A2 positive melanoma, unresectable melanoma, or metastatic melanoma) or Merkel cell carcinoma. In some embodiments, the cancer is a renal cancer, such as Renal Cell Carcinoma (RCC) (e.g., metastatic renal cell carcinoma). In some embodiments, the cancer is breast cancer, such as metastatic breast cancer or stage IV breast cancer, such as Triple Negative Breast Cancer (TNBC). In some embodiments, the cancer is a virus-related cancer. In some embodiments, the cancer is anal canal cancer (e.g., anal canal squamous cell carcinoma). In some embodiments, the cancer is cervical cancer (e.g., cervical squamous cell carcinoma). 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 a head and neck cancer (e.g., HPV positive and negative head and neck Squamous Cell Carcinoma (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 proficiency colorectal cancer, or mismatch repair deficient colorectal cancer.

In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is a leukemia, such as acute myelogenous leukemia, chronic myelogenous 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., recurrent or refractory HL or DLBCL). In some embodiments, the cancer is a myeloma, e.g., multiple myeloma.

Administration of the pharmaceutical compositions described herein may be performed in any convenient manner (e.g., injection, ingestion, infusion, inhalation, implantation, or transplantation). In some embodiments, the pharmaceutical compositions described herein are administered by injection or infusion. The pharmaceutical compositions described herein may be administered to a patient arterially, subcutaneously, intravenously, intradermally, intratumorally, intraganglionally, 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 a subject at a site of inflammation, a localized disease, a lymph node, an organ, a tumor, or an infection site.

The subject to be treated with the methods described herein can be a mammal (e.g., primate, mouse, humanized mouse, rat, non-human mammal, livestock, such as dog, cat, cow, or horse), and preferably a human (e.g., a patient suffering from or at risk of suffering from 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 cancer. In some embodiments, the subject is not undergoing treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy. In another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy. In certain embodiments, the subject has undergone surgery to remove cancerous or pre-cancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in a non-operable region of the body, such as in vital tissue or in a region where surgery would pose a substantial risk of injury to the subject.

Additive agent

The present disclosure provides, inter alia, one or more additional agents (e.g., 2, 3, 4, 5, or more additional agents) that can be administered in combination therapy with an anti-KIR 3DL3 antibody or antigen-binding fragment thereof described herein. As used herein, an additive may be or include any known treatment for a particular disease, disorder, or condition (e.g., cancer). For example, the additive may be or include one or more of the following: chemotherapeutic agents, immune checkpoint inhibitors, gene expression modulators, immune modulating interleukins, immune modulating chemokines, hormonal therapies, cell-based therapies, cancer vaccines, epigenetic modifiers (e.g., histone Deacetylase (HDAC) modifiers), immune modulating drugs, immune modulating antibodies, nutritional supplements, hyperthermia treatments, photodynamic therapies, surgery, radiation, or transplantation.

In some embodiments, the chemotherapeutic agent comprises 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 (HDAC), one or more topoisomerase inhibitors (e.g., one or more inhibitors of topoisomerase I or II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, or a combination thereof.

In some embodiments, the chemotherapeutic agent comprises or is one or more of the following: actinomycin, all-trans retinoic acid, orestatin, azacytidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunomycin, docetaxel, deoxyfluorouridine, doxorubicin, epirubicin, epothilone, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and/or their analogues (e.g., DM 1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, rituximab, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, dittansine or vinblastine. In some embodiments, the chemotherapeutic agent comprises or is an antibody-drug conjugate (ADC). In some embodiments, the ADC comprises or is hLL 1-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 (gemtuzumab ozogamicin), rituximab Shan Kangwei statin (brentuximab vedotin), trastuzumab maytansinone (trastuzumab emtansine), etomizumab ozogamicin (inotuzumab ozogamicin), erbitux Mo Shankang vildagliptin (glembatumomab 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 ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vomitozumab Ma Foduo statin (vorsetuzumab mafodotin), luo Fozhu monoclonal antibody minostatin (lorvotuzumab mertansine), or a combination thereof.

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 receptor 、TIM-1、TIM-3、TIM-4、LAG-3、BTLA、SIRPalpha(CD47)、CD48、2B4(CD244)、B7.1、B7.2、ILT-2、ILT-4、TIGIT、HHLA2、TMIDG2、KIR3DL3 and A2aR.

In some embodiments, the gene expression modulator comprises or is one or more of an inhibitory nucleic acid, a CRISPR/Cas guide system, a TALEN, or a ZFN. Inhibitory nucleic acids can comprise RNAi molecules (e.g., double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), micrornas (miRNA), short interfering RNAs (siRNA), short hairpin RNAs (shRNA), and triplex-forming oligonucleotides (TFO)). Modulators of gene expression may also comprise modified forms of any of the foregoing RNA molecules, and thus comprise synthetic chemically modified RNAs. In some embodiments, the CRISPR/Cas guide system can comprise Cas9, cas12 (e.g., cas12 a), or Cas 13.

In some embodiments, the immunomodulatory interleukin comprises or is one or more of the following: IL-2, IL-6, IL-7, IL-12, IL-17 or IL-23. In some embodiments, the immunomodulatory chemokines comprise or are one or more of the following: CCL3, CCL26, and CXCL7.

In some embodiments, the immunomodulatory drug comprises or is an immune cell growth inhibitory drug, a glucocorticoid, a cytostatic agent, an immunophilin, and modulators thereof (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporine (cyclosporine), pimecrolimus, abbe's limus, guanrolimus, li Dafu limus, 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, anti-thymocyte globulin, anti-lymphocyte globulin, thalidomide, lenalidomide pentoxifylline, bupropion, curcumin, catechin, opioids, IMPDH inhibitors, mycophenolic acid, myricetin, fingolimod, NF-xB inhibitors, raloxifene, drotregin alfa, dinomilast, NF-kB signaling cascade inhibitors, disulfiram, olmesartan, dithiocarbamate, proteasome inhibitors, bortezomib, MG132, prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, fraapine, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethyl epoxyquinolone (DHMEQ), I3C (indole-3-methanol)/DIM (diindolmethane) (13C/DIM), bay11-7082, luteolin, cell penetrating peptide SN-50, ikba super repressor overexpression, NFKB decoy Oligodeoxynucleotide (ODN) or derivatives or analogues of any of the foregoing.

In some embodiments, the immunomodulatory antibody comprises or is one or more of the following: antibodies that bind CD40, toll-like receptor (TLR), OX40, GITR, CD27 or 4-1BB, T cell bispecific antibodies, anti-IL-2 receptor antibodies, anti-CD 3 antibodies, OKT3 (moluzumab), oxybutymab, telbizumab, wegener-mab, anti-CD 4 antibodies, celecoxib, keliximab, zamu mab, anti-CD 11 antibodies, efalizumab, anti-CD 18 antibodies, ellizumab, luo Weizhu mab, anti-CD 20 antibodies, alfutizumab, ometizumab, ofuzumab, palivizumab, rituximab, anti-CD 23 antibodies, lu Xishan antibodies, anti-CD 40 antibodies, tenectimab, tolizumab, anti-CD 40L antibodies, lu Lizhu monoclonal antibodies, anti-CD 62L antibodies, alemtuzumab, anti-CD 80 antibodies, gancicb, anti-CD 147 antibodies, ganciclibizumab, bizumab, bikino (bls) and B lymphocyte stimulation factor (ys); inhibitory antibodies, belimumab, CTLA4-Ig fusion proteins, abacavir, berac, anti-CTLA 4 antibodies, ipilimumab, trimomumab, anti-eosinophil chemokine 1 antibodies, bai Ti mumab, anti-a 4 integrin antibodies, natalizumab, anti-IL-6R antibodies, tozumab, anti-LFA-1 antibodies, ondimomab, anti-CD 25 antibodies, basiliximab, daclizumab, enomomab, anti-CD 5 antibodies, zo Li Moshan antibodies, anti-CD 2 antibodies, cetrimab, nerimumab, famimumab, alisuzumab, atropimumab, cetrimab, attitumumab, rituximab, goruzumab, golimumab, bizumab (lebrilizumab), ma Simo mab, mozumab, pegamab, raylimumab, rayleimumab, luo Weizhu mab, tamarind mab, altimomab, valiximab, vipamomab, albesieadditional, alfasin, cilobuzepine, IL-1 receptor antagonists, anakinra, anti-IL-5 antibodies, meperimab, igE inhibitors, omab, IL12 inhibitors, IL23 inhibitors, or ulipristinab.

In some embodiments, the hormone therapy may be or include tamoxifen, raloxifene, leuprorelin, bicalutamide, granisetron, flutamide, or a combination thereof. In some embodiments, the cell-based therapy comprises or is a chimeric antigen receptor T (CAR-T) cell, CAR-NK cell, TCR-transduced T cell, dendritic cell, tumor-infiltrating lymphocyte (TIL), natural Killer (NK) cell, irradiated autologous or allogeneic tumor cell, or a combination thereof. In some embodiments, the hyperthermia treatment comprises either local hyperthermia (e.g., external, intraluminal, or interstitial hyperthermia), regional hyperthermia (e.g., deep tissue hyperthermia, regional perfusion, or continuous thermal peritoneal perfusion), or whole-body hyperthermia. In some embodiments, photodynamic therapy comprises or is administered a photosensitizer, such as hematoporphyrin and its derivatives, verteporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxyhypocrellin a, 2BA-2-DMHA, or a combination thereof. In some embodiments, the surgery includes or is surgery to remove cancerous or precancerous tissue. In some embodiments, the graft comprises or is a stem cell graft or an organ graft.

In some embodiments, the nutritional supplement includes or is one or more of vitamin a, vitamin E, vitamin C, and the like (see, e.g., U.S. patent nos. 4,981,844 and 5,230,902, and PCT publication WO 2004/004483).

In some embodiments, the additive is administered prior to, substantially simultaneously with, or after administration of an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein. In some embodiments, administration of an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein and an additive results in a greater degree of improvement in a disease, disorder, or condition (e.g., cancer) than that produced by a bispecific antibody molecule, or antigen-binding fragment thereof, or additive, described herein alone. The difference between the combined effect and the effect of each agent alone may be a statistically significant difference. In some embodiments, the combined effect may be a synergistic effect. In some embodiments, the combined administration of an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein with an additive allows for administration of the additive at reduced doses, reduced numbers of doses, and/or reduced dosing frequency compared to standard dosing regimens (e.g., approved dosing regimens for the additive).

Conjugates with therapeutic agents

The present disclosure provides, inter alia, anti-KIR 3DL3 antibodies or antigen-binding fragments thereof conjugated to one or more therapeutic agents. In some embodiments, the therapeutic agent comprises or is a cytotoxic agent, a drug, and/or a radioisotope. When conjugated with a cytotoxic agent, such conjugates may be referred to as "immunotoxins". Cytotoxic agents include any agent that is detrimental to (e.g., can kill) cells. Examples of cytotoxic agents include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecac, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs of any of the foregoing.

The anti-KIR 3DL3 antibodies or antigen-binding fragments thereof described herein may be conjugated to one or more therapeutic agents (e.g., one or more drugs) including, but not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and/or 5-fluorouracil dacarbazine), topoisomerase I inhibitors (e.g., delutekang), alkylating agents (e.g., nitrogen mustard, thiotepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and/or cis-dichlorodiamiplatin (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (previously known as daunorubicin) and/or doxorubicin), antibiotics (e.g., actinomycin D, bleomycin, photomycin, and/or amicin (mitomycin)) and antimuscarines (e.g., AMC or vincristine). The anti-KIR 3DL3 antibodies described herein, or antigen-binding fragments thereof, can be conjugated to one or more radioisotopes (e.g., radioiodine) to produce a cytotoxic radiopharmaceutical for use in treating a disease, disorder, or condition described herein, such as a cancer described herein.

Pharmaceutical composition

The present disclosure provides, inter alia, pharmaceutical compositions comprising a population of immune effector cells (e.g., NK cells or T cells) as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

The present disclosure also provides, inter alia, pharmaceutical compositions comprising at least one KIR3DL3 inhibitor in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises an anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises a modified population of immune effector cells described herein and at least one KIR3DL3 inhibitor described herein.

When indicated as "therapeutically effective amount", "immunologically effective amount", "anti-immune response effective amount" or "immune response suppressing effective amount", the precise amount of a pharmaceutical composition comprising a population of modified immune effector cells (e.g., NK cells or T cells) described herein and/or a pharmaceutical composition comprising at least one KIR3DL3 inhibitor (e.g., an anti-KIR 3DL3 antibody or antigen-binding fragment thereof) described herein can be determined by a physician taking into account individual differences in age, weight, immune response and condition of the patient (subject).

The pharmaceutical compositions described herein may comprise a buffer, including neutral buffered saline or Phosphate Buffered Saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextran or mannitol; a protein, polypeptide, or amino acid (e.g., glycine); an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and (3) a preservative. In some embodiments, the pharmaceutical composition is substantially free of contaminants, e.g., free of detectable levels of contaminants (e.g., endotoxins).

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

The pharmaceutical compositions described herein may take a variety of forms. These forms 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. The preferred composition may be an injectable or infusible solution. The pharmaceutical compositions described herein may be formulated for intravenous, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intraarterial, or intraperitoneal administration.

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 preferred embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous or intravenous injection or infusion. The pharmaceutical compositions described herein may be formulated for administration by using infusion techniques generally known in immunotherapy (see, e.g., rosenberg et al, new Eng.J.of Med.319:1676,1988, incorporated herein by reference in its entirety).

As used herein, the terms "parenteral administration (PARENTERAL ADMINISTRATION)" and "parenteral administration (ADMINISTERED PARENTERALLY)" refer to modes of administration that are typically performed by injection or infusion in addition to enteral and topical administration, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrasternal injection and infusion.

Pharmaceutical compositions comprising modified immune effector cells (e.g., NK cells or T cells) described herein can be administered in an amount of about 10 ⁴ to about 10 ⁹ cells/kg body weight (e.g., about 10 ⁵ to about 10 ⁶ cells/kg body weight, Including all integer values within those ranges). In some embodiments, the dose of modified immune effector cells (e.g., NK cells or T cells) described herein comprises at least about 1x 10 ⁶, about 1.1x 10 ⁶, about 2x 10 ⁶, about 3.6x 10 ⁶, About 5x 10 ⁶, about 1x 10 ⁷, about 1.8x 10 ⁷, about 2x 10 ⁷, About 5x 10 ⁷, about 1x 10 ⁸, about 2x 10 ⁸, about 5x 10 ⁸, About 1x 10 ⁹, about 2x 10 ⁹, or about 5x 10 ⁹ cells. The pharmaceutical compositions described herein may also be administered multiple times in doses. One skilled in the art can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient's signs of disease, disorder or condition and adjusting the treatment accordingly.

In some embodiments, the pharmaceutical compositions described herein are administered in combination (e.g., before, concurrently with, or after) bone marrow transplantation or lymphocyte ablation therapy using a chemotherapeutic agent (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or rituximab (Rituxan)). In certain embodiments, the subject is subjected to standard treatment with high doses of chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives infusion of one or more of the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical compositions described herein may be administered before or after surgery.

The dose of any of the foregoing therapies to be administered to a subject will vary with the disease, disorder or condition being treated and based on the particular subject. Dose scaling for human administration may be performed according to accepted practices in the art.

Kit for detecting a substance in a sample

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, a kit may include one or more containers comprising a pharmaceutical composition comprising at least one anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, described herein, and instructions for use and/or administration.

The present disclosure provides, inter alia, kits 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 kits may comprise one or more containers comprising a first pharmaceutical composition comprising at least one immune cell activator described herein and a pharmaceutically acceptable carrier and a second pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein and a pharmaceutically acceptable carrier. In some embodiments, the kits described herein comprise one or more anti-KIR 3DL3 antibodies, or antigen-binding fragments thereof, described herein, and instructions for use and/or administration.

The present disclosure provides, inter alia, kits comprising a modified population of immune effector cells and 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 modified population of immune effector cells described herein and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising at least one KIR3DL3 inhibitor described herein and a pharmaceutically acceptable carrier. In some embodiments, such kits comprise at least one KIR3DL3 antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.

In some embodiments, the kit includes instructions for any of the methods described herein. The instructions may include a description of administering the first and second pharmaceutical compositions to a subject to achieve a desired activity in the subject. The kit may further include instructions for selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions comprise a description of administering the first and second pharmaceutical compositions to a subject in need of treatment.

Instructions relating to the first and second pharmaceutical compositions described herein generally include information regarding the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is for treating, delaying the onset of, and/or alleviating a disease, disorder, or condition in a subject.

The kits provided herein are in suitable packaging. Suitable packages include, but are not limited to, vials, bottles, cans, flexible packages, and the like. Packages for use in combination with specific devices, such as infusion devices, are also contemplated. The kit may have a sterile access port (for example, the container may be an iv bag or vial having a stopper that is pierceable by a hypodermic injection needle). The container may also have a sterile inlet.

The kit optionally may provide additional components, such as buffers and interpretation information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the above-described kit.

Incorporated by reference

All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless defined otherwise, 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

The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. And should not be construed as limiting the scope or content of the present disclosure in any way.

Example 1: initial monoclonal antibody combined with human KIR3DL3

To explore the therapeutic potential of HHLA2-KIR3DL3 blockade, we generated a first monoclonal antibody called NPX267 (Hu 26E10z7p7 in table 1) that binds human killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 3 (KIR 3DL 3) with high affinity. KIR3DL3 is a member of the killer cell Ig-like (KIR) receptor family and is expressed by both NK cells and T cells. KIR3DL3 has recently been shown to be a co-inhibitory receptor for the B7 ligand human endogenous retrovirus H long terminal repeat associated protein 2 (HHLA 2) (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).

KIR3DL3 expressed on T and NK cells in tumor microenvironment inhibited the immune response after binding to HHLA2 (fig. 1A and 1B). Upon HHLA 2-induced KIR3DL3 activation, SHP-1 and SHP-2 are recruited to the cytoplasmic immunoreceptor tyrosine based inhibition motif (ITIM) of KIR3DL3 and downstream activation signals are inactivated. As a result, the activity of T cells and NK cells is inhibited. HHLA2 is expressed in normal tissues, but is highly expressed in many cancers and is often associated with poor patient outcome. In Renal Cell Carcinoma (RCC), HHLA2 expression is not normally co-expressed with PD-L1. However, co-expression of HHLA and PD-L1 in tumors from RCC patients is associated with a poorer progression-free survival than patients with tumors expressing only PD-L1. Thus, KIR3DL3-HHLA axis represents a new immune checkpoint pathway, and blocking KIR3DL3 signaling may be a promising strategy to promote anti-tumor immunity.

NPX267 is a humanized IgG4 monoclonal antibody that was demonstrated to specifically bind KIR3DL3 (fig. 2). Monovalent binding affinity of NPX267 to recombinant KIR3DL3 protein was determined by SPR using a Biacore instrument; KIR3DL3 protein was immobilized on CM5 chip and NPX267 was run as analyte. NPX267 has an affinity of 679pM for human KIR3DL 3. Off-target binding was not found in the screening of more than 5800 plasma membrane proteins.

NPX267 was demonstrated to be able to specifically bind to KIR3DL3 expressed on 300.19-KIR3DL3 cells, NK92MI cells and primary human NK cells (FIGS. 3A-3C). 300.19-KIR3DL3 cells were treated with NPX267 or IgG4 isotype control antibody at a concentration ranging from 10mg/mL to 0.0005mg/mL for 30 min (FIG. 3A). Flow cytometry analysis to detect primary antibody binding using an anti-human PE secondary antibody. The mean and standard deviation of duplicate data points are shown. Data from duplicate runs were entered GRAPHPAD PRISM into the software and EC ₅₀ was determined using a 4-parameter nonlinear regression equation.

NK92 MI cells were treated with NPX267 in a concentration range of 10mg/mL to 0.0005mg/mL for 30min (FIG. 3B). Flow cytometry analysis to detect primary antibody binding using an anti-human PE secondary antibody. Mean and standard deviation of triplicate data points are shown.

For primary human NK cells, human whole blood cells were treated with NPX267 at a concentration in the range of 10mg/mL to 0.00016351mg/mL for 30min (FIG. 3C). Erythrocytes were lysed and the cells were stained with a mixture of anti-human CD3, anti-human CD56 and anti-human IgG4 PE. Flow cytometry analysis to detect primary antibody binding using an anti-human PE secondary antibody.

NPX267 was demonstrated to bind KIR3DL3 on tumor-infiltrating CD56 ⁺ NK cells (fig. 4). Cryopreserved human dissociated tumor cells were thawed and separated into NPX276 and Fluorescence Minus One (FMO) conditions. Cells were stained with Viability dye, CD45-BV421, epCAM-FITC and CD56-PE antibody mixture for 30min +/-NPX267-APC. Samples were immediately collected on a flow cytometer. The NPX267+ gate was determined from FMO samples of each donor. KIR3DL3 expression ranged from 6% to 15% of infiltrating cd56+ NK cells.

NPX267 proved to block KIR3DL3 binding to HHLA (fig. 5). 300.19-KIR3DL3 cells were treated with NPX267 or IgG4 isotype control antibody at a concentration ranging from 10mg/mL to 0.0005mg/mL for 30 min. 5mg/mL of recombinant biotinylated HHLA2 was added to the cells and incubated for 30 minutes. Cells were treated with 1:250 APC-streptavidin for 30 minutes to detect recombinant biotinylation HHLA2 bound to the cells. gMFI of APC-streptavidin on 300.19-KIR3DL3 cells was measured by flow cytometry and percent inhibition was calculated. Samples treated with FACS buffer alone, recombinant biotinylated HHLA protein and APC-streptavidin were used to establish the maximum binding signal (0% inhibition), while samples treated with biotinylated HHLA2 without APC-streptavidin added were used to set the minimum binding signal (100% inhibition). NPX267 blocks the binding of recombinant HHLA-Fc to its inhibitory receptor KIR3DL3 in a dose-dependent manner.

NPX267 was demonstrated to block HHLA 2-mediated inhibitory activity in a T cell reporter assay (fig. 6). HHLA2/TCR/CHO cells were seeded into 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 hour. Tissue culture medium was removed from HHLA/TCR/CHO cells and Jurkat/IL-2/KIR3DL3 cells pre-compounded with experimental antibodies and anti-CD 28 agonist antibodies were added for 5-6 hours. Luciferase assays were performed using a one-step luciferase assay system. Luminescence was measured using a photometer (BioTek Synergy ^TM enzyme-labeled instrument). All treatment conditions were performed in triplicate. Luminescence intensity data was analyzed using GRAPHPAD PRISM software. In the absence of compound, the luminescence intensity (L _t) in each dataset was defined as 1. The luminescence induction fold 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 cell, lt = luminescence intensity in the absence of compound. The induction fold values were plotted in GRAPHPAD PRISM software and IC ₅₀ was determined using a 4 parameter nonlinear regression equation.

NPX267 was demonstrated to enhance NK cell killing of HHLA-expressing tumor cells (fig. 7A and 7B). NK92MI effector cells were seeded in round bottom 96 well plates and treated with 10mg/mL NPX267 for 30 min. After treatment, NK92MI cells were mixed with K562 cells engineered to express HHLA2 at an effector to target ratio of 1:1 and incubated for 3 hours at 37C and 5% CO 2. 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 in a dose-dependent manner by up to a factor of two compared to IgG4 isotype control (fig. 7A).

Kir3dl3+ human NK cells were incubated with 10mg/mL NPX267 or IgG4 isotype control for 30min and then added to CELLTRACE VIOLET labeled HCC827 cells at a ratio of 5 NK cells to 1 HCC827 cells for 6 hours. Cells were stained with 7-AAD. Non-viable HCC827 cells (CELLTRACE VIOLET +7-AAD+) were detected using flow cytometry. Specific lysis was calculated by specific lysis = (CELLTRACE VIOLET +7-aad+ cell number)/(CELLTRACE VIOLET + cell number) ×100. The mean and standard deviation of triplicate data points for each condition are shown. P values were calculated using a double sided Student T-test. NPX267 increased target cell death in two different donors with different KIR3DL3 levels (fig. 7B).

NPX267 is a humanized version of 26E10, which was demonstrated to be able to enhance NK cell-mediated antitumor activity in an in vivo HCC827 model (fig. 8). Luciferase-labelled HCC827 cells (4 x10 ⁶) were intraperitoneally (ip) injected into NSG mice (n=6/group) of 6-8 weeks of age. When tumors were established, mice were ip injected with KIR3DL3+ NK cells (1X 10 ⁷), 1 μg rhIL-2, 1 μg rhIL-15, and 200 μg NPX267 (or mIgG 1) every other day for a total of 5 injections. Tumor growth was assessed by imaging.

Taken together, the data presented herein demonstrate that NPX267 blocks the binding of HHLA2 to KIR3DL3 on primary human NK and T cells in a dose-dependent manner. The ability of NPX267 to block KIR3DL 3-mediated inhibition of T cell activation was assessed using a T cell reporter system and primary CD8 ⁺ T cell function assay. Blocking KIR3DL3 with NPX267 inhibited HHLA 2-mediated inhibition of T cell activation in a dose-dependent manner. The blocking of the anti-tumor activity of KIR3DL3 with NPX267 was also demonstrated in NK cell mediated cytotoxicity assays. NPX267 treatment enhanced the ability of human NK cell lines and primary NK cells to kill HHLA- ⁺ tumor cells in vitro. Finally, blocking HHLA 2-mediated KIR3DL3 signaling by NPX267 enhances anti-tumor immunity in a humanized mouse model carrying HHLA2 ⁺ human tumor.

Taken together, these data demonstrate that KIR3DL3-HHLA2 pathway is a novel immune checkpoint axis that promotes tumor escape by attenuating the innate and adaptive anti-tumor immune response. NPX267 is an initial KIR3DL3 blocking antibody that enhances anti-tumor immunity against HHLA2, 2 ⁺ tumors and represents a promising approach to treat certain diseases, disorders or conditions, particularly cancer.

Equivalent scheme

Those skilled in the art will appreciate that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only, and any invention described in this disclosure is further described in detail by the appended claims.

Those skilled in the art will appreciate typical standard deviations or errors attributable to the values obtained in the assays or other methods described herein. Publications, web addresses, and other references cited herein to describe the background of the invention and provide additional details regarding its practice are hereby incorporated by reference in their entirety.

Claims

1. A method of treating a subject having a disease, disorder or condition comprising administering a modified population of immune effector cells,

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 modified population of immune effector cells.

2. A method of treating a subject having a disease, disorder, or condition, comprising:

(i) Administering a modified population of 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 modified population of immune effector cells, and

(Ii) Administering at least one KIR3DL3 inhibitor to the subject.

3. The method of claim 1 or 2, wherein the at least one KIR3DL3 inhibitor is or comprises an anti-KIR 3DL3 antibody or antigen-binding fragment thereof, miRNA, shRNA, siRNA, CRISPR/Cas guide system, TALENs, ZFNs, and/or demethylating agent.

4. The method of claim 3, wherein the antigen binding fragment comprises scFv, fab, fab ', F (ab') 2, fc, or nanobody.

5. The method of claim 3 or 4, wherein the anti-KIR 3DL3 antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising one, two or three VH CDR sequences each having at least about 90% identity to a VH CDR of table 1; and/or

(B) A light chain variable region (VL) comprising one, two, or three VL CDR sequences each having at least about 90% identity to a VL CDR of table 1.

6. A method according to claim 3, wherein the demethylating agent comprises or is 5-Aza-2-deoxycytidine (Aza), 5-azacytidine, 1- β -D-arabinofuranosyl-5-azacytidine, or dihydro-5-azacytidine.

7. The method of any one of claims 1-6, wherein the immune cell activator results in increased T cell proliferation and/or endogenous expression of at least one cytokine.

8. The method of any one of claims 1-7, wherein the immune cell activator comprises or is a cytokine agent.

9. The method of claim 8, wherein the cytokine agent is or comprises IL-2, IL-15, IL-12, IL-17, IL-18 and/or IL-21, ifnγ and/or tnfα.

10. The method of claim 9, wherein the IL-2 binds IL-2rα, IL-2rβ, or IL-2rγ.

11. The method of claim 9 or 10, wherein the IL-2 expands only T cells and does not substantially expand tregs.

12. The method of claim 8, wherein the cytokine agent is or comprises an inhibitor of a cytokine signaling inhibitor (SOCS) protein.

13. The method of any one of claims 1-7, wherein the immune cell activator comprises or is a co-stimulatory antibody or antigen binding fragment thereof, a small molecule, a polypeptide, a glycoprotein or a foreign cell.

14. The method of claim 13, wherein the co-stimulatory antibody or antigen binding fragment thereof or co-stimulatory small molecule binds 4-1BB, CD3, CD40, CD28, OX40, GITR, CTLA-4, PD-1, PD-L2, TIM-3, TGF- β or LAG-3, CD39 or CD73.

15. The method of claim 14, wherein the co-stimulatory antibody or antigen binding fragment thereof comprises or is OKT3.

16. The method of claim 13, wherein the co-stimulatory polypeptide comprises or is a soluble HHLA Fc fusion polypeptide.

17. The method of claim 13, wherein the co-stimulatory glycoprotein comprises or is fibronectin or a fragment thereof.

18. The method of claim 13, wherein the exogenous cell comprises or is an artificial antigen presenting cell.

19. The method of any one of claims 1-18, wherein the immune effector cells are isolated from Peripheral Blood Mononuclear Cells (PBMCs) or tumor cells.

20. The method of any one of claims 1-19, wherein the modified immune effector cell comprises or is an NK cell and/or a T cell.

21. The method of claim 20, wherein the T cells comprise or are cd4+ T cells and/or cd8+ T cells.

22. The method of any one of claims 1-21, wherein the modified immune effector cell comprises at least one CAR.

23. The method of any one of claims 1-22, wherein the modified immune effector cell is administered to the subject in less than about 3 hours of contact with the at least one KIR3DL3 inhibitor.

24. The method of claim 23, wherein the modified immune effector cell is 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 the at least one KIR3DL3 inhibitor.

25. The method of any one of claims 1-24, wherein the modified population of immune effector cells and/or the KIR3DL3 inhibitor is administered parenterally.

26. The method of claim 25, wherein parenteral administration is or comprises subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion.

27. The method of any one of claims 1-26, wherein the method comprises sequentially administering the modified population of immune effector cells and the at least one KIR3DL3 inhibitor.

28. The method of claim 27, wherein:

(i) Administering the modified population of immune effector cells prior to administering the at least one KIR3DL3 inhibitor; or alternatively

(Ii) Administering the modified population of immune effector cells after administering the at least one KIR3DL3 inhibitor.

29. The method of any one of claims 1-26, comprising co-administering the modified population of immune effector cells and the at least one KIR3DL3 inhibitor.

30. The method of claim 29, wherein the method comprises co-administration by injection.

31. The method of any one of claims 1-30, wherein the subject has cancer.

32. The method of claim 31, wherein the subject has a solid tumor.

33. The method of claim 32, wherein the solid tumor is or comprises one or more of: renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer.

34. The method of claim 31, wherein the subject has hematological cancer.

35. The method of claim 34, wherein the hematological cancer comprises or is leukemia or lymphoma.

36. The method of claim 35, wherein the leukemia comprises or is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia.

37. The method of claim 35, wherein the lymphoma comprises or is Hodgkin's Lymphoma (HL), non-hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).

38. The method of any one of claims 31-37, wherein the subject has a cancer that is resistant to treatment comprising a cytokine agent.

39. The method of any one of claims 1-38, further comprising determining expression of TMIGD2 and/or KIR3DL3 of the modified immune effector cells.

40. The method of any one of claims 1-39, further comprising determining activation of the immune effector cell.

41. The method of any one of claims 1-40, further comprising determining the expression of CD25, CD69, CD137, CD16, CD56, CD96, CD226, KIR2DL5, and/or NKG 2D.

42. The method of any one of claims 1-41, further comprising formulating the modified population of immune cells into a composition for administration to a subject.

43. A method of preparing a modified population of immune effector cells, the method comprising:

(i) Contacting a 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 a modified population of immune effector cells.

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

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

46. A kit comprising at least one immune cell activator, at least one KIR3DL3 inhibitor, and instructions for use and/or administration.

47. A kit comprising a modified population of immune effector cells and at least one KIR3DL3 inhibitor and instructions for use and/or administration,

Wherein the immune effector cell is contacted with at least one immune cell activator.

48. An anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, which is or comprises:

49. The anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of claim 48, which is or comprises:

(a) VH comprising one, two or three VH CDR sequences each having at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VH CDR of table 1; and/or

(B) VL comprising one, two, or three VL CDR sequences each having at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a VL CDR of table 1.

50. The anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of claim 48 or 49, which is or comprises:

(a) VH having at least about 90% or greater identity to VH of table 1; and/or

(B) VL having at least about 90% or greater identity to VL of table 1.

51. The anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of claim 50, which is or comprises:

(a) VH having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to VH of table 1; and/or

(B) VL having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to VL of table 1.

52. The anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of any one of claims 48-51, which 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.

53. The anti-KIR 3DL3 antibody, or antigen-binding fragment thereof, of claim 52, which is or comprises:

(a) A heavy chain having at least about 95%, 96%, 97%, 98%, 99%, 99.5% or more 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 more identity to a light chain of table 1.

54. A nucleic acid encoding the anti-KIR 3DL3 antibody of any one of claims 48-53, or an antigen-binding fragment thereof.

55. An expression vector comprising the nucleic acid of claim 54.

56. A host cell comprising or expressing the anti-KIR 3DL3 antibody of any one of claims 48-53, or an antigen-binding fragment thereof, comprising the nucleic acid of claim 54, or comprising the expression vector of claim 55.

57. A pharmaceutical composition comprising at least one anti-KIR 3DL3 antibody or antigen-binding fragment thereof of any one of the preceding claims, and a pharmaceutically acceptable carrier, diluent or excipient.

58. A method of treating a subject having a disease, disorder, or condition, comprising: the pharmaceutical composition of claim 57 in a therapeutically effective amount.

59. A method of modulating an immune response in a subject, comprising: the pharmaceutical composition of claim 57 in a therapeutically effective amount.

60. The method of claim 58 or 59, wherein the subject has or is at risk of developing cancer.

61. The method of claim 60, wherein the subject has a solid tumor or hematological cancer.

62. The method of claim 61, wherein the solid tumor is or comprises one or more of: renal cancer, bone cancer, skin cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, lung cancer, ovarian cancer, liver cancer, bile duct cancer, or thyroid cancer.

63. The method of claim 61, wherein the hematological cancer comprises or is leukemia or lymphoma.

64. The method of claim 63, wherein the leukemia comprises or is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia.

65. The method of claim 63, wherein the lymphoma comprises or is Hodgkin's Lymphoma (HL), non-hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B-cell lymphoma (DLBCL).