Patent Application
20250019435
This application contains an ST.26 compliant Sequence Listing, which was submitted in xml format via Patent Center and is hereby incorporated by reference in its entirety. The .xml copy, created on Oct. 28, 2022 is named 129807.8027.W000 Sequence Listing.xml and is 64 KB in size.
Human killer-cell immunoglobulin-like receptors (KIRs) regulate Natural killer (NK) cells' activity by recognizing self-HLA class I molecules (Wagtmann 1995; Vilches 2002). KIR2DL5 is a member of the KIR family, but its biological functions are largely unknown (Winter 1998; Frazier 2013; Vilches 2000; Cisneros 2016; Gomez-Lozano 2002; Du 2008).
Poliovirus receptor (PVR, also known as CD155) is a member of the nectin/nectin-like family, which mediates cell adhesion, invasion and migration, and proliferation (Verschueren 2020; Husain 2019; Shilts 2022; Takai 2008; Kucan Brlic 2019). PVR overexpression induces tumor cell immune escape and is associated with a poor prognosis and enhanced tumor progression (Triki 2019; Carlsten 2007; Castriconi 2004; Masson 2001). Besides its tumor-intrinsic roles, PVR participates in multiple immunoregulatory events through interaction with the stimulatory receptor DNAX accessory molecule 1 (DNAM-1, also known as CD226) and the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD96 (Bottino 2003; Yu 2009; Chan 2014). Certain immunotherapies targeting the TIGIT/PVR axis as a potential cancer therapy are in clinical trials (Bendell 2020; Niu 2022; Cohen 2021; Wainberg 2021; Rodriguez-Abreu 2020; Ge 2021). Alternative approaches targeting other PVR pathways could contribute to improved outcomes. Accordingly, the present technology provides therapeutic strategies targeting the KIR2DL5/PVR pathway in the tumor microenvironment (TME) to satisfy an urgent need in the field.
Provided herein in certain embodiments are methods of modifying immune cell activity by altering KIR2DL5 expression and/or activity.
In one aspect, the present disclosure provides a method of increasing immune cell function in a subject comprising administering to the subject one or more agents that decrease KIR2DL5 expression and/or activity.
In another aspect, the present disclosure provides a method of treating an infectious disease in a subject in need thereof comprising administering to the subject one or more agents that decrease KIR2DL5 expression and/or activity.
In yet another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering one or more agents that decrease KIR2DL5 expression and/or activity.
In some embodiments, the one or more agents prevent or reduce KIR2DL5 binding to poliovirus receptor (PVR). In certain of these embodiments, the one or more agents binds KIR2DL5 at or near its binding site for PVR. In certain of these embodiments, the one or more agents bind PVR at or near its binding site for KIR2DL5.
In some embodiments, binding of the one or more agents to PVR does not block PVR binding to TIGIT, DNAM-1, and CD96.
In some embodiments, the one or more agents is selected from a peptide, polypeptide, or small molecule. In certain of these embodiments, the polypeptide is an antibody or a fusion protein comprising said antibody. In some embodiments, the antibody is a monoclonal antibody.
In yet another embodiment, the antibody is an antagonist antibody. In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a high chain variable region (VH) comprising an amino acid sequence encoded by SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, or SEQ ID NO:30.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a VH region comprising an amino acid sequence encoded by a nucleotide sequence that is at least 80% identical to SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, or SEQ ID NO:30.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a VH region comprising an amino acid of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, or SEQ ID NO:31.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a VH region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, or SEQ ID NO:31.
In another embodiment, the antibody or fusion protein comprising said antibody comprises a light chain variable region (LH) comprising an amino acid sequence encoded by SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a LH region comprising an amino acid sequence encoded by a nucleotide sequence that is at least 80% identical to SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, or SEQ ID NO:32.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a LH region comprising an amino acid of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, or SEQ ID NO:33.
In some embodiments, the antibody or fusion protein comprising said antibody comprises a LH region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, or SEQ ID NO:33.
In some embodiments, the infectious disease is caused by a pathogen. In certain of these embodiments, the pathogen is selected from a virus, bacterium, prion, fungus, parasite, or combination thereof. In some embodiments, the virus is selected the group consisting of human immunodeficiency viruses, influenza viruses, papillomaviruses, coronaviruses, hepatitis viruses, and herpesviruses. In some embodiments, the bacterium is Mycobacterium tuberculosis. In some embodiments, the fungus is Pneumocystis jirovecii (PJP).
In some embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell lymphoma, B-cell lymphoma, T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, T-cell lymphoma, Hodgkin's disease, B-cell non-Hodgkin's lymphoma, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative conditions, mucosa-associated lymphoid tissue (MALT) lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.
In yet another embodiment, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, lung cancer, kidney cancer, gastric cancer, gallbladder cancer, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers, combinations of the cancers, and metastatic lesions of the cancers.
In some embodiments, the cancer is a human hematologic malignancy. In certain of these embodiments, the human hematologic malignancy is selected from myeloid neoplasm, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML, acute leukemias of ambiguous lineage, myeloproliferative neoplasm, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndromes (MDS), myeloproliferative/myelodysplastic syndromes, chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, myelodysplastic syndromes (MDS), refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated del (5q), unclassifiable MDS, myeloproliferative/myelodysplastic syndromes, chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, unclassifiable myeloproliferative/myelodysplatic syndromes, lymphoid neoplasms, precursor lymphoid neoplasms, B lymphoblastic leukemia, B lymphoblastic lymphoma, T lymphoblastic leukemia, T lymphoblastic lymphoma, mature B-cell neoplasms, diffuse large B-cell lymphoma, primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma/leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, mantle cell lymphoma, marginal zone lymphomas, post-transplant lymphoproliferative disorders, HIV-associated lymphomas, primary effusion lymphoma, intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma, or solitary plasmacytomas (solitary bone and extramedullary).
In some embodiments, the cancer is selected from the group consisting of bladder cancer, kidney cancer, breast cancer, lung cancer, liver cancer, brain cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, uterine cancer, and stomach cancer.
In some embodiments, the cancer is a metastatic cancer.
In some embodiments, the method further comprises administering the subject to one or more additional cancer therapies selected from chemotherapy, radiation therapy, immunotherapy, surgery, and a combination thereof.
In another aspect, the present disclosure provides a method of decreasing immune cell function in a subject comprising administering to the subject one or more agents that increase KIR2DL5 expression and/or activity.
In some aspects, the present disclosure provides a method of treating an autoimmune disease in a subject comprising administering to the subject one or more agents that increase KIR2DL5 expression and/or activity to the subject.
In yet another aspect, the present disclosure provides a method of decreasing transplant rejection in a subject comprising administering to the subject one or more agents that increase KIR2DL5 expression and/or activity to the subject.
In some embodiments, the one or more agents is selected from the group consisting of a peptide, polypeptide, and a small molecule. In certain of these embodiments, the polypeptide is a fusion protein or an antibody. In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody increases activity of KIR2DL5. In some embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody.
In some embodiments, the autoimmune disease is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), alopecia areata, antiphospholipid syndrome, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lipoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticarial, autoimmune uveitis, Behçet's disease, celiac disease, Chagas disease, cold agglutinin disease, Crohn's disease, dermatomyositis, diabetes mellitus type 1, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Grave's syndrome, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, lupus erythematosus, Miller-Fisher syndrome, mixed connective tissue disease, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, relapsing polychondritis, rheumatoid arthritis, rheumatic fever, Sjögren's syndrome, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, Wegener's granulomatosis, and adult rheumatoid arthritis.
In some embodiments, the transplant is a stem cell transplant, bone marrow transplant, or combination thereof. In certain of these embodiments, the transplant is selected from the group consisting of a kidney transplant, a lung transplant, a heart transplant, a pancreas transplant, a cornea transplant, or a liver transplant.
The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.
The natural killer cell protein KIR2DL5 is a type I transmembrane molecule containing an N-terminal signal peptide, an ectodomain composed of tandem D0-D2 domains, a transmembrane region, and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). The amino acid sequence of KIR2DL5 is set forth in SEQ ID NO:1.
SEQ ID NO:1: MSLMVISMACVGFFLLQGAWTHEGGQDKPLLSAWPSAVVPRGGHVTLLCRSRLGFTIF SLYKEDGVPVPELYNKIFWKSILMGPVTPAHAGTYRCRGSHPRSPIEWSAPSNPLVIVVT GLFGKPSLSAQPGPTVRTGENVTLSCSSRSSFDMYHLSREGRAHEPRLPAVPSVNGTFQA DFPLGPATHGGTYTCFGSLHDSPYEWSDPSDPLLVSVTGNSSSSSSSPTEPSSKTGIRRHL HILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQ LDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTAL SQNRVASSHVPAAGI (1-21: signal peptide; 22-239: extracellular domain; 42-102: DO; 137-200: D2; 240-259: transmembrane; 260-374: cytoplasmic tail; 296-301: ITIM; 326-331: ITSM; see also NP_065396.1).
KIR2DL5 has NCBI Accession Number and Ensembl Gene Number of NG_005994.1 (NM_020535.3, NP_065396.1) and ENSG00000274143.1, respectively. The NCBI Accession number(s) and Ensembl Gene Number(s) provided herein were accessed on Oct. 28, 2022.
KIR2DL5 was recently identified as a binding partner for poliovirus receptor (PVR) via a high-throughput in vitro screen of IgG superfamily (IgSF) (Husain 2019; Wojtowicz 2020). However, the biology of the KIR2DL5-PVR pathway is largely unknown (Beziat 2017).
As disclosed herein, KIR2DL5 is an inhibitory receptor of the immune system. Specifically, it was found that (1) KIR2DL5 and the PVR receptors TIGIT, DNAM-1, and CD96 can simultaneously bind to nonidentical sites on PVR; (2) KIR2DL5 is expressed on the surface of human adaptive immune cells and innate immune cells; (3) KIR2DL5 inhibits NK cell function and mediates PVR+ tumor immune resistance; and (5) KIR2DL5 blockade promotes anti-tumor immunity. Based on these results, methods and compositions are provided herein for increasing immune cell activity by inhibiting KIR2DL5 expression and/or activity, and for decreasing immune cell activity by increasing KIR2DL5 expression and/or activity.
KIR2DL5 is polymorphic and is represented by 2DL5A*001 and 2DL5A*005 (Cisneros 2016). While most KIR2DL5B alleles are epigenetically silent because of a distinctive substitution in a promoter RUNX binding site, 2DL5B*003 and 2DL5B*00602 alleles with intact RUNX binding sites are predicted to be transcribed and expressed on the cell surface (Du 2008). These 2 alleles have an identical DO domain to KIR2DL5A*001 through which F8B30 recognized KIR2DL5, contains only 4 polymorphic sites: T46S, R52H, G97S, and P112S (IPD-KIR Database, Release 2.9.0) (Robinson 2010).
The terms “treat,” “treating,” and “treatment” as used herein with regard to a condition refer to alleviating the condition partially or entirely; slowing the progression or development of the condition; eliminating, reducing, or slowing the development of one or more symptoms associated with the condition; or increasing progression-free or overall survival of the condition.
The terms “prevent,” “preventing,” and “prevention” as used herein with regard to a condition refers to averting the onset of the condition or decreasing the likelihood of occurrence or recurrence of the condition, including in a subject that may be predisposed to the condition but has not yet been diagnosed as having the condition.
The term “infectious disease” may refer to any disease caused by an infectious organism such as a virus, bacteria, parasite, and/or fungus.
The term “antibody” as used herein refers to an immunoglobulin molecule or an immunologically active portion thereof that binds to a specific antigen, e.g., a cancer cell antigen, viral antigen, or microbial antigen. In those embodiments where the targeting moiety is an antibody and the antibody is a full-length immunoglobulin molecule, the antibody comprises two heavy chains and two light chains, with each heavy and light chain containing three complementary determining regions (CDRs). In those embodiments where the targeting moiety is an antibody and the antibody is an immunologically active portion of an immunoglobulin molecule, the antibody may be, for example, a Fab, Fab′, Fv, F(ab′)2, disulfide-linked Fv, scFv, single domain antibody (dAb), diabody, triabody, tetrabody, or linear antibody. Antibodies used as targeting moieties may be, for example, natural antibodies, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies, bispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, or fragments thereof that retain the ability to bind a specific antigen.
A “subject” as used herein refers to a mammalian subject, preferably a human.
Based on the results disclosed herein showing that KIR2DL5 inhibits human immune cell function, provided herein in certain embodiments are methods of increasing human immune cell function in a subject by decreasing KIR2DL5 expression and/or activity. Since increased immune cell function results in increased identification and removal of pathogens, methods are further provided for treating infectious disease in a subject by decreasing KIR2DL5 expression and/or activity. Similarly, since increased immune cell function may result in increased cancer cell killing, methods are provided for treating cancer in a subject by decreasing KIR2DL5 expression and/or activity.
In certain embodiments of the methods provided herein, KIR2DL5 activity is decreased in a subject by administering one or more agents that prevent or reduce KIR2DL5 binding to PVR. In certain of these embodiments, the agents bind KIR2DL5 and prevent or reduce its binding interaction with PVR, for example by binding KIR2DL5 at or near its binding site for PVR. In other embodiments, the agents bind PVR and prevent or reduce its binding interaction with KIR2DL5, for example by binding PVR at or near its binding site for KIR2DL5. In certain embodiments, agents that decrease KIR2DL5 activity by binding PVR and preventing or reducing KIR2DL5/PVR binding also block binding of PVR to one or more of its other known receptors, including TIGIT, DNAM-1, and CD96. In other embodiments, the agents prevent or reduce binding between KIR2DL5 and PVR while allowing PVR to bind one or more of its other known receptors. In certain embodiments of the methods provided herein, the agents that prevent or reduce KIR2DL5 binding to PVR are peptides, polypeptides, or small molecules. Suitable polypeptides include, but are not limited to, antibodies that specifically bind KIR2DL5 or PVR, truncated forms of KIR2DL5 or PVR (e.g., extracellular domain of KIR2DL5 or PVR or a portion thereof), and fusion polypeptides comprising antibodies or truncated forms of KIR2DL5 or PVR.
In certain embodiments of the methods provided herein, KIR2DL5 activity and/or expression is decreased in a subject by administering one or more agents that inhibit one or more pathways that upregulate KIR2DL5 expression. This inhibition may occur at any step in the pathway, for example by inhibiting the interaction between a surface receptor and its ligand, inhibiting the interaction between two or more intracellular proteins, blocking a KIR2DL5 promoter region, or the like.
In certain embodiments of the methods provided herein, KIR2DL5 activity and/or expression is decreased in a subject by altering a nucleotide sequence in the KIR2DL5 gene or one or more of its corresponding regulatory domains, e.g., promoters or enhancers. For example, in certain embodiments one or more nucleotide substitutions, insertions, or deletions may be introduced into the KIR2DL5 gene or its corresponding regulatory domains using a CRISPR/Cas (e.g., CRISPR/Cas9) system.
In some embodiments, methods are provided for treating an infectious disease in a subject in need thereof by decreasing KIR2DL5 expression and/or activity as disclosed herein. In certain of these embodiments, the infectious disease is caused by a pathogen. The pathogen can be one or more of a virus, bacterium, prion, fungus, and parasite. In some embodiments, the virus is selected from the group consisting of human immunodeficiency viruses, influenza viruses, papillomaviruses, coronaviruses, hepatitis viruses, or herpesviruses. In some embodiments, the bacterium is Mycobacterium tuberculosis. In certain embodiments, the fungus is Pneumocystis jirovecii (PJP).
In some embodiments, methods are provided for treating cancer in a subject in need thereof by decreasing KIR2DL5 expression and/or activity as disclosed herein. In certain of these embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell lymphoma, B-cell lymphoma, T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B-cell prolymphocytic leukemia, T-cell lymphoma, Hodgkin's disease, B-cell non-Hodgkin's lymphoma. blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, or preleukemia. In other embodiments, the cancer the cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, lung cancer, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers, combinations of the cancers, and metastatic lesions of the cancers. In other embodiments, the cancer is a human hematologic malignancy.
In certain embodiments, the human hematologic malignancy is selected from myeloid neoplasm, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related AML, acute leukemias of ambiguous lineage, myeloproliferative neoplasm, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndromes (MDS), myeloproliferative/myelodysplastic syndromes, chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, myelodysplastic syndromes (MDS), refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated del (5q), unclassifiable MDS, myeloproliferative/myelodysplastic syndromes, chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, unclassifiable myeloproliferative/myelodysplatic syndromes, lymphoid neoplasms, precursor lymphoid neoplasms, B lymphoblastic leukemia, B lymphoblastic lymphoma, T lymphoblastic leukemia, T lymphoblastic lymphoma, mature B-cell neoplasms, diffuse large B-cell lymphoma, primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, Burkitt lymphoma/leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, mantle cell lymphoma, marginal zone lymphomas, post-transplant lymphoproliferative disorders, HIV-associated lymphomas, primary effusion lymphoma, intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma, or solitary plasmacytomas (solitary bone and extramedullary).
In certain embodiments of the methods provided herein, the agents that decrease KIR2DL5 activity and/or expression are antibodies, immunogenic fragments thereof, or antibody fragments thereof that specifically bind KIR2DL5, or fusion proteins comprising such antibodies. In certain of these embodiments, the antibodies are monoclonal antibodies. In certain embodiments, the antibodies are chimeric antibodies, humanized antibodies, or fully human antibodies.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a variable heavy (VH) chain sequence comprising one or more of the CDR sequences of antibody B2A18 disclosed herein, i.e., residues 45-54, 69-85, and 116-131 of SEQ ID NO:3. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:3 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a variable light (VL) chain sequence comprising one or more of the CDR sequences of antibody B2A18 disclosed herein, i.e., residues 24-32, 50-56, and 89-97 of SEQ ID NO:5. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:5 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:4 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:3 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:5 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody B7B23 disclosed herein, i.e., residues 45-54, 69-85, and 116-125 of SEQ ID NO:7. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:7 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody B7B23 disclosed herein, i.e., residues 47-63, 79-85, and 118-126 of SEQ ID NO:9. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:9 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:7 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:9 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody B11B4 disclosed herein, i.e., residues 45-54, 69-87, and 116-127 of SEQ ID NO:11. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:11 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:10 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:10.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody B11B4 disclosed herein, i.e., residues 46-60, 76-82, and 115-123 of SEQ ID NO:13. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:13 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:11 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:11 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:13 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody B19C11 disclosed herein, i.e., residues 44-54, 69-84, and 115-129 of SEQ ID NO:15. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:15 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:14 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody B19C11 disclosed herein, i.e., residues 24-38, 54-60, and 93-101 of SEQ ID NO:17. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:17 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:16 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:15 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:17 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody B33C12 disclosed herein, i.e., residues 45-54, 69-87, and 116-127 of SEQ ID NO:19. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:19 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:18 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:18.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody B33C12 disclosed herein, i.e., residues 44-58, 74-80, and 113-121 of SEQ ID NO:21. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:21 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:20.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:19 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:19 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:21 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody E12B11 disclosed herein, i.e., residues 45-54, 69-85, and 116-131 of SEQ ID NO:23. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:23 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:22 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:22.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody E12B11 disclosed herein, i.e., residues 24-34, 50-56, and 89-97 of SEQ ID NO:25. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:25 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:24 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:24.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:23 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:23 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:25 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody F8B30 disclosed herein, i.e., residues 45-54, 69-85, and 116-125 of SEQ ID NO:27. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:27 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:27. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:26 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:26.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody F8B30 disclosed herein, i.e., residues 24-34, 50-56, and 89-97 of SEQ ID NO:29. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:29 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:29. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:28.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:27 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:27 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:29 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:29.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain sequence comprising one or more of the CDR sequences of antibody G11B22 disclosed herein, i.e., residues 45-54, 69-85, and 116-131 of SEQ ID NO:31. In certain of these embodiments, the VH chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:31 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:31. In certain embodiments, the VH chain is encoded by the nucleotide sequence of SEQ ID NO:30 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:30.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VL chain sequence comprising one or more of the CDR sequences of antibody G11B22 disclosed herein, i.e., residues 24-34, 50-56, and 89-97 of SEQ ID NO:33. In certain of these embodiments, the VL chain comprises, consists of, or consists essentially of the amino acid of SEQ ID NO:33 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:33. In certain embodiments, the VL chain is encoded by the nucleotide sequence of SEQ ID NO:32 or a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:32.
In certain embodiments of the methods provided herein wherein the agents that decrease KIR2DL5 activity and/or expression are KIR2DL5 antibodies, immunogenic fragments thereof, or antibody fragments thereof, which comprise a VH chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:31 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:31 and a VL chain comprising, consisting of, or consisting essentially of the amino acid of SEQ ID NO:33 or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:33.
Based on the results disclosed herein showing that KIR2DL5 inhibits human immune cell function, further provided herein in certain embodiments are methods of decreasing human immune cell function in a subject by increasing KIR2DL5 expression and/or activity. Since immune cell hyperactivity may be associated with autoimmune disease, methods are further provided for treating an autoimmune disease in a subject by increasing KIR2DL5 expression and/or activity. Similarly, decreasing immune cell activity may be helpful in the context of transplantation, where it is desirable to suppress the immune system to prevent transplant rejection. Accordingly, also provided are methods for decreasing transplant rejection by increasing KIR2DL5 expression and/or activity.
In certain embodiments of the methods provided herein, KIR2DL5 activity and/or expression is increased in a subject by administering one or more agents that are KIR2DL5 or PVR agonists. In certain embodiments, the agents increase the binding affinity of KIR2DL5 for PVR. In certain embodiments, the agents mimic KIR2DL5 by binding and activating PVR.
In certain embodiments of the methods provided herein, KIR2DL5 activity and/or expression is increased by administering one or more agents that increase the activity of one or more pathways that upregulate KIR2DL5 expression. This increased activity may occur at any step in the pathway, for example by activating a surface receptor, increasing the interaction between two or more intracellular proteins, activating a KIR2DL5 promoter region, or the like.
Suitable agents for increasing KIR2DL5 activity and/or expression included, but are not limited to, peptides, polypeptides (e.g., antibodies or fusion proteins), or small molecules.
In some embodiments, methods are provided for treating an autoimmune disorder in a subject in need thereof by increasing KIR2DL5 activity and/or expression as disclosed herein. In certain of these embodiments, the autoimmune disease or disorder is selected from the group consisting of acute disseminated encephalomyelitis (ADEM), alopecia areata, antiphospholipid syndrome, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lipoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticarial, autoimmune uveitis, Behçet's disease, celiac disease, Chagas disease, cold agglutinin disease, Crohn's disease, dermatomyositis, diabetes mellitus type 1, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Grave's syndrome, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, lupus erythematosus, Miller-Fisher syndrome, mixed connective tissue disease, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, relapsing polychondritis, rheumatoid arthritis, rheumatic fever, Sjögren's syndrome, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis. In certain embodiments, the condition is adult rheumatoid arthritis.
In some embodiments, methods are provided for decreasing transplant rejection in a subject in need thereof by increasing KIR2DL5 activity and/or expression. In certain of these embodiments, the transplant is selected from a stem cell transplant or a bone marrow transplant. In some embodiments, the transplant is selected from the group consisting of a kidney transplant, a lung transplant, a heart transplant, a pancreas transplant, a cornea transplant, or a liver transplant. In some embodiments, KIR2DL5 activity and/or expression is increased in advance of transplantation, i.e., one or more hours, days, or weeks prior to transplantation; at the time of transplantation; and/or after transplantation.
Provided herein in certain embodiments are agents for use in the disclosed methods, namely agents that either decrease or increase KIR2DL5 activity and/or expression. These agents may be small molecules, peptides, or polypeptides, including antibodies and fusion proteins, as disclosed herein. Also provided herein are formulations, including pharmaceutical formulations, comprising such agents, as well as kits comprising any of the disclosed agents or formulations.
The foregoing and the following working examples are merely intended to illustrate various embodiments of the present invention. The specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.
In this example, it was determined that PVR-Ig protein binds KIR2DL5 expressed on 3T3 cells in a dose-dependent manner, but does not bind KIR2DL4, a close homolog of KIR2DL5 (
Specifically, a cell-based binding assay was performed by incubating PVR-Ig fusion protein with KIR2DL5- or KIR2DL4-expressing 3T3 cells. Conversely, KIR2DL5 was selectively bound by PVR, but not by CD112 (also known as nectin-2), another ligand for TIGIT and DNAM-1 in the nectin/nectin-like family (
In competition studies, DNAM-1, TIGIT, and CD96 receptors did not block the interaction of PVR with KIR2DL5 (
The deletion of either DO or D2 alone abrogated binding to PVR (
A KIR2DL5 DO-Ig fusion protein was generated by fusing the KIR2DL5 DO coding region (H22-A128) to a human IgG1 Fc tag, and a KIR2DL5 DO-D2-Ig fusion protein was generated by fusing the KIR2DL5 D0D2 coding region (H22-H240) to a human IgG1 Fc tag using previously reported methods (Zhao 2013). The fusion proteins were expressed in a S2 system and then purified. Mice were immunized with KIR2DL5 DO(H22-A128)-Ig fusion protein and hybridomas were generated by standard techniques from splenocytes fused to NSO myeloma cells.
The VH and VL sequences of eight mAb clones are set forth in Table 1 (CDRs underlined, FRs italicized. Binding affinity as determined by biolayer interferometry for these eight clones is set forth in Table 2.
cctgaggtgaagaagcctggagagacagtcaagatctcctgcaaggcttct
gggtacaccttcacaaagtatggaatga
ac
tgggtgaagcaggctccaggaaagggtttaaagtggatgggc
tggataaacaccaacactggagagtcaacatatg
ctgaagacttcaaggga
cggtttgccttctctttggaaacctctgccaacactgcctttttgcagatcaacaacctcaaaaa
tgaggacacggctgcatatttctgt
gcaagatggggcccatacggtagtagcctttactatggtatggactactggggtcaa
MNWVKQAPGKGLKWMGWINTNTGESTYAEDFKGRFAFSLETSANTAFLQINN
LKNEDTAAYFC
ARWGPYGSSLYYGMDY
WGQGTSVTVS (SEQ ID NO: 3;
gacatccagatgacacaatcttcatcctccttttctatatctctaggagacagagtcaccattacttgc
agggcaagtgag
gacttatataatcga
ttagcctggtatcagcagaaaccaggaaatgctcctaggctcttaatatct
ggtgcaaccagtttgg
aaact
ggggttccttcaagattcagtggcagtggatctgggaaggatttcactctcagcattcccagtgttcagactgaag
atgttggtacttactactgt
caacagtatcggtatactccgtggacg
ttcggaggaggcaccacgctgaat (SEQ ID
DIQMTQSSSSFSISLGDRVTITC
RASEDLYNR
LAWYQQKPGNAPRLLIS
GATSLET
GVPSRFSGSGSGKDFTLSIPSVQTEDVGTYYC
QQYRYTPWT
FGGGTTLN (SEQ ID
gctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttct
ggctacacattcactgattatactatgcac
t
gggtgaagcagagtcatgcaaagagtctagagtggattgga
gttattagtccttactatggtgatgctagctacaaccag
aagttcaagggc
aaggccacaatgactgtagacaaatcctccagcacagcctatatggaacttgccagactgacatct
gaagattctgccatctattactgt
gcaagagggttactacgtgggtttgcttac
tggggccaagggactctggtcactgtct
ct (SEQ ID NO: 6; CDRH1: 133-162; CDRH2: 205-255; CDRH3: 346-375)
H
WVKQSHAKSLEWIG
VISPYYGDASYNQKFKG
KATMTVDKSSSTAYMELARLTS
EDSAIYYC
ARGLLRGFAY
WGQGTLVTVS (SEQ ID NO: 7; CDRH1: 45-54;
cagtctccatcctccaaggatatgtcagtaggacagaaggtcactatgaggtgc
aagtccagtaagagccttttaaatag
tagcaatcaaaagaaatatttggcc
tggtaccagcagaaaccaggacagtctcctaaacttctggtatac
tttgcatccatt
agggaatct
ggggtccctgatcgcttcataggcagtggatctgggacagatttcactcttaccatcagcagtgtgcaggtt
gaagacctggcagattacttctgt
cagcaacattatagcactccgtggacg
ttcggtggaggc (SEQ ID NO: 8;
LNSSNQKKYLA
WYQQKPGQSPKLLVY
FASIRES
GVPDRFIGSGSGTDFTLTISSV
QVEDLADYFC
QQHYSTPWT
FGGG (SEQ ID NO: 9; CDRL1: 47-63; CDRL2: 79-
gtgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggctttt
ggctacaccttcacttcctttccaatagag
tg
gatgaagcagaatcatgggaagagcctagagtggattgga
aattttcatccttataatgatgatactaagtacaatgaaaa
attcaacggcaaggcc
aaattgactgcagaaacatcctctaaaacagtctatttggagctcagccgattaacatctgatg
actgcagtgtttattactgt
gcaagagggagtgattattcgtttggtttggactac
tggggtcaaggaacctcagtcaccgtc
tcctca (SEQ ID NO: 10; CDRH1: 133-162; CDRH2: 205-261; CDRH3: 346-381)
WMKQNHGKSLEWIG
NFHPYNDDTKYNEKFNGKA
KLTAETSSKTVYLELSRLTSD
DCSVYYC
ARGSDYSFGLDY
WGQGTSVTVSS (SEQ ID NO: 11; CDRH1: 45-54;
aatctccagcttctttggttgtgtctctagggcagagggccaccatatcttgc
agagccagtgaaagtgttgatagttatggc
tatagttttatgcac
tggtaccagcagaaaccaggacagccacccaaactcctcatttat
cgtgcatccaacctaaaatct
gggatccctgccaggttcagtggcagtgggtgtaggacagacttcaccctcaccattaatcctgtggaggctgatgatgt
tgcaacctattactgt
cagcaaagtaatgaggatccttggacg
ttcggtggaggcaccaaagctggaaat (SEQ ID
GYSFMH
WYQQKPGQPPKLLIY
RASNLKS
GIPARFSGSGCRTDFTLTINPVEADDV
ATYYC
QQSNEDPWT
FGGGTKAGN (SEQ ID NO: 13; CDRL1: 46-60; CDRL2: 76-
ctggtgaaaccttctcagtctctgtccctcacctgcactgtcact
ggctactcaatcaccagtgattatgcctggaac
tggat
ccggcagtttccaggaaacaaactggagtggatgggc
tacataagcaacaatggtcgcgctaggtataatccatctctca
aaagt
cgaatctctatcactcgagacacattcaagaaccagttcttcctgcagttgaattctgtgactactgaggacaca
gccacatattactgt
gcaagagaggcctcgcatgatggttccttctggtacttcgatgtc
tggggcgcagggaccacggtc
accgtctct (SEQ ID NO: 14; CDRH1: 130-162; CDRH2: 205-252; CDRH3: 343-387)
WIRQFPGNKLEWMG
YISNNGRARYNPSLKS
RISITRDTFKNQFFLQLNSVTTEDT
ATYYC
AREASHDGSFWYFDV
WGAGTTVTVS (SEQ ID NO: 15; CDRH1: 44-54;
gacagtgtgttgacccaatctccagcttgtttggttgtgtgtatagggcagagggccaccatatcttgc
agagccagtgaa
agtgttgatagttatggctatagttttatgcat
tggtaccagcagaaaccaggacagccacccaaactcctcatttat
cgtg
catccaacctagaatgt
gggatccctgccaggttcagtggcagggggtctaggacggacttcaccctcaccattactcct
gtggagggtgatgatgttgcaacctattactgt
cagcaaagtaatgaggatcctcggacg
ttcggtggaggcaccaagct
ggaaatcaaa (SEQ ID NO: 16; CDRL1: 70-114; CDRL2: 160-180; CDRL3: 277-303)
DSVLTQSPACLVVCIGQRATISC
RASESVDSYGYSFMH
WYQQKPGQPPKLLIY
RA
SNLEC
GIPARFSGRGSRTDFTLTITPVEGDDVATYYC
QQSNEDPRTFGGGTKLEIK
ctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggctttt
ggctacaccttcacttcctttccaatagag
tg
gatgaagcagaatcatgggaagagcctagagtggattgga
aattttcatccttctaatgatgatactaagtacaatgaaaa
attcaacggcaaggcc
aaattgactgcagaaacatcctctaaaacagtctatttggagctcagccgattaacatctgatg
actctgctgtttattactgt
gcaagagggagtgattattcctttgctttggactac
tggggtcaaggaacctcagtcaccgtctc
ctca (SEQ ID NO: 18; CDRH1: 133-162; CDRH2: 205-261; CDRH1: 346-381)
WMKQNHGKSLEWIG
NFHPSNDDTKYNEKFNGKA
KLTAETSSKTVYLELSRLTSD
DSAVYYC
ARGSDYSFALDY
WGQGTSVTVSS (SEQ ID NO: 19; CDRH1: 45-54;
gcttctttggctgtgtctctagggcagagggccaccatatcctgc
agagccagtgaaagtgttgatagttatggctatagtttt
atgcac
tggtaccagcagaaaccaggacagccacccaaactcctcatctat
cgtgcatccaacctaaaatct
gggatcc
ctgccaggttcagtggcagtgggtctaggacagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacc
tattactgt
cagcaaagtaatgaggatccttggacg
ttcggtggaggcaccaagctggaaatcaaa (SEQ ID
SFMH
WYQQKPGQPPKLLIY
RASNLKS
GIPARFSGSGSRTDFTLTINPVEADDVAT
YYC
QQSNEDPWT
FGGGTKLEIK (SEQ ID NO: 21; CDRL1: 44-58; CDRL2: 74-80;
cctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttct
ggatataccttcacaaactatggaatga
ac
tgggtgaagcaggctccaggaaagggtttaaagtggatgggc
tggataaacaccaacactggagagacaacatatg
ctgaagaggtcaaggga
cggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaa
atgaggacacggctacatatttctgt
gcaagatggggcccatacggtagtagcctttatttttctatggactac
tggggtcaa
ggaacctcagtcaccgtctcctca (SEQ ID NO: 22; CDRH1: 133-162; CDRH2: 205-255;
MN
WVKQAPGKGLKWMG
WINTNTGETTYAEEVKG
RFAFSLETSASTAYLQINNL
KNEDTATYFC
ARWGPYGSSLYFSMDY
WGQGTSVTVSS (SEQ ID NO: 23;
gatatccagatgacacaatcttcatcctccttttctgtatctctaggagacagactcaccattacttgc
aaggcaagtgagg
acatatataatcggttagcc
tggtatcaacagaaaccaggaaatgctcctaggctcttaatatat
ggtgcaaccagtttgg
aaagt
ggggttccttcaagattcagtggcagtggatctggaaaggattacactctcagcattcccagttttcagagagaa
gatggtggtagcaacttatgt
caacagtatcggaatagagcgtggacg
ttcggaggagggaccaagctggaaataaa
acgg (SEQ ID NO: 24; CDRL1: 70-102; CDRL2: 148-168; CDRL3: 265-291)
DIQMTQSSSSFSVSLGDRLTITC
KASEDIYNRLA
WYQQKPGNAPRLLIY
GATSLES
GVPSRFSGSGSGKDYTLSIPSFQREDGGSNLC
QQYRNRAWT
FGGGTKLEIKR
cctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttct
ggttataccttcacagactattcaatgca
c
tgggtgaagcaggctccaggaaagggtttaaagtggatgggc
tggataaacactgagactggtgggccaacatatgc
cgatgacttcaaggga
cggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacaacctcaaaaat
gaggacacggctacatatttctgt
agtagagatgtcgacctctactttgactac
tggggccaaggcaccactctcacagtc
tct (SEQ ID NO: 26; CDRH1: 133-162; CDRH2: 205-255; CDRH3: 346-375)
H
WVKQAPGKGLKWMG
WINTETGGPTYADDFKG
RFAFSLETSATTAYLQINNLK
NEDTATYFC
SRDVDLYFDY
WGQGTTLTVS (SEQ ID NO: 27; CDRH1: 45-54;
gacatccagatgactcagtctccagcctccctatctgcatctgtgggagaaactgtcaccatcacatgt
cgaacaagtga
gaatatttacagttatttagca
tggtatcagcagaaacagggaaaatctcctcagctcctggtctat
aatgcaaaaaccctg
gtagaa
ggtgtgccatcgaggttcagtggcagtggatcaggcacacagttttctgtgaagatcaacagcctgcagcctg
aagattttgggaattattactgt
caacatcattatgggattccgttcacg
ttcggaggagggaccaaactagaaataaaa
DIQMTQSPASLSASVGETVTITC
RTSENIYSYLA
WYQQKQGKSPQLLVY
NAKTLV
E
GVPSRFSGSGSGTQFSVKINSLQPEDFGNYYC
QHHYGIPFT
FGGGTKLEIK (SEQ
cctgaggtgaagaagcctggagagacagtcaagatctcctgcaaggcttct
gggtataccttcacaaagtatggaatga
ac
tgggtgaagcaggctccaggaaagggtttaaagtggatgggc
tggataaacaccaacactggagagccaacatatg
ctgaagagttcaaggga
cggtttgccttctctttggaaacctctgccagcactgcctttttgcagatcaacaacctcaaaa
atgaggacacggctgcatatttctgt
gcaagatggggcccatacggtagtagcctttactatgctatggactac
tggggtca
aggaacctcagtcaccgtctct (SEQ ID NO: 30; CDRH1: 133-162; CDRH2: 205-255;
MN
WVKQAPGKGLKWMG
WINTNTGEPTYAEEFKG
RFAFSLETSASTAFLQINNL
KNEDTAAYFCARWGPYGSSLYYAMDYWGQGTSVTVS (SEQ ID NO: 31;
gacatccagatgacacaatcttcatcctccttttctgtatctgtaggagacagagtcaccattacttgc
agggcaagtgag
gacatatataatcggttagcc
tggtatcagcagaaaccaggaaatgctcctaggctcttaatatct
ggtgcaaccagtttg
gaaact
ggggttccttcaagattcagtggcagtggatctgggaaggattacactctcagcattcccagtgttcagagaga
agatggaggtagcaacttatgt
cagccatcacggagtagaccgtgcacg
ttcggaggaggcaccaagctgaaatcaa
agcga (SEQ ID NO: 32; CDRL1: 70-102; CDRL2: 148-168; CDRL3: 265-291)
DIQMTQSSSSFSVSVGDRVTITC
RASEDIYNRLA
WYQQKPGNAPRLLIS
GATSLET
GVPSRFSGSGSGKDYTLSIPSVQREDGGSNLC
QPSRSRPCT
FGGGTKLKSKR (SEQ
As shown in
Notably,
The four DO domain polymorphism variants, namely, T46S, R52H, G97S, and P112S, were generated by mutating KIR2DL5A*001. As shown in
Based on the performance of the mAbs of the present technology over UP-R1 for KIR2DL5 recognition (see e.g., Example 2), F8B30 was used to redefine the KIR2DL5 expression pattern in human immune cells. KIR2DL5 protein was expressed on both innate (NK and γδ T cells) and adaptive (CD8+ T cells) immune cells from human peripheral blood (
By FACS analysis of PBMC with the anti-KIR2DL5 mAbs of Example 2, it was found that KIR2DL5 is widely expressed on the cell surface of innate immune cells (NK cells, γδT cells) and adaptive immune cells (CD8 Tcells, CD4 T cells) (
According to the mRNA expression pattern (
Primary KIR2DL5+ NK cells were sorted out and an NK cell-based redirected cytotoxicity assay was performed as reported previously (Wei 2021). Co-engagement of CD16 with KIR2DL5, but not with CD56, significantly inhibited lysis of P815 (
To explore the effect of KIR2DL5-PVR engagement on NK-mediated tumor cell lysis, human tumor lines A427 (solid tumor) and Jurkat (hematologic malignancy) expressing endogenous PVR were treated with CRISPR-Cas9 to knock-out PVR (PVRko A427, PVRko Jurkat) or scramble negative control. These cells were used as targets and co-cultured with primary KIR2DL5+ NK cells and anti-KIR2DL5 blocking mAb F8B30 or control mIgG1 (
To validate whether KIR2DL5 directly inhibits primary NK cell functions, KIR2DL5+ NK cells from human PBMCs was sorted out, which confirmed stable KIR2DL5 expression after activation and expansion (
The effect of the KIR2DL5-PVR engagement on NK-mediated tumor cell lysis was examined. Primary NK cells with KIR2DL5 (
To investigate whether KIR2DL5-PVR interaction mediated inhibitory synapse formation, primary KIR2DL5+ NK cells was incubated with Raji cells expressing PVR-YFP (PVR/Raji) or control-YFP (Control Raji) fusion protein (
The impact of direct blockade of KIR2DL5 on NK cell functions against PVR+ human tumors was examined. As shown in
Therapeutic efficacy of KIR2DL5 blockade was evaluated with primary NK cells in vivo in three humanized mouse models. NSG mice were subcutaneously engrafted with A427 and then treated with expanded primary KIR2DL5+ NK cells intratumorally, as well as anti-KIR2DL5 blocking mAb F8B30 or mIgG1. F8B30 significantly reduced tumor growth (
Using a more physiologically relevant human lung cancer model, mice were inoculated with A427 cells intravenously and then reconstituted with primary KIR2DL5+ NK cells and treated with F8B30 or mIgG1. Tumor growth in the lung was significantly inhibited (
Next, efficacy was evaluated in the hematologic malignancy Jurkat xenograft tumor model. NSG mice were inoculated with Jurkat cells intravenously and then reconstituted with primary KIR2DL5+ NK cells and treated with F8B30 or mIgG1. Tumor growth in vivo was significantly inhibited (
Specifically, upon incubation with anti-KIR2DL5 blocking mAb F8B30, KIR2DL5+ NK cells manifested more potent cytotoxicity, degranulation (CD107a), and functional cytokine (IFN-γ and TNF-α) production after coculturing with PVR+ A427 (
The enhancement of NK cell function by KIR2DL5 blockade recapitulated in vivo was also investigated. Humanized nonobese diabetic (NOD) was used since murine does not express a KIR2DL5 homolog. Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse models. A subcutaneous tumor model was initially used, in which NSG mice were engrafted with A427 cells and then reconstituted with KIR2DL5+ primary NK cells intratumorally, followed by F8B30 or isotype control treatment (
Next, the antitumor efficacy of F8B30 in a more physiologically relevant lung tumor model was tested. NSG mice were inoculated i.v. with luciferase+ A427 tumor cells (A427-luc2) and treated with KIR2DL5+ primary NK cells and F8B30 or mIgG1 (
Taken together, these results demonstrate that KIR2DL5-PVR blockade restores the effector function of immune cells and promotes anti-tumor immunity in vitro and in vivo.
KIR2DL5-induced downstream signaling pathways were investigated in human NK cells. Upon KIR2DL5 signaling initiation, a substantially reduced activation of Vav1, ERK1/2, RSK, and NF-kB was observed in CD16-stimulated KIR2DL5+ primary NK cells (
As shown in
A receptor cross-linking assay was conducted to initiate KIR2DL5 signaling in CD16-stimulated primary NK cells and then subjected them to a human phospho-kinase array. Compared with CD16 alone, coengagement of KIR2DL5 with CD16 displayed a reduced phosphorylation level of multiple kinases, including ERK1/2 and p90RSK (
To further understand the KIR2DL5/PVR pathway within the human tumor microenvironment, data sets from the Gene Expression Omnibus database and BloodSpot databases were analyzed. It was found that KIR2DL5A mRNA was upregulated in several human solid tumors and hematopoietic malignancies by comparison with respective normal tissues (
To further explore the KIR2DL5/PVR pathway in various human cancers, immunohistochemistry (IHC) staining for KIR2DL5 was initially tried, but none of the antibodies worked. RNAScope in situ hybridization was used (Niu, 2022) to examine KIR2DL5 mRNA expression on human tumor tissue microarrays (TMAs) with KIR2DL5-specific probes. The probe set for KIR2DL5A specifically stained KIR2DL5+ NK cells, but not KIR2DL5− PBMCs (
Mice. BALB/c mice were purchased from Charles River Laboratory. NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJ (NSG) and NSG-IL-15 mice were purchased from The Jackson Laboratory. Mice were used between 6 and 8 weeks of age. All mice were bred and maintained in a specific pathogen-free facility with a 12-hour light/12-hour dark cycle at Albert Einstein College of Medicine (Bronx, New York, USA).
Cell lines. Human cell lines used herein include Phoenix-ampho, retrovirus producer line (ATCC, CRL-3213); HEK293T, lentivirus producer line (a gift from Wenjun Guo, Department of Cell Biology, Albert Einstein College of Medicine); K562, human chronic myelogenous leukemia (ATCC, CCL-243); Jurkat, a human T lymphoblastic leukemia cell line (ATCC, TIB-152); Raji, human B cell lymphoma (ATCC, CCL-86); and A427, human lung adenocarcinoma (a gift from Haiying Cheng, Department of Cell Biology, Albert Einstein College of Medicine). These cell lines were cultured in either EMEM, DMEM, or RPMI 1640 (Gibco) medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Mouse cell lines used herein were mouse fibroblast line NIH 3T3 (ATCC, CRL-1658), mouse mast cell line P815 (ATCC, TIB-64), and mouse myeloma cell line NSO (a gift from Matthew D. Scharff, Department of Cell Biology, Albert Einstein College of Medicine). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were cultured at 37° C. in a humidified atmosphere containing 5% CO2.
Human phospho-kinase arrays. The phosphorylation profiles of downstream kinases of the PVR/KIR2DL5 pathway were determined by use of a human phospho-kinase array (R&D Systems). Briefly, KIR2DL5+ primary NK cells (5×106) were preincubated with 10 μg/mL isotype control mIgG1 or anti-KIR2DL5 mAbs (clone F8B10) in the presence of anti-CD16 (5 μg/mL) for 30 minutes on ice. After washing with medium, primary NK cells were cross-linked with 25 μg/mL goat anti-mouse IgG (minimal x-reactivity) (BioLegend) at 37° C. water bath for 2 minutes. Cells were immediately transferred to ice to stop the reaction and then lysed with cell lysis buffer, followed by analysis of the relative levels of protein phosphorylation according to the manufacturer's instructions.
Production and purification of human fusion proteins. KIR2DL5-Ig was generated in an inducible secreted serum-free Drosophila expression system as described previously (Wei 2021; Zhao 2013). Briefly, the coding region of the extracellular domain without signal peptide of KIR2DL5 was fused to a human IgG1 Fc tag in a pMT/BiP vector. Construct was cotransfected with a blasticidin-resistant plasmid into Drosophila Schneider 2 (S2) cells by the calcium phosphate transfection kit (Invitrogen). The stably transfected S2 cells were selected and expanded in Schneider's Drosophila Medium (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL blasticidin (Gold Biotechnology). The S2 cells were induced to secrete fusion proteins in Express Five serum-free medium (Life Technologies) in the presence of 0.75 mM CuSO4. Proteins were purified using Protein G resin (GenScript) columns.
Generation of stable cell lines. Molecules expressed in NIH 3T3 and Raji cells were introduced by retrovirus transduction. Retrovirus was produced in Phoenix-ampho cells transfected with pCMV-VSV-G and MSCV-YFP containing the gene of interest using jetPRIME reagents (Polyplus Transfection).
Molecules expressed in A427, Jurkat, and K562 were introduced by lentiviral transduction. Lentivirus was produced in HEK293T cells transfected with pCMVR8.74, pCMV-VSV-G, and a lentiviral back-bone vector containing the gene of interest using jetPRIME reagents (Polyplus Transfection). Virus-containing supernatant was harvested 48-72 hours after transfection and filtered through a 0.45 μm filter. Cells were spin-infected at 2000 g for 120 minutes at 37° C. in the presence of 5 μg/mL Polybrene (Merck Millipore) and 1-2 mL virus supernatant. Transduced cells were sorted using a BD FACSAria Fusion Cell Sorter (BD Biosciences).
Fusion protein cell binding assays. PVR-Ig, CD112-Ig, or hIgG (R&D Systems) was incubated with corresponding 3T3 cells on ice for 45 minutes, followed by incubation with APC- or PE-conjugated anti-human IgG Fc antibody (1:100; clone HP6017, BioLegend) on ice for 30 minutes. Cells were then acquired on an LSR II flow cytometer (BD Biosciences). In the anti-KIR2DL5 mAb blocking assay, KIR2DL5/3T3 cells were preincubated with a serial concentration of anti-KIR2DL5 mAb F8B30 or mIgG1 on ice for 30 minutes. After washing, the cells were then incubated with 20 μg/mL PVR-Ig or hIgG on ice for 45 minutes, followed by incubation with APC anti-human IgG Fc antibody on ice for 30 minutes. In the PVR receptor competition binding assay, PVR-YFP/3T3 cells were preincubated with recombinant human DNAM-1-His (R&D Systems), TIGIT-His (R&D Systems), or CD96-His (Thermo Fisher Scientific) tag proteins at indicated concentrations at room temperature (RT) for 40 minutes. KIR2DL5-Ig (20 μg/mL) protein was then incubated with PVR-YFP/3T3 cells on ice for 45 minutes, followed by PE anti-human IgG Fc (1:200; BioLegend) on ice for 30 minutes. In the reverse direction, PVR-Ig protein (20 μg/mL) was preincubated with indicated concentrations of His-tagged protein and then stained KIR2DL5/3T3 cells on ice for 45 minutes, followed by PE anti-human IgG Fc on ice for 30 minutes. Cells were then acquired on an LSR II (BD Biosciences).
Intercellular conjugation assay. PVR/3T3 and HHLA2/3T3 cells were prelabeled with eFluor 450 (eBioscience) while KIR2DL5/3T3 and KIR3DL3/3T3 were prelabeled with PKH26 (Sigma-Aldrich) to distinguish from each other. PVR/3T3 or HHLA2/3T3 cells (2×105) were then incubated with KIR2DL5/3T3 or KIR3DL3/3T3 (2×105) at 37° C. for 45 minutes. In the mAb blocking assay, PVR/3T3 cells were coincubated with KIR2DL5/3T3 or KIR3DL3/3T3 cells in the presence of the indicated anti-KIR2DL5 mAbs or mIgG1 (10 μg/mL). After washing, cells were acquired on an LSR II (BD Biosciences) to analyze intercellular conjugation.
Generation of mAbs against KIR2DL5. Mouse anti-KIR2DL5 mAbs were generated by hybridoma techniques as described previously (Wei 2021; Zhao 2013). Briefly, splenocytes from KIR2DL5-Ig-immunized BALB/c mice were fused with NSO myeloma cells. Eight clones that specifically recognized KIR2DL5 were selected by high-throughput flow cytometry. Hybridoma cells were cultured in CELLine 350 Bioreactor Flask (DWK Life Sciences). Antibodies were purified from hybridoma supernatant by Protein G resin (GenScript) columns. The purity and integrity of antibodies were determined by SDS-PAGE and FACS. Clone F8B30 was conjugated with PE by SiteClick R-PE Antibody Labeling Kit (Invitrogen) for the following analysis.
Biolayer interferometry. The affinities of anti-KIR2DL5 mAbs were analyzed by biolayer interferometry using an Octet RED96 system (ForteBio, Pall LLC). Briefly, anti-human Fc capture biosensors (ForteBio, Pall LLC) were preloaded with KIR2DL5-Ig and then dipped into a solution containing mAb at 2-fold serial dilutions (from 200 to 1.5 μg/mL). Data were analyzed using Forte Pall (Port Washington, New York, USA) software 9.0. The global data fitting to a 1:1 binding model was used to estimate values for the Kon (association rate constant), Koff (dissociation rate constant), and KD (equilibrium dissociation constant).
Immunophenotyping by flow cytometry. Monoclonal antibodies (clone 26E10) against KIR3DL3 were purified in-house (Wei 2021). The following fluorophore-conjugated antibodies were used (all antibodies from BioLegend unless otherwise indicated) (see Table 5): CD3 (clone UCHT1, BD Biosciences), CD4 (clone RPA-T4), CD8 (clone RPA-T8, BD Biosciences), CD16 (clone 3G8, BD Biosciences), CD19 (clone SJ25C1), CD56 (clone 5.1H11), anti-human CD57 (clone QA17A04), TCR γδ (clone B1), CCR7 (clone G043H7), CD45RA (clone HI100), CD155 (clone SKII.4), DNAM-1 (clone 11A8), TIGIT (clone A15153G), CD96 (clone NK92.39), CD107a (clone H4A3), IFN-γ (clone B27), TNF-α (clone MAb11), CD57 (clone HNK-1), KLRG1 (clone SA231A2), KIR3DL2 (clone 539304, R&D), KIR2DL1/S1/S3/S5 (clone HP-MA4), KIR2DL2/3 (clone DX27), KIR2DL4 (clone mAb 33), KIR2DL5 (clone UP-R1), NKG2D (clone 1D11), NKG2C (clone 134591, R&D), NKG2A (clone 131411, BD Biosciences), 2B4 (clone C1.7), NKp46 (clone 9E2), NKp44 (clone p44-8, BD Biosciences), NKp30 (clone p30-15, BD Biosciences).
Human PBMCs were stained with Zombie Violet Fixable Viability Kit (BioLegend) and then incubated with FcR blocking reagents (Miltenyi Biotec). For surface marker staining, cells were incubated with specific antibodies for 30-45 minutes at 4° C. For CD107a and intracellular cytokine staining, cells were incubated with anti-CD107a in the presence of 5 μg/mL brefeldin A and 2.5 μg/mL monensin (BioLegend) for 5 hours. Cells were then fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences) according to the manufacturer's instructions, followed by staining with intracellular antibodies for 30-45 minutes at 4° C. All samples were acquired on an LSR II (BD Biosciences) or Aurora (Cytek) and were analyzed using FlowJo software (BD Biosciences). DownSample and t-distributed stochastic neighbor embedding (t-SNE) plugins in FlowJo and ggplot2 package in R were used to generate t-SNE plots.
Isolation and culture of human NK cells. Human PBMCs were isolated from the buffy coats of healthy donors purchased from New York Blood Center, using Ficoll-Hypaque (GE Healthcare) density gradient separation. Human KIR2DL5+ primary NK cell were sorted by FACS and then expanded by culturing with autologous PBMCs as feeder cells (irradiated at 30 Gy, feeder cells: NK cells=20:1) in OpTimizer (Invitrogen) supplemented with 5% human AB serum (Sigma-Aldrich), 1% L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, anti-CD3 OKT3 (10 ng/mL; BioLegend), recombinant human IL-2 (40 ng/mL; BioLegend), and IL-15 (10 ng/mL; BioLegend). Five or six days later, NK cells were further expanded in the same medium without anti-CD3 and feeder cells.
Primary NK cell transduction. KIR2DL5 wild type and variants of ITIM/ITSM expressed on the surface of KIR2DL5− primary NK cells were introduced by lentiviral transduction. Lentivirus was produced in HEK293T cells cotransfected with psPAX, pMD2.G, and a lentiviral backbone pSin vector (a gift from the Alec Zhang laboratory, Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA) containing the full-length gene sequence of KIR2DL5A*001 using jetPRIME reagents (Polyplus Transfection). Virus-containing supernatant was harvested 48-72 hours after transfection and filtered through a 0.45 m filter. Non-tissue-culture-treated plates were coated with retronectin, and virus supernatant was then incubated on the surface of plates at 2,000 g for 120 minutes at 37° C. NK cells were subsequently spun down at 1000 g for 10 minutes at 37° C. Transduced NK cells were sorted using a BD FACSAria Fusion Cell Sorter (BD Biosciences).
Cytotoxicity assay. Cytotoxicity assays were performed through a flow-based assay. Briefly, target cells were labeled with PKH26 (Sigma-Aldrich) for 2 minutes at 37° C. For mAb blocking assay, primary NK cells were preincubated with 20 μg/mL of mIgG1, anti-KIR2DL5 mAb (clone F8B30), anti-TIGIT mAbs (clone MBSA43, eBioscience), or indicated combination for 30 minutes before coculture with target cells.
In CD16-induced redirected cytotoxicity assays, anti-human CD16 mAbs (clone 3G8) were used to activate NK cells through cross-linking CD16. Briefly, P815 cells were preincubated with 0.5 μg/mL of anti-human CD16 and 2 μg/mL of mIgG1, anti-KIR2DL5 mAb (clone F8B30), or anti-CD56 (clone 5.1H11) for 15 minutes at RT. Target cells were coincubated with effector NK cells in 96-well round-bottom plates at indicated E/T ratios for 4-6 hours at 37° C. Supernatants from redirected cytotoxicity assays were collected after 24 hours of coculture for Human Cytokine 65-Plex Assay (Eve Technologies). 7-AAD was used to differentiate dead cells from live cells. The standard formula of 100×PKH26+7-AAD+ cells/PKH26+ cells % was used to calculate specific lysis percentages.
NK-Raji conjugation assay. KIR2DL5+ primary NK or transduced NK cells (5×105) with KIR2DL5 WT, Y298F, Y328F, or Y298/328F mutants were coincubated with 5×105 PVR-YFP/Raji or YFP/Raji cells in a 50 mL tube at 37° C. for 40 minutes. Cell mixtures were then loaded onto poly-L-lysine-precoated slides and fixed with 4% formaldehyde at RT for 15 minutes. After blocking with 5% normal goat serum at RT for 1 hour, cells were stained with 20 g/mL anti-KIR2DL5 antibodies (a mixture of 8 homemade clones) at 4° C. overnight and then with goat anti-mIgG (H+L) Alexa Flour 647 (Invitrogen) at RT for 2 hours. The cells were permeabilized by 0.1% Triton X-100 at RT for 15 minutes and stained with Alexa Flour Plus 405 Phalloidin (Life Technologies) for 1 hour at RT. The slides were then mounted by Gold Antifade Mountant without DAPI (Life Technologies). The mean pixel intensity of synapse and non-synapse was respectively measured and statistically analyzed. Images were acquired by Leica SP8 confocal microscope and processed by ImageJ (NIH).
Plasmid construction and site-directed mutagenesis. The plasmid encoding KIR2DL5 was purchased from Molecular Cytogenetics Core of Albert Einstein College of Medicine, and the fragment of KIR2DL5 was inserted into MSCV-YFP vector. The mutagenesis was carried out using New England Biolabs Q5 Site Directed Mutagenesis Kit.
The mutants of KIR2DL5 were constructed using the following primers: deleted DO forward, GGTCTATTTGGGAAACCTTCACTCTCAG (SEQ ID NO:34); deleted DO reverse, TGTCCAGGCCCCCTGCAG (SEQ ID NO:35); deleted D2 forward, GGAAACTCTTCAAGTAGTTCATC (SEQ ID NO:36); deleted D2 reverse, TGTGACCACGATCACCAG (SEQ ID NO:37); N173D forward, GCCCAGCGTCGATGGAACATTCC (SEQ ID NO:38); N173D reverse, ACTGCAGGGAGCCTAGGTT (SEQ ID NO:39); N173D/G195S for 2DL5A*005 forward, CACATGCTTCAGCTCTCTCCATGAC (SEQ ID NO:40); N173D/G195S for 2DL5A*005 reverse, TAGGTCCCTCCGTGGGTG (SEQ ID NO:41); I6V forward, GCTCATGGTCGTCAGCATGGCGT (SEQ ID NO:42); I6V reverse, GACATAGATCTAATCCGGCGC (SEQ ID NO:43); I6V/T21P for 2DL5B*00602 forward, GGGGGCCTGGCCACATGAGGGTG (SEQ ID NO:44); I6V/T21P for 2DL5B*00602 reverse, TGCAGCAAGAAGAACCCAACACAC (SEQ ID NO:45); I6V/T21P/V116M for 2DL5B*003 forward, CCTGGTGATCATGGTCACAGGTC ((SEQ ID NO:46); I6V/T21P/V116M for 2DL5B*003 reverse, GGGTTGCTGGGTGCTGAC (SEQ ID NO:47); T46S forward, GGACATGTGAGTCTTCTGTGTCGC (SEQ ID NO:48); T46S reverse, TCCTCGAGGCACCACAGC (SEQ ID NO:49); R52H forward, TGTCGCTCTCATCTTGGGTTTAC (SEQ ID NO:50); R52H reverse, CAGAAGAGTCACATGTCC (SEQ ID NO:51); G97S forward, CAGATGTCGGAGTTCACACCCAC (SEQ ID NO:52); G97S reverse, TAGGTCCCTGCGTGTGCA (SEQ ID NO:53); P112S forward, ACCCAGCAACTCCCTGGTGAT (SEQ ID NO:54); P112S reverse, GCTGACCACTCAATGGGG (SEQ ID NO:55); Y298F forward primer, GGAGGTGACATTTGCACAGTTGG (SEQ ID NO:56); Y298F reverse primer, TGAGGGTCTTGATCATCAG (SEQ ID NO:57); Y328F forward primer, TACCACCATGTTCATGGAACTTC (SEQ ID NO:58); Y328F reverse primer, TCTGTTGGAGGTGTCTTG (SEQ ID NO:59).
The following primers were used to construct pSin-KIR2DL5 WT vector and mutants:
The restriction enzyme sites were Bsu36I and SpeI.
Lentiviral CRIPR/Cas9-induced deletion of PVR. The scramble control sgRNA and PVR-targeting sgRNA were designed using GPP sgRNA Designer (Doench 2016) (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). Oligonucleotides were annealed in T4 DNA-ligase buffer (New England Biolabs), cloned into lentiCRISPR version 2 (Addgene, 52961).
The sgRNA sequences were as follows: scrambled control sgRNA: 5′-GCACTACCAGAGCTAACTCA-3′ (SEQ ID NO:62); PVR targeting sgRNA no. 1: 5′-GATGTTCGGGTTGCGCGTAG-3′ (SEQ ID NO:63); PVR targeting sgRNA no. 2: 5′-TTGAGGGCACCAATATCCAG-3′ (SEQ ID NO:64).
All these constructs are not predicted to target any known sequences in the human genome. The lentiviruses were produced as described above. A427 and K562 were transduced with viral supernatant and then selected by puromycin (2 μg/mL) for 3 days. Stable knockout of PVR (PVR KO) was confirmed by flow cytometry analysis.
Coimmunoprecipitation and immunoblotting. NK92 cells or primary NK cells pretreated with or without 1 mM pervanadate (New England BioLabs) were lysed in Pierce immunoprecipitation lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Proteins from whole-cell lysis were further incubated with anti-KIR2DL5 antibodies and Dynabeads protein G (Thermo Fisher Scientific) for further immunoprecipitation. To analyze phosphorylation status, after receptor cross-linking, the cells were lysed in radioimmunoprecipitation lysis buffer (50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktail. Samples were separated on SDS-PAGE gels (GenScript) and transferred onto nitrocellulose membranes (Bio-Rad) for protein detection.
The following antibodies were used: anti-phospho-tyrosine 4G10 (1:1,000; Sigma-Aldrich), anti-SHP-1 (1:500; Cell Signaling Technology [CST]), anti-SHP-2 (1:500; CST), anti-Vav1 (1:2,000; CST), anti-phospho-Vav1 Tyr160 (1:2,000; Invitrogen), anti-ERK1/2 (1:2,000; CST), anti-phospho-ERK1/2 Thr202/Tyr204 (1:1000; BioLegend), anti-p90RSK (1:1,000; CST), anti-phospho-p90RSK Thr359/Ser363 (1:1000; CST), anti-phospho-NF-κB p65 Ser536 (1:1,000; CST), anti-3-actin (1:2,000; Santa Cruz Biotechnology), HRP-conjugated goat anti-mouse (1:1,0000; Jackson ImmunoResearch), rabbit anti-goat (1:1,0000; Jackson ImmunoResearch), and goat anti-rabbit (1:2,000; CST) secondary antibodies and enhanced chemiluminescent substrate (ECL; Bio-Rad).
RNAScope in ISH and imaging. RNAScope ISH for KIR2DL5 and CD45 mRNA expression in FFPE human tumor tissue microarrays (TMAs; US Biomax) was performed with RNAScope 2.5 HD Reagent kit (Advanced Cell Diagnostics) per the manufacturer's instructions (Wang 2012). Briefly, TMA slides were deparaffinized, subjected to antigen retrieval using citrate buffer for 15 minutes at a boiling temperature, and then treated with 10 μg/mL protease at 40° C. for 30 minutes. Probes were hybridized for 2 hours at 42° C. followed by signal amplification. For fluorescent detection, the label probe sets for KIR2DL5 and CD45 were conjugated to Opal 570 and 690 nm (Akoyo Biosciences), respectively. Assays were typically performed in parallel with positive (UBC) and negative (bacterial gene dapB) controls to assess both tissue RNA integrity and background signals. The slides were scanned by a 3DHistech P250 high-capacity slide scanner by 3 channels with filter settings for DAPI, FITC, and Cy7. Staining was analyzed with Volocity software by a trained researcher.
IHC staining and imaging. The same cohorts of TMAs used in RNAScope ISH were deparaffinized, followed by antigen retrieval with citrate unmasking buffer (CST) in a steamer for 20 minutes at a sub-boiling temperature (95° C.-98° C.). Slides were then blocked by 3% hydrogen peroxidase solution at RT for 10 minutes and subsequently by 10% normal goat serum at RT for 1 hour. A rabbit anti-PVR (clone D8A5G, CST) mAb was used at a dilution of 1:200 for overnight incubation at 4° C. The slides were then incubated with boost detection reagent (HRP, CST) at RT for 30 minutes, followed by SignalStain DAB (CST) and hematoxylin nuclear counterstaining. Positive and negative controls (FFPE cell blocks) were included in each staining.
Xenograft models of human cancers. For the subcutaneous A427 tumor model, 6- to 8-week-old NSG or NSG-hIL-15 mice were inoculated s.c. with 3×106 A427 cells on the hind flanks. Three or five days later, mice were randomized into 2 groups (n=6 or 8) and treated with KIR2DL5+ primary NK cells (1×107) and 200 μg anti-KIR2DL5 mAb (clone F8B30) or isotype control (mIgG1) intratumorally twice (once every 3 days). Tumors were measured by caliper, and tumor volume was calculated as (width2×length)/2.
For the intravenous A427 tumor model, NSG mice were injected intravenously (i.v.) with 1×106 luciferase-expressing A427 cells (A427-luc2). One day later, mice underwent bioluminescence imaging (BLI) and were allocated to 2 groups (n=5) based on similar average photon flux (photons/second). Mice were then treated i.v. with KIR2DL5+ primary NK cells (1×107) and 200 μg F8B30 or mIgG1 twice (once every 3 days). Lung tumor growth was monitored by BLI weekly, and mice were euthanized when the total flux reached to 1×108 photons/second.
For the intravenous Jurkat tumor model, NSG mice were injected i.v. with 5×105 luciferase-expressing Jurkat cells (Jurkat-luc2). Four days later, mice were allocated to 2 groups (n=4 or 6) based on similar average photon flux (photons/second), and treated i.v. with KIR2DL5+ primary NK cells (1×107) and 200 μg F8B30 or mIgG1 twice (once every 3 days).
Tumor growth was monitored by BLI, and mice were euthanized when the total flux reached to 1×1010 photons/second. For all BLI, D-luciferin (150 mg/kg; Gold Biotechnology) was administered by intraperitoneal injection to mice for 10 minutes before imaging. The data were analyzed with Living Image 3.0 software.
Data availability and statistical analysis. Previously published Gene Expression Omnibus (GEO) data that were reanalyzed here are available under accession codes GSE7904, GSE19069, and GSE39612.
Statistical analyses were performed in GraphPad Prism, version 9.0 (GraphPad Software) using appropriate tests as indicated in the figure legends (unpaired 2-tailed t test, paired 2-tailed t test, 1-way ANOVA followed by Tukey's or Dunnett's multiple-comparison test, 2-way ANOVA followed by Šidák's multiple-comparison test, multiple t test, and log-rank test for Kaplan-Meier survival curves). The data are expressed as mean±SEM of n=3 or more determinations. A P value of less than 0.05 was considered statistically significant.
Discussion Human KIRs are critical regulators of NK cell function and are important for immunological tolerance and tumor surveillance (Pende 2019). KIR2DL5 is the most recently identified KIR molecule (Estefania 2007). A nectin/nectin-like family protein, PVR, was recently identified as a binding partner for KIR2DL5 (Verschueren 2020; Husain 2019).
The present examples demonstrate that KIR2DL5 suppresses primary NK cell cytotoxicity against multiple solid and hematopoietic tumor cells in a PVR-dependent manner. KIR2DL5-induced inhibitory signaling in primary NK cells. Blockade of KIR2DL5 with blocking mAbs of the present technology significantly enhanced NK-mediated antitumor immunity both in vitro and in vivo, demonstrating blockade of the KIR2DL5/PVR pathway as an immunotherapy for treating human cancers.
The identification of KIR2DL5 as an inhibitory receptor of PVR adds KIR2DL5 into a complex regulatory network composed of the other 2 inhibitory receptors, TIGIT and CD96, and 1 activating receptor, DNAM-1, for PVR. Unlike TIGIT and CD96, which share a common binding site with DNAM-1 on PVR (Yu 2009), KIR2DL5 bound to a non-identical site on PVR and did not compete with those 3 receptors for PVR binding, suggesting a distinct mechanism by which KIR2DL5 exerts an inhibitory effect through engagement with PVR. KIR2DL5 mediated PVR+ tumor immune resistance to NK cell killing. Furthermore, KIR2DL5-mediated inhibition on NK cytotoxicity was abolished upon depletion of PVR on tumor cells. These findings support PVR as a primary ligand for KIR2DL5 to induce NK cell suppression and tumor immune evasion.
Allelic polymorphism significantly influences cell surface expression, antibody recognition, and ligand avidity of KIRs (Carr 2005; Campbell 2011). Distinct from UP-R1, which required both DO and D2 domain for KIR2DL5 recognition, anti-KIR2DL5 mAb F8B30 of the present technology bound to KIR2DL5 through the DO domain, suggesting that they recognize different epitopes on KIR2DL5. Besides 2DL5A*001 and DO variants, F8B30 also detected surface-expressed 2DL5A*005, the second most common 2DL5A allele in the human population, while UP-R1 failed to do so. Furthermore, PVR displayed a different binding capacity to different KIR2DL5 alleles. In comparison with 2DL5A*001, 2DL5B*00602 was moderately bound by PVR, while surface-expressed 2D5A*005 and 2DL5B*003 were not bound by PVR.
Crosstalk between NK cells and dendritic cells (DCs) via cytokines or direct cell-contact stimuli results in activation and cytokine production by both cell types, contributing to the coordination of innate and adaptive immune responses (Cooper 2004; Walzer 2005). The present technology demonstrates that KIR2DL5 significantly decreases production of a broad spectrum of cytokines and chemokines by NK cells, such as IFN-γ, TNF-α, and GM-CSF, which might subsequently impair NK cell-induced DC maturation and activation. PVR is highly expressed not only by tumor cells but also by some immune cell subsets, including DCs. TIGIT induces PVR phosphorylation and signaling in DCs, resulting in increased IL-10 and decreased IL-12 production by DCs (Yu 2009). DC-released IL-12 induces IFN-γ production and potentiate the cytotoxicity of NK cells (Biron 1999).
ITIM and ITSM sequences found in many inhibitory receptors are critical in transducing negative signaling through recruiting phosphatases, such as SHP-1 or SHP-2, upon tyrosine phosphorylation (Daeron 2008; Long 2008). The present tyrosine mutation study showed that both ITIM and ITSM were essential for KIR2DL5-mediated NK cell inhibition. KIR2DL5 recruited both SHP-1 and SHP-2 in primary human NK cells. Notably, both phosphorylated ITIM and ITSM contributed to KIR2DL5 association with SHP-1. KIR2DL5 association with SHP-2 completely relied on phosphorylated ITIM, but not ITSM. ITIM/SHP-1/SHP-2 and ITSM/SHP-1 inhibited the Vav1/ERK1/2/p90RSK and downstream NF-1B signaling pathway. These findings revealed the molecular basis for KIR2DL5-mediated suppression on NK cells.
Preclinical studies have demonstrated that the TIGIT/PVR axis is an attractive cancer immunotherapy target owing to its roles in modulating CD8+ T cell and NK cell responses (Andrews 2019; Yu 2009; Stanietsky 2009). However, TIGIT blockade monotherapy shows minimal effects on controlling tumor growth. Whereas dual blockade of TIGIT and PD-1/PD-L1 shows promising results in some experimental tumor models (Hung 2018; Johnston 2014; Dixon 2018) and in multiple trials (Bendell 2020; Niu 2022; Cohen 2021; Wainberg 2021; Rodriguez-Abreu 2020), combination of the anti-TIGIT antibody tiragolumab and the PD-L1 inhibitor atezolizumab failed to improve progression-free survival in a phase III extensive-stage small cell lung cancer trial (ClinicalTrials.gov NCT04256421).
As disclosed herein, the noncompetitive binding of KIR2DL5 and TIGIT to PVR suggested that both receptors can function simultaneously and independently and that blockade of the TIGIT/PVR axis would still leave the KIR2DL5/PVR pathway intact. TIGIT blockade had a minimal effect on NK cell cytotoxicity, whereas KIR2DL5 blockade markedly restored the cytolytic activity of NK cells. Thus, the existence of KIR2DL5-mediated inhibition on NK cells in the TME represents a substantial obstacle to the success of the blockade of TIGIT. KIR2DL5+ immune cells infiltrated in various human cancers that highly expressed PVR. Blockade of KIR2DL5 effectively inhibited tumor growth and improved mouse survival across multiple humanized mouse models.
In summary, the findings disclosed herein unraveled the cellular and molecular mechanisms underlying the inhibitory function of the KIR2DL5/PVR pathway, supporting that blockade of the immunosuppressive KIR2DL5/PVR axis alone or in combination with other therapies is a new therapeutic strategy.
This application claims priority to U.S. Provisional Application No. 63/263,710, filed on Nov. 8, 2021, the disclosure of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/079401 | 11/7/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63263710 | Nov 2021 | US |
