Chimeric antigen receptors targeting HLA-G positive cancers

Abstract

Methods and compositions relating to targeting HLA-G expressing cells are provided. [0010] The embodiments of the present disclosure include methods and compositions related to targeting HLA-G expressing cells with specific engineered receptors. In certain embodiments, NK cells are specifically engineered to bind HLA-G with specific chimeric antigen receptor constructs. In certain embodiments, the vector expressing the HLA-G targeting CAR also expresses a specific suicide gene and / or one or more specific cytokines.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 193,515, filed May 26, 2021, which is incorporated by reference herein in its entirety.

[0002] Embodiments of the present disclosure include the fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine. [Background technology]

[0003] Genetic reprogramming of natural killer (NK) cells for adoptive cancer immunotherapy has clinically relevant applications and advantages, such as 1) innate antitumor surveillance without the need for prior sensitization, 2) allogeneic efficacy without graft-versus-host reactivity, and 3) direct cell-mediated cytotoxicity and cytolysis of target tumors. The development of human NK cells and the acquisition of self-tolerance, alloreactivity, and effector functions is an adaptive process of licensing, calibrating, and arming. At the molecular level, specific activating and inhibitory receptors direct NK cell function by aggregating, balancing, and integrating extracellular signals into distinct effector functions. NK cell functional activity and responsiveness to foreign stimuli follow a "rheostat" model of continuing education and are therefore amenable to reprogramming. Genetic modification to direct the effector function of NK cells is an effective way to harness their cytotoxic capacity to kill tumor cells.

[0004] HLA-G is a non-classical HLA class I molecule that is expressed only in the placenta in healthy adults. HLA-G interacts with receptors on NK cells, T cells, B cells, monocytes, and dendritic cells, inhibiting their function and creating a state of immune tolerance between the mother and fetus. HLA-G is overexpressed in many hematological and solid tumors. Cancer cells aberrantly express HLA-G and escape immune surveillance by engaging with the inhibitory receptors KIR2DL4, leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) on NK cells.

[0005] The present disclosure relates, in certain embodiments, to methods and compositions for the genetic engineering of cells, including human NK cells, for cell therapy targeting cancers, including HLA-G positive tumors. Summary of the Invention

[0006] Embodiments of the present disclosure encompass methods and compositions related to engineered cell receptors, including chimeric antigen receptors (CARs) that target HLA-G (e.g., also known as HLA class I histocompatibility antigen, alpha chain G, HLA G antigen, or MHC class I antigen G). In certain embodiments, the engineered receptors that target HLA-G are in the form of polynucleotides, polypeptides, and / or are included on the surface of any type of cell, including immune cells. In certain cases, the cells are immune cells, and in certain embodiments, the immune cells are NK cells, NKT cells, invariant NKT cells, gamma delta T cells, alpha beta T cells, regulatory T cells, B cells, macrophages, mesenchymal stromal cells (MSCs), dendritic cells, and the like, from any source. In some embodiments, the immune cells are NK cells. In certain embodiments, reprogrammed NK cells from umbilical cord blood (CB-NK) are encompassed to target cancers that express HLA-G molecules.

[0007] HLA-G is utilized as the target antigen for the disclosed method and composition embodiments, at least in part, because it is expressed on many cancers, including breast cancer, colorectal cancer, cervical cancer, esophageal cancer, Ewing's cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, lung cancer, nasopharyngeal carcinoma, ovarian cancer, and thyroid cancer.In addition, HLA-G overexpression is involved in cancer immune evasion, and HLA-G can be considered as an immune checkpoint.Therefore, its targeting can be used to overcome this important mechanism of tumor immune evasion.

[0008] The present disclosure includes several novel CAR molecules, which in particular cases include fusions of scFvs targeting human HLA-G (including, for example, scFvs from antibody clones such as G233, 26-2H11, MEM-G / 1, MEM-G / 9, MEM-G / 11, MEM-G / 13, 1B8, 5E6H7, 1-2C3, 16G1, 5A6G7, 87G, or 3C / G4), or the extracellular domains from KIR2DL4, LILRB1, or LILRB2, with one or more activation signaling endodomains, and optionally incorporating CD3zeta alone or in combination with costimulatory or adaptor signaling domains (e.g., from NKG2D, OX-40, CD27, 41BB, CD28, DAP10, DAP12, and / or 2B4). In certain cases, allogeneic CB-NK cells are retrovirally transduced to express HLA-G CAR. In certain embodiments, the immune cells of the present disclosure that contain HLA-G CAR molecules also express one or more proteins that support their survival and proliferation. In certain cases, the immune cells are engineered to express one or more cytokines that promote the expansion and persistence of the cells. In specific cases, the one or more cytokines are interleukin 15 (IL-15), IL-2, IL-7, IL-12, IL-18, IL-21, and / or IL-23. In certain embodiments, the vector encoding the CAR also encodes a cytokine, each of which is ultimately produced as a separate polypeptide. In other embodiments, the CAR and the cytokine are encoded on separate vectors.

[0009] Certain embodiments of the present disclosure allow for the use of pre-made immune cells, including at least NK cells, that are allogeneic to the recipient individual, target any type of HLA-G positive cell, and may or may not be transduced to express one or more cytokines, such as IL-15, IL-2, IL-21, IL-12, IL-23, IL-7, and / or IL-18.

[0010] In certain embodiments of the present disclosure, the expression of one or more endogenous genes in immune cells is modified, for example, expression can be partially or completely reduced.Modification can occur by any means, but in certain embodiments, the expression of one or more genes is modified, for example, by reducing expression level, which can occur by any suitable means, including at least CRISPR.Just as an example, the endogenous genes can be selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, CTLA-4, TDAG8, CD38, and combinations thereof.

[0011] The embodiments of the present disclosure include a polynucleotide that encodes an anti-HLA-G chimeric antigen receptor (CAR) and may also encode one or more cytokines. In certain embodiments, the polynucleotide encodes a CAR that includes a KIR2DL4 extracellular domain, a transmembrane domain, and an intracellular domain that is not a KIR2DL4 intracellular domain. In some embodiments, the CAR includes all of the extracellular domain of the KIR2DL4 protein. Alternatively, the CAR includes an HLA-G binding portion of the extracellular domain of the KIR2DL4 protein. In certain embodiments, the KIR2DL4 extracellular domain includes SEQ ID NO:9. In certain embodiments, the KIR2DL4 extracellular domain is encoded by SEQ ID NO:7. In some embodiments, the KIR2DL4 extracellular domain includes SEQ ID NO:10. In some cases, the KIR2DL4 extracellular domain is a codon-optimized KIR2DL4 extracellular domain. In certain embodiments, the KIR2DL4 extracellular domain is encoded by SEQ ID NO:8. In some embodiments, the CAR comprises a portion of the KIR2DL4 intracellular domain, for example a portion of the KIR2DL4 intracellular domain that can mediate an activating signal.

[0012] Additional embodiments of the present disclosure include polynucleotides that encode anti-HLA-G CARs and may also encode one or more cytokines. In certain embodiments, the polynucleotides encode CARs that include the LILRB1 extracellular domain, the transmembrane domain, and an intracellular domain that is not the LILRB1 intracellular domain. In some embodiments, the CARs include all of the extracellular domain of the LILRB1 protein. Alternatively, the CARs include an HLA-G binding portion of the extracellular domain of the LILRB1 protein. In some embodiments, the CARs include a portion of the KIR2DL4 intracellular domain, such as a portion of the LILRB1 intracellular domain that can mediate an activation signal. Further embodiments of the present disclosure include polynucleotides that encode anti-HLA-G CARs, where the CARs include the LILRB2 extracellular domain, the transmembrane domain, and an intracellular domain that is not the LILRB2 intracellular domain. In some embodiments, the CARs include all of the extracellular domain of the LILRB2 protein. Alternatively, the CARs include an HLA-G binding portion of the extracellular domain of the LILRB2 protein. In some embodiments, the CAR comprises a portion of the KIR2DL4 intracellular domain, such as a portion of the LILRB2 intracellular domain that can mediate an activation signal.

[0013] Any transmembrane domain may be, for example, a transmembrane domain from CD28, the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the zeta chain of the T cell receptor, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, DAP12, a killer immunoglobulin-like receptor (KIR), for example, KIR2DL2, LILRB1, LILRB2, or any combination thereof. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. The CD28 transmembrane domain may include SEQ ID NO: 12. In certain cases, the CD28 transmembrane domain is encoded by SEQ ID NO: 11. In some embodiments, the transmembrane domain is a CD8 transmembrane domain. The CD8 transmembrane domain may comprise SEQ ID NO: 14. In certain cases, the CD28 transmembrane domain is encoded by SEQ ID NO: 13.

[0014] Any intracellular domain may be, for example, an intracellular domain from CD3 zeta, CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, or NKp80, or any combination thereof. In some embodiments, the intracellular domain is a CD3 zeta intracellular domain. The CD3 zeta intracellular domain may comprise SEQ ID NO: 16 or SEQ ID NO: 18. In a specific case, the CD3 zeta intracellular domain is encoded by SEQ ID NO: 15 or SEQ ID NO: 17. In some embodiments, the intracellular domain is a CD28 intracellular domain. The CD28 intracellular domain may comprise SEQ ID NO: 24. In a specific case, the CD28 intracellular domain is encoded by SEQ ID NO: 23. A CAR may comprise two or more intracellular domains or three or more intracellular domains. In certain embodiments, the two or more intracellular domains include a CD3 zeta intracellular domain and an additional intracellular domain selected from CD28, DAP10, DAP12, 4-1BB, NKG2D, and 2B4 intracellular domains. In certain cases, the two or more intracellular domains include a CD3 zeta intracellular domain and a CD28 intracellular domain.

[0015] In some embodiments, the CAR further comprises a signal peptide. In certain embodiments, the signal peptide is derived from CD8, CD27, granulocyte macrophage colony-stimulating factor receptor (GMSCF-R), Ig heavy chain, KIR (e.g. KIR2DL4), CD3, or CD4. In some embodiments, the signal peptide is a CD8 signal peptide. The CD8 signal peptide may comprise SEQ ID NO:6. In some embodiments, the signal peptide is encoded by SEQ ID NO:5. In certain embodiments, the CAR does not comprise a signal peptide.

[0016] In certain embodiments, the polynucleotide encoding the CAR of the present disclosure further encodes an additional polypeptide of interest. The sequence encoding the additional polypeptide of interest and the sequence encoding the CAR may be separated on the polynucleotide by an E2A element, such as an E2A element. In certain embodiments, one or more polypeptides of interest are utilized, such as therapeutic proteins and / or proteins that enhance the activity, expansion, and / or persistence of cells. In some embodiments, the additional polypeptide of interest is a suicide gene, a cytokine, and / or a human or viral protein that enhances proliferation, expansion, and / or metabolic fitness. In certain embodiments, the additional polypeptide of interest is a cytokine, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7. In certain embodiments, the cytokine is IL-15.

[0017] Aspects of the present disclosure relate to a polypeptide encoding a CAR comprising a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 2. In some embodiments, the CAR comprises SEQ ID NO:2. In some embodiments, the polynucleotide comprises a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises SEQ ID NO:1.

[0018] Additional aspects of the disclosure relate to a polypeptide encoding a CAR comprising a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 4. In some embodiments, the CAR comprises SEQ ID NO: 4. In some embodiments, the polynucleotide has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity.

[0019] Also provided herein is the vector that comprises the polynucleotide of the present disclosure.The vector contemplated herein includes viral vector (e.g., adenovirus vector, adeno-associated virus vector, lentivirus vector and retrovirus vector) and non-viral vector (e.g., plasmid).

[0020] The embodiments of the present disclosure include any type of immune cell, including any polynucleotide and / or polypeptide encompassed herein. In certain embodiments, the immune cell is a NK cell, a T cell, a gamma delta T cell, an alpha beta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, a dendritic cell, or a mixture thereof. When the immune cell is a NK cell, the NK cell can be derived from umbilical cord blood (including pooled umbilical cord blood units), peripheral blood, induced pluripotent stem cells, bone marrow, and / or a cell line. In certain aspects, the NK cell line is the NK-92 cell line, another NK cell line derived from a tumor, or a healthy NK cell or progenitor cell.

[0021] In certain embodiments, the immune cells are NK cells, e.g., derived from cord blood, e.g., from cord blood mononuclear cells. NK cells, in certain cases, are CD56 + The NK cells may be NK cells. The NK cells may express one or more exogenously provided cytokines, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, IL-7, or a combination thereof. Certain embodiments include populations of any type of immune cells of the present disclosure, and the cells may be present in any type of suitable medium or suitable carrier.

[0022] For example, encompassed herein are methods of treating or preventing any type of cancer by administering cells expressing a particular anti-HLA-G CAR in a therapeutically effective amount that ameliorate or prevent the cancer, reduce the risk of the cancer, reduce the severity of the cancer, prevent metastasis or the risk thereof, or delay the onset of the cancer.

[0023] In some embodiments, disclosed is a method of killing any kind of HLA-G positive cells in an individual, comprising administering to the individual an effective amount of cells having any polynucleotide and / or polypeptide of the present disclosure (e.g., HLA-G CAR of the present disclosure). In certain embodiments, the cells are NK cells, T cells, gamma delta T cells, alpha beta T cells, invariant NKT (iNKT) cells, B cells, macrophages, mesenchymal stromal cells (MSCs), or dendritic cells. The NK cells can be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, or cell lines. The NK cells can be derived from umbilical cord blood mononuclear cells. In some cases, the HLA-G positive cells are cancer cells, for example, from hematopoietic cancer or solid tumors. The cells can be allogeneic or autologous to the individual, which may or may not be human. The cells may be administered to an individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesional, intracranially, percutaneously, subcutaneously, topically, by perfusion, into the tumor microenvironment, or combinations thereof.

[0024] In certain embodiments of the method, the cells may be administered to the individual one or more times. The period between administration of the cells to the individual may be 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or more than a year. The method may further comprise administering to the individual an effective amount of an additional therapy, such as surgery, radiation, gene therapy, immunotherapy, and / or hormone therapy. The additional therapy may optionally comprise one or more antibodies or antibody-based agents. In some embodiments of the method, the method may further comprise identifying HLA-G positive cells in the individual.

[0025] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for any measuring or quantitating method.

[0026] The use of the words "a" or "an" when used in conjunction with the term "comprising" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0027] The phrase "and / or" means "and" or "or." By way of example, A, B, and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" operates as an inclusive.

[0028] The words "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0029] The compositions and methods of use thereof can "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consist essentially of" any of the disclosed components or steps limit the claims to particular materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0030] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Further, the compositions of the invention can be used to achieve the methods of the invention.

[0031] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure so that the detailed description of the invention that follows may be better understood. Additional features and advantages will be described hereinafter which form the subject of the claims. It should be understood by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed to be characteristic of the design disclosed herein, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. [Brief description of the drawings]

[0032] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0033] [Figure 1] 1 shows expression of HLA-G in various cancer cell types as measured by flow cytometry, including breast cancer (MDAMB231 and BCX010), acute myeloid leukemia (OCI-3), multiple myeloma (MM1), and glioblastoma cells (GCS267, GCS20, GCS8-11).

[0034] [Diagram 2]1 shows expression of CB-NK cells transduced with an anti-HLA-G CAR (KIR2DL4 CAR) of the present disclosure. The KIRD2DL4 CAR peak is on the far right.

[0035] [Diagram 3] Figure 1 shows the results of intracellular cytokine staining of OCI-AML13 cells cultured with KIR2LD4 CAR NK cells of the present disclosure (CD8-KIR2DL4-TMD-3Z and CD8-KIR2DL4-CD28-3Z) or non-transduced control NK cells (NT). Increased TNFα, INFγ, and CD107a expression was observed in KIR2LD4 CAR NK cells compared to controls.

[0036] [Figure 4] Measured 51Cr release from OCI-AML13 cells cultured with KIR2LD4 CAR NK cells of the present disclosure (CD8-KIR2DL4-TMD-3Z and CD8-KIR2DL4-CD28-3Z) or non-transduced control NK cells (NT) is shown. Increased Cr release was observed with KIR2LD4 CAR NK cells compared to the control (bottom line).

[0037] [Diagram 5] Shows HEK293T cell transfection efficiency for an example HLA-G CAR construct.

[0038] [Figure 6] Shown is the transduction efficiency for HLA-G CAR NK cells.

[0039] [Figure 7] We demonstrate that HLA-G CAR cells efficiently kill OCI-AML 3 cells.

[0040] [Figure 8A-8B] Showing that HLA-G CAR NK cells efficiently kill GSC20 spheroids.

[0041] [Figure 9] Showing that HLA-G CAR NK cells release TNF-α in response to tumor targets.

[0042] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can be made by those skilled in the art without departing from the present invention. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] I. Definition Examples Following long-standing patent law practice, the words "a" and "an" when used in conjunction with the word comprising, herein, including in the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0044] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the recited step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0045] Throughout this specification, reference to "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "an embodiment," "additional embodiment," or "further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0046] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0047] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0048] The term "engineered" as used herein refers to entities produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, engineered entities are synthetic and contain elements that do not occur in nature or that are constructed by the methods utilized in this disclosure.

[0049] The term "isolated" as used herein refers to a molecule or biological or cellular material that is substantially free from other materials. In one embodiment, the term "isolated" refers to a nucleic acid such as DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, that is separated from other DNA or RNA, or proteins or polypeptides, or cells or organelles, or tissues or organs, respectively, as they exist in a natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, culture medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. Furthermore, "isolated nucleic acid" means to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to include both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues, and is meant to include both cultured and engineered cells or tissues.

[0050] As used herein, "prevent" and similar terms such as "prevent", "preventing" refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0051] The term "sample" as used herein generally refers to a biological sample. A sample can be taken from an individual's tissue or cells. In some examples, a sample consists of or is derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spot, cultured cells, discarded tissue. A sample may be separated from its source prior to collection. Non-limiting examples include blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, stool, tears, sweat, or mucosal excretions, and other bodily fluids separated from a primary source prior to collection. In some examples, a sample is separated from its primary source (cells, tissues, bodily fluids such as blood, environmental samples, etc.) during sample preparation. A sample may or may not be purified or concentrated from its primary source. In some cases, the primary source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or concentrated for nucleic acids or treated with RNase. The sample may include intact, fragmented, or partially disaggregated tissues or cells.

[0052] The term "subject" as used herein generally refers to an individual having a biological sample being processed or analyzed, and in certain cases, an individual having or suspected of having cancer. A subject may be any living organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), household pets (e.g., dogs, cats, rodents), horses, and transgenic non-human animals. A subject may be, for example, a patient who has or is suspected of having a disease (sometimes referred to as a pathology), such as a benign or malignant neoplasm, or cancer. A subject may be undergoing or having been treated. A subject may be asymptomatic. A subject may be a healthy individual, but may wish to prevent cancer. The terms "individual" are used interchangeably, at least in some cases. A "subject" or "individual" as used herein may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may be administered one or more medical compositions via the Internet.An individual may include any age of human or non-human animal, and thus includes both adults and juveniles (i.e., children) and infants, and also includes individuals in utero.This term is not intended to imply the need for medical treatment, and thus an individual may participate in an experiment, whether it is clinical or supports basic science research, whether voluntarily or involuntarily.

[0053] As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or pathological condition of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either the reduction or amelioration of symptoms of the disease or condition, or the delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or the symptoms associated therewith.

[0054] The present disclosure relates to methods and compositions directed to the treatment of HLA-G positive cancers, particularly utilizing adoptive cell therapy targeting HLA-G positive cancer cells. In certain embodiments, any kind of genetically engineered mammalian immune cells, including at least human NK cells, are generated that target HLA-G positive cancers. The present disclosure encompasses any kind of genetically engineered receptors for HLA-G, including chimeric antigen receptors (CARs). In certain aspects, several novel expression constructs are provided, including retroviral constructs that express the HLA-G targeting extracellular domains used in CARs, including KIR2DL4 extracellular domains, and optionally also express one or more cytokines, such as IL-15, to support cell survival and proliferation. In some embodiments, the CAR is a fusion of the extracellular domain of KIR2DL4 (the natural ligand for HLA-G) and one or more additional domains not derived from KIR2DL4 (e.g., transmembrane domain, intracellular domain). II. Engineered Receptors

[0055] The immune cells of the present disclosure can be engineered to express one or more antigen-binding receptors that target HLA-G, such as engineered CARs or other engineered TCRs. For example, the immune cells can be immune cells that have been modified to express CARs and / or TCRs with antigen specificity for HLA-G. Other CARs and / or TCRs can be expressed by the same cell as the HLA-G antigen receptor-expressing cell and can be directed to different antigens. In some embodiments, the immune cells are engineered to express HLA-G-specific CARs or HLA-G-specific TCRs by knocking in the CAR or TCR using CRISPR / Cas technology.

[0056] Suitable cell modification methods are known in the art. For example, see Sambrook and Ausubel, supra. For example, using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009, cells can be transduced to express CAR or TCR with antigen specificity for cancer antigen.

[0057] In some embodiments, the cell comprises one or more nucleic acids introduced through genetic engineering, encoding one or more antigen targeting receptors (at least one of which is for HLA-G), and the genetically engineered product of such nucleic acid.In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell that is not normally found, e.g., in the cell to be manipulated and / or the organism from which such cell is derived.In some embodiments, the nucleic acid is not naturally occurring, e.g., a nucleic acid that is not found in nature (e.g., chimera).

[0058] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods of engineering and introducing such receptors into cells are described in, for example, WO 2000 / 14257, WO 2013 / 126726, WO 2012 / 129514, WO 2014 / 031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication No. 2002 / 131960, U.S. Patent Application Publication No. 2013 / 287748, U.S. Patent Application Publication No. 2013 / 0149337, U.S. Patent No. 6,451,995, and the like. , U.S. Pat. Nos. 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, as well as in EP 2 537 416 A1, and / or those described by Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some embodiments, engineered antigen receptors include CARs described in U.S. Pat. No. 7,446,190 and those described in WO 2014 / 055668. A. Chimeric Antigen Receptor

[0059] In certain embodiments, an HLA-G specific CAR is utilized, which comprises at least an extracellular domain comprising a) one or more intracellular signaling domains, b) a transmembrane domain, and c) at least one antigen binding region that targets HLA-G, including specific binding to HLA-G. In some embodiments, the antigen binding region is an antibody or a functional fragment thereof. In other cases, the antigen binding region of the CAR is not an antibody or a functional fragment thereof (e.g., a ligand for HLA-G, such as KIR2DL4, LILRB1, or LILRB2). In some embodiments, the antigen binding region of the CAR is a KIR2DL4 extracellular domain or an antigen binding portion thereof. In some embodiments, the antigen binding region of the CAR is a LILRB1 extracellular domain or an antigen binding portion thereof. In some embodiments, the antigen binding region of the CAR is a LILRB2 extracellular domain or an antigen binding portion thereof. In some embodiments, an HLA-G specific CAR binds only to HLA-G, while in other cases, the CAR as a single polypeptide is bispecific by containing two or more antigen binding domains, one of which binds to HLA-G and the other of which binds to another, non-identical antigen.

[0060] In some embodiments, engineered antigen receptors include CARs, including activating or stimulating CARs, or costimulatory CARs (see WO2014 / 055668). CARs generally comprise an extracellular antigen (or ligand) binding domain that is linked to one or more intracellular signaling components, in some embodiments, via a linker and / or a transmembrane domain. Such molecules typically mimic or approximate the signaling through a natural antigen receptor, the signaling through such receptor in combination with a costimulatory receptor, and / or the signaling through a costimulatory receptor alone.

[0061] It is believed that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stable transfection of cells by electroporation with naked DNA are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector in a suitable orientation for expression.

[0062] Alternatively, chimeric CAR constructs can be introduced into immune cells using viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors).Vector suitable for use according to the method of the present disclosure is non-replicative in immune cells.Many virus-based vectors are known, such as HIV, SV40, EBV, HSV, or BPV-based vectors, whose virus copy number maintained in cells is low enough to maintain cell viability.

[0063] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding HLA-G specific CAR polypeptides, including CARs that are humanized to reduce immunogenicity (hCARs), including at least one intracellular signaling domain, a transmembrane domain, and an extracellular domain that includes one or more signaling motifs. In certain embodiments, HLA-G specific CARs can recognize epitopes that include shared spaces between one or more antigens. In certain embodiments, the binding region can include the complementarity determining region of a monoclonal antibody, the variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0064] It is contemplated that the human HLA-G CAR nucleic acid may be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length HLA-G specific CAR cDNA or coding region. The antigen binding region or domain is the V of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody. H Chain and V L The fragments may include fragments of the chains, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. The fragments may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an HLA-G-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0065] The configuration can be a multimer, such as a diabody or a multimer. The multimer is most likely formed by cross-pairing of the variable parts of the light and heavy chains into a diabody. The hinge part of the construct can have many options, from being completely deleted, to maintaining the first cysteine, to a proline instead of a serine substitution, to being truncated to the first cysteine. The Fc part can be deleted. Any protein that is stable and / or dimerizes can serve this purpose. Only one of the Fc domains can be used, for example, either the CH2 or CH3 domain from a human immunoglobulin. The hinge, CH2 and CH3 regions of a human immunoglobulin that have been modified to improve dimerization can also be used. Only the hinge part of an immunoglobulin can also be used. A portion of CD8 alpha can also be used.

[0066] In some embodiments, HLA-G specific CARs are constructed with specificity for HLA-G, such as HLA-G expressed on disease cell types. Thus, CARs typically contain one or more HLA-G binding molecules, such as one or more antigen-binding fragments, domains, antibody variable domains, and / or any type of antibody molecule, in their extracellular portion. An example of human HLA-G nucleic acid is in the GenBank® database of the National Center for Biotechnology Information under accession number NM_002127. An example of human HLA-G polypeptide is in GenBank® under accession number NP_002118. A person skilled in the art can generate antibodies comprising scFvs against HLA-G, based at least on knowledge of polypeptides and routine practice, but many anti-HLA-G scFvs and monoclonal antibodies already exist in the art. In some embodiments, the HLA-G specific scFv is an scFV derived from one or more antibody clones.

[0067] In some embodiments, the HLA-G specific CAR comprises an antigen-binding portion of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). In certain embodiments, the antibody or functional fragment thereof is or is derived from G233, 26-2H11, MEM-G / 1, MEM-G / 9, MEM-G / 11, MEM-G / 13, 1B8, 5E6H7, 1-2C3, 16G1, 5A6G7, 87G, or 3C / G4. The antibody may also be generated de novo against HLA-G, and the scFv sequence may be obtained or derived from such a de novo antibody.

[0068] In certain embodiments, the anti-HLA-G CAR comprises an extracellular domain that is or comprises a ligand for HLA-G. In certain embodiments, the anti-HLA-G CAR comprises an extracellular domain from KIR2DL4, LILRB1, LILR2, or a fragment or mimic thereof. In some embodiments, the anti-HLA-G CAR comprises a KIR2DL4 extracellular domain or an antigen-binding portion thereof. In some embodiments, the anti-HLA-G CAR comprises an extracellular domain comprising SEQ ID NO:4.

[0069] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or can be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns are known to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0070] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally binds to one of the domains in the CAR is used. In some examples, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain binding to the transmembrane domain of the same or different surface membrane protein, and to minimize interaction with other members of the receptor complex. The transmembrane domain in some embodiments is derived from either natural or synthetic sources. When the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. The transmembrane region may be derived from (i.e., at least includes) the alpha, beta, or zeta chain of the T cell receptor, CD28, DAP12, DAP10, NKG2D, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, KIR such as KIR2DL4, GITR / CD357, and the like. Additionally, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0071] In some embodiments, the HLA-G CAR nucleic acid comprises sequences encoding other costimulatory receptors, such as transmembrane domains and one or more intracellular signaling domains. In addition to the primary T cell activation signal, such as that initiated by CD3ζ and / or FcεRIγ, additional stimulatory signals for immune effector cell proliferation and effector function after the chimeric receptor binds to the target antigen can be utilized. For example, some or all of the human costimulatory receptors can be utilized for enhanced cell activation, which can help improve in vivo persistence and improve the therapeutic success of adoptive immunotherapy. Examples include costimulatory domains from molecules such as DAP12, DAP10, NKG2D, CD2, CD28, CD27, 4-1BB, (CD137), OX40, ICOS, (CD278), CD30, HVEM, CD40, LFA-1 (CD11a / CD18), ICAM-1, and / or portions of the KIR2DL4 cytoplasmic domain that can induce an activating signal, although in alternative specific embodiments any one of these listed may be excluded from use in the CAR.

[0072] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells, such as NK cells, includes (i) non-viral gene transfer using an electroporation device (e.g., nucleofector), (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations), (iii) CARs with variable length extracellular domains linking an HLA-G recognition domain to the cell surface, and, in some cases, (iv) CARs. + These include K562-derived artificial antigen-presenting cells (aAPCs) that enable robust and numerical expansion of immune cells ( Singh et al., 2008 ; Singh et al., 2011 ). B. Examples of Specific CAR Embodiments

[0073] In certain embodiments, certain HLA-G CAR molecules are encompassed herein. In some cases, the HLA-G binding domain of the CAR is an scFv, and any scFv that binds to HLA-G can be utilized herein. In some cases, the HLA-G binding domain of the CAR is a binding domain derived from a ligand of HLA-G (e.g., KIR2DL4, LILRB1, or LILRB2), and any domain that binds to HLA-G can be utilized herein. When an anti-HLA-G scFv is utilized in the extracellular domain of the CAR, the variable heavy and variable light chains of the scFv can be in any order in the N-terminal to C-terminal direction. For example, the variable heavy chain can be N-terminal to the variable light chain, or vice versa. The scFv and / or ligand that binds to HLA-G in the CAR may or may not be codon-optimized. In certain embodiments, a vector encodes an HLA-G-specific CAR and also encodes one or more other molecules. For example, a vector can encode an HLA-G specific CAR and may also encode another protein of interest, such as another engineered antigen receptor, a suicide gene, and / or a particular cytokine.

[0074] On the same molecule, an HLA-G specific CAR may include one or more antigen-specific extracellular domains, a specific hinge, a specific transmembrane domain, one or more specific costimulatory domains, and one or more specific activation signals. For example, when multiple antigen-specific extracellular domains are utilized to target two different antigens, one of which is HLA-G, there may be a linker between the two antigen-specific extracellular domains.

[0075] In certain embodiments of certain CAR molecules, CARs may utilize DAP10, DAP12, 4-1BB, NKG2D, or other costimulatory domains (which may be referred to herein as cytoplasmic domains). In some cases, CD3 zeta is utilized without any costimulatory domain. In certain embodiments of certain CAR molecules, CARs may utilize any suitable transmembrane domain, for example, from DAP12, DAP10, 4-1BB, 2B4, OX40, CD27, NKG2D, CD8, or CD28.

[0076] In certain embodiments, there are expression constructs that contain sequences encoding engineered specific HLA-G specific receptors. In certain embodiments, any HLA-G CAR can contain one of the following: (a) a CD8 signal peptide, a KIR2DL4 extracellular domain, a CD28 transmembrane domain, a CD3 zeta intracellular domain; or (b) a CD8 signal peptide, a KIR2DL4 extracellular domain, a CD8 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

[0077] Examples of specific sequence embodiments are provided below. 1. Antigen-specific extracellular domain

[0078] In certain embodiments, the antigen-specific extracellular domain comprises the KIR2DL4 extracellular domain and / or the anti-HLA-G antigen binding region of an HLA-G specific antibody. Examples of specific sequence embodiments are provided below.

[0079] In a specific embodiment, the KIR2DL4 extracellular domain nucleotide sequence is utilized as follows: TGGGCACACGTGGGTGGTCAGGACAAGCCCTTCTGCTCTGCCTGGCCCAGCGCTGTGGTGCCTCAAGGAGGACACGTGACTCTTCGGTGTCACTATCGTCGTGGGTTCAACATCTTCACGCTGTACAAGAAAGATGGGGTCCCTGTCCCTGAGCTCTACAACAGA ATATTCTGGAACAGTTTCCTCATTAGCCCTGTGACCCCAGCACACGCAGGGACCTACAGATGTCGAGGTTTTCACCCGCACTCCCCCACTGAGTGGTCGGCACCCAGCAACCCCCTGGTGATCATGGTCACAGGTCTATATGAGAAACCTTCGCTTACAGCCCGGC TGGGCCCCACGGTTCGCGCAGGAGAGAACGTGACCTTGTCCTGCAGCTCCCAGAGCTCCTTTGACATCTACCATCTATCCAGGGAGGGGGAAGCCCATGAACTTAGGCTCCCTGCAGTGCCCAGCATCAATGGAACATTCCAGGCCGACTTCCCTCTGGGTCCTGC CACCCACGGAGAGACCTACAGATGCTTCGGCTCTTTCCATGGATCTCCCTACGAGTGGTCAGACCCGAGTGACCCACTGCCTGTTTCTGTCACAGGAAACCCTTCTAGTAGTTGGCCTTCACCCACTGAACCAAGCTTCAAAACTGGTATCGCCAGACACCTGCAT (Sequence number: 7)

[0080] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:7, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:7.

[0081] In certain embodiments, a codon-optimized KIR2DL4 extracellular domain nucleotide sequence is utilized as follows: TGGGCTCACGTTGGCGGCCAGGATAAGCCTTTTTGTTCTGCCTGGCCTAGCGCCGTGGTTCCTCAAGGTGGACACGTGACCCTGCGGTGTCACTACAGACGGGGCTTCAACATCTTCACCCTGTACAAGAAAGACGGCGTGCCCGTGCCTGAGCTGTACAACAGA ATCTTCTGGAACAGCTTCCTGATCAGCCCCGTGACACCAGCTCACGCCGGCACATACAGATGCAGAGGCTTTCACCCTCACAGCCCCACAGAGTGGTCCGCTCCATCTAACCCTCTGGTCATCATGGTCACCGGCCTGTACGAGAAGCCTAGCCTGACAGCTAGAC TGGGCCCTACAGTTAGAGCCGGCGAGAATGTGACCCTGTCCTGTAGCAGCCAGAGCAGCTTCGACATCTACCACCTGTCTAGAGAGGGCGAAGCCCACGAACTGAGACTGCCTGCCGTGCCTAGCATCAATGGCACCTTCCAGGCCGATTTTCCACTGGGACCTGC CACACACGGCGAGACTTACAGATGCTTTGGCAGCTTCCACGGCAGCCCTTACGAGTGGTCTGATCCTAGCGATCCTCTGCCTGTGTCCGTGACAGGCAATCCTAGCAGCAGCTGGCCTTCTCCAACCGAGCCTAGCTTTAAGACCGGAATCGCCCGGCATCTGCAC (Sequence number: 8)

[0082] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:8, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:8.

[0083] An example of a KIR2DL4 extracellular domain amino acid sequence is as follows: WAHVGGQDKPFCSAWPSAVVPQGGHVTLRCHYRRGFNIFTLYKKDGVPVPELYNRIFWNSFLISPVTPAHAGTYRCRGFHPHSPTEWSAPSNPLVIMVTGLYEKPSLTARLGPTVRAGENVTLSCSSQSSFDIYHLSREGEAHELRLPAVPSINGTFQADFPLGPATHGETYRCFGSFHGSPYEWSDPSDPLPVSVTGNPSSSWPSPTEPSFKTGIARHLH (SEQ ID NO: 9)

[0084] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:9, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:9.

[0085] Additional examples of KIR2DL4 extracellular domain amino acid sequences are as follows: WAHVGGQDKPFCSAWPSAVVPQGGHVTLRCHYRRGFNIFTLYKKDGVPVPELYNRIFWNSFLISPVTPAHAGTYRCRGFHPHSPTEWSAPSNPLVIMVTGLYEKPSLTARPGPTVRAGENVTLSCSSQSSFDIYHLSREGEAHELRLPAVPSINGTFQADFPLGPATHGETYRCFGSFHGSPYEWSDPSDPLPVSVTGNPSSSWPSPTEPSFKTGIARHLH (SEQ ID NO: 10)

[0086] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:9, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:9.

[0087] In specific examples, the region of KIR2DL4 utilized in the CAR molecule comprises, consists of, or consists essentially of the following amino acids: 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93 of SEQ ID NO: 9 or 10 (SEQ ID NO: 9 or SEQ ID NO: 10) in the Sequence Listing ,1-94,1-95,1-96,1-97,1-98,1-99,1-100,1-101,1-102,1-103,1-104,1-105,1-106,1-107,1-108,1-109,1-110,1-111,1-112,1-113,1-114,1-11 5,1-116,1-117,1-118,1-119,1-120,1-121,1-122,1-123,1-124,1-125,1-126,1-127,1-128,1-129,1-130,1-131,1-132,1-133,1-134,1-135,1-1 36,1-137,1-138,1-139,1-140,1-141,1-142,1-143,1-144,1-145,1-146,1-147,1-148,1-149,1-150,1-151,1-152,1-153,1-154,1-155,1-156,1- 157,1-158,1-159,1-160,1-161,1-162,1-163,1-164,1-165,1-166,1-167,1-168,1-169,1-170,1-171,1-172,1-173,1-174,1-175,1-176,1-177,1 -178,1-179,1-180,1-181,1-182,1-183,1-184,1-185,1-186,1-187,1-188,1-189,1-190,1-191,1-192,1-193,1-194,1-195,1-196,1-197,1-198, 1-199,1-200,1-201,1-202,1-203,1-204,1-205,1-206,1-207,1-208,1-209,1-210,1-211,1-212,1-213,1-214,1-215,1-216,1-217,1-218,1-219,1-220, or all (1-221); in certain embodiments, such amino acids in these ranges are contiguous. In some embodiments, a region of SEQ ID NO:9 or SEQ ID NO:10 is utilized that has a truncation at the N-terminus, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids from the N-terminus. In certain aspects, there is a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids at the N-terminus and a truncation at the C-terminus.

[0088] The amino acid sequence of the extracellular domain of LILRB1 is as follows: GHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPS PVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETL TLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 48)

[0089] The amino acid sequence of the extracellular domain of LILRB2 is as follows: QTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGV (SEQ ID NO: 49)

[0090] In a specific embodiment, the antigen-specific extracellular domain comprises an anti-HLA-G antigen-binding region of an HLA-G specific antibody, which may or may not be an scFv.

[0091] In a specific embodiment, the antigen-specific extracellular domain comprises an antibody of the HLA-G monoclonal antibody MEM-G11. In such a case, the MEM-G / 11 sequence comprises the following light chain sequence, including variable and constant regions:

[0092] DIVLTQSPASLDVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQSPKLLIYLASNRESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:50), where the constant regions are underlined. In a specific embodiment, such a light chain sequence may comprise CDR1 of RASKSVSTSGYSYMH (SEQ ID NO:51); CDR2 of LASNRES (SEQ ID NO:52); and CDR3 of QHSREFPT (SEQ ID NO:53).

[0093] The MEM-G / 11 variable light chain sequence comprises:

[0094] DIVLTQSPASLDVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQSPKLLIYLASNRESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPTFGAGTKLELK (SEQ ID NO:54).

[0095] In a specific embodiment, the polynucleotide encoding the MEM-G / 11 variable light chain sequence comprises: Gatattgtgctgaccgagcccggcgagcctggatgtgagcctgggccagcgcgcgaccattagctgccgcgcgagcaaaagcgtgagcaccagcggctatagctatatgcattggtatcagcagaaaccgggccagagcccgaaactgctgatttatctggcg agcaaccgcgaaagcggcgtgccggcgcgctttagcggcagcggcagcggcaccgattttaccctgaacattcatccggtggaagaagaagatgcggcgacctattattgccagcatagccgcgaatttccgacctttggcgcgggcaccaaactggaactgaaa (Sequence number: 55)

[0096] In a specific embodiment, the MEM-G / 11 sequence may comprise the following heavy chain sequence, including variable and constant regions:

[0097] KVQLVESGGGLVKPGGSLKLSCSASGFPFSDYYMYWVRQTPEKRLEWVATISDDDDYTYYPDSMKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCSRGIYYGSSPFAYWG QGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 56) Here, the constant regions are underlined. In a specific embodiment, such a heavy chain sequence may comprise CDR1 of GFPFSDY (SEQ ID NO:57); CDR2 of SDDDDY (SEQ ID NO:58); and CDR3 of GIYYGSSPFAY (SEQ ID NO:59).

[0098] The MEM-G / 11 variable heavy chain sequence may comprise: KVQLVESGGGLVKPGGSLKLSCSASGFPFSDYYMYWVRQTPEKRLEWVATISDDDDYTYYPDSMKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCSRGIYYGSSPFAYWGQGTLVTVSA (SEQ ID NO:60). In a specific embodiment, a polynucleotide encoding a MEM-G / 11 variable heavy chain sequence may comprise: Aaagtgcagctggtggaaagcggcggcggcctggtgaaaccgggcggcagcctgaaactgagctgcagcgcgagcggctttccgtttagcgattattatgtattgggtgcgccagaccccggaaaaacgcctggaatgggtggcgaccattagcgatgatgatgattatacctattat ccggatagcatgaaaggccgctttaccattagccgcgataacgcgaaaaacaacctgtatctgcagatgagcagcctgaaaagcgaagataccgcgatgtattattgcagccgcggcatttattatggcagcagcccgtttgcgtattggggccagggcaccctggtgaccgtgagcgcg (SEQ ID NO: 61).

[0099] In a specific embodiment, the antigen-specific extracellular domain consists of the antibody of the HLA-G monoclonal antibody of the 87G clone. In such a case, the sequence may consist of the following light chain sequence, including the variable and constant regions:

[0100] ETTVTQSPASLSVATGEKVTIRCITSTDIDDDMNWYQQKPGEPKLLISEDNILRPGVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQSDNMPLTFGGGTRLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:62), where the constant region is underlined. In certain embodiments, such a light chain sequence can comprise CDR1 of ITSTDIDDDMN (SEQ ID NO:63); CDR2 of EDNILRP (SEQ ID NO:64); and CDR3 of LQSDNMPLT (SEQ ID NO:65).

[0101] The 87G variable light chain sequence may comprise:

[0102] ETTVTQSPASLSVATGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEDNILRPGVPSRFSSSGYGTDFVFTIENTLSEDVADYYCLQSDNMPLTFGGGTRLEIK (SEQ ID NO:66). Polynucleotides that may encode the 87G variable light chain sequence include:

[0103] Gaaaccaccgtgacccagagcccggcgagcctgagcgtggcgaccggcgaaaaagtgaccattcgctgcattaccagcaccgatattgatgatgatatgaactggtatcagcagaaaccgggcgaaccgccgaaactgctgattagcgaagataacattc tgcgcccgggcgtgccgagccgctttagcagcagcggctatggcaccgattttgtgtttaccattgaaaacaccctgagcgaagatgtggcggattattattgcctgcagagcgataacatgccgctgacctttggcggcggcacccgcctggaaattaaa (Sequence number: 67).

[0104] In a specific example, the sequence may consist of the following heavy chain sequence including the variable and constant regions:

[0105] EVKLVESGGSLVQPGGSLKLSCAASGFSFSSYTMSWVRQTPKKRLEWVAYVSNGAGTTYYPDSLKGRFTISRDNAKNTLHLLMTSLKSEDTAIYYCARHYYGSYHFDYWGQGT TLIVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPC PPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK (SEQ ID NO:68), where the constant regions are underlined. In a specific embodiment, such a heavy chain sequence may comprise CDR1 of GFSFSSY (SEQ ID NO:69); CDR2 of SNGAGT (SEQ ID NO:70); and CDR3 of HYYGSYHFDY (SEQ ID NO:71).

[0106] In some embodiments, the 87G variable heavy chain sequence comprises:

[0107] EVKLVESGGSLVQPGGSLKLSCAASGFSFSSYTMSWVRQTPKKRLEWVAYVSNGAGTTYYPDSLKGRFTISRDNAKNTLHLLMTSLKSEDTAIYYCARHYYGSYHFDYWGQGTTLIVSS (SEQ ID NO:72). In certain embodiments, a polynucleotide encoding the 87G variable heavy chain sequence comprises:

[0108] Gaagtgaaactggtggaaagcggcggcagcctggtgcagccgggcggcagcctgaaactgagctgcgcggcgagcggctttagctttagcagctataccatgagctgggtgcgccagaccccgaaaaaacgcctggaatgggtggcgtatgtgagcaacggcgcgggcaccacctatt atccggatagcctgaaaggccgctttaccattagccgcgataacgcgaaaaacaccctgcatctgctgatgaccagcctgaaaagcgaagataccgcgatttattattgcgcgcgccattattatggcagctatcattttgattattggggccagggcaccaccctgattgtgagcagc (Sequence number: 73)

[0109] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:55, 61, 67, or 73, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:55, 61, 67, or 73, respectively.

[0110] Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:50-54, 56-60, 62-66, or 68-72, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:50-54, 56-60, 62-66, or 68-72, respectively. 2. Transmembrane domain

[0111] Any suitable transmembrane domain can be used in the HLA-G specific CAR of the present disclosure.Examples include at least the transmembrane domain from DAP10, DAP12, CD28, NKG2D, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 or CD154, the transmembrane domain from T cell receptor a or b chain, the transmembrane domain from CD3 zeta chain, the transmembrane domain from ICOS, their functional derivatives, and their combinations.Specifically, the transmembrane domain from DAP10, DAP12, CD28, CD8 or NKG2D is used. In some embodiments, a transmembrane domain from a Killer Immunoglobulin-Like Receptor (KIR) is utilized, such as a transmembrane domain from an inhibitory KIR (e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3) or an activating KIR (e.g., KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1). Examples of specific transmembrane domain sequences are provided below:

[0112] CD28 transmembrane domain sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 11)

[0113] The amino acid sequence of the CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 12)

[0114] CD8 transmembrane domain sequence: accacaacaccagcacctagacctccaactccagctcctacaatcgccagccagcctctgtctctgaggcctgaagcttgtagacctgctgctggcggagccgtgcataccagaggactggatttcgcctgcgatatctacatctgggcccctctggctggaacatgtggcgtgctgctgctgagcctcgtgatcaca (Sequence number: 13)

[0115] CD8 transmembrane domain amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 14)

[0116] 4-1BB transmembrane domain sequence: ATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTT (SEQ ID NO: 25)

[0117] 4-1BB transmembrane domain amino acid sequence: IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO:26)

[0118] DAP10 transmembrane domain sequence: CTCCTGGCAGGCCTCGTGGCTGCTGATGCGGTGGCATCGCTGCTCATCGTGGGGGCGGTGTTC (SEQ ID NO: 27)

[0119] DAP10 transmembrane domain amino acid sequence: LLAGLVAADAVASLLIVGAVF (SEQ ID NO:28)

[0120] DAP12 transmembrane domain sequence: GGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTG (SEQ ID NO: 29)

[0121] DAP12 transmembrane domain amino acid sequence: GVLAGIVMGDLVLTVLIALAV (SEQ ID NO:30)

[0122] NKG2D transmembrane domain sequence: GCGGTGATGATTATTTTTCGCATTGGCATGGCGGTGGCGATTTTTTGCTGCTTTTTTTTTCCG (SEQ ID NO: 31)

[0123] NKG2D transmembrane domain amino acid sequence: AVMIIFRIGMAVAIFCCFFFP (SEQ ID NO:32)

[0124] Any polynucleotide encompassed by this disclosure can consist of SEQ ID NO:11, 13, 25, 27, 29, or 31, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:11, 13, 25, 27, 29, or 31. Any polypeptide encompassed by this disclosure can comprise SEQ ID NO:12, 14, 16, 28, 30, or 32, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more identical to SEQ ID NO:12, 14, 16, 28, 30, or 32. 3. Intracellular domain

[0125] One or more intracellular domains (which may also be referred to herein as signal activation domains or costimulatory domains, as appropriate) may or may not be utilized in a particular anti-HLA-G CAR of the present disclosure. Specific examples include intracellular domains from CD3 zeta, 4-1BB, NKG2D, OX-40, CD27, DAP10, DAP12, B7-1 / CD80, CD28, 2B4, 4-1BBL, B7-2 / CD86, CTLA-4, B7-H1 / PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, PDCD6, BTLA, or combinations thereof. In some embodiments, the intracellular domain of a CAR of the present disclosure is not the KIR2DL4 intracellular domain.

[0126] Examples of specific intracellular domains that can be used in the CARs of the present disclosure are shown below:

[0127] An example of the CD3 zeta intracellular domain sequence: acgcgtaagaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaaaagacgtggccgggaccctgagatggggggaaagccgagaaggaagaagaaccctcaggaa ggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcgga (Sequence number: 15)

[0128] An example of a CD3 zeta intracellular domain amino acid sequence:

[0129] TRKKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 16)

[0130] Example of CD3 zeta intracellular domain sequence: AAACGGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGA (Sequence number: 17)

[0131] An example of a CD3 zeta intracellular domain amino acid sequence:

[0132] KRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 18)

[0133] 4-1BB intracellular domain sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 33)

[0134] 4-1BB intracellular domain amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:34)

[0135] DAP10 intracellular domain sequence: CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 35)

[0136] DAP10 intracellular domain amino acid sequence: LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO:36)

[0137] DAP12 intracellular domain sequence: TACTTCCTGGGCCGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA (SEQ ID NO: 37)

[0138] DAP12 intracellular domain amino acid sequence: YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 38)

[0139] NKG2D intracellular domain sequence: AGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATG (SEQ ID NO: 39)

[0140] NKG2D intracellular domain amino acid sequence: SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGM (SEQ ID NO:40)

[0141] Polynucleotides encompassed by this disclosure may include: SEQ ID NO: 15, 17, 33, 35, 37, or 39, or a sequence which is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO: 15, 17, 33, 35, 37, or 39. Any polypeptide encompassed by this disclosure may include: SEQ ID NO: 16, 18, 34, 36, 38, or 40, or a sequence which is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO: 16, 18, 34, 36, 38, or 40.

[0142] 4. Hinge

[0143] In some embodiments of the CAR, there is a hinge region between one or more extracellular antigen binding domains and the transmembrane domain. In specific embodiments, the hinge has a particular length, such as, for example, 10-20, 10-15, 11-20, 11-15, 12-20, 12-15, or 15-20 amino acids. The hinge can be any suitable hinge, and optionally includes a hinge from IgG, or CD28. In specific embodiments, the hinge is a small flexible polypeptide that connects the CH2-CH3 domain and the CH1 domain of IgG Fc. For example, CH2-CH3 hinges (partial or complete) of various IgG subclasses (IgG1-4, modified or unmodified) can be utilized. However, in some cases, rather than the entire CH2-CH3 hinge, a portion of the hinge is utilized (such as the CH3 alone or a portion of the CH3 alone). In certain embodiments, the CH2-CH3 hinge from IgG1 is utilized, optionally the entire CH2-CH3 hinge is utilized (all 229 amino acids), optionally only the CH3 hinge (119 amino acids), or optionally a short hinge (12 amino acids).

[0144] In certain cases, the identity or length of the spacer and / or hinge can be altered to optimize the efficiency of the CAR. See, e.g., Hudecek et al. (2014) and Jonnalagadda et al. (2015). In certain embodiments, an HLA-G CAR utilizes, for example, an IgG4 hinge + CH3 or utilizes a CD8a stalk.

[0145] Thus, in specific embodiments, the IgG hinge region utilized is typically IgG1 or IgG4, and in some cases, the CAR comprises the CH2-CH3 domain of IgG Fc. The use of IgG Fc domains can provide flexibility to the CAR, making it less immunogenic, easier to detect CAR expression with anti-Fc reagents, and allowing one or more CH2 or CH3 modules to be removed to accommodate different spacer lengths. However, in one embodiment, mutations of certain spacers to avoid FcγR binding, for example to avoid soluble and cell surface Fc gamma receptor binding, may improve CAR+ T cell engraftment and anti-tumor efficacy, yet maintain activity to mediate antigen-specific lysis. For example, an IgG4-Fc spacer with modified CH2 region can be used. For example, the CH2 region may be mutated, including point mutations and / or deletions. Specific modifications have been demonstrated at two sites within the CH2 region (L235E; N297Q) and / or incorporate CH2 deletions (Jonnalagadda et al, 2015). In specific embodiments, the IgG4 hinge-CH2-CH3 domain (229 aa in length) or the hinge domain alone (12 aa in length) can be employed (Hudececk et al., 2015).

[0146] In specific embodiments, the hinge is IgG, CD28, CD-8α, 4-1BB, 0X40, CD3 zeta, CD3 zeta chain from the T cell receptor a chain or b chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, or CD154.

[0147] Examples of specific sequences of hinges that can be utilized include at least the following:

[0148] IgG Hinge sequence: GTACGGTCACTGTCTCTTCACAGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATT (SEQ ID NO: 41)

[0149] IgG Hinge amino acid sequence: TVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL (SEQ ID NO: 42)

[0150] CD28 Hinge sequence: ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 43)

[0151] CD28 Hinge amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:44)

[0152] Any polynucleotide encompassed by this disclosure may comprise SEQ ID NO:41 or 43, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:41 or 43. Any polypeptide encompassed by this disclosure may comprise SEQ ID NO:42 or 44, or a sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent identical to SEQ ID NO:42 or 44. 5. Other proteins

[0153] In some embodiments, one or more other proteins are utilized with the anti-HLA-G CAR of the present disclosure. The one or more other proteins may be utilized for any reason, including promoting the efficacy of the CAR itself and / or any type of cell expressing the CAR. In some cases, the other protein facilitates treatment of an individual receiving a cell expressing the CAR as a treatment, regardless of whether the other protein(s) directly or indirectly affect the activity of the CAR or the cell. In some cases, the other protein is a suicide gene, one or more cytokines, or both. In certain embodiments, the one or more other proteins are produced from a vector and are ultimately produced as two separate polypeptides. For example, the anti-HLA-G CAR and the other protein(s) may be separated, for example, by a 2A sequence or an IRES.

[0154] In certain embodiments, a cytokine such as IL-15 is utilized in conjunction with an anti-HLA-G CAR.

[0155] An example of an IL-15 sequence is as follows:

[0156] IL-15 sequence: GCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGA CTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGC AAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGACAATT (Sequence number: 21)

[0157] IL-15 amino acid sequence: ISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 22)

[0158] If it is intended that CAR and another protein in the same vector be produced into two distinct polypeptides, a specific 2A sequence may be utilized.

[0159] In one example, the E2A sequence is used as follows: CAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCC (SEQ ID NO: 19)

[0160] The E2A amino acid sequence may be: QCTNYALLKLAGDVESNPGP (SEQ ID NO:20)

[0161] Other 2A examples may utilize:

[0162] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO:45)

[0163] P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO:46)

[0164] F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:47)

[0165] The present disclosure also encompasses specific CAR molecules, including expression in any type of immune effector cell.

[0166] In one example, an anti-HLA-G CAR is utilized, which includes a CD8 signal peptide, a KIR2DL4 extracellular domain, a CD28 transmembrane domain, and a CD3 zeta intracellular domain. In the vector, the CAR can be expressed together with IL-15, which can be separated from the CAR by a 2A sequence. In a specific example, such a CAR and IL-15 construct can have the following base sequence: CD8SP-KIR2DL4EC_CD28 TMD-CD3 zeta_IL15

[0167] The amino acid sequence corresponding to CD8SP-KIR2DL4EC_CD28 TMD-CD3zeta_IL15 is as follows: MALPVTALLLPLALLLHAARPWAHVGGQDKPFCSAWPSAVVPQGGHVTLRCHYRRGFNIFTLYKKDGVPVPELYNRIFWNSFLISPVTPAHAGTYRCRGFHPHSPTEWSAPSNPLVIMVTGLYEKPSLTARLGPTVRAGEN VTLSCSSQSSFDIYHLSREGEAHELRLPAVPSINGTFQADFPLGPATHGETYRCFGSFHGSPYEWSDPSDPLPVSVTGNPSSSWPSPTEPSFKTGIARHLHFWVLVVVGGVLACYSLLVTVAFIIFWVTRKKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 2)

[0168] In one example, an anti-HLA-G CAR is utilized that contains a CD8 signal peptide, a codon-optimized KIR2DL4 extracellular domain, a CD8 transmembrane domain, a CD3 zeta intracellular domain, and a CD28 costimulatory domain. In the vector, the CAR can be expressed together with IL-15, which can be separated from the CAR by a 2A sequence. In a specific example, such a CAR and IL-15 construct can have the following base sequence: CD8SPcoKIR2L4_EC_CD8tmd28Z15

[0169] The amino acid sequence corresponding to CD8SPcoKIR2L4_EC_CD8tmd28Z15 is as follows: MGMALPVTALLLPLALLLHAARPWAHVGGQDKPFCSAWPSAVVPQGGHVTLRCHYRRGFNIFTLYKKDGVPVPELYNRIFWNSFLISPVTPAHAGTYRCRGFHPHSPTEWSAPSNPLVIMVTGLYEKPSLTARLGPTVRAGENVTLSCSSQ SSFDIYHLSREGEAHELRLPAVPSINGTFQADFPLGPATHGETYRCFGSFHGSPYEWSDPSDPLPVSVTGNPSSSWPSPTEPSFKTGIARHLHTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 4)

[0170] In some embodiments, a specific CAR is employed that utilizes an anti-HLA-G antibody. In a particular embodiment, the construct encoding the CAR includes a CAR that includes inducible caspase 9, MEM-G / 11 antibody (where the variable heavy chain is at the N-terminus of the variable light chain), CD28 hinge, CD28 costimulatory domain, and CD3 zeta, and the expression construct also expresses IL-15. The separate polypeptides may be separated by a 2A sequence. An exemplary sequence of such a construct is as follows: iC9MEMVHVL28HingeCD28CD3ZIL15

[0171]

[0172] Examples of iC9MEMVHVL28HingeCD28CD3ZIL15 polypeptides include the following:

[0173] In a specific example, the construct encoding the CAR includes a CAR that includes inducible caspase 9, MEM-G / 11 antibody (where the variable heavy chain is N-terminal to the variable light chain), CD28 hinge, DAP10 costimulatory domain, and CD3 zeta, and the expression construct also expresses IL-15. The separate polypeptides may be separated by a 2A sequence. An exemplary sequence for such a construct is as follows: iC9MEMVHVL28HingeDAP10CD3ZIL15

[0174]

[0175] An example of a polypeptide encoded by iC9MEMVHVL28HingeDAP10CD3ZIL15 can be:

[0176] In a specific example, the construct encoding the CAR consists of inducible caspase 9, a CAR consisting of the 87G antibody (where the variable heavy chain is N-terminal to the variable light chain), a CD28 hinge, a CD28 costimulatory domain, and CD3 zeta, and the expression construct also expresses IL-15. The separate polypeptides may be separated by a 2A sequence. An exemplary sequence for such a construct is as follows: iC9-87GVHVL28HingeCD28CD3ZIL15

[0177]

[0178] An example of a polypeptide encoded by iC9-87GVHVL28HingeCD28CD3ZIL15 can include:

[0179] In a specific example, the construct encoding the CAR includes a CAR that includes inducible caspase 9, 87G antibody (where the variable heavy chain is N-terminal to the variable light chain), CD28 hinge, DAP10 costimulatory domain, and CD3 zeta, and the expression construct also expresses IL-15. The separate polypeptides may be separated by a 2A sequence. An exemplary sequence of such a construct is as follows: iC9-87GVHVL28HingeDAP10CD3ZIL15

[0180]

[0181] An example of a polypeptide encoded by iC9-87GVHVL28HingeDAP10CD3ZIL15 can include: CT cell receptor (TCR)

[0182] In some embodiments, the engineered HLA-G targeted antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell, or one or more parts thereof. "T cell receptor" or "TCR" refers to a molecule that contains variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and can specifically bind to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is an αβ form.

[0183] Typically, TCRs, which exist in αβ and γδ forms, are generally structurally similar, but the T cells expressing them may have different anatomical locations or functions. TCRs may be found on the surface of cells or in soluble forms. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of the TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full length TCRs, including αβ or γδ forms of the TCR.

[0184] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a particular antigenic peptide bound to an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding portion" or "antigen-binding fragment" of a TCR, which may be used interchangeably, refers to a molecule that contains a portion of the structural domain of a TCR but binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, such as the variable α and β chains of the TCR, to form a binding site for binding to a particular MHC-peptide complex, e.g., where each chain generally contains three complementarity determining regions.

[0185] In some embodiments, the variable domains of the TCR chains combine to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins. This confers antigen recognition and determines peptide specificity by forming the binding site of the TCR molecule, and determines peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognition of processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0186] In some embodiments, the TCR chain contains a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., α chain, β chain) contains two immunoglobulin domains, an N-terminal variable domain (e.g., V aor Vp; typically, Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.), and one constant domain adjacent to the cell membrane (e.g., the α chain constant domain or C a , typically amino acids 117-259 based on Kabat, and a beta chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane proximal constant domains, and two membrane distal variable domains that contain the CDRs. The constant domain of the TCR domain contains a short linking sequence in which cysteine ​​residues form disulfide bonds to link the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the alpha and beta chains such that the TCR contains two disulfide bonds within the constant domains.

[0187] In some embodiments, TCR chains may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, TCR chains contain a cytoplasmic tail. In some cases, this structure allows TCR to bind to other molecules such as CD3. For example, TCRs that contain a constant domain with a transmembrane region may anchor the protein in the cell membrane and bind to the invariant subunit of the CD3 signaling device or complex.

[0188] In general, CD3 is a multiprotein complex that may have three different chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, the complex may contain a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to bind to the positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, although each CD3ζ chain has three. In general, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and are responsible for transmitting signals from the TCR into the cell. The CD3 chain and the ζ chain, together with the TCR, form what is known as the T cell receptor complex.

[0189] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β or γ and δ chains) linked by a disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR may be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, for example, from cells derived from T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells may be obtained from in vivo isolated cells. In some embodiments, high affinity T cell clones may be isolated from a patient and the TCRs isolated. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, TCR clones against target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against target antigens (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, TCRs or antigen-binding portions thereof can be synthetically generated from knowledge of the sequence of the TCR. III. Cytokines

[0190] One or more cytokines may be utilized with one or more engineered HLA-G targeting receptors, such as HLA-G specific CARs. In some cases, one or more cytokines are present on the same vector molecule as the engineered receptor, while in other cases, they are present on a separate vector molecule. In certain embodiments, one or more cytokines are co-expressed from the same vector as the engineered receptor. One or more cytokines may be generated as a separate polypeptide from the HLA-G specific receptor. As an example, interleukin-15 (IL-15) is utilized. IL-15 may be used because, for example, it is tissue restricted and is only observed in serum or systemically at some level under pathological conditions. IL-15 has several attributes that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors through the relief of functional inhibition of tumor-resident cells, and inhibits activation-induced cell death. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. As an example, the one or more cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, or a combination thereof. NK cells that express IL-15 can be utilized and are capable of continued supportive cytokine signaling, which is useful for survival after infusion.

[0191] In certain embodiments, the NK cells express one or more exogenously provided cytokines. The cytokines can be exogenously provided to the NK cells because they are expressed from an expression vector in the cells and / or provided in the culture medium of the cells. Alternatively, the endogenous cytokines in the cells are upregulated by regulatory engineering of the expression of the endogenous cytokine, such as genetic engineering at the promoter site of the cytokine. When the cytokine is provided to the cells on an expression construct, the cytokine can be encoded from the same vector as the suicide gene. The cytokine can be expressed as a polypeptide molecule separate from the suicide gene and as a polypeptide separate from the engineered receptor of the cell. In some embodiments, the present disclosure relates to the co-use of CAR and / or TCR vectors with IL-15, particularly in NK cells. IV. Suicide Genes

[0192] In certain embodiments, suicide genes are utilized with any type of cell therapy to control its use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells as needed. The HLA-G targeted cells of the present disclosure modified to have a vector encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that results in the transfer of the gene product to a compound that kills the host cell upon administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that is targeted by an agent (such as an antibody) that targets the suicide gene product, if desired.

[0193] Examples of suicide gene / prodrug combinations that can be used include Herpes Simplex Virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. E. coli purine nucleoside phosphorylase, a suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be used. Other examples of suicide genes used in prodrug therapy include the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0194] Exemplary suicide genes also include CD20, CD52, EGFRv3, or inducible caspase 9. In one embodiment, a truncated version of EGFR variant III (EGFRv3) can be used as a suicide antigen that can be removed by cetuximab. Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-alpha, gamma-lyase (MET), and thymidine phosphorylase (TP).

[0195] In certain embodiments, the vector encoding the HLA-G targeted CAR or any vector in the NK cell encompassed herein comprises one or more suicide genes.The suicide gene may or may not be on the same vector as the HLA-G targeted CAR.If the suicide gene is on the same vector as the HLA-G targeted CAR, the suicide gene and the CAR may be separated, for example, by an IRES or 2A element. V. Vector

[0196] HLA-G targeting CAR can be delivered to recipient immune cells by any suitable vector, including viral vector or non-viral vector.Examples of viral vector include at least retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.Examples of non-viral vector include at least plasmid, transposon, lipid, nanoparticle, etc.

[0197] When immune cells are transduced with a vector encoding an HLA-G targeting receptor and also require the transduction of another gene, such as a suicide gene and / or a cytokine and / or an optional therapeutic gene product, into the cells, the HLA-G targeting receptor, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be included in the same vector. In some cases, the HLA-G targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene are expressed from the same vector molecule, such as the same viral vector molecule. In such cases, the expression of the HLA-G targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene may or may not be regulated by the same regulatory element. When the HLA-G targeting CAR, the suicide gene, the cytokine, and the optional therapeutic gene are on the same vector, they may or may not be expressed as separate polypeptides. When they are expressed as separate polypeptides, they may be separated on the vector, for example, by a 2A element or an IRES element (or both types may be used on the same vector, one or more times). A. General embodiment

[0198] Those of skill in the art would be well-versed in constructing vectors by standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference) for expression of the antigen receptors of the present disclosure. 1. Adjustment element

[0199] The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence (5' to 3' direction). The promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells can be composed of multiple genetic elements. The cellular machinery can collect and incorporate the regulatory information transmitted by each element, allowing different genes to evolve different, often complex patterns of transcriptional regulation. Promoters used in connection with the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the production of cancer treatments, the promoter can be effective under conditions of hypoxia. 2. Promoter / Enhancer

[0200] The expression constructs provided herein include promoters for driving the expression of antigen receptors and other cistron gene products. Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, separate elements that cover the start site itself help to fix the location of start. Additional promoter elements regulate the frequency of transcription initiation. Typically, this is located in the region upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of DNA to promote expression of the encoded RNA.

[0201] Spacing between promoter elements is frequently flexible enough that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0202] A promoter may be one that is naturally associated with a nucleic acid sequence, such as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence and located either downstream or upstream of said sequence. Certain advantages may also be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0203] Of course, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, which is incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as being advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0204] Additionally, expression can be driven using any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Data Base EPDB, via the World Wide Web at epd.isb-sibi.ch / ). Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0205] Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus (RSV) early promoter, eukaryotic promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter, and linked response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and response element promoter near minimal TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in GenBank®, Accession No. X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat No. ATCC 45007). In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I, or MHC class II promoter. However, any other promoter useful for driving expression of therapeutic genes can be used in the practice of the present disclosure.

[0206] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that have the potential to increase the activity of a promoter and act in cis and regardless of its orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily limited to such long distances, since it may function in close proximity to a given promoter. 3. Initiation Signals and Linked Expression

[0207] Also, specific initiation signals can be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. Such signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals including the ATG initiation codon. Those skilled in the art can easily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. Exogenous translation control signals and initiation codons can be either natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements.

[0208] In certain embodiments, an internal ribosome entry site (IRES) element is used to produce multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRES from mammalian messages have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES resulting in a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0209] As detailed elsewhere herein, certain 2A sequence elements can be used to provide for linked or co-expression of genes in constructs provided in this disclosure. For example, a truncation sequence can be used to link open reading frames to form a single cistron, thereby allowing genes to be co-expressed. Exemplary truncation sequences include Equine Rhinitis A Virus (E2A) or F2A (Foot and Mouth Disease Virus 2A) or "2A-like" sequences (e.g., Thosea asigna Virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In certain embodiments, in a single vector, multiple 2A sequences are not identical, but in alternative embodiments, the same vector utilizes two or more identical 2A sequences. Examples of 2A sequences are described in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety. 4. Origin of replication

[0210] To propagate the vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP described above, or a genetically engineered oriP with similar or enhanced function in programming, which is a specific nucleic acid sequence from which replication is initiated. Alternatively, the origin of replication of other extrachromosomally replicating viruses described above, or an autonomously replicating sequence (ARS), may be used. 5. Selectable and Screenable Markers

[0211] In some embodiments, NK cells containing the HLA-G targeting receptor construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will confer an identifiable change to the cell that allows easy identification of cells containing the expression vector. In general, a selection marker is one that confers a property that allows selection. A positive selection marker is one whose presence allows selection, and a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0212] In general, the inclusion of a drug selection marker aids in cloning and identification of transformants; for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screenable markers such as GFP, whose basis is colorimetric analysis. Other screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized as negative selection markers. Also, those skilled in the art will likely know how to use immunological markers in combination with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art. B. Multicistronic Vectors

[0213] In certain embodiments, the HLA-G targeting receptor, the optional suicide gene, the optional cytokine, and / or the optional therapeutic gene are expressed from a multicistronic vector (the term "cistron" as used herein refers to a nucleic acid sequence from which a gene product can be generated). In certain embodiments, the multicistronic vector encodes the HLA-G targeting receptor, the suicide gene, and at least one cytokine and / or an engineered receptor, such as a T cell receptor, and / or an additional non-HLA-G targeting CAR. Optionally, the multicistronic vector encodes at least one HLA-G targeting CAR, at least one TNF-alpha variant, and at least one cytokine. The cytokine can be a specific type of cytokine, such as human or mouse, or any species. In certain cases, the cytokine is IL15, IL12, IL2, IL18, and / or IL21.

[0214] In certain embodiments, the present disclosure provides a flexible modular system utilizing polycistronic vectors capable of expressing multiple cistrons at substantially the same level (the term "module" as used herein refers to a cistron or a component of a cistron, allowing its interchangeability, such as by removal and replacement of the entire cistron or each of the components of the cistron, for example, by using standard recombinant techniques). The system can be used for cell engineering to allow combinatorial expression (including overexpression) of multiple genes. In certain embodiments, one or more of the genes expressed by the vector includes one, two, or more antigen receptors. The multiple genes can include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vector can further include: (1) one or more reporters, such as fluorescent or enzymatic reporters for cell assays and animal imaging, and the like; (2) one or more cytokines or other signaling molecules; and / or (3) a suicide gene.

[0215] In certain cases, the vector may contain at least four cistrons separated by any kind of cleavage site, such as 2A cleavage sites. The vector may or may not be based on Moloney Murine Leukemia Virus (MoMLV or MMLV) containing 3' and 5' LTR with psi packaging sequence in pUC19 backbone. The vector may contain four or more cistrons with three or more 2A cleavage sites and multiple ORFs for gene exchange. The system allows for combinatorial overexpression of multiple genes (seven or more) in some embodiments flanked by restriction sites for rapid integration by subcloning, and also contains at least three 2A self-cleavage sites. Thus, the system allows for the expression of multiple CARs, TCRs, signaling molecules, cytokines, cytokine receptors, and / or homing receptors. The system may also be used for other viral and non-viral vectors, including but not limited to lentivirus, adenovirus AAV, and non-viral plasmids.

[0216] The modularity of the system also allows for efficient subcloning of genes into each of the four cistrons within the polycistronic expression vector, and swapping of genes for rapid testing, etc. Strategically positioned restriction sites within the polycistronic expression vector allow genes to be swapped efficiently.

[0217] Embodiments of the present disclosure encompass systems that utilize polycistronic vectors in which at least a portion of the vector is modular, for example, by allowing for the removal and replacement of one or more cistrons (or components of one or more cistrons), such as by utilizing one or more restriction enzyme sites whose identities and locations are specifically selected to facilitate modular use of the vector. Vectors also have embodiments that confer the advantage that the vector expresses separate gene products in substantially equimolar concentrations, with multiple cistrons being translated into a single polypeptide and processed into separate polypeptides.

[0218] The vectors of the present disclosure are configured such that modularity can be altered for one or more cistrons of the vector and / or one or more components of one or more particular cistrons. The vectors can be designed to take advantage of unique restriction enzyme sites flanking the ends of one or more cistrons and / or flanking the ends of one or more components of a particular cistron.

[0219] The embodiments of the present disclosure include polycistronic vectors that include at least two, at least three, or at least four cistrons, each flanked by one or more restriction enzyme sites, with at least one cistron encoding at least one antigen receptor. In some cases, two, three, four, or more cistrons are translated into a single polypeptide and cleaved into separate polypeptides, while in other cases, multiple cistrons are translated into a single polypeptide and cleaved into separate polypeptides. Adjacent cistrons on a vector can be separated by a self-cleaving site, such as a 2A self-cleaving site. In some cases, each cistron expresses a separate polypeptide from the vector. In certain cases, adjacent cistrons on a vector are separated by an IRES element.

[0220] In certain embodiments, the present disclosure provides a system for cell engineering that allows for combinatorial expression, including overexpression, of multiple cistrons, which may include, for example, one, two, or more antigen receptors. In certain embodiments, the use of polycistronic vectors described herein allows the vectors to generate equimolar levels of multigene products from the same mRNA. Multigenes may include, but are not limited to, CARs, TCRs, cytokines, chemokines, homing receptors, CRISPR / Cas9-mediated gene mutations, decoy receptors, cytokine receptors, chimeric cytokine receptors, and the like. The vectors may further include one or more fluorescent or enzymatic reporters for cell assays, animal imaging, and the like. The vectors may also include suicide gene products for the termination of cells carrying the vector when the cells are no longer needed or become harmful to the host to which it is provided.

[0221] In certain embodiments, the vector is a viral vector (e.g., a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector) or a non-viral vector. The vector may include Moloney Murine Leukemia Virus (MMLV) 5'LTR, 3'LTR, and / or psi packaging elements. In certain cases, the psi packaging is integrated between the 5'LTR and the antigen receptor coding sequence. The vector may or may not include a pUC19 sequence. In some embodiments of the vector, at least one cistron encodes a cytokine (e.g., IL-15, IL-7, IL-21, IL-23, IL-18, IL-12, or IL-2), a chemokine, a cytokine receptor, and / or a homing receptor.

[0222] If 2A cleavage sites are utilized in the vector, the 2A cleavage sites may include a P2A, T2A, E2A, and / or F2A site.

[0223] The restriction enzyme site may be of any type and may contain any number of bases in its recognition site, for example, 4-8 bases; the number of bases in the recognition site may be at least 4, 5, 6, 7, 8, or more. The site when cleaved may generate blunt cut ends or sticky ends. The restriction enzyme may be, for example, type I, type II, type III, or type IV. Restriction enzyme sites may be obtained from available databases, for example, the Integrated relational Enzyme database (IntEnz) or BRENDA (The Comprehensive Enzyme Information System).

[0224] An exemplary vector may be circular, and by convention, position 1 (the 12 o'clock position at the top of the circle, with the remaining sequences in a clockwise direction) is set at the start of the 5'LTR.

[0225] In embodiments in which a self-cleaving 2A peptide is utilized, the 2A peptide can be a viral oligopeptide 18-22 amino acids (aa) long that mediates the "cleavage" of the polypeptide during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome, and various viral 2As are commonly named after the viruses from which they are derived. The first 2A discovered was F2A (foot and mouth disease virus), and subsequently E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (Thosea asigna virus 2A) were also identified. The mechanism of 2A-mediated "self-cleavage" was discovered to be ribosomal skipping, which skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A.

[0226] In certain cases, the vector can be a gamma-retroviral transfer vector. Retroviral transfer vectors can include a backbone based on a plasmid, such as pUC19 plasmid (large fragment (2.63 kb) between the restriction enzyme sites of HindIII and EcoRI). The backbone can have viral components derived from Moloney Murine Leukemia Virus (MoMLV), including 5'LTR, psi packaging sequence, and 3'LTR. LTRs are long terminal repeat sequences found on both sides of retroviral proviruses, and in the case of transfer vectors, they enclose the genetic cargo of interest, such as HLA-G targeting CAR and related components. Also, the psi packaging sequence, which is the target site for packaging by the nucleocapsid, is integrated in cis and sandwiched between the 5'LTR and the CAR coding sequence. Thus, the basic structure of an example transfer vector can be constructed as follows: pUC19 sequence-5'LTR-psi packaging sequence-genetic cargo of interest-3'LTR-pUC19 sequence. The system can also be used with other viral and non-viral vectors, including but not limited to lentiviruses, adenoviruses AAV, and non-viral plasmids. VI.Cells

[0227] The present disclosure encompasses any type of immune cell or stem cell with at least one vector encoding an HLA-G targeting receptor and may also encode at least one cytokine and / or at least one suicide gene. In some cases, the various vectors encode a CAR versus encoding a suicide gene and / or cytokine. Immune cells, including NK cells, can be derived from umbilical cord blood (including pooled umbilical cord blood from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. NK cells can be derived from cell lines, such as, for example, but not limited to, NK-92 cells. NK cells are characterized by CD56 + They may be cord blood mononuclear cells, such as NK cells.

[0228] The present disclosure encompasses any type of immune cell or other cell, including conventional T cells, gamma-delta T cells, NK T and invariant NKT cells, regulatory T cells, macrophages, B cells, dendritic cells, mesenchymal stromal cells (MSCs), or mixtures thereof.

[0229] In some cases, the cells are expanded in the presence of an effective amount of universal antigen presenting cells (UAPC), including any suitable ratio. The cells can be cultured with UAPC at a ratio of 10:1 to 1:10; 9:1 to 1:9; 8:1 to 1:8; 7:1 to 1:7; 6:1 to 1:6; 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 2:1 to 1:2; or 1:1, including, for example, a ratio of 1:2. In some cases, the NK cells were expanded in the presence of IL-2 at a concentration of, for example, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 100-500, 100-400, 100-300, 100-200, 200-500, 200-400, 200-300, 300-500, 300-400, or 400-500 U / mL.

[0230] After genetic modification with the vector, the NK cells may be infused immediately or stored. In certain embodiments, after genetic modification, the cells may be expanded ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, 5 days or more after gene introduction into the cells. In further embodiments, the transfectants are cloned (clones showing the presence of a single integrated or episomally maintained expression cassette or plasmid) and the expression of the HLA-G targeted CAR is expanded ex vivo. The clones selected for expansion demonstrate the ability to specifically recognize and lyse HLA-G expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-21, IL-23, etc.). The recombinant immune cells may be expanded by stimulation with artificial antigen presenting cells. In further embodiments, the genetically modified cells may be cryopreserved.

[0231] The embodiments of the present disclosure include cells that express one or more HLA-G targeted CARs and one or more suicide genes encompassed herein. In certain embodiments, the NK cells comprise recombinant nucleic acids encoding one or more HLA-G targeted CARs and one or more engineered, non-secretable, membrane-bound TNF-alpha variant polypeptides. In certain embodiments, in addition to expressing one or more HLA-G targeted CARs and TNF-alpha variant polypeptides, the cells also comprise nucleic acids encoding one or more therapeutic gene products.

[0232] The cells may be obtained directly from an individual or from a repository or other storage facility. Cells as a therapy may be autologous or allogeneic to the individual to whom the cells are provided as a therapy.

[0233] The cells may be derived from an individual in need of treatment for a medical condition and, after engineering to express the HLA-G targeted CAR, optional suicide gene, optional cytokine, and optional therapeutic gene product (e.g., using standard techniques for transduction and expansion for adoptive cell therapy), may be returned to the individual from whom they were originally sourced. In some cases, the cells are stored for later use in the individual or another individual.

[0234] The immune cells may be included within a population of cells, the population being predominantly transduced with one or more HLA-G targeted receptors and / or one or more suicide genes and / or one or more cytokines. The cell population may include 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of immune cells transduced with one or more HLA-G targeted receptors and / or one or more suicide genes and / or one or more cytokines. The one or more HLA-G targeted receptors and / or the one or more suicide genes and / or the one or more cytokines may be separate polypeptides.

[0235] Immune cells can be generated with one or more HLA-G targeted receptors and / or one or more suicide genes and / or one or more cytokines for the purpose of being modular with respect to a particular purpose. For example, cells expressing HLA-G targeted CARs and / or one or more suicide genes and / or one or more cytokines (or nucleic acids encoding variants are distributed for subsequent transduction) can be generated, for example, for commercial distribution, and users can modify the cells to express one or more other genes of interest (including therapeutic genes) depending on the intended purpose. For example, an individual interested in treating HLA-G positive cells, including HLA-G positive cancers, can obtain or generate suicide gene expressing cells (or heterologous cytokine expressing cells) and modify them to express receptors including HLA-G specific scFvs, or vice versa.

[0236] In certain embodiments, NK cells can be utilized to modify the genome of transduced NK cells that express one or more HLA-G targeting CARs and / or one or more suicide genes and / or one or more cytokines.Genome can be modified in any manner, but in certain embodiments, genome is modified by, for example, CRISPR gene editing.Genome of cell can be modified to enhance the effectiveness of cell for any purpose. VII. Gene editing of HLA-G specific CAR cells

[0237] In certain embodiments, at least the cell that comprises the engineered HLA-G specific receptor is genetically edited to modify the expression of one or more endogenous genes in the cell.In certain cases, the HLA-G specific CAR cell is modified to have a reduced expression level of one or more endogenous genes, including the inhibition (which may be referred to as knockout) of the expression of one or more endogenous genes.Such cells may or may not be expanded.

[0238] In certain cases, one or more endogenous genes of the HLA-G-specific CAR cells are modified, e.g., expression is disrupted, expression is partially or completely reduced. In certain cases, one or more genes are knocked down or knocked out using the process of the present disclosure. In certain cases, multiple genes are knocked down or knocked out, which may or may not occur at the same step in their generation. The gene edited in the HLA-G-specific CAR cells can be of any type, but in certain embodiments, the gene is a gene whose gene product inhibits the activity and / or proliferation of the HLA-G-specific CAR cells (one example is HLA-G-specific CAR NK cells, such as those derived from umbilical cord blood). In certain cases, the gene edited in the HLA-G-specific CAR cells allows the HLA-G-specific CAR cells to act more effectively in the tumor microenvironment. In certain cases, the genes are one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and CD38. In certain embodiments, the TGFBR2, CISH, and / or CD38 genes are knocked out or knocked down in the HLA-G-specific CAR cells.

[0239] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonucleases (RGENs). For example, modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In some embodiments, CpF1 is utilized instead of Cas9. In general, "CRISPR system" refers to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences ("direct repeats", encompassing tracrRNA-processed portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci.

[0240] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner, and a Cas protein (e.g., Cas9) that has nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0241] In some embodiments, Cas nuclease and gRNA (including a fusion of a crRNA specific for a target sequence and a fixed tracrRNA) are introduced into cells. In general, a target site at the 5' end of the gRNA targets the Cas nuclease to a target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems feature elements that promote the formation of a CRISPR complex at the site of the target sequence. Typically, a "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.

[0242] The CRISPR system can induce a double-strand break (DSB) at the target site, followed by destruction or modification, as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each guided by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced when a nick is introduced simultaneously. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0243] The target sequence may comprise any polynucleotide, for example DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, for example in an organelle of a cell. In general, the sequence or template that can be used for recombination into the target locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, the exogenous template polynucleotide may be referred to as editing template. In some embodiments, the recombination is homologous recombination.

[0244] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). Also, the tracr sequence may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence), but may form part of a CRISPR complex, for example, by hybridization with all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (such as at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned) to hybridize and participate in the formation of a CRISPR complex.

[0245] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell, such that the expression of the elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Also, the components can be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0246] The vector may include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, such as a Cas protein, non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, and the like. , Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, Cpf1 (Cas12a), their homologs, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0247] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some cases, Cpf1 (Cas12a) may be used as an endonuclease instead of Cas9. The CRISPR enzyme may direct the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). In some embodiments, Cas9 nickases can be used in combination with guide sequences, such as two guide sequences, that target the sense and antisense strands of a DNA target, respectively, allowing both strands to be nicked and used to induce NHEJ or HDR.

[0248] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species show a particular bias for a particular codon of a particular amino acid. Codon bias (the difference in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon to be translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0249] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of CRISPR complex to the target sequence.In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0250] Optimal alignment can be determined by use of any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0251] CRISPR enzyme can be part of a fusion protein that contains one or more heterologous protein domains. CRISPR enzyme fusion protein can contain any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences that code for proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins that contain CRISPR enzymes are described in US Patent Publication No. 2011 / 0059502, which is incorporated herein by reference. VIII. Treatment Methods

[0252] In various embodiments, diseased cells or other cells expressing endogenous HLA-G on their surface are targeted to improve a medical condition in an individual with a medical condition, or to reduce the risk of a medical condition in an individual, or to delay the severity and / or onset.In certain cases, cancer cells expressing endogenous HLA-G are targeted to kill cancer cells.

[0253] The HLA-G targeted CAR constructs, nucleic acid sequences, vectors, immune cells, etc. contemplated herein, and / or pharmaceutical compositions comprising them, are used to prevent, treat, or ameliorate cancerous diseases, such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the present disclosure may be particularly useful for preventing, ameliorating, and / or treating cancers, including, for example, cancers that express HLA-G and may or may not be solid tumors.

[0254] The immune cells in which the HLA-G targeted receptor is utilized may be, in certain embodiments, NK cells, T cells, gamma delta T cells, alpha beta T cells, or NKT or invariant NKT (iNKT), or invariant NKT cells engineered for cell therapy for mammals. When the cells are NK cells, the NK cell therapy may be of any type, and the NK cells may be of any type. In certain embodiments, the cells are NK cells engineered to express one or more HLA-G targeted CARs and / or one or more suicide genes and / or one or more cytokines. In certain embodiments, the cells are NK cells transduced with HLA-G targeted CARs.

[0255] In certain embodiments, the present disclosure contemplates, in part, HLA-G CAR expressing cells, HLA-G targeted CAR constructs, HLA-G targeted CAR nucleic acid molecules, and HLA-G targeted CAR vectors that can be administered alone or in any combination using standard vectors and / or gene delivery systems, and in at least some embodiments, with a pharma- ceutically acceptable carrier or excipient. In certain embodiments, after administration, the nucleic acid molecule or vector can be stably integrated into the subject's genome.

[0256] In certain embodiments, viral vectors can be used that are specific to certain cells or tissues and persist in NK cells.Suitable pharmaceutical carriers and excipients are well known in the art.The composition prepared according to the present disclosure can be used for the prevention or treatment or delay of the above-mentioned specified diseases.

[0257] Furthermore, the present disclosure relates to a method for the prevention, treatment, or amelioration of a neoplastic disease, comprising administering to a subject in need thereof an effective amount of cells expressing an HLA-G-targeted CAR, nucleic acid sequence, vector as contemplated herein and / or produced by a process as contemplated herein.

[0258] Exemplary possible indications for administration of compositions of HLA-G targeted CAR cells are cancerous diseases, including, for example, tumor diseases, including B-cell malignancies, multiple myeloma, breast cancer, glioblastoma, renal cancer, pancreatic cancer, or lung cancer. Exemplary indications for administration of compositions of HLA-G targeted CAR cells are cancerous diseases, including any malignant tumor expressing HLA-G. Administration of compositions of the present disclosure is useful for all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0259] The present disclosure further includes co-administration protocols with other compounds, such as bispecific antibody constructs, targeted toxins, or other compounds that act through immune cells.The clinical regimen for co-administration of the compounds of the present invention may include co-administration at the same time, before, or after administration of other components.Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0260] The embodiments relate to kits comprising an HLA-G targeted CAR construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and / or a host cell (such as an immune cell) as defined herein. It is also contemplated that the kits of the present disclosure comprise a pharmaceutical composition as described herein above, alone or in combination with additional agents to be administered to an individual in need of medical treatment or intervention. A. Pharmaceutical Compositions

[0261] Also provided herein are pharmaceutical compositions and formulations comprising the transduced NK cells and a pharma- ceutically acceptable carrier. The transduced cells may be contained in a medium suitable for transfer into an individual and / or a medium suitable for storage, such as cryopreservation, including prior to transfer into an individual.

[0262] The pharmaceutical compositions and formulations described herein comprise an active ingredient (such as cells) having a desired purity in the form of a lyophilized formulation or an aqueous solution, optionally in one or more pharma- ceutical acceptable carriers (see Remington's Pharmaceutical Sciences 22). ndPharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) soluble ... (less than 1000) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Therapy

[0263] In certain embodiments, the compositions and methods of the present embodiment include immune cell populations (including NK cell populations) combined with at least one additional therapy.Additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, oncolytic virus, or combinations of the above.Additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0264] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic agent, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents known in the art.

[0265] In certain embodiments, in addition to the inventive cell therapy of the present disclosure, the individual may have been, is being, and / or will be receiving certain additional therapies for cancer, including one or more of surgery, radiation, immunotherapy (other than the cell therapy of the present disclosure), hormone therapy, gene therapy, chemotherapy, and the like.

[0266] Immune cell therapy may be administered before, during, after, or in various combinations with additional cancer therapy. Administration may be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments where immune cell therapy is administered to a patient separately from additional therapeutic agents, one skilled in the art would generally ensure that a significant period does not lapse between each delivery time so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is believed that the antibody therapy and the anti-cancer therapy may be administered to a patient within about 12 to 24 or 72 hours of each other, more specifically within about 6 to 12 hours of each other. In some situations, it may be desirable to extend the treatment period significantly when several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0267] Various combinations can be used. In the following examples, the immune cell therapy is "A" and the anti-cancer therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0268] Administration of any compound or cell therapy of the present embodiments to a patient will follow standard protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from combination therapy. 1.Chemotherapy

[0269] A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. The agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0270] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine); acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 2). liptophysin 8; dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II (calicheamicin gammaII and calicheamicin omegaII); dynemicins (including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarmicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxo rubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, promycin, and folic acid analogs. teropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenals such as mitotane and trilo Stans; folic acid supplements, such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;Nitraerine; pentostatin; phenameth; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; Arabinosides ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the foregoing. 2. Radiation therapy

[0271] Other agents that cause DNA damage and have been widely used include gamma radiation, X-rays, and / or what is commonly known as the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. 3. Immunotherapy

[0272] Those skilled in the art will understand that additional immunotherapy of CAR can be used alone or in conjunction with the method of the embodiment, or in combination. Any antibody encompassed herein can be used for therapeutic molecules other than CAR, including bispecific or multispecific antibodies. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells or agents and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. Immune effectors can be, for example, antibodies specific for some markers on the surface of tumor cells. The antibody alone can function as an effector of therapy, or it can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to function as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0273] Antibody-drug conjugates have emerged as a revolutionary approach to the development of cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells enriched with antigen levels. Targeted delivery of the drug also minimizes exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The FDA's approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has validated the approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become more and more mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are up-regulation / high levels of expression in tumor cells and robust internalization.

[0274] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. There are many tumor markers, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0275] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945; and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0276] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints make signals (e.g., costimulatory molecules) stronger or weaker. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0277] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or an antibody, such as a human antibody (e.g., WO 2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies. Alternative and / or equivalent names known to those skilled in the art may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0278] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain embodiments, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Publication No. 2014 / 0294898, U.S. Patent Application Publication No. 2014 / 022021, and U.S. Patent Application Publication No. 2011 / 0008369, all of which are incorporated herein by reference.

[0279] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0280] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0281] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0282] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, any art-recognized anti-CTLA-4 antibody can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156; Hurwit Zeta l. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 may be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference.Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used.For example, humanized CTLA-4 antibodies are described in WO 2001 / 014424, WO 2000 / 037504, and U.S. Patent No. 8,017,114 (all of which are incorporated herein by reference).

[0283] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0284] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. No. 5,844,905, U.S. Pat. No. 5,885,796, and WO 1995 / 001994 and WO 1998 / 042752 (all of which are incorporated by reference herein), and immunoadhesins such as those described in U.S. Pat. No. 8,329,867 (which is incorporated by reference herein). 4.Surgery

[0285] Approximately 60% of people with cancer will undergo some kind of surgery, including preventive, diagnostic, or staging, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy. Tumor resection refers to the physical removal of at least a part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0286] When part or all of the cancerous cells, tissue, or tumor is removed, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application of the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also vary in dosage. 5. Other drugs

[0287] It is believed that other agents can be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are believed to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. In addition, it is believed that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. IX. Proteins

[0288] As used herein, a "protein" or "polypeptide" refers to a molecule that comprises at least three amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used. However, in many embodiments of the present disclosure, modified proteins or polypeptides are used to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function, but may otherwise retain a wild type activity or function, such as immunogenicity.

[0289] When a protein is specifically mentioned herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or a protein that, optionally, has any signal sequence removed. A protein may be directly isolated from a natural organism, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence that encodes a polypeptide (e.g., an antibody or fragment thereof). The term "recombinant" may be used with a polypeptide, or with the name of a particular polypeptide, generally referring to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is a product of replication of such a molecule.

[0290] In certain embodiments, the size of a protein or polypeptide (wild-type or modified) may include, but is not limited to, the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, shortened from their wild-type counterparts, and modified by fusing or attaching heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, to enhance immunogenicity, for purification purposes, etc.) As used herein, the term "domain" refers to any discrete functional or structural unit of a protein or polypeptide, generally a sequence of amino acids having a structure or function recognizable to one of skill in the art.

[0291] A polypeptide, protein, or a polynucleotide encoding such a polypeptide or protein of the present disclosure may include: or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more (or an inducible range thereof) mutant amino acid or nucleic acid substitutions with any one of SEQ ID NOs: 1 to 81; or is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or a range derivable therein) similar, identical, or homologous to Up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids or derived ranges thereof.

[0292] In some embodiments, the protein, polypeptide, or polynucleotide may include: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range) consecutive amino acids or nucleotides.

[0293] In some embodiments, a polypeptide, protein, or polynucleotide may include at least, at most, or exactly the following: Any one of SEQ ID NOs: 1 to 81 in the sequence listing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range thereof) contiguous amino acids or nucleotides which are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, respectively, of SEQ ID NOs: 1-81,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or derivable range) similar, identical, or homologous to any one of SEQ ID NOs: S:1-81.

[0294] In one embodiment, a nucleic acid molecule or polypeptide starting at the following position in any one of SEQ ID NOs: 1 to 49: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000, and at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 (or a derivable range) consecutive amino acids or nucleotides.

[0295] Nucleotide and protein, polypeptide and peptide sequences of various genes have been disclosed previously and can be found in known computerized databases.Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (ncbi.nlm.nih.gov / on the World Wide Web) and The Universal Protein Resource (UniProt; uniprot.org on the World Wide Web).The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or known to those skilled in the art.

[0296] It is contemplated that in the compositions of the present disclosure, there is about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition is about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein). X. Kits of the Present Disclosure

[0297] Any of the compositions described herein may be included in the kit. In a non-limiting example, cells, reagents for generating cells, vectors, and reagents for generating vectors and / or components thereof may be included in the kit. In certain embodiments, NK cells may be included in the kit, which may or may not express HLA-G targeting receptors, optional cytokines, or optional suicide genes. Such kits may or may not have one or more reagents for engineering cells. Such reagents include, for example, small molecules, proteins, nucleic acids, antibodies, buffers, primers, nucleotides, salts, and / or combinations thereof. Nucleotides encoding one or more HLA-G targeting CARs, suicide gene products, and / or cytokines may be included in the kit. Proteins such as cytokines or antibodies, including monoclonal antibodies, may be included in the kit. Nucleotides encoding components of engineered CAR receptors may be included in the kit, including reagents for generating said components.

[0298] In certain embodiments, the kit also includes the NK cell therapy of the present disclosure and another cancer treatment. In some cases, the kit also includes a second cancer treatment, such as, for example, chemotherapy, hormone therapy, and / or immunotherapy, in addition to the cell therapy embodiment. The kit can be tailored to the specific cancer of the individual and can include the respective second cancer treatment for the individual.

[0299] The kit may include a properly aliquoted composition of the present disclosure. The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and preferably properly aliquoted. If there are multiple components in the kit, the kit may also generally contain a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The kit of the present invention also typically includes a means for containing the composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held. EXAMPLES

[0300] XI. Working Examples The following examples are included to demonstrate specific embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that work well in the practice of the present invention and can therefore be considered to constitute specific modes for its implementation. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Example 1 HLA-G-targeted chimeric antigen receptor

[0301] In addition to the production of the cytokine IL-15, expression constructs were generated for the production of HLA-G targeted chimeric antigen receptors (CARs). [Table 1]

[0302] Cord blood-derived natural killer (CB-NK) cells were transduced with the different constructs to confirm CAR expression (Figure 2). CAR activity was tested against the HLA-G positive AML cell line OCI-AML13. Intracellular cytokine staining for TNFα, IFNγ, and CD107a (Figure 3) and 51 Activity was confirmed by Cr release assay (Figure 4).

[0303] In Figure 5, HEK293T cells were transfected with two HLA-G CAR constructs, MG and GG, derived from MEM-G / 11 and 87G clone monoclonal antibodies against human HLA-G, respectively. Both constructs showed positive expression on HEK293T membranes when stained with Alexa-Fluor 647 affinity purified F(ab')2 fragment goat anti-human IgG (H+L) antibody. Cord blood-derived NK cells were transduced with HLA-G CAR constructs (MG) (two experiments - MG1 and MG22) or not transduced (NT). Viral transduction efficiency was checked via staining of the IgG hinge region with Alexa-Fluor 647 affinity purified F(ab')2 fragment goat anti-human IgG (H+L) antibody. Both showed positive staining compared to NT (Figure 6). In Figure 7, HLA-G CAR NK cells and non-transduced (NT) NK cells were co-cultured with OCI-AML3 cells at a ratio of 1:1 (E:T) for 22 hours and imaged by an Incucyte system. Figure 7 provides a graph depicting the decrease in mean fluorescence intensity of tumor targets over time. The top line is untransduced cells; the MG1 line is the bottom line at the end of the period. HLA-G CAR NK cells kill more efficiently compared to NT-NK cells. In Figures 8A and 8B, HLA-G CAR (MG clone)-expressing NK cells and non-transduced (NT) NK cells were co-cultured with GSC20 spheroids at a ratio of 4:1 (E:T) for 73 hours and imaged by an Incucyte system. Figure 8A provides a graph depicting the decrease in mean fluorescence intensity of GSC20 spheroids expressing mCherry over time co-cultured with HLA-G CAR-transduced CB-NK cells (MG1 and MG2-2 repeats) compared to NT-NK cells. The line for CB31 NT (4:1) is the top line. Figure 8B provides representative images of GSC20 spheroids co-cultured with NT-NK and HLA-G CAR constructs. HLA-G CAR construct (MG)-expressing NK cells and non-transduced (NT) NK cells were co-cultured with OCI-AML3 cells or GSC-20 cells at 1:1 and 2:1 (E:T) ratios for 4 h and stained for CD56, CD3, and TNF-α.Cells carrying the MG construct show significant secretion of TNF-α compared to NT.

[0304] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

**Claim 1** A polynucleotide encoding an anti-HLA-G chimeric antigen receptor (CAR), wherein the CAR comprises (a) the extracellular domain of KIR2DL4 or the anti-HLA-G antigen-binding region of an HLA-G-specific antibody, (b) a transmembrane domain, and (c) an intracellular domain other than the intracellular domain of KIR2DL4. **Claim 2** The polynucleotide according to claim 1, wherein the CAR comprises the anti-HLA-G antigen-binding region of an HLA-G-specific antibody. **Claim 3** The CAR comprises (a) a CD8 signal peptide, the extracellular domain of KIR2DL4, the transmembrane domain of CD28, and the intracellular domain of CD3 zeta; or (b) a CD8 signal peptide, a codon-optimized extracellular domain of KIR2DL4, the transmembrane domain of CD8, and the intracellular domain of CD3 zeta. The polynucleotide according to any one of claims 1 to 2. **Claim 4** The polynucleotide according to any one of claims 1 to 3, further encoding an additional polypeptide of interest. **Claim 5** The polynucleotide according to claim 4, wherein the additional polypeptide of interest is a therapeutic protein or a protein that enhances the activity, expansion, and / or persistence of cells. **Claim 6** The polynucleotide according to any one of claims 4 to 5, wherein the additional polypeptide of interest is a suicide gene, cytokine, or human or viral protein that enhances proliferation, expansion, and / or metabolic fitness. **Claim 7** The polynucleotide according to any one of claims 4 to 6, wherein the additional polypeptide of interest is a cytokine. **Claim 8** The polynucleotide according to claim 7, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7. **Claim 9** The polynucleotide according to claim 8, wherein the cytokine is IL-15. **Claim 10** A vector comprising the polynucleotide according to any one of claims 1 to 9. **Claim 11** The vector according to claim 10, which is a viral vector. **Claim 12** An immune cell comprising the polynucleotide according to any one of claims 1 to 9 or the vector according to any one of claims 10 to 11. **Claim 13** The immune cell according to claim 12, wherein the immune cell is a natural killer (NK) cell, a T cell, a gamma delta T cell, an alpha beta T cell, an invariant NK T (iNKT) cell, a B cell, a macrophage, a mesenchymal stromal cell, or a dendritic cell.

14. The immune cell according to claim 13, which is an NK cell.

15. The immune cell according to claim 14, wherein the NK cell is derived from umbilical cord blood mononuclear cells.

16. The immune cell according to any one of claims 14 to 15, wherein the NK cell expresses a recombinant cytokine.

17. The immune cell according to claim 16, wherein the cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or IL-23.

18. The immune cell according to claim 17, wherein the cytokine is IL-15.

19. A population of immune cells comprising the immune cell according to any one of claims 14 to 18.

20. A composition for killing HLA-G positive cells in an individual, the composition comprising cells having the polynucleotide according to any one of claims 1 to 9.

21. The composition according to claim 20, wherein the cells having the polynucleotide are immune cells.

22. The composition according to claim 21, wherein the immune cell is an NK cell, a T cell, a gamma delta T cell, an alpha beta T cell, an invariant NK T (iNKT) cell, a B cell, a macrophage, a dendritic cell, or a mixture thereof.

23. The composition according to claim 22, wherein the immune cell comprises an NK cell, and the NK cell is derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, hematopoietic stem cells, bone marrow, cell lines, or a mixture thereof.

24. The composition according to any one of claims 20 to 23, wherein the individual has cancer.

25. The composition according to claim 24, wherein the individual has breast cancer, acute myeloid leukemia, multiple myeloma, or glioblastoma.

26. A polynucleotide composition comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO:

80.

27. A polypeptide composition comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO:

81.

28. An antibody-drug conjugate in which the antibody portion is encoded by SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67, or SEQ ID NO:

73.

29. An antibody-drug conjugate in which the antibody portion comprises a sequence comprising one or more of SEQ ID NOs: 50-54, 56-60, 62-66, and 68-72.

30. The antibody-drug conjugate according to claim 28 or 29, wherein the antibody is conjugated to a toxin, a chemotherapeutic agent, a radionuclide, a small molecule, and / or a ricin A chain.

31. The antibody-drug conjugate according to claim 30, wherein the toxin is cholera toxin and / or pertussis toxin.

32. A bispecific or multispecific antibody comprising an HLA-G specific antibody.

33. The antibody according to claim 32, wherein the anti-HLA-G antigen-binding region of the HLA-G specific antibody comprises an scFv derived from an antibody clone selected from the group consisting of G233, 26-2H11, MEM-G / 1, MEM-G / 9, MEM-G / 11, MEM-G / 13, 1B8, 5E6H7, 1-2C3, 16G1, 5A6G7, 87G, and 3C / G4.

34. The antibody according to any one of claims 32 to 33, comprising a CD3 specific antibody.

35. A cell comprising the antibody-drug conjugate according to any one of claims 28 to 31 and / or the antibody according to any one of claims 32 to 34.

36. The cell according to claim 35, wherein the cell is an immune effector cell.

37. The immune effector cell according to claim 36, wherein the immune effector cell is an NK cell, an NK T cell, an invariant NK T cell, a gamma delta T cell, an alpha beta T cell, a regulatory T cell, a B cell, a macrophage, a mesenchymal stromal cell (MSC), a dendritic cell, or a mixture thereof.

38. The cell according to claim 36 or 37, contained in a pharmaceutically acceptable excipient.

39. A composition for treating or preventing cancer in an individual, the composition comprising the cell according to any one of claims 36 to 38.

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