CD27-extracellular domain CAR targeting CD70-positive tumors

Abstract

Provided are methods and compositions that utilize anti-CD70 chimeric antigen receptors (CARs) that lack an antibody or antibody fragment as part of the receptor that includes a portion of the antigen binding region of the CAR. [0003] Embodiments of the present disclosure encompass methods and compositions that utilize anti-CD70 chimeric antigen receptors (CARs) that lack an antibody or antibody fragment as part of the receptor, including a portion of the antigen binding region of the CAR. In certain embodiments, instead of the CAR utilizing an antibody that binds to CD70, the CAR molecule utilizes CD27, which is the receptor for the ligand CD70. In specific embodiments, the CAR is composed of a truncated version of CD27 rather than full-length CD27. Specifically, the CAR lacks the CD27 transmembrane domain.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 141,016, filed January 25, 2021, and U.S. Provisional Application No. 63 / 270,414, filed October 21, 2021, both of which are incorporated by reference in their entireties herein.

[0002] (Sequence Listing) This application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 18, 2022, is named "MDAC.P1261WO_ST25 SL.txt" and is 209,719 bytes in size.

[0003] Embodiments of the present disclosure include the fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine. [Background technology]

[0004] Adoptive cancer immunotherapy through genetic reprogramming of natural killer (NK) cells has clinically relevant applications and advantages, including 1) innate antitumor surveillance without the need for prior sensitization, 2) allogeneic effects 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, calibration, 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 reprogrammable. Genetic modifications that direct the effector function of NK cells are an effective way to harness their cytotoxic potential to kill tumor cells.

[0005] CD70, a ligand for the cytokine receptor CD27, is expressed in hematological malignancies such as acute myeloid leukemia (AML) and lymphoma, as well as many solid cancers, including kidney cancer, bladder cancer, lung cancer, triple-negative breast cancer, renal cell carcinoma, pancreatic cancer, and melanoma, and is only transiently found on activated T lymphocytes, B lymphocytes, and dendritic cells, making it an attractive "pan-cancer antigen." Unlike other AML targets, CD70 is not expressed on normal hematopoietic stem cells and is therefore particularly advantageous as a target for immunotherapy of AML, as it is unlikely that recipients will experience prolonged cytoreduction and require hematopoietic stem cell transplantation following CAR therapy, in contrast to most current clinical CAR-T therapies for AML.The present disclosure provides a solution to a long-felt need in the art of cancer therapy for CD70-positive cancers. Summary of the Invention

[0006] The present disclosure is directed to methods and compositions for the treatment or prevention of cancers with CD70-expressing cells. The present disclosure relates to certain anti-CD70 chimeric antigen receptors (CARs) and their use for targeting any type of CD70-positive cancer. In certain embodiments, the antigen-binding domain that targets CD70, including when specifically binding CD70, is not an antibody. In certain embodiments, the anti-CD70 CAR is devoid of any type of antibody, including scFv, that binds to CD70. In certain embodiments, the extracellular domain of the anti-CD70 CAR does not include scFv or other types of antibodies.

[0007] In certain embodiments, the antigen binding domain that targets CD70 is a natural receptor for CD70, such as the receptor CD27. In certain aspects, a part or all of CD27 is employed in the CAR molecule. In an aspect of the present disclosure, the antigen binding domain present in the anti-CD70 CAR molecule consists of a part or all of the extracellular domain of CD27, and in certain aspects, the CAR molecule utilizes or does not utilize the transmembrane domain of CD27. In some cases, the CD28 transmembrane domain is employed instead of the CD27 transmembrane domain, even though the CD27 extracellular domain is used in the CAR. A specific intracellular domain may be employed in the CAR, such as CD3zeta alone or a combination of CD3zeta and DAP10 or DAP12, and in some cases, the intracellular domain does not include that of 4-1BB.

[0008] The embodiments of the present disclosure include any polynucleotide encoding part or all of an anti-CD70 CAR. Specific embodiments encompass polynucleotides encoding an anti-CD70 CAR, including an antigen-binding domain, a transmembrane domain, and at least one intracellular domain (including one or more costimulatory domains), where the CAR comprises an antigen-binding domain that does not include an antibody. Vectors carrying expression constructs encoding anti-CD70 CARs, and optionally other genes, are contemplated. The vectors may or may not be viral. Cells carrying the vectors are also contemplated, including any type of immune cell, such as at least NK cells and T cells. In some cases, the cells are commercially available cells, including NK cells, which may or may not have already been engineered to express one or more heterologous genes, such as one or more exogenous cytokines.

[0009] Methods of treating or preventing any type of cancer are encompassed herein, including by administering cells expressing a particular anti-CD70 CAR in a therapeutically effective amount to ameliorate or prevent cancer, or to reduce the risk of cancer, reduce the severity of cancer, prevent or the risk of metastasis, or delay the onset of cancer.

[0010] Embodiments of the present disclosure include methods of targeting CD70-expressing cells, and in certain embodiments, the cells are cancerous, while in other methods, the CD70-expressing cells are immune regulatory cells such as Tregs.

[0011] The present disclosure includes methods for engineering human NK cells (including those derived from umbilical cord blood (CB)) to target CD70+ve tumors with CD27-ectodomain CARs. Encompassed herein are various novel CAR constructs that fuse the extracellular binding portion of CD27 (CD27eCAR, or CD27 EC) with an activating signaling endodomain, either alone or in combination with costimulatory or adaptor signaling domains such as 4-1BB, CD28, DAP10, or DAP12, cytokine genes such as IL15, IL-12, IL-18, or IL-21, to support NK cell survival and proliferation. A series of in vitro studies have confirmed the activity of CD27eCAR / IL-15-transduced CB-NK cells against AML and lymphoma targets.

[0012] Embodiments of the present disclosure include polynucleotides encoding anti-CD70 chimeric antigen receptors (CARs) comprising a signal peptide, an anti-CD70 antigen binding domain, a transmembrane domain, and at least one intracellular domain, where the anti-CD70 antigen binding domain does not comprise an antibody, and optionally the transmembrane domain of the CAR is not a CD27 transmembrane domain. In specific embodiments, the anti-CD70 antigen binding domain may be configured and / or codon optimized in the extracellular domain of CD27. In certain embodiments, the antigen binding domain comprises, consists of, or consists essentially of SEQ ID NO:2. In some embodiments, the antigen binding domain and the transmembrane domain comprise, consist of, or consist essentially of SEQ ID NO:1. The sequence encoding the transmembrane domain may be codon-optimized, and / or the transmembrane domain is any of CD28, alpha chain of the T cell receptor, beta chain of the T cell receptor, 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, or DAP12. In certain embodiments, the CD28 transmembrane domain comprises, consists of, or consists essentially of SEQ ID NO:3. The CAR may comprise two or more, or three or more intracellular domains. The intracellular domain may comprise any ITAM-containing signaling domain. The intracellular domain may comprise CD3zeta. The intracellular domain may be an intracellular costimulatory domain, such as one selected from the group consisting of CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, NKp80, and combinations thereof.

[0013] In some embodiments, the signal peptide is from CD27 or granulocyte macrophage colony stimulating factor receptor (GMSCF-R), and the sequence of the CD27 signal peptide comprises, consists of, or consists essentially of SEQ ID NO:6. The signal peptide may be from CD27, the anti-CD70 antigen binding domain may be from CD27, the transmembrane domain may be from CD27, and the intracellular domain is from CD27. The signal peptide comprises, consists of, or consists essentially of SEQ ID NO:6. The CAR comprises, consists of, or consists essentially of SEQ ID NO:4. In some embodiments, the transmembrane domain comprises, consists of, or consists essentially of SEQ ID NO:3 or SEQ ID NO:7. In certain embodiments, the intracellular domain comprises one or more of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. The CAR comprises, consists of, or consists essentially of SEQ ID NO:5. The CAR comprises, consists of, or consists essentially of SEQ ID NO:1.

[0014] In certain embodiments, the polynucleotide encodes a CAR, wherein the CAR comprises one or more of the following: (a) CD27 signal peptide (SP), CD27 extracellular domain (EC), CD27 transmembrane domain (TMD), DAP12 intracellular domain (ICD), and CD3zeta; (b) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, DAP12 ICD; (c) CD27 SP, CD27 EC, CD28 TMD, DAP12 ICD, CD3zeta; (d) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, DAP12 ICD, and CD3zeta; (e) CD27 SP, CD27 EC, CD27 TMD, natural killer group 2 member D (NKG2D) ICD, and CD3zeta; (f) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, NKG2D ICD, and CD3zeta; (g) CD27 SP, CD27 EC, CD28 TMD, natural killer group 2 member D (NKG2D) ICD, and CD3zeta; (h) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, NKG2D ICD, and CD3zeta; (i) CD27 SP, CD27 EC, CD27 TMD, 4-1BB ICD, and CD3zeta; (j) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, 4-1BB ICD, and CD3zeta; (k) CD27 SP, CD27 EC, CD28 TMD, 4-1BB ICD, and CD3zeta; (l) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, 4-1BB ICD, and CD3zeta; (m) CD27 SP, CD27 EC, CD27 TMD, DAP10 ICD, and CD3zeta; (n) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, DAP10 ICD, and CD3zeta; (o) CD27 SP, CD27 EC, CD28 TMD, DAP10 ICD, and CD3zeta; (p)GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, DAP10 ICD, and CD3zeta; (q) CD27 SP, CD27 full length (FL), CD27 TMD, CD27 ICD, and CD3zeta; (r) SP of GMSCF-R, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, and CD3zeta; (s) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, CD28 ICD, and CD3zeta; (t) SP of GMSCF-R, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, CD28 ICD, and CD3zeta; (u) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, 4-1BB ICD, and CD3zeta; (v) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, 4-1BB ICD, and CD3zeta; (w) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD, and CD3zeta; (x) GMSCF-R SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD, and CD3zeta; (y) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP12 ICD, and CD3zeta; (z) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, DAP12 ICD, and CD3zeta; (aa) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, NKG2D ICD, and CD3zeta; (bb) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, NKG2D ICD, and CD3zeta; (cc) CD27 SP, CD27 EC, CD27 TMD, and CD3zeta; (dd) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, and CD3zeta; (ee) CD27 SP, CD27 EC, CD28 TMD, and CD3zeta; (ff) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD28 TMD, and CD3zeta; (gg) CD27 SP, CD27 EC, CD27 TMD, CD28 ICD, and CD3zeta; (hh) SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, CD28 ICD, and CD3zeta of GMSCF-R; (ii) CD27 SP, CD27 EC, CD28 TMD, CD28 ICD, and CD3zeta; or (jj) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, and CD3zeta.

[0015] The embodiments of the present disclosure include cells comprising any of the polynucleotides encompassed herein. The cells may or may not be immune cells. Examples of immune cells include natural killer (NK) cells, T cells, gamma delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, MSCs, or dendritic cells. When the cells are NK cells, they may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic stem cells (hematopoietic stem cells), cell lines, or mixtures thereof. The NK cell lines may be NK-92 cell lines, other NK cell lines derived from tumors or healthy NK cells, or progenitor cells. In specific embodiments, the NK cells are derived from umbilical cord blood mononuclear cells. The NK cells may be CD56+ NK cells. In some cases, the NK cells express one or more exogenously provided cytokines, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or combinations thereof.

[0016] An embodiment of the present disclosure includes a population of immune cells of the present disclosure, said cells being present in a suitable medium. Specifically, the immune cells are NK cells. The immune cells may or may not be obtained from cryopreservation.

[0017] In certain embodiments, there is a method of killing CD70 positive cells in an individual, comprising administering to the individual a therapeutically effective amount of cells carrying any polynucleotide encompassed herein. The cells may be NK cells, T cells, gamma delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, gamma delta T cells, or dendritic cells. The NK cells may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic stem cells, or cell lines. The NK cells may be derived from umbilical cord blood mononuclear cells. In some cases, the CD70 positive cells are not cancer cells, but rather T regulatory cells. The individual has or is suspected of having a cancer that expresses CD70. The individual has or is suspected of having, for example, acute myeloid leukemia, lymphoma, lung cancer, renal cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, pancreatic cancer, or a combination thereof. The cells may be allogeneic or autologous with respect to the individual. The individual may be a human. In some embodiments, the cells are 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.

[0018] In certain embodiments of the methods encompassed herein, the methods further comprise providing to the individual an effective amount of an additional therapy, such as surgery, radiation therapy, gene therapy, immunotherapy, and / or hormone therapy. In certain embodiments, the additional therapy comprises one or more antibodies.

[0019] In certain embodiments, the cells are administered to the individual by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intravesically, intracranially, percutaneously, subcutaneously, topically, perfusion, within the tumor microenvironment, or combinations thereof. In certain embodiments, the method further comprises identifying CD70 positive cells in the individual.

[0020] The foregoing has outlined, rather broadly, the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will be described below which form the subject matter of the claims herein. It should be understood by those skilled in the art that the ideas 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 defined by the appended claims. The novel features believed characteristic of the design disclosed herein, both as to 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 figures. It is to be expressly understood, however, that each figure is provided for the purpose of illustration and description only, and is not intended as a definition of the limits of the present disclosure.

[0021] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0022] [Figure 1] 1A-1B show CD70 expression on AML patient samples using Tsne plots.

[0023] [Diagram 2] Figures 2A-2E show various anti-CD70 CAR constructs.

[0024] The following table provides information about the truncated (tr) and full-length (fl) CD27 constructs encompassed herein and their respective reference numbers used in the data. S.No refers to the serial number. [Table 1] [Table 2]

[0025] For this table, information regarding the truncated (tr) and full length (fl) CD27 constructs and their respective identification numbers used in the figures is as follows:

[0026] GSP: GMCSF receptor signal peptide

[0027] co: codon optimization

[0028] CD28 tmd.CD28 transmembrane domain

[0029] CD27 tmd.CD27 transmembrane domain

[0030] 3z: CD3 zeta signal

[0031] CD28 ic.CD28 intracellular domain

[0032] Natural killer group 2 member D (NKG2D) ic.NKG2D intracellular domain

[0033] Dap12 icd.DAP12 intracellular domain

[0034] Dap10 icd.DAP12 intracellular domain

[0035] 41bb icd 41bb intracellular domain

[0036] [Diagram 3] FIG. 3 shows the transfection efficiency of truncated (tr) and full-length (fl) CD27 constructs in 293T cells.

[0037] [Figure 4] FIG. 4 shows the transduction efficiency of truncated (tr) and full-length (fl) CD27 constructs in cord blood-derived NK cells.

[0038] [Diagram 5]FIG. 5A reveals CD70 expression on the surface of tumor targets, CD70-positive Raji cells. FIG. 5B reveals CD70 expression on the surface of tumor targets, CD70-positive Karpas cells. FIG. 5C reveals the killing activity of cord blood natural killer (CB-NK) cells transduced with various trCD27 constructs against CD70-positive Raji cells and CD70-positive Karpas cells using a 4-hour Annexin V assay. FIG. 5C shows that various trCD70-transduced CB-NK cells kill both Raji cells (black bars) and Karpas cells (gray bars) better when compared to NT (non-transduced) CBNK cells, IL15 (construct with only IL15 cytokine gene) CAR-transduced CBNK cells, or CD70-IL15 (construct with CD70 scFv together with IL15) CAR-transduced CBNK cells.

[0039] [Figure 6] Figure 6 reveals CD107a degranulation (a marker for NK cell cytotoxic activity) by CB-NK cells transduced with different trCD27 constructs against Raji and Karpas cells. trCD27 CAR-transduced CBNK cells were co-cultured with cancer cells (Raji and Karpas) at a 1:1 ratio in the presence of CD107a antibody for 6 hours. After incubation, CD107a expression is assessed by flow cytometry. In a group of three bars, the left (green) bar is a control with CB-NK cells not co-cultured with cancer cells, the middle (black) bar is trCD70-transduced CB-NK cells co-cultured with Raji cells, and the right (grey) bar is trCD70-transduced CB-NK cells co-cultured with Karpas cells. Most of the trCD27-transduced CB-NK cells exhibited enhanced cytotoxic activity against Raji and Karpas cells when compared with NT CB-NK cells, IL15 CAR-transduced CB-NK cells, or CD70-IL15 CAR-transduced CB-NK cells.

[0040] [Figure 7] FIG. 7A shows the killing activity of CB-NK cells transduced with various trCD27 constructs against Raji cells using Incucyte live imaging. FIG. 7B shows the killing activity of CB-NK cells transduced with various trCD27 constructs against Karpas cells using Incucyte live imaging. trCD27 CAR-transduced CBNK cells were co-cultured with Raji or Karpas cells at a 1:1 ratio, and real-time cytotoxicity of NK cells against Raji and Karpas cells was measured every hour over a 12-hour period. trCD27-transduced CB-NK cells showed enhanced cytotoxic activity against Raji and Karpas cells when compared to NT CB-NK cells, IL15 CAR-transduced CB-NK cells, or CD70-IL15 CAR-transduced CB-NK cells. Cancer cells cultured in the absence of CBNK cells (cancer cells alone) were used as a control.

[0041] [Figure 8] Figure 8A shows the in vivo anti-tumor activity of CB-NK cells transduced with various trCD27 constructs against Raji cells with high CD70 expression. Figure 8B shows the in vivo anti-tumor activity of CB-NK cells transduced with various trCD27 constructs against Raji cells with high CD70 expression. Figure 8A shows bioluminescence imaging of Raji tumors after injection of CB-NK cells transduced with various trCD27 CARs. Figure 8B shows the survival curve after injection of CB-NK cells transduced with various trCD27 CARs.

[0042] [Figure 9]Figure 9A shows the in vivo antitumor activity of CB-NK cells transduced with various trCD27 constructs against THP-1 cells with high CD70 expression. Figure 9B shows the in vivo antitumor activity of CB-NK cells transduced with various trCD27 constructs against THP-1 cells with high CD70 expression. Figure 9A shows bioluminescence imaging of THP-1 tumors after injection of CB-NK cells transduced with various trCD27 CARs. Figure 9B shows the survival curve after injection of CB-NK cells transduced with various trCD27 CARs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Consistent with long-standing patent law practice, the words "a" and "an" mean "one or more" when used herein in conjunction with the word comprising, including in the claims. 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 may be implemented with respect to any other method or composition described herein, and that various embodiments may be combined.

[0044] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of the specified steps or elements or group of steps or elements, but not the exclusion of any other steps or elements or group of steps or elements. By "consisting of", it is meant to include and be limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that other elements may not be present. By "consisting essentially of", it is meant to include whatever elements are recited following this phrase, and to be limited to other elements that neither interfere with nor contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may be present or absent depending on whether they affect the activity or function of the recited elements.

[0045] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a certain embodiment," "an additional embodiment," "a further embodiment," or combinations thereof means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, such 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 components 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 intended that x, y, or z may be specifically excluded from an embodiment.

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

[0048] The term "engineered," as used herein, refers to entities that result from the hand of man, including cells, nucleic acids, polypeptides, and vectors. In at least some cases, engineered entities contain elements that are synthetic and do not occur in nature or are not constructed in the manner utilized in this disclosure. In specific embodiments, vectors are engineered by recombinant nucleic acid techniques and cells are engineered by transfection or transduction with the engineered vector.

[0049] As used herein, "prevent" and similar terms (e.g., "prevented," "preventing," etc.) refer to an approach to preventing a disease or condition (e.g., cancer) or inhibiting a disease or condition (e.g., cancer) or reducing the likelihood of the onset or recurrence of a disease or condition (e.g., cancer). Such approaches also refer 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.

[0050] The term "sample" as used herein generally refers to a biological sample. A sample may be obtained from tissue or cells from an individual. In some instances, a sample may include or be derived from tissue biopsies, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, discarded tissue. A sample may be isolated from a source prior to collection. Non-limiting examples include blood (including but not limited to umbilical cord blood), serum, plasma, cerebrospinal fluid, pleural fluid, amniotic fluid, lymphatic fluid, saliva, urine, stool, tears, sweat, bone marrow or mucosal discharge, and other bodily fluids isolated from a primary source prior to collection. In some instances, a sample is isolated from its primary source (cells, tissues, bodily fluids (e.g., blood, etc.), environmental samples, etc.) during sample preparation. A sample may or may not be purified or otherwise concentrated from its primary source. In some instances, a 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 enriched for nucleic acids or may be treated with RNase. The sample may contain intact or minced or partially degraded tissues or cells.

[0051] The term "subject" as used herein generally refers to an individual having a biological sample undergoing processing or analysis, and in particular, has or is suspected of having cancer. A subject can be any biological 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, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be a patient, such as a patient having or suspected of having a disease (which may be referred to as a medical condition) (e.g., benign or malignant neoplasms, i.e., cancer, etc.). A subject may be currently undergoing or have undergone treatment. A subject may be asymptomatic. A subject may be a healthy individual, but an individual who desires to prevent cancer. The term "individual" may be used interchangeably in at least some cases. A "subject" or "individual" as used herein may or may not be housed in a medical facility and may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may include humans or non-human animals of any age, and thus includes both adults and juveniles (i.e., children) and infants, as well as individuals in utero. The term is not intended to imply the need for medical treatment, and thus an individual may be a part of an experiment, whether clinical or in support of basic scientific research, either voluntarily or involuntarily.

[0052] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition (e.g., cancer) being treated. Treatment can involve either the reduction or amelioration of symptoms of the disease or condition, or the slowing of the progression of the disease or condition, as necessary. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition or its associated symptoms.

[0053] The present disclosure includes targeting CD70-expressing cancers using anti-CD70 CARs, and in some cases, the CARs are expressed on the surface of NK cells derived from umbilical cord blood. A number of novel CAR molecules are described herein, which are created by fusing the extracellular binding portion of CD27 (CD27eCAR) with a signaling endodomain for activation that incorporates CD3zeta, either alone or in combination with a costimulatory or adaptor signaling domain (e.g., 4-1BB, CD28, DAP10, or DAP12, etc.), and, in at least some cases, with a cytokine gene (e.g., IL-15, IL-12, IL-18, or IL-21, etc.). In some embodiments, the CAR is a fusion of the extracellular domain of CD27 (the natural ligand for CD70) with specificity for the human CD70 antigen. In addition, in some cases, codon optimization has been utilized to improve the surface expression of CD27 on the surface of transduced cells. In some cases, the vector containing the expression construct encoding the anti-CD70 CAR also carries cytokine genes, such as for the production of human interleukin 15 (IL-15), IL-12, IL-18 or IL-21, to aid in the survival and maintenance of cells that receive the vector, including, by way of example, NK cells.

[0054] I. Chimeric Antigen Receptors (CARs) The immune cells of the present disclosure may be engineered to express one or more antigen-binding receptors (such as engineered CARs) that target one or more antigens (e.g., CD70). For example, the immune cells may be immune cells that are modified to express a CAR with antigen specificity for CD70. Other CARs may be expressed by the same cell as the CD70 antigen receptor expressing cell, and may be directed to different antigens. In some aspects, the immune cells are engineered to express a CD70-specific CAR, for example, by knocking in the CAR using CRISPR / Cas technology.

[0055] Various suitable methods for modifying cells are known in the art. For example, see Sambrook and Ausubel (supra). For example, cells can be transduced to express CAR with antigen specificity for cancer antigens using the transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0056] In some embodiments, the cells contain one or more engineered nucleic acids encoding one or more antigen targeting receptors (at least one of which may be directed against CD70), and engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, such as a nucleic acid not normally found in the cell being manipulated and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature (e.g., a chimera).

[0057] The present disclosure encompasses CD70-specific CARs that include a) an extracellular domain that includes an antigen-binding region that targets CD70, including specifically binding to CD70; b) a transmembrane domain; and c) one or more intracellular domains, including an intracellular signaling domain. In some aspects, an antigen-specific binding or recognition component is linked to the transmembrane domain and one or more intracellular signaling domains. In some embodiments, a CAR includes a transmembrane domain that is fused to the extracellular domain of the CAR that includes the antigen-binding region or antigen-binding domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used, but in other cases, such a transmembrane domain is not used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other components of the receptor complex.

[0058] The present disclosure relates to anti-CD70 CARs that include an antigen-binding domain that does not use an antibody or an antibody fragment. In certain embodiments, instead of an anti-CD70 CAR that uses an antibody or an antibody fragment as an antigen-binding domain, the CAR uses a part or the whole of CD27 in the CAR, including using the extracellular domain of CD27 as the antigen-binding domain of the CAR in some cases.

[0059] For reference, the human (Homo sapiens) CD27 molecule (CD27) on chromosome 12 is provided by the National Center for Biotechnology Information (NCBI) GenBank® Accession No. NG_031995.1, which is incorporated herein by reference in its entirety. The nucleotide sequence encoding the entire CD27 protein comprises the following sequence:

[0060] (SEQ ID NO:9)

[0061] Any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO:9 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:9.

[0062] An example of a complete wild-type CD27 protein sequence is NCBI GenBank® Accession No. P26842 (which is also identical to the amino acid sequence in Accession No. NG_031995.1): 1 MARPHPWWLC VLGTLVGLSA TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAA 61 QCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA NAECACRNGW QCRDKECTEC 121 DPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM QTLADFRQLP ARTLSTHWPP 181 QRSLCSSDFI RILVIFSGMF LVFTLAGALF LHQRRKYRSN KGESPVEPAE PCHYSCPREE 241 EGSTIPIQED YRKPEPACSP

[0063] (SEQ ID NO:4)

[0064] In certain embodiments, a part or the whole of SEQ ID NO: 4 is used in the CAR. In certain embodiments, the full-length wild-type (or codon-optimized) CD27 protein is used in the CAR, while in other embodiments, a truncated version of the wild-type CD27 protein is used as the antigen-binding domain in the CAR. In a specific example, a part or the whole of the extracellular domain of CD27 is used, so that the truncated CD27 used as the antigen-binding domain is a truncated version of CD27 that is truncated at the C-terminus of the native protein.

[0065] The nucleotide sequence encoding the truncated form of the wild-type CD27 protein comprises the following sequence:

[0066] GCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGA TCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTC GCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAG ATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGC (SEQ ID NO: 63)

[0067] Any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO:63 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:63.

[0068] Polypeptide sequences encoding truncated forms of the wild-type CD27 protein include the following sequences:

[0069] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO: 5)

[0070] Any polypeptide encompassed by the present disclosure may utilize SEQ ID NO:5 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:5.

[0071] In the specific case where full-length CD27 protein is used in CAR molecule, no additional transmembrane domain is used.In the specific case where full-length CD27 protein is used in CAR molecule, in this case, full-length CD27 protein contains its own intracellular signaling domain, so additional intracellular signaling domain may or may not be used in CAR.In the specific case where full-length CD27 protein is used in CAR molecule, even though full-length CD27 protein contains its own signal peptide, additional signal peptide may or may not be used.In certain embodiments, CD3 zeta (and / or DAP10, DAP12, 2B4, CD28, NKG2D, 41BB, or any ITAM-containing signaling domain) is used in CAR.

[0072] In one example, the region of CD27 utilized as the antigen binding domain in the CAR molecule comprises, or consists of, or consists essentially of amino acids 1-211 of SEQ ID NO: 4 (including the TMD of CD27). In another example, the region of CD27 utilized as the antigen binding domain in the CAR molecule comprises, or consists of, or consists essentially of amino acids 1-191 of SEQ ID NO: 4 (lacking the TMD of CD27), or consists of amino acids 1-191 of SEQ ID NO: 4 (lacking the TMD of CD27), or consists essentially of amino acids 1-191 of SEQ ID NO: 4 (lacking the TMD of CD27). In specific examples, the region of CD27 utilized in the CAR molecule comprises, consists of, or consists essentially of 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 of SEQ ID NO:4. 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, 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, 1-115, 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, 1~136, 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 ,1~214,1~215,1~216,1~217,1~218,1~219,1~220,1~221,1~222,1~223,1~224,1~225,1~226,1~227,1~228,1~229,1~230,1~231,1~232,1~233,1~234,1~235,1~236,1~237,1~238,1~239,1~240,1 in specific embodiments, such amino acids in these ranges are contiguous. In some embodiments, a region of SEQ ID NO: 4 having an N-terminal truncation, for example, 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 from the N-terminus is utilized. In certain cases, 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 there is also a truncation at the C-terminus, such as at the truncated ends as shown in the list above.

[0073] In certain embodiments, any region of the CAR molecule is codon-optimized. In some embodiments, the antigen-binding domain comprises any of the illustrative CD27 sequences encompassed herein, but is codon-optimized. In specific cases, the antigen-binding domain comprises a C-terminal truncated CD27 (e.g., extracellular domain) and is also codon-optimized. In certain aspects, the antigen-binding domain comprises, or consists of, or consists essentially of amino acids 1-191 of CD27, and is also codon-optimized. In other aspects, the antigen-binding domain and the transmembrane domain comprise, or consist of, or consist essentially of amino acids 1-211 of CD27, and are also codon-optimized.

[0074] In some cases, specific components of CAR molecules are utilized. In certain embodiments, CARs contain specific transmembrane domains. Examples include transmembrane domains from CD28 or CD27. In some embodiments, the transmembrane domain of CD27 or the transmembrane domain of CD28 is not utilized in CARs, whereas in alternative embodiments, the transmembrane domain of CD27 is utilized in CARs. In certain embodiments, signal peptides are used for CARs, examples include the signal peptides of CD27 or GMCSF-R, or both may be used. Examples of intracellular signaling domains for CARs include at least CD27, CD28, DAP10, DAP12, NKG2D, 4-1BB, or combinations thereof.

[0075] In some cases, the signal peptide of CD27 is utilized in the CAR (MARPHPWWLCVLGTLVGLS (SEQ ID NO: 6); ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCA (SEQ ID NO: 61)), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 6 or SEQ ID NO: 61 is utilized. In some cases, the signal peptide of GMCSF-R is utilized in the CAR (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 14); ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 62)), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 14 or SEQ ID NO: 62 is utilized.

[0076] The CAR may or may not include the TM of CD27 (ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 7); ATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCAT (SEQ ID NO: 66)) or may or may not include a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 7 or SEQ ID NO: 66.

[0077] The CAR may or may not include the ICD of CD27, which is QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 8), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 8.

[0078] In some embodiments, the ICD of CD3 zeta is RVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKPRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 10), CGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATG AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAC (SEQ ID NO: 67), which sequence may be used in the CAR, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 10 or SEQ ID NO: 67 may be used.

[0079] In some embodiments, the ICD of CD28 (RSKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS; SEQ ID NO:11) is utilized in the CAR molecule, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:11 is utilized.

[0080] In some embodiments, the ICD of 4-1BB (KRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEEGGCEL (SEQ ID NO: 12); AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 68)) is utilized in the CAR molecule, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 12 or SEQ ID NO: 68 is utilized.

[0081] In some embodiments, the ICD of DAP10 (LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 72); CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 69)) is used in the CAR molecule, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 69 or SEQ ID NO: 72 is used.

[0082] In some embodiments, the ICD of DAP12 (YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 74); TACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAA (SEQ ID NO: 71)) is used in the CAR molecule, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 71 or SEQ ID NO: 74 is used.

[0083] In some embodiments, the ICD of NKG2D (SANERCKSKVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGM (SEQ ID NO: 73); AGCGCGAACGAACGCTGCAAAAGCAAAGTGGTGCCGTGCCGCCAGAAACAGTGGCGCACCAGCTTTGATAGCAAAAAACTGGATCTGAACTATAACCATTTTGAAAGCATGGAATGGAGCCATCGCAGCCGCCGCGGCCGCATTTGGGGCATG (SEQ ID NO: 70)) is used in the CAR molecule, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 70 or SEQ ID NO: 73 is used.

[0084] In certain embodiments, a codon-optimized CD27 is utilized, an example being as follows:

[0085] ATG GCG AGA CCA CAC CCT TGG TGG CTG TGC GTA CTC GGC ACA CTT GTA GGT CTG TCC GCT ACA CCG GCT CCG AAG TCC TGC CCG GAG CGG CAT TAT TGG GCA CAG GGC AAG TTG TGT TGT CAA ATG TGT GAG CCG GGA ACC TTT CTC GTG AAG GAT TGC GAT CAG CAT CGG AAG GCC GCG CAG TGC GAC CCA TGT ATA CCA GGG GTC TCA TTT TCC CCA GAT CAC CAT ACG AGG CCG CAC TGT GAG TCT TGC AGG CAT TGT AAT TCC GGC TTG TTG GTC CGC AAC TGT ACT ATT ACT GCG AAT GCA GAG TGT GCT TGT AGA AAC GGA TGG CAG TGC AGG GAC AAA GAA TGT ACG GAG TGT GAT CCA CTG CCT AAC CCC AGT CTT ACA GCA AGA TCT TCA CAG GCC CTC AGC CCG CAT CCT CAA CCA ACA CAT CTT CCT TAC GTG TCA GAA ATG TTG GAG GCG CGA ACC GCA GGC CAT ATG CAG ACC CTG GCG GAC TTT CGG CAG CTG CCA GCA CGC ACA CTT AGT ACA CAC TGG CCA CCA CAA CGC AGC TTG TGC TCT TCC GAT TTC ATC CGC ATA CTG GTC ATC TTT TCT GGA ATG TTC CTT GTG TTC ACC CTG GCA GGA GCC CTG TTC CTT CAC CAG AGA CGC AAG TAC AGG TCA AAC AAG GGT GAG AGC CCC GTT GAA CCC GCA GAG CCG TGT AGA TAC TCA TGT CCT AGA GAA GAA GAG GGC TCT ACT ATC CCT ATT CAG GAA GATTAT AGA AAA CCC GAA CCC GCG TGC AGC CCC (SEQ ID NO: 13)

[0086] Any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO:13 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:13.

[0087] In certain embodiments, a codon-optimized extracellular domain of CD27 is utilized, one example being as follows:

[0088] GCTACACCGGCTCCGAAGTCCTGCCCGGAGCGGCATTATTGGGCACAGGGCAAGTTGTGTTGTCAAATGTTGAGCCGGGAACCTTTCTCGTGAAGGATTGCGATCAGCATCGGAAGGCCGCGCAGTGCGA CCCATGTATACCAGGGGTCTCATTTTCCCCAGATCACCATACGAGGCCGCACTGTGAGTCTTGCAGGCATTGTAATTCCGGCTTGTTGGTCCGCAACTGTACTATTACTGCGAATGCAGAGTGTGCTTGTA GAAACGGATGGCAGTGCAGGGACAAAGAATGTACGGAGTGTGATCCACTGCCTAACCCCAGTCTTACAGCAAGATCTTCACAGGCCTCAGCCCGCATCCTCAACCAACACATCTTCCTTACGTGTCAGAA ATGTTGGAGGCGCGAACCGCAGGCCATATGCAGACCCTGGCGGACTTTCGGCAGCTGCCAGCACGCACACTTAGTACACACTGGCCACCACAACGCAGCTTGTGCTCTTCCGATTTCATCCGC (SEQ ID NO: 64)

[0089] Any polynucleotide encompassed by the present disclosure may utilize SEQ ID NO:64 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:64.

[0090] 2A-2E provide specific examples of CAR constructs that include certain components. These are specific examples where the CAR construct may include full-length CD27 (FL; all 260 amino acids). In alternative cases, when full-length CD27 is not utilized, the extracellular domain of CD27 (CD27 EC) may be utilized, which is residues 20-191 of CD27. In certain embodiments, when a truncated CD27 is utilized in the CAR, the intracellular costimulatory domain (ICD) of CD27 may not be utilized in the CAR, and / or the transmembrane domain (TMD) of CD27 may not be utilized in the CAR.

[0091] In FIG. 2A, example (1) of an anti-CD70 CAR includes a signal peptide (SP) of CD27, full-length CD27 (FL), TMD of CD27, ICD of CD27, and CD3 zeta. Example (2) of FIG. 2A includes a signal peptide of granulocyte-macrophage colony-stimulating factor receptor (GMSCF-R), codon-optimized (co) full-length CD27, codon-optimized transmembrane domain of CD27, codon-optimized ICD of CD27, and CD3 zeta. Example (3) of FIG. 2A includes a SP of CD27, full-length CD27, TMD of CD27, ICD of CD27, ICD of CD28, and CD3 zeta. Example (4) of FIG. 2A includes a SP of GMSCF-R, codon-optimized full-length CD27, codon-optimized TMD of full-length CD27, codon-optimized ICD of CD27, ICD of CD28, and CD3 zeta. Example (5) in Figure 2A includes the SP of CD27, full length CD27, the TMD of CD27, the ICD of CD27, the ICD of 4-1BB, and CD3 zeta. Example (6) includes the SP of GMSCF-R, the codon-optimized full length CD27, the codon-optimized TMD of CD27, the codon-optimized ICD of CD27, the ICD of 4-1BB, and CD3 zeta.

[0092] Example (7) in FIG. 2B includes the SP of CD27, full-length CD27, the TMD of CD27, the ICD of CD27, the ICD of DAP10, and CD3 zeta. Example (8) in FIG. 2B includes the SP of GMSCF-R, the codon-optimized full-length CD27, the codon-optimized TMD of CD27, the codon-optimized ICD of CD27, the ICD of DAP10, and CD3 zeta. Example (9) in FIG. 2B includes the SP of CD27, full-length CD27, the TMD of CD27, the ICD of CD27, the ICD of DAP12, and CD3 zeta. Example (10) in FIG. 2B includes the SP of GMSCF-R, the codon-optimized full-length CD27, the codon-optimized TMD of CD27, the codon-optimized ICD of CD27, the ICD of DAP12, and CD3 zeta. Example (11) in Figure 2B includes the SP of CD27, full length CD27, TMD of CD27, ICD of CD27, ICD of NKG2D, and CD3 zeta. Example (12) in Figure 2B includes the SP of GMSCF-R, codon-optimized full length CD27, codon-optimized TMD of CD27, codon-optimized ICD of CD27, ICD of NKG2D, and CD3 zeta.

[0093] Example (13) in FIG. 2C includes the SP of CD27, the extracellular domain (EC) of CD27, the TMD of CD27, and CD3 zeta. Example (14) in FIG. 2C includes the SP of GMSCF-R, the codon-optimized EC of CD27, the codon-optimized TMD, and CD3 zeta. Example (15) in FIG. 2C includes the SP of CD27, the EC of CD27, the TMD of CD28, and CD3 zeta. Example (16) in FIG. 2C includes the SP of GMSCF-R, the codon-optimized EC of CD27, the codon-optimized TMD of CD28, and CD3 zeta. Example (17) in FIG. 2C includes the SP of CD27, the EC of CD27, the TMD of CD27, the ICD of CD28, and CD3 zeta. Example (18) in Figure 2C includes the GMSCF-R SP, the codon-optimized CD27 EC, the codon-optimized CD27 TMD, the CD28 ICD, and CD3 zeta. Example (19) in Figure 2C includes the CD27 SP, the CD27 EC, the CD28 TMD, the CD28 ICD, and CD3 zeta. Example (20) in Figure 2C includes the GMSCF-R SP, the codon-optimized CD27 EC, the CD28 TMD, the CD28 ICD, and CD3 zeta.

[0094] Example (21) in FIG. 2D includes the CD27 SP, CD27 EC, CD27 TMD, 4-1BB ICD, and CD3 zeta. Example (22) in FIG. 2D includes the GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, 4-1BB ICD, and CD3 zeta. Example (23) in FIG. 2D includes the CD27 SP, CD27 EC, CD28 TMD, 4-1BB ICD, and CD3 zeta. Example (24) in FIG. 2D includes the GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, 4-1BB ICD, and CD3 zeta. Example (25) in FIG. 2D includes the CD27 SP, CD27 EC, CD27 TMD, DAP10 ICD, and CD3 zeta. Example (26) in Figure 2D includes the SP of GMSCF-R, the codon-optimized EC of CD27, the codon-optimized TMD of CD27, the ICD of DAP10, and CD3 zeta. Example (27) in Figure 2D includes the SP of CD27, the EC of CD27, the costimulatory domain of CD28, the ICD of DAP10, and CD3 zeta. Example (28) in Figure 2D includes the SP of GMSCF-R, the codon-optimized EC of CD27, the TMD of CD28, the ICD of DAP10, and CD3 zeta.

[0095] Example (29) in Figure 2E includes the SP of CD27, the EC of CD27, the TMD of CD27, the ICD of DAP12, and CD3 zeta. Example (30) in Figure 2E includes the SP of GMSCF-R, the codon-optimized EC of CD27, the codon-optimized TMD of CD27, the ICD of DAP12, and CD3 zeta. Example (31) in Figure 2E includes the SP of CD27, the EC of CD27, the TMD of CD28, the ICD of DAP12, and CD3 zeta. Example (32) in Figure 2E includes the SP of GMSCF-R, the codon-optimized EC of CD27, the TMD of CD28, the ICD of DAP12, and CD3 zeta. Example (33) in Figure 2E includes the SP of CD27, the EC of CD27, the TMD of CD27, the ICD of NKG2D, and CD3 zeta. Example (34) in Figure 2E includes the GMSCF-R SP, the codon-optimized CD27 EC, the CD28 TMD, the NKG2D ICD, and CD3 zeta. Example (35) in Figure 2E includes the CD27 SP, the CD27 EC, the CD28 TMD, the NKG2D ICD, and CD3 zeta. Example (36) in Figure 2E includes the GMSCF-R SP, the codon-optimized CD27 EC, the CD28 TMD, the NKG2D ICD, and CD3 zeta.

[0096] One example of an anti-CD70 CAR of the disclosure comprises SEQ ID NO:1 below, which comprises the extracellular domain of CD27 (non-underlined in SEQ ID NO:1 below) operably linked to the transmembrane domain of CD28 (underlined in SEQ ID NO:1 below) and the signal peptide of CD27 (bold in SEQ ID NO:1 below):

[0097] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 1)

[0098] The CD27 extracellular domain of SEQ ID NO:1 comprises the following sequence as SEQ ID NO:2: MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR

[0099] The CD28 transmembrane domain of SEQ ID NO:1 comprises the following sequence as SEQ ID NO:3: FWVLVVVGGVLACYSLLVTVAFIIFWV (TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO:65)).

[0100] The CD27 signal peptide of SEQ ID NO:1 comprises the following sequence as SEQ ID NO:6: MARPHPWWLCVLGTLVGLS (ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCA (SEQ ID NO:61)).

[0101] The present disclosure provides cells, including immune cells (e.g., NK cells, etc.), that receive a vector encoding at least one CAR, which may be, for example, first generation, second generation, or third or subsequent generation. A CAR may be bispecific for two or more different antigens, one of which is CD70, or may not be bispecific. In certain embodiments, a CAR is not a bivalent tandem CAR. A CAR may include one or more costimulatory domains. For example, each costimulatory domain may include one or more costimulatory domains, for example, of a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, any ITAM-containing signaling domain, or combinations thereof. In a specific embodiment, a CAR includes CD3 zeta. In certain embodiments, the CAR lacks one or more specific costimulatory domains, for example, the CAR may lack 4-1BB and / or may lack CD28.

[0102] In certain embodiments, the CAR polypeptide in the cell comprises an extracellular spacer domain that links the antigen binding domain and the transmembrane domain. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, the hinge of CD8-alpha, CD28, an artificial spacer made from a polypeptide (e.g., Gly3, etc.), or the CH1 domain, CH3 domain of IgGs (e.g., human IgG1 or IgG4, etc.). In particular cases, the extracellular spacer domain may comprise (i) the hinge, CH2 and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8-alpha, (v) the hinge, CH2 and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vii) the hinge and CH2 of IgG1, (viii) the hinge region of CD28, or a combination thereof.

[0103] In a specific embodiment, the hinge is derived from IgG1, and in certain aspects, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0104] In a specific embodiment, the hinge is derived from CD28, and in certain aspects, the CAR polypeptide comprises a particular CD28 hinge amino acid sequence or is encoded by a particular CD28 hinge nucleic acid sequence.

[0105] The transmembrane domain, in some embodiments, is derived from either a natural source or a synthetic source. If the source is natural, the domain is in some aspects derived from any membrane-bound or transmembrane protein. Transmembrane regions include transmembrane regions derived from the alpha, beta or zeta chains of the T cell receptor, CD28, 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, and DAP molecules (such as, for example, DAP10 or DAP12) (i.e., ... The transmembrane region includes at least the transmembrane region(s) of the alpha, beta or zeta chain of a cell receptor, CD28, 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 and DAP molecules (such as DAP10 or DAP12). Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, synthetic transmembrane domains contain primarily hydrophobic residues (such as leucine and valine). In some aspects, a triplet of phenylalanine, tryptophan and valine may be found at each end of the synthetic transmembrane domain.

[0106] In certain embodiments, the CD70-specific CAR may be co-expressed with one or more cytokines to improve persistence when low amounts of tumor-associated antigens are present. For example, the CAR may be co-expressed with one or more cytokines, such as, for example, IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, IL-7, GMCSF, or a combination thereof.

[0107] In a particular embodiment, the anti-CD70 CAR may have the following nucleotide sequence:

[0108] CD27tr28tdmCD3zIL15:

[0109] ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACC AGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGC TGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGG CTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (Sequence number: 15)

[0110] The corresponding amino acid sequence of CD27tr28tdmCD3zIL15 is as follows:

[0111] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 38)

[0112] CD27Tr28tmd41BBicd3zIL15:

[0113]

[0114] The corresponding amino acid sequence of CD27Tr28tmd41BBicd3zIL15 is as follows:

[0115] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 39)

[0116] GSPco27Tr28tmd41BBicCD3zIL15:

[0117]

[0118] The corresponding amino acid sequence of GSPco27Tr28tmd41BBicCD3zIL15 is as follows:

[0119] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 40)

[0120] CD27Tr28tmdDAP10icd3z15:

[0121]

[0122] The corresponding amino acid sequence of CD27Tr28tmdDAP10icd3z15 is as follows:

[0123] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKD CDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 41)

[0124] GSPco27Tr28tmdDAP10IL15:

[0125]

[0126] The corresponding amino acid sequence of GSPco27Tr28tmdDAP10IL15 is as follows:

[0127] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLV KDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAF IIFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 42)

[0128] CD27Tr28tmdDAP12icd3z15:

[0129]

[0130] The corresponding amino acid sequence of CD27Tr28tmdDAP12icd3z15 is as follows:

[0131] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKA AQCDPCIPGVFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRG RGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 43)

[0132] GSPco27Tr28tmddap12icd15:

[0133]

[0134] The corresponding amino acid sequence of GSPco27Tr28tmddap12icd15 is as follows:

[0135] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPR GRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 44)

[0136] CD27Tr28tmdNKG2Dic3z15:

[0137]

[0138] The corresponding amino acid sequence of CD27Tr28tmdNKG2Dic3z15 is as follows:

[0139] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKA AQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKSKV VPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 45)

[0140] GSPco27Tr28tmdNKG2Dicd3z15:

[0141]

[0142] The corresponding amino acid sequence of GSPco27Tr28tmdNKG2Dicd3z15 is as follows:

[0143] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKS KVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 46)

[0144] CD27Tr41BBicd3z15:

[0145]

[0146] The corresponding amino acid sequence of CD27Tr41BBicd3z15 is as follows:

[0147] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 47)

[0148] GSPco27Tr41BBicd3z15:

[0149]

[0150] The corresponding amino acid sequence of GSPco27Tr41BBicd3z15 is as follows:

[0151] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDC DQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 48)

[0152] CD27TrCD3ZIL15:

[0153]

[0154] The corresponding amino acid sequence of CD27TrCD3ZIL15 is as follows:

[0155] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCE PGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGM FLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 49)

[0156] GSPco27Tr3z15:

[0157]

[0158] The corresponding amino acid sequence of GSPco27Tr3z15 is as follows:

[0159] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQM CEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSG MFLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 50)

[0160] CD27TrCD28icd3z15:

[0161]

[0162] The corresponding amino acid sequence of CD27TrCD28icd3z15 is as follows:

[0163] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 51)

[0164] GSPco27Tr28CD3z15:

[0165]

[0166] The corresponding amino acid sequence of GSPco27Tr28CD3z15 is as follows:

[0167] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKD CDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 52)

[0168] CD27TrCD28tmdicd3z15:

[0169]

[0170] The corresponding amino acid sequence of CD27TrCD28tmdicd3z15 is as follows:

[0171] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 53)

[0172] GSPco27Tr28tmdicCD3z15:

[0173]

[0174] The corresponding amino acid sequence of GSPco27Tr28tmdicCD3z15 is as follows:

[0175] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 54)

[0176] CD27TrDAP10icd3z15:

[0177]

[0178] The corresponding amino acid sequence of CD27TrDAP10icd3z15 is as follows:

[0179] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLV KDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFL HLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 55)

[0180] GSPco27FLdap10icd3z15:

[0181]

[0182] The corresponding amino acid sequence of GSPco27FLdap10icd3z15 is as follows:

[0183] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTF LVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALF LHLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 56)

[0184] CD27TrDAP12icd3z15:

[0185]

[0186] The corresponding amino acid sequence of CD27TrDAP12icd3z15 is as follows:

[0187] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRGA AEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 57)

[0188] GSPco27FLdap12icd3z15:

[0189]

[0190] The corresponding amino acid sequence of GSPco27FLdap12icd3z15 is as follows:

[0191] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQ HRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRG AAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 58)

[0192] CD27TrNKG2Dic3z15:

[0193]

[0194] The corresponding amino acid sequence of CD27TrNKG2Dic3z15 is as follows:

[0195] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVVPC RQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 59)

[0196] GSPco27TrNKG2Dicd3z15:

[0197]

[0198] The corresponding amino acid sequence of GSPco27TrNKG2Dicd3z15 is as follows:

[0199] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQ HRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVV PCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 60)

[0200] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA or 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 have been found to stabilize mRNA. It may also be more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0201] It is intended that the chimeric construct can be introduced into any kind of immune cell as naked DNA or in a suitable vector.Various methods are known in the art for stably transfecting cells by electroporation using naked DNA.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 the appropriate orientation for expression.

[0202] Alternatively, viral vectors (e.g., retroviral, adenoviral, adeno-associated or lentiviral vectors) can be used to introduce chimeric constructs into immune cells. The vectors suitable for use according to the method of the present disclosure are non-replicative in immune cells. Numerous virus-based vectors are known, in which the copy number of the virus maintained in cells is low enough to maintain cell viability, such as HIV, SV40, EBV, HSV or BPV-based vectors. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

[0203] II. Cytokines In some embodiments, cells expressing an anti-CD70 CAR are engineered to express one or more heterologous cytokines and / or to upregulate normal expression of one or more heterologous cytokines. The cells may or may not be subject to transduction or transfection for the one or more cytokines in the same vector as other genes.

[0204] One or more cytokines may be co-expressed from the vector as separate polypeptides from the antigen receptor. For example, interleukin-15 (IL-15) is tissue-restricted and is found in serum at any level or systemically only under pathological conditions. IL-15 has several desirable attributes for adoptive therapy. IL-15 is a homeostatic cytokine that induces natural killer cell development and cell proliferation, promotes eradication of established tumors by relieving functional inhibition of tumor-resident cells, and inhibits activation-induced cell death (AICD). 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 for human applications. NK cells expressing IL-15 are capable of uninterrupted supportive cytokine signaling that is useful for their survival after infusion.

[0205] In a specific embodiment, the cells express one or more exogenously provided cytokines. In one example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, GMCSF, or a combination thereof. In another example, the cytokine is IL-21. In another example, the cytokine is IL-12. The cytokine may be exogenously provided to the NK cell as it is expressed from an expression vector in the cell. In an alternative case, an endogenous cytokine in the cell is upregulated upon manipulating the regulation of expression of the endogenous cytokine, such as by genetic modification at the promoter site(s) of the cytokine. When the cytokine is provided to the cell in an expression construct, the cytokine may be encoded from the same vector as the suicide gene and / or the anti-CD70 CAR. In some embodiments, the disclosure relates to co-using a CAR with IL-15. In some embodiments, the disclosure relates to co-using a CAR with IL-21. In some embodiments, the disclosure relates to co-using a CAR with IL-12.

[0206] III. Suicide Genes In certain embodiments, suicide genes are utilized in conjunction with anti-CD70 cell therapy to inhibit the use of anti-CD70 cell therapy and to provide for the termination of cell therapy in desired situations and / or at desired times. Suicide genes are used in transduced cells for the purpose of inducing death of the transduced cells when necessary. Cells of the present disclosure that are modified to receive vectors 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, upon administration of a prodrug or other drug, results in the transition of the gene product into a compound that kills the host cell. In other embodiments, the suicide gene encodes a gene product that, when desired, is targeted by a drug (such as, for example, an antibody) that targets the suicide gene product.

[0207] In some cases, when an individual undergoing cell therapy and / or an individual who has undergone cell therapy exhibits one or more symptoms of one or more adverse events, such as (for example) cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity, or is deemed to be at risk, including imminent, for having one or more symptoms, the cell therapy may be conditional on utilizing one or more suicide genes, whatever the type of suicide gene. The use of the suicide gene may be part of a planned protocol for treatment, or may be used only when a need for its use is recognized. In some cases, the cell therapy is terminated by the use of an agent(s) that targets the suicide gene or a gene product therefrom, since the cell therapy is no longer needed.

[0208] The use of suicide genes may be initiated when at least one adverse event begins for an individual, and the adverse event may be recognized by any means, including during routine monitoring, which may or may not continue from the beginning of cell therapy. The adverse event(s) may be detected during consultation and / or examination. If an individual has cytokine release syndrome (which may also be indicated as cytokine storm), the individual may have, for example, elevated inflammatory cytokine(s) (just as examples: interferon-gamma, granulocyte macrophage colony stimulating factor, IL-10, IL-6 and TNF-alpha); fever; fatigue; hypotension; hypoxia, tachycardia; nausea; capillary leakage; cardiac / renal / liver dysfunction; or a combination thereof. If an individual has neurotoxicity, the individual may have confusion, delirium, hypoplasia and / or epileptic seizures. In some cases, individuals are tested for markers associated with the onset and / or severity of cytokine release syndrome (eg, C-reactive protein, IL-6, TNF-alpha and / or ferritin, etc.).

[0209] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009, which is incorporated herein by reference in its entirety), which may be affected by delivery of antibodies that bind to the TNF-alpha mutant. Examples of suicide gene / prodrug combinations that may 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 thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Purine nucleoside phosphorylase from E. coli (a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine) may be utilized. Other suicide genes include, by way of example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0210] In certain embodiments, the vector encoding the CD70-targeted CAR, i.e., the vectors in NK cells encompassed herein, contain one or more suicide genes. The suicide gene may or may not be present in the same vector as the CD70-targeted CAR. If the suicide gene is present in the same vector as the CD70-targeted CAR, the suicide gene and the CAR may be separated, for example, by an IRES element or a 2A element.

[0211] IV. Pharmaceutical Compositions The pharmaceutical composition of the present disclosure comprises an effective amount of cells expressing anti-CD70 CAR dissolved or dispersed in a pharma- ceutical acceptable carrier. The phrase "pharmaceutical, or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered appropriately to an animal (e.g., human, etc.). The preparation of pharmaceutical compositions comprising cells expressing anti-CD70 CAR will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams and Wilkins, 2005; which is incorporated herein by reference). Moreover, it will be understood that for administration to an animal (e.g., human), the preparation must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0212] As used herein, "pharmaceutical acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, such types of materials, and combinations thereof, as would be known to one skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Mack Printing Company, 1990, pp. 1289-1329); incorporated herein by reference). Except where any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0213] Pharmaceutical compositions may contain different types of carriers depending on whether they are to be administered in solid, liquid or aerosol form, and depending on whether they need to be sterile for routes of administration such as injection. The presently disclosed compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion to directly bathe target cells, via catheter, via lavage, in creams, in lipid compositions (e.g., liposomes), or by other methods, or any combination of the foregoing, as would be known to one skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Mack Printing Company, 1990); which is incorporated herein by reference).

[0214] The cells expressing anti-CD70 CAR may be formulated in the composition in free base form, neutral form or salt form. Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with free amino groups of the proteinaceous composition, or with inorganic acids (e.g., hydrochloric acid or phosphoric acid, etc.), or with organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, etc.), or organic bases such as isopropylamine, trimethylamine, histidine or procaine. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as dosage forms formulated for parenteral administration (such as, for example, injectable solutions or aerosols for pulmonary delivery) or dosage forms formulated for gastrointestinal administration (such as, for example, drug release capsules).

[0215] Further, in accordance with the present disclosure, the compositions of the present disclosure suitable for administration are provided in a pharma- ceutically acceptable carrier, with or without an inert diluent. The carrier must be assimilable, and includes liquid carriers, semi-solid carriers (i.e., pastes), or solid carriers. Except where any conventional vehicle, agent, diluent, or carrier is harmful to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for use in carrying out the method of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, bulking agents, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of the action of microorganisms can be provided by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0216] In accordance with the present disclosure, the compositions are combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine for those skilled in the art.

[0217] In a specific embodiment of the present disclosure, the composition is combined with a semi-solid carrier or a solid carrier or mixed thoroughly.Mixing can be carried out in any convenient manner, such as by grinding.Stabilizers can also be added in the mixing process to protect the composition from loss of therapeutic activity, i.e., from denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids (such as glycine and lysine), carbohydrates (such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0218] In further embodiments, the disclosure may relate to the use of pharmaceutical lipid vehicle compositions comprising cells expressing anti-CD70 CAR and, optionally, an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a wide variety of substances that are characteristically insoluble in water and extractable by organic solvents. This wide variety of compounds is widely known to those skilled in the art, and as the term "lipid" is used herein, lipids are not limited to any particular structure. Examples include various compounds containing various long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or manufactured by man). However, lipids are usually biological substances. Various biological lipids are widely known in the art, including, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether- and ester-linked fatty acids, and polymerizable lipids, and combinations thereof. It will be understood that compounds other than those specifically described herein that would be understood by those of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

[0219] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles.For example, cells expressing anti-CD70 CAR may be dispersed in a solution containing lipid, may be dissolved with lipid, may be emulsified with lipid, may be mixed with lipid, may be combined with lipid, may be covalently bound to lipid, may be contained as a suspension in lipid, may be contained with micelles or liposomes, may be complexed with micelles or liposomes, or may otherwise be associated with lipid or lipid structures by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.

[0220] The actual dosage of the composition of the present disclosure administered to an animal patient can be determined by various physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic disease of the patient, and based on the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the frequency of administration of an effective amount may vary depending on the subject's response. The person responsible for administration will in any case determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0221] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, between about 2% and about 75%, or between about 25% and about 60% by weight of the unit, and any range derivable therein. Of course, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Various factors, such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, as well as other pharmacological considerations, will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable.

[0222] In other non-limiting examples, dosages can also include, per administration, about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, to about 1000 mg / kg / body weight or more, and any range derivable therein. In non-limiting examples of ranges that can be derived from the numbers recited herein, ranges such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, can be administered based on the above numbers.

[0223] A. Compositions and Formulations for the Gastrointestinal Tract In certain embodiments of the present disclosure, the cells expressing anti-CD70 CAR are formulated to be administered via the gastrointestinal route. The gastrointestinal route includes all possible routes of administration in which the composition is in direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, bucally, rectally or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or may be enclosed in hard or soft gelatin capsules, or may be compressed into tablets, or may be directly incorporated with the food of the meal.

[0224] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, each of which is specifically incorporated by reference herein in its entirety). Tablets, troches, pills, capsules, and the like may also contain the following: binders (e.g., gum tragacanth, gum arabic, corn starch, gelatin, or combinations thereof), excipients (e.g., dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof), disintegrants (e.g., corn starch, potato starch, alginic acid, or combinations thereof), lubricants (e.g., magnesium stearate), sweeteners (e.g., sucrose, lactose, saccharin, or combinations thereof), flavoring agents (e.g., peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like). When the dosage unit form is a capsule, the capsule may contain a liquid carrier in addition to materials of the above types. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. When the dosage form is a capsule, the capsule may contain various carriers (e.g., liquid carriers, etc.) in addition to the above-mentioned types of materials. Gelatin capsules, tablets or pills may be enteric coated. The enteric coating prevents the composition from denaturing in the stomach or upper intestine, where the pH is acidic. See, for example, U.S. Patent No. 5,629,001. When the small intestine is reached, the basic pH in the small intestine dissolves the coating, allowing the composition to be released and absorbed by specialized cells (e.g., epithelial intestinal cells and Peyer's patch M cells).Syrup of elixir may contain active compound, sucrose as sweetener, methylparaben and propylparaben as preservatives, dye, and flavoring agent (such as cherry or orange flavor). Of course, any material used in the preparation of any dosage unit form must be pharma- ceutically pure and substantially non-toxic in the amounts used. In addition, active compound may be incorporated into sustained-release preparations and formulations.

[0225] For oral administration, the composition of the present disclosure may alternatively be incorporated with one or more excipients in the form of mouthwash, dentifrice, oral tablet, oral spray or sublingual oral administration formulation.For example, a mouthwash may be prepared that incorporates the active ingredient in the required amount in a suitable solvent (such as sodium borate solution (Dobell's solution)).Alternatively, the active ingredient may be incorporated in an oral solution (such as an oral solution containing sodium borate, glycerin and potassium bicarbonate), or dispersed in a dentifrice, or may be added in a therapeutically effective amount to a composition that may include water, binding agents, abrasives, flavoring agents, foaming agents and humectants.Alternatively, the composition may be in tablet or solution form that may be placed under the tongue or otherwise disintegrate in the mouth.

[0226] Further formulations suitable for other modes of gastrointestinal administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually with a medicinal agent, for insertion into the rectum. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. In general, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, about 0.5% to about 10% of the active ingredient, preferably about 1% to about 2%.

[0227] B. Parenteral Compositions and Formulations In further embodiments, the composition may be administered via parenteral route.As used herein, the term "parenteral" includes a route that bypasses the digestive tract.Specifically, the pharmaceutical composition disclosed herein may be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally.See U.S. Patent Nos. 6,613,308, 5,466,468, 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety).

[0228] Solutions of the active compounds as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as, for example, hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy injectability exists. The form must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms, such as, for example, bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent (e.g., lecithin, and the like), by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent (e.g., sugar or sodium chloride, and the like). Prolonged absorption of the injectable composition can be brought about by the use in the composition of agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0229] For parenteral administration in aqueous solution, for example, the solution must be suitably buffered if necessary, and the liquid diluent must first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, various sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, a dosage may be dissolved in an isotonic NaCl solution and added to a subcutaneous infusion, or may be injected at the proposed injection site, either of which is possible (see, for example, "Remington's Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0230] Sterile injectable solution is prepared by incorporating the active compound in the required amount in a suitable solvent with various other ingredients as listed above as required, and then performing filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients as required from the ingredients listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying techniques that produce a powder of active ingredient and any additional desired ingredients from their previously sterile filtered solutions.Powdered composition is combined with liquid carrier (such as water or physiological saline solution) with or without stabilizer.

[0231] C. Other Pharmaceutical Compositions and Formulations In other specific embodiments of the invention, the active compound cells expressing an anti-CD70 CAR may be formulated for administration via a variety of different routes, for example, for topical administration (i.e., transdermal administration), mucosal administration (intranasal administration, vaginal administration, etc.) and / or inhalation.

[0232] Pharmaceutical compositions for topical administration may include active compounds formulated for medicinal application (e.g., ointments, pastes, creams, or powders, etc.). Ointments include all oil-based compositions for topical application based on adsorption, emulsification, and water solubility, while creams and lotions are such compositions that contain only an emulsifying base. Topically administered pharmaceutical drugs may contain a penetration enhancer to facilitate the adsorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidones, and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorbent ointment base, emulsifying ointment base, or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide a homogeneous mixture. Transdermal administration of the present invention may also involve the use of a "patch." For example, a patch may deliver one or more active substances at a predetermined rate and in a continuous manner over a period of time.

[0233] In certain embodiments, pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation and / or other aerosol delivery vehicles.Methods for delivering compositions directly to the lungs via intranasal aerosol sprays are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212 (each of which is specifically incorporated herein by reference in its entirety).Similarly, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and the delivery of lysophosphatidylglycerol compounds (U.S. Patent No. 5,725,871, specifically incorporated herein by reference in its entirety) are also widely known in the pharmaceutical arts.Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045 (specifically incorporated herein by reference in its entirety).

[0234] The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. A typical aerosol of the present invention for inhalation will consist of a suspension of the active ingredient in a liquid propellant or in a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. The administration of the aerosol will vary according to the age, weight, and severity and response of the subject to symptoms.

[0235] V. Combination Therapy In certain embodiments, the compositions and methods of the present embodiments involve a cancer treatment that is additional to the composition comprising the cells expressing an anti-CD70 CAR. The additional treatment may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination of the above. The additional treatment may be in the form of adjuvant or neoadjuvant therapy.

[0236] In some embodiments, the additional treatment is administration of a small molecule enzyme inhibitor(s) or metastasis inhibitor(s). In some embodiments, the additional treatment 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). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent(s). The additional treatment may be one or more chemotherapeutic agents known in the art.

[0237] Immune cell therapy (in addition to the cell therapy of the present disclosure) may be administered before, during, after, or in various combinations with additional cancer therapy (such as, for example, immune checkpoint therapy). These administrations may be at intervals ranging from simultaneously, to minutes, to days, to weeks. In embodiments in which immune cell therapy is administered to a patient separately from the composition(s) of the present disclosure, it will generally be ensured that no meaningful time period expires between the time of each delivery, such that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the patient may be administered the immunotherapy and the disclosed composition within about 12 to 24 hours or 72 hours of each other, more specifically within about 6 to 12 hours of each other. In some circumstances, it may be desirable to significantly extend the period for treatment, in which case several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) will lapse between each administration.

[0238] Administration of any of the compounds or cell therapies of the present embodiments to a patient will follow general 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 that may result from the combination therapy.

[0239] The term "cancer" as used herein may be used to describe solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, the cancer may occur in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer is a stage I cancer. In some embodiments, the cancer is a stage II cancer. In some embodiments, the cancer is a stage III cancer. In some embodiments, the cancer is a stage IV cancer.

[0240] The cancer may be of the following histological types, including, but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma. carcinoma);transitional cell carcinoma;papillary transitional cell carcinoma;adenocarcinoma;gastrinoma, malignant;bile duct carcinoma;hepatocellular carcinoma;mixed hepatocellular and bile duct carcinoma;trabecular adenocarcinoma;adenoid cystic carcinoma;adenocarcinoma in adenomatous polyps;adenocarcinoma, familial polyposis coli;solid cancer;carcinoid tumor, malignant;branchio-alveolar adenocarcinoma;papillary adenocarcinoma;chromophobe carcinoma;acidophil carcinoma;oxyphilic adenocarcinoma;basophilic carcinoma;clear cell adenocarcinoma;granular cell carcinoma;follicular adenocarcinoma;papillary follicular adenocarcinoma;nonencapsulated sclerosing carcinoma;adrenal cortical carcinoma;endometrioid carcinoma carcinoma);skin adnexal cancer;apocrine gland carcinoma;sebaceous gland carcinoma;earwax gland carcinoma;mucoepidermoid carcinoma;cyst gland carcinoma;papillary cyst gland carcinoma;papillary serous cyst gland carcinoma;mucinous cyst gland carcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease, breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglioma, malignant;extramammary paraganglioma, malignant;pheochromocytoma;glomus angiosarcoma;malignant melanocytoma melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic odontosarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Anaplastic neuroectodermal;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular Cell tumors, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin's;Side granuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis fungoides;Other specified non-Hodgkin's lymphoma;Malignant histiocytoma;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lympho-sarcoma cell leukemia;Myeloid leukemia:Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myeloid sarcoma;And Hairy cell leukemia.;

[0241] A. Chemotherapy A wide variety of chemotherapeutic agents may be used according to the present embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply compounds or compositions administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity in cells, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on whether they can directly crosslink DNA, cause intercalation into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0242] Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridine compounds, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine compounds and methylamelamines, such as altretamine, triethylenemelamine, trimethylolpropane ... triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8). dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyins; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 11); dynemicins, such as dynemicin A; bisphosphonates such as clodronate; esperamicins;Also included are neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, icin), 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potofilomycin anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin and trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamperidin ... 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; antiadrenals such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformithine; Elliptinium acetate; Epothilones; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids, such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pe antostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine ;mitobronitol;mitolactol;pipobroman;gacytosine;arabinoside ("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;a minopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, as well as pharmaceutical acceptable salts, acids, or derivatives of any of the above;

[0243] B. Radiation therapy Other factors that cause DNA damage and are widely used include what is commonly known as gamma radiation, X-rays, and / or guided delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam radiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV radiation. Presumably, all of these factors affect a wide range of damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens over prolonged periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the radioisotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0244] C. Immunotherapy Those skilled in the art will understand that additional immunotherapies (outside the scope of the disclosed cell therapy) can be used in combination with or in conjunction with the method of the present embodiment. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone may serve as the effector of therapy. Alternatively, the antibody may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with the tumor cell target. The various effector cells include cytotoxic T cells and NK cells that differ from those with knockdown or knockout of TGF-beta R2.

[0245] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) comprise a monoclonal antibody (MAb) covalently linked to a cell-killing drug. In this approach, the high specificity of the MAb for its antigen target is combined with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells that bear elevated levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, thereby 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 proven this approach effective. 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 increasingly mature, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this endeavor, and the generation of MAbs for targeting. Two criteria for ADC targets are upregulated / high expression levels in tumor cells and unaffected internalization.

[0246] In one aspect of immunotherapy, tumor cells must have some marker that is amenable to targeting, i.e., some marker that is not present on the majority of other cells. Many tumor markers exist, 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 stimulating effects. There are also immune stimulating molecules, which include cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, etc.), chemokines (e.g., MIP-1, MCP-1, IL-8, etc.), and growth factors (e.g., FLT3 ligand, etc.).

[0247] Examples of immunotherapies currently under consideration 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 any type of interferon, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1999); 998); 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.

[0248] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints can either enhance (e.g., costimulatory molecules) or weaken signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B / 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), immunoglobulin and mucin domains of T cells 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.

[0249] D.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, where 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 therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microsurgery (Mohs surgery).

[0250] When part or all of cancerous cells, tissues or tumors are removed, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application to the area with additional anti-cancer treatment. Such treatment may be repeated, for example, every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, or every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. These treatments may also be of various dosages.

[0251] E. Other drugs It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of treatment. These additional agents include agents that affect cell surface receptor upregulation and GAP binding, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. An increase in intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (such as antibody c225) may be used in combination with certain aspects of the present embodiment to improve the efficacy of treatment.

[0252] VI. Vector CD70 targeting CAR can be delivered to recipient cells, including NK cells, by any suitable vector, including by viral vector or non-viral vector.Examples of viral vector include at least retroviral vector, lentiviral vector, adenoviral vector, or adeno-associated viral vector.Examples of non-viral vector include at least plasmid, transposon, lipid, nanoparticle, liposome, and combinations thereof.

[0253] In cases where NK cells are transduced with a vector encoding a CD70-targeted CAR and transduction of another gene or genes, such as a suicide gene and / or a cytokine and / or an optional therapeutic gene product, into the cells is also required, the CD70-targeted CAR, the optional suicide gene, the optional cytokine(s) may or may not be included in the same vector or together with the same vector. In some cases, the CD70-targeted CAR, the suicide gene, and / or the cytokine(s) are expressed from the same vector molecule (e.g., the same viral vector molecule, etc.). In such cases, the expression of the CD70-targeted CAR, the suicide gene, and / or the cytokine(s) may or may not be regulated by the same regulatory element(s). When the CD70-targeted CAR, the suicide gene, and / or the cytokine are present in the same vector, they may or may not be expressed as separate polypeptides. When they are expressed as separate polypeptides, they may be separated in the vector by, for example, a 2A element or an IRES element (or both types may be used one or more times in the same vector).

[0254] Those of skill in the art will be well equipped to construct vectors for expression of the antigen receptors of the present disclosure by standard recombinant techniques (e.g., Sambrook et al. (2001) and Ausubel et al. (1996), both of which are incorporated herein by reference).

[0255] A. Regulatory Elements The expression cassette contained in the vector useful in the present disclosure specifically contains (in the 5' to 3' direction) a eukaryotic transcriptional promoter operably linked to the protein coding sequence, a splice signal with intervening sequences, and a transcription termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein coding genes in eukaryotic cells may be composed of multiple genetic elements. The cellular machinery can collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to develop distinct and often complex patterns of transcriptional regulation. Promoters used in the context of the present disclosure include, for example, constitutive promoters, inducible promoters, and tissue-specific promoters. When the vector is utilized for the creation of cancer treatments, the promoter may be effective under hypoxic conditions.

[0256] B. Promoters / Enhancers The expression constructs provided herein include promoters for driving the expression of antigen receptors and other cistron gene products. Promoters generally contain a sequence that functions to position the start site for RNA synthesis. The best known example of this sequence is the TATA box, but in some promoters that lack a TATA box, such as the promoters for mammalian terminal deoxynucleotidyl transferase genes and the promoters for the SV40 late genes, discrete elements that overlap the start site itself serve to position initiation. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region upstream of the start site, although many 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 and promotes expression of the encoded RNA.

[0257] Spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either 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.

[0258] A promoter may be one that is naturally associated with a nucleic acid sequence, 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 being "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of the nucleic acid sequence. Alternatively, certain advantages may 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 even from prokaryotic or eukaryotic cells, as well as promoters or enhancers that are not "naturally occurring", i.e., promoters or enhancers 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 synthetically producing promoter and enhancer nucleic acid sequences, a variety of 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 within non-nuclear organelles, such as mitochondria and chloroplasts, may be used as well.

[0259] 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 generally understand that various promoters, various enhancers, and various cell type combinations are used 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 levels of expression of the introduced DNA segment, such as being advantageous in large-scale production of recombinant proteins and / or recombinant peptides. The promoter may be heterologous or endogenous.

[0260] Additionally, any promoter / enhancer combination (e.g., from the Eukaryotic Promoter Database EPDB via the World Wide Web at epd.isb-sib.ch / ) could also be used to drive expression. 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 either as part of the delivery complex or as an additional gene expression construct.

[0261] 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, etc.; eukaryotic cell promoters, such as beta actin promoter, GADPH promoter, metallothionein promoter, etc.; and chained response element promoters, such as cyclic AMP response element promoter (cre), serum response element promoter (sre), phorbol ester promoter (TPA), and minimal TATA box proximal response element promoter (tre), etc. It is also possible to use the human growth hormone promoter sequence (e.g., human growth hormone minimal promoter described in GenBank®, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoter (which is available from ATCC; catalog number ATCC45007). 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 a therapeutic gene is applicable to the practice of the present disclosure.

[0262] In certain aspects, the methods of the present disclosure also relate to enhancer sequences, i.e. nucleic acid sequences that increase the activity of a promoter and have the potential to 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 enhancers can also function in the immediate vicinity of a given promoter.

[0263] C. Initiation Signals and Linked Expression Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. Those skilled in the art will be able to easily determine this and provide the necessary signals. It is widely known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be either natural or synthetic. Expression efficiency may be enhanced by including appropriate transcriptional enhancer elements.

[0264] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of translation that relies on 5' methylated Cap and allow translation to begin at an internal site. Various IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well as IRES from mammalian messages. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, but each separated by an IRES, resulting in a polycistronic message. IRES elements allow each open reading frame to be accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using only one promoter / enhancer to transcribe only one message.

[0265] As detailed elsewhere herein, certain 2A sequence elements could be used to produce linked or co-expression of genes in the constructs provided in this disclosure. For example, truncation sequences could be used to co-express multiple genes by linking multiple open reading frames to form a single cistron. One exemplary truncation sequence is Equine Rhinitis A virus (E2A) or F2A (foot and mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A) or Porcine Teschovirus-1 (P2A). In a specific embodiment, in one vector, multiple 2A sequences are not identical, although in alternative embodiments, the same vector utilizes two or more of the same 2A sequences. Various examples of 2A sequences are provided in US Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety.

[0266] D. Origin of replication To propagate the vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), for example, a nucleic acid sequence corresponding to the oriP of EBV as described above, or an engineered oriP with a similar or enhanced function in programming (these are specific nucleic acid sequences from which replication is initiated). Alternatively, the origin of replication of other extrachromosomally replicating viruses as described above, or an autonomously replicating sequence (ARS), may be used.

[0267] E. Selectable and Screenable Markers In some embodiments, cells such as NK cells that contain the anti-CD70 CAR expression construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would confer an identifiable change to the cell, thereby allowing easy identification of cells that contain the expression vector. In general, a selection marker is one that confers a property that allows for selection. A positive selection marker is one whose presence allows for its selection, while a negative selection marker is one whose presence prevents its selection. One example of a positive selection marker is a drug resistance marker.

[0268] Usually, 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 phenotypes that allow for the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screening markers based on colorimetric analysis (e.g., GFP, etc.). Alternatively, screening markers as negative selection markers, such as thymidine kinase (tk) of herpes simplex virus, or chloramphenicol acetyltransferase (CAT), may be utilized. Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used does not appear to be important, so long as it is capable of being expressed simultaneously with the nucleic acid that codes for the gene product. Further examples of selection and screening markers are well known to those skilled in the art.

[0269] VII.Cells The present disclosure encompasses any type of immune cell that receives a vector that encodes a CD70-targeted CAR and may also encode at least one cytokine and at least one suicide gene. In some cases, the various vectors encode the CAR versus the suicide gene and / or cytokine. In certain cases, the cells are NK cells, although conventional T cells, NK cells, gamma-delta T cells, NKT cells and invariant NK T cells, regulatory T cells, macrophages, B cells, tumor-infiltrating lymphocytes or mixtures thereof may be used. The NK cells may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs) or bone marrow. The NK cells may be derived, for example, from a cell line, such as, but not limited to, NK-92 cells. The NK cells may be cord blood mononuclear cells, such as, for example, CD56+ NK cells.

[0270] In some cases, the cell is not an immortalized cell line, but is a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As an example, the cell is a T lymphocyte obtained from an individual. As another example, the cell is a cytotoxic cell obtained from an individual. As another example, the cell is a stem cell (e.g., a peripheral blood stem cell) or a progenitor cell obtained from an individual.

[0271] In certain embodiments, the cells of the present disclosure may be specially formulated and / or the cells of the present disclosure may be cultured in a particular medium. The cells may be formulated in such a manner that they are suitable for delivery to a recipient without adverse effects.

[0272] In certain aspects, the medium may be prepared using a medium used for culturing animal cells as the basal medium for the cells, for example, any of AIM V medium, X-VIVO-15 medium, NeuroBasal medium, EGM2 medium, TeSR medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI-1640 medium and Fischer medium, as well as any combination thereof, but the medium may not be particularly limited thereto, as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0273] The medium may be serum-containing or serum-free, or xenogeneic component-free. In order to prevent contamination by xenogeneic animal-derived components, serum may be derived from the same animal as the animal of the stem cell. Serum-free medium refers to a medium that does not contain raw or unpurified serum, and therefore may include a medium that contains purified blood-derived components or animal tissue-derived components (e.g., growth factors, etc.).

[0274] The medium may or may not contain a serum replacement. Serum replacements may include albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes (e.g., recombinant albumin or humanized albumin, etc.), vegetable starch, dextran, and protein hydrolysates, etc.), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum replacements may be prepared, for example, by the method disclosed in International Publication No. 98 / 30679, which is incorporated herein in its entirety. In the replacement, any commercially available product may be used more conveniently. Commercially available products include knockout serum replacement (KSR), Chemically-defined lipid concentrated (Gibco), and Glutamax (Gibco).

[0275] In certain embodiments, the medium may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins, such as biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A (acetate); proteins, such as BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components, such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or T3 (triiodo-I-thyronine). In specific embodiments, one or more of these may be explicitly excluded.

[0276] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12; or the medium comprises a combination thereof or a salt thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or a combination thereof. In some embodiments, the medium comprises one or more of the following: B-27® supplement, xeno-free B-27® supplement, GS21™ supplement, or a combination thereof. In some embodiments, the medium comprises or further comprises amino acids, simple sugars, inorganic ions.In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof. In some embodiments, the medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain embodiments, the medium includes or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement. supplements, xenogeneic B-27® supplements, GS21™ supplements, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be explicitly excluded.

[0277] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as, for example, non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, of which, in specific embodiments, one or more may be explicitly excluded.

[0278] One or more of the media components may be added at a concentration of at least, at most, or approximately 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0279] In a specific embodiment, the cells of the present disclosure are specially formulated. The cells may or may not be formulated as a cell suspension. In a specific case, the cells are formulated in a single dose form. The cells may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., at 5% DMSO). The cell formulation may include albumin, including human albumin, and a specific formulation includes 2.5% human albumin. The cells may be specially formulated for intravenous administration; for example, the cells are formulated for intravenous administration for less than 1 hour. In a specific embodiment, the cells are in a formulated cell suspension that is stable at room temperature for 1 hour, 2 hours, 3 hours, or 4 hours or more from the time of thawing.

[0280] In some cases, the immune cells are expanded prior to use and / or manufacture. In some cases, the immune cells are NK cells that have been expanded in the presence of an effective amount of antigen presenting cells, including universal antigen presenting cells (UAPCs), including at any suitable ratio. The cells may be cultured with UAPCs at a ratio of, for example, 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 at a ratio of 1:2. In some cases, the NK cells are expanded in the presence of IL-2, e.g., at a concentration of 10-500 U / mL, 10-400 U / mL, 10-300 U / mL, 10-200 U / mL, 10-100 U / mL, 10-50 U / mL, 100-500 U / mL, 100-400 U / mL, 100-300 U / mL, 100-200 U / mL, 200-500 U / mL, 200-400 U / mL, 200-300 U / mL, 300-500 U / mL, 300-400 U / mL, or 400-500 U / mL.

[0281] After genetic modification with the vector(s), the NK cells may be infused immediately or may be stored. In certain aspects, after genetic modification, the cells may be expanded ex vivo for days, weeks, or months as a bulk population within about 1, 2, 3, 4, 5, or more days after gene transfer into the cells. In further aspects, the transfectants are cloned and clones that demonstrate the presence of a single integrated or episomally maintained expression cassette or plasmid and expression of the CD70-targeted CAR are expanded ex vivo. The clones selected for expansion demonstrate the ability to specifically recognize and lyse CD70-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, and IL-21, etc.). The recombinant immune cells may be expanded by stimulation with artificial antigen-presenting cells. In a further aspect, the genetically modified cells may be cryopreserved.

[0282] Various embodiments of the present disclosure include cells expressing one or more CD70-targeted CARs as encompassed herein. In a specific embodiment, the NK cells comprise recombinant nucleic acids encoding one or more CD70-targeted CARs and one or more engineered non-secretable membrane-bound TNF-alpha variant polypeptides. In a specific embodiment, in addition to expressing one or more CD70-targeted CARs, the cells also comprise nucleic acids encoding one or more therapeutic gene products.

[0283] The cells may be obtained directly from the individual or may be obtained from a repository or other storage facility. Cells as a therapy may be autologous or allogeneic with respect to the individual to whom the cells are given as a therapy.

[0284] The cells may be obtained from an individual in need of treatment for a medical condition, and after cell engineering to express a CD70-targeting CAR, the cells may be returned to the individual from whom they were originally obtained. In some cases, the cells are stored for later use for the individual or another individual. In such cases, the cells may or may not be further modified prior to use.

[0285] Of the NK cells that receive the CD70-targeted CAR, the NK cells may be included in a population of cells, and that population may have a majority that are transduced with one or more CD70-targeted CARs. The cell population may comprise 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% cells transduced with one or more CD70-targeted CARs.

[0286] NK cells may be produced with one or more CD70-targeted CARs for the purpose of being modular with respect to a specific purpose. For example, cells expressing a CD70-targeted CAR may be produced, including for commercial distribution, and the user may modify the cells to express one or more other genes of interest (including therapeutic genes) depending on the intended purpose(s). For example, a party interested in treating CD70-positive cancer may modify the cells to express a second CAR for the same or a different antigen. Other modifications to the cells may include modifications that tailor the cells to the needs of the individual receiving the cells, including the specific needs for the particular type of cancer the individual suffers from.

[0287] VIII. Gene editing of cells In certain embodiments, cells comprising an anti-CD70 CAR are genetically edited to modify the expression of one or more endogenous genes in the cells. In specific cases, the cells are modified to have a reduced expression level of one or more endogenous genes, including inhibition of expression of one or more endogenous genes (which may be referred to as a knockout). Such cells may or may not be expanded.

[0288] In certain cases, one or more endogenous genes of a cell are modified, such as disrupted in expression, in which case expression is partially or completely reduced. In specific cases, one or more genes are knocked down or knocked out using the process of the present disclosure. In specific cases, multiple genes are knocked down or knocked out, which may or may not be performed at the same step in their production. The gene edited in a cell can be any kind of gene, but in specific embodiments, such a gene is a gene whose gene product inhibits the activity and / or proliferation of a cell, including, as an example, antigen-specific, e.g., CD70-specific CAR NK cells (such as such cells derived from umbilical cord blood). In specific cases, the gene edited in an antigen-specific CAR cell, e.g., CD70-specific CAR cell, allows the antigen-specific CAR cell, e.g., CD70-specific CAR cell, to act more effectively in the tumor microenvironment. In a specific case, the gene is 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 and CD7. In a specific embodiment, the TGFBR2 gene is knocked out or knocked down in an antigen-specific CAR cell, for example, a CD70-specific CAR cell.

[0289] In some embodiments, gene editing is carried out using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonuclease (RGEN).For example, the modification can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein.In some embodiments, CpF1 can be used instead of Cas9. In general, a "CRISPR system" collectively refers to transcripts and other elements involved in expression or directing activity of CRISPR-associated ("Cas") genes (including sequences encoding Cas genes), tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeat sequences" in the context of an endogenous CRISPR system, and partial direct repeat sequences processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and / or other sequences and transcripts from a CRISPR locus.

[0290] 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 a nuclease function (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, for example, from a particular organism that contains an endogenous CRISPR system (e.g., Streptococcus pyogenes).

[0291] In some embodiments, Cas nuclease and gRNA (comprising a fusion of a target sequence-specific crRNA and a given tracrRNA) are introduced into cells. In general, a target site at the 5' end of gRNA targets Cas nuclease to its target site, e.g., a gene, by complementary base pairing. Target sites can be selected based on the location immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, 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. In general, "target sequence" generally refers to a sequence that the guide sequence is designed to have complementarity with, and hybridization of the target sequence with the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not necessary, as long as there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

[0292] The CRISPR system can induce a double-strand break (DSB) at the target site followed by a disruption 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. For example, to improve specificity, a pair of nickases can be used, each of which is guided by a different pair of gRNAs targeting sequences, and 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 transcriptional activator, to affect gene expression.

[0293] The target sequence may comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in a cell organelle. In general, the sequence or template that can be used for recombination into the targeted locus that comprises the target sequence is referred to as "editing template" or "editing polynucleotide" or "editing sequence". In some embodiments, an exogenous template polynucleotide may be referred to as an editing template. In some embodiments, the recombination is a homologous recombination.

[0294] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more of the target sequence). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more, or more than about 20 nucleotides, about 26 nucleotides, about 32 nucleotides, about 45 nucleotides, about 48 nucleotides, about 54 nucleotides, about 63 nucleotides, about 67 nucleotides, about 85 nucleotides or more of the wild-type tracr sequence) may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence (e.g., 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.

[0295] One or more vectors can be introduced into a cell to drive the expression of one or more elements of the CRISPR system, so that the expression of those elements directs the formation of a CRISPR complex at one or more target sites. Also, components can be delivered to a cell as protein and / or RNA. For example, Cas enzyme, guide sequence linked to tracr mate sequence, and 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 providing 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, 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.

[0296] 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), homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database under the accession number Q99ZW2.

[0297] The CRISPR enzyme can be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some embodiments, Cpf1 (Cas12a) can be used instead of Cas9. The CRISPR enzyme can 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 complementary strand of the target sequence. The vector can encode a mutated CRISPR enzyme compared to the corresponding wild-type enzyme, where the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide that contains the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I 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, the Cas9 nickase can be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of the DNA target, respectively. This combination can be used to nick both strands and induce NHEJ or HDR.

[0298] 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 can be a cell of a particular organism (e.g., a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate) or a cell derived from those organisms. 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 that host cell, while maintaining the native amino acid sequence. Different species show a particular bias for certain codons 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 being translated and the availability of a particular transfer RNA (tRNA) molecule. The predominance of the selected tRNA in a cell usually reflects the most frequently used codon in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a given organism.

[0299] 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 a corresponding target sequence is about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or more, or more than about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or more when optimally aligned using a suitable alignment algorithm.

[0300] Optimal alignment can be determined using any suitable algorithm for aligning sequences, including, but not limited to, 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).

[0301] 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 termination 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 encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, 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 containing CRISPR enzymes are described in US Patent Publication No. 20110059502, which is incorporated herein by reference.

[0302] IX. Treatment Method In various embodiments, the CD70-targeting CAR constructs, nucleic acid sequences, vectors, and host cells, etc., as 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 in preventing, ameliorating, and / or treating cancer, including, for example, cancers that express CD70 and may or may not be solid tumors. Individuals may utilize the treatment methods of the present disclosure, for example, as a first treatment, or after (or with) another treatment, such as after HSCT. Immunotherapy methods may be tailored to the needs of individuals with cancer based on the type and / or stage of the cancer, and in at least some cases, immunotherapy may be changed for individuals during the course of treatment.

[0303] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of cells produced by the method of the present disclosure. In one embodiment, a medical disease or disorder is treated by the transfer of a cell population produced by the method herein, the cell population inducing an immune response. In certain embodiments of the present disclosure, cancer is treated by the transfer of a cell population produced by the method of the present disclosure, the cell population inducing an immune response. Provided herein is a method for treating cancer or slowing the progression of cancer in an individual, comprising administering an effective amount of CD70-specific cell therapy to the individual. The method may be applied to treat solid or hematological cancers.

[0304] Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in solid tumors or hematological tumors, etc. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, and pancreatic cancer.

[0305] In certain embodiments of the present disclosure, immune cells expressing anti-CD70 CAR are delivered to an individual in need thereof, such as an individual with cancer. The cells then boost the individual's immune system to attack cancer cells. In some cases, the individual is given the immune cells in one or more doses. If the individual is given the immune cells in more than one dose, the period between doses must be sufficient to allow time for propagation in the individual, and in specific embodiments, the period between doses is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more.

[0306] The NK cells in which the CD70-targeted CAR is utilized may, in certain embodiments, be NK, T, or invariant NKT cells engineered for cell therapy for a mammal. In such cases where the cells are NK cells, the NK cell therapy may be of any type and the NK cells may be of any type. In a specific embodiment, the cells are NK cells engineered to express one or more CD70-targeted CARs and given to an individual in a therapeutically effective amount (ranging from 103 to 1010) that ameliorates at least one symptom associated with the CD70-expressing cells in the individual. In a specific embodiment, the cells are NK cells transduced with a CD70-targeted CAR. The therapeutically effective amount may range from 10 3 ~10 pieces 10 pieces, 10 3 ~10 pieces 9 pieces, 10 3 ~10 pieces 8 pieces, 10 3 ~10 pieces7 pieces, 10 3 ~10 pieces 6 pieces, 10 3 ~10 pieces 5 pieces, 10 3 ~10 pieces 4 pieces, 10 4 ~10 pieces 10 pieces, 10 4 ~10 pieces 9 pieces, 10 4 ~10 pieces 8 pieces, 10 4 ~10 pieces 7 pieces, 10 4 ~10 pieces 6 pieces, 10 4 ~10 pieces 5 pieces, 10 5 ~10 pieces 10 pieces, 10 5 ~10 pieces 9 pieces, 10 5 ~10 pieces 8 pieces, 10 5 ~10 pieces 7 pieces, 10 5 ~10 pieces 6 pieces, 10 6 ~10 pieces 10 pieces, 10 6 ~10 pieces 9 pieces, 10 6 ~10 pieces 8 pieces, 10 6 ~10 pieces 7 pieces, 10 7 ~10 pieces 10 pieces, 10 7 ~10 pieces 9 pieces, 10 7 ~10 pieces 8 pieces, 10 8 ~10 pieces 10 pieces, 10 8 ~10 pieces 9 Pieces or 10 9 ~10 pieces 10 Thus, in certain embodiments, an individual with a CD70-positive cancer is given one or multiple therapeutically effective amounts of cells, including NK cells, expressing one or more CD70-targeted CARs.

[0307] In certain embodiments, the present disclosure contemplates, in part, cells expressing CD70 CAR, CD70-targeted CAR constructs, CD70-targeted CAR nucleic acid molecules, and CD70-targeted CAR vectors that can be administered using standard vectors and / or gene delivery systems, either alone or in any combination, and in at least some aspects together with a pharma- ceutically acceptable carrier or excipient. In certain embodiments, after administration, the nucleic acid molecule or vector may be stably integrated into the subject's genome.

[0308] In a specific embodiment, a viral vector may be used that is specific for a certain cell or tissue and persists in, for example, NK cells.Suitable pharmaceutical carriers and excipients are widely known in the art.The composition prepared according to the present disclosure can be used for preventing or treating or delaying the above-identified diseases.

[0309] 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 a cell expressing a CD70-targeting CAR, a nucleic acid sequence, a vector as contemplated herein and / or produced by a process as contemplated herein.

[0310] Possible indications for administering the exemplary CD70-targeted CAR cell composition(s) include cancerous diseases, including neoplastic diseases, including, for example, B-cell malignancies, multiple myeloma, breast cancer, or lung cancer. Exemplary indications for administering the CD70-targeted CAR cell composition(s) include cancerous diseases, including any malignancy that expresses CD70. Administration of the composition(s) of the present disclosure is useful for all stages and types of cancer, including, for example, minimal residual disease, early cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0311] Therapeutically effective amounts of the produced cells can be administered by many routes, including parenteral administration, e.g., intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intratumoral injection, intrathecal injection, intraventricular injection, via a reservoir, intraarticular injection, or by infusion.

[0312] A therapeutically effective amount of generated cells for use in adoptive cell therapy is that amount that achieves a desired effect in the subject being treated. For example, this can be the amount of immune cells necessary to inhibit the development of or cause the regression of cancer.

[0313] The cell population produced can be administered in a treatment regimen consistent with the disease, for example, in one or several doses over a period of one to several days to improve the disease state, or in regular doses over a long period of time to suppress disease progression and prevent disease recurrence. The exact dose to be used in the formulation will also depend on the route of administration and the seriousness of the disease or disorder, and must be determined according to the judgment of the physician and each patient's circumstances. The therapeutically effective amount of cells will depend on the subject being treated, the severity and type of the affliction, and the mode of administration. In some embodiments, the dose that may be used in the treatment of a human subject is about 1 ml. 2 At least 1 × 10 T cells per 3 Pieces, at least 1 × 10 4 pieces, 3.8×10 4 Pieces, at least 3.8 x 10 5 Pieces, at least 3.8 x 10 6 Pieces, at least 3.8 x 10 7 Pieces, at least 3.8 x 10 8 Pieces, at least 3.8 x 10 9 pieces, or at least 3.8 × 10 10 In certain embodiments, the dose used in treating a human subject ranges from 1 ml to 100 ml. 2 Approximately 3.8 × 10 T cells per 9 From 3.8 x 10 10In a further embodiment, the therapeutically effective amount of T cells ranges from about 5×10 6 Approximately 7.5 × 10 cells / kg body weight 8 The number of cells / kg body weight can vary, for example, up to about 2 x 10 cells / kg body weight. 7 Approximately 5 × 10 cells 8 up to 10 cells or approximately 5 x 10 cells per kg of body weight 7 Approximately 2 x 10 cells 8 The amount of T cells can vary up to 1000 cells.The exact amount of T cells can be easily determined by those skilled in the art based on the age, weight, sex and physiological condition of the subject.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0314] The present disclosure further includes co-administration protocols with other compounds that act through immune cells (e.g., bispecific antibody constructs, targeted toxins, or other compounds). Clinical treatment regimens for co-administration of the compound(s) of the present invention may include simultaneous co-administration, co-administration before or after administration of the other component. Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.

[0315] Various embodiments relate to kits comprising a CD70-targeted CAR construct as defined herein, a nucleic acid sequence as defined herein, a vector as defined herein, and / or a host as defined herein. The kits of the present disclosure are also intended to include pharmaceutical compositions as described herein above, either alone or in combination with additional pharmaceutical agents to be administered to an individual in need of medical treatment or intervention.

[0316] X. Kits of the Present Disclosure Any of the compositions described herein may be included in a kit. In a non-limiting example, the kit includes an anti-CD70 CAR molecule, a cell containing it, and / or a reagent for producing it, any of which may be included in a suitable container means in the kit of the present disclosure. The kit may include immune cells, including NK cells, vectors, expression construct polynucleotides for insertion into the vector (viral or not), and CD27 sequences of any kind and as encompassed herein, etc. Primers for amplifying any polynucleotide may be included. In some cases, the kit includes cryopreserved cells, including NK cells. Any reagent for transfection or transduction of cells may be included.

[0317] The compositions of the kit may be packaged either in aqueous medium or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which one or more components may be placed, preferably in suitable aliquots. When more than one component is present in the kit, the kit will 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 kits of the present disclosure will also typically include a means for containing the anti-CD70 CAR molecule, cells comprising same, and / or reagents for producing same in close confinement for commercial sale. Such containers may include injection molded or blow molded plastic containers into which the desired vials are retained.

[0318] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, in which case a sterile aqueous solution is particularly envisaged. The composition may also be formulated into a syringeable composition. In such a case, the container means may itself be a syringe, pipette and / or other similar device, from which the formulation may be applied to an infected area of ​​the body, may be injected into an animal body, and / or may even be added to and / or mixed with other components of the kit.

[0319] However, the components of the kit may be provided as a dry powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0320] Regardless of the number and / or type of containers, the kit of the present disclosure may also include and / or be packaged with an instrument to assist in the injection / administration and / or placement of the final composition into the animal's body. Such an instrument may be a syringe, a pipette, a forceps, and / or any such medically approved delivery vehicle. In some embodiments, various reagents or devices or containers are included in the kit for ex vivo use. EXAMPLES

[0321] The following examples are included to demonstrate preferred 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 found by the inventors to function well in the practice of the present invention, and therefore may be considered to constitute preferred modes for its practice. 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 that many changes can still produce the same or similar results without departing from the spirit and scope of the present invention.

[0322] Example 1 CD27-containing CARs to target CD70-positive tumors We first confirmed CD70 expression on AML patient samples. Tsne plots from mass cytometry data showed high expression of CD70 on primary AML samples (n=54), but not on healthy CB CD34+ cells (n=10) (Figure 1A). As shown in Figure 1B, Tsne plots also showed various myeloid markers on lineage neg cells derived from CB samples (upper CB population from Figure 1A) and AML samples (lower AML population from Figure 1A). CD70 expression in AML samples was similar to other well-characterized myeloid markers.

[0323] Figure 3 reveals that TrCD27 constructs showed higher transfection efficiency compared to flCD27 constructs. 293T cells were transfected with various CD27 constructs as shown in the figure using GeneJuice as transfection reagent. Transfection efficiency was determined by examining the surface expression of CD27 in 293T cells after virus recovery using flow cytometry for CD27 constructs. Non-transduced (NT) cells were used as control. We also included a construct containing only IL15 (i.e., IL15) and a CD70scFv CAR construct containing only one heavy and light chain, each derived from the sequence of a commercial CD70 antibody, together with IL15 (i.e., CD70-IL15). Surface expression of IgG was used to determine the transfection efficiency for the latter two constructs.

[0324] Figure 4 reveals that the TrCD27 construct showed higher transduction efficiency compared to the flCD27 construct. Retroviral supernatant collected from the transfection experiment (Figure 3) was used to transduce cord blood-derived NK (CBNK) cells, and RetroNectin was used to increase the transduction efficiency. 48 hours after transduction, the transduction efficiency was measured by examining the surface expression of CD27 on CBNK cells using flow cytometry. Supernatant from non-transduced (NT) cells was used as a control. For the IL15 CAR construct and the CD70 CAR-IL15 CAR construct, the surface expression of IgG on CBNK cells was used to determine the transduction efficiency. It was emphasized to focus on the trCD27 construct, since the flCD27 construct under these conditions shows poor transfection and transduction efficiency.

[0325] In Figures 5A-C, CBNK cells transduced with trCD27 constructs induced apoptosis of cancer cells. Annexin V-LIVE / DEAD™ Fixable Aqua staining assay was performed to examine whether CBNK cells transduced with various trCD27 constructs could induce the death of CD70-expressing cancer cells. Raji cells and Karpas cells were used as targets because both have relatively high surface expression of CD70 (Figures 5A and 5B, respectively). NK cells transduced with TrCD27 constructs were co-cultured with cancer cells at a 1:1 ratio for 4 hours, and Annexin V-LIVE / DEAD™ Fixable Aqua staining was performed. Various trCD70-transduced CBNK cells (e.g., #3, #13, #15, #17, #19, #20, etc.) exhibited superior killing of both Raji cells (black bars) and Karpas cells (gray bars) when compared to NT CBNK cells, IL15 CAR-transduced CBNK cells, or CD70-IL15 CAR-transduced CBNK cells (Figure 5C).

[0326] In Figure 6, CB-NK cells transduced with trCD27 constructs showed enhanced cytotoxic activity against cancer cells. The degranulation marker CD107a is a widely accepted marker for the cytotoxic activity of NK cells. trCD27 CAR-transduced CBNK cells were co-cultured with cancer cells (Raji and Karpas) at a 1:1 ratio in the presence of CD107a antibody for 6 hours. After incubation, CD107a expression is assessed by flow cytometry. Most of the trCD70-transduced CB-NK cells showed enhanced cytotoxic activity against Raji cells (black bars in the middle) and Karpas cells (gray bars on the right) when compared with NT CB-NK cells, IL15 CAR-transduced CB-NK cells, or CD70-IL15 CAR-transduced CB-NK cells. CB-NK cells without co-culture with cancer cells show minimal basal expression levels of CD107a (green bars on the left).

[0327] Figures 7A-7B reveal that CB-NK cells transduced with trCD27 constructs exhibit enhanced cytotoxic activity against cancer cells. To analyze the real-time cytotoxic activity of trCD27 CAR-transduced CBNK cells against cancer cells, an IncuCyte cytotoxicity assay was performed. trCD27 CAR-transduced CB-NK cells were co-cultured with Raji cells (Figure 7A) or Karpas cells (Figure 7B) at a 1:1 ratio, and the real-time cytotoxicity of NK cells against Raji cells and Karpas cells was measured every hour over a 12-hour period. All of the trCD70 CAR-transduced CB-NK cells exhibited enhanced cytotoxic activity against Raji cells (left panel) and Karpas cells (right panel) when compared to NT CB-NK cells, IL15 CAR-transduced CB-NK cells, or CD70-IL15 CAR-transduced CB-NK cells.

[0328] Figure 8A-8B reveal that CB-NK cells transduced with trCD27 CAR have effective anti-tumor activity against CD70-positive Raji cells in vivo. NSG mice were implanted with firefly luciferase-labeled Raji cells (Raji-FFluc) with high CD70 expression. Mice were injected with 20,000 Raji cells / mouse and 5M CBNK cells / mouse. Bioluminescence imaging of tumors showed that CB-NK cells transduced with trCD27 CAR could reduce tumor burden caused by CD70-high Raji cells (Figure 8A). In addition, survival curves showed a significant survival benefit for NSG mice with Raji when injected with a single dose of CB-NK cells transduced with various trCD27 CARs when compared with non-transduced (NT) cells and cells transduced with IL15 CAR or CD70-IL15 CAR (Figure 8B). The trCD27 #23 CAR construct showed the most significant survival benefit: p=0.002 (Raji+NT vs. Raji+CD27 #23); p=0.0018 (Raji+IL15 vs. Raji+CD27 #23); p=0.001 (Raji+CD70-IL15 vs. Raji+CD27 #23). The Mantel-Cox test was used to estimate p-values.

[0329] Figure 9A-9B reveal that CB-NK cells transduced with trCD27 CAR have effective antitumor activity against CD70-positive AML cells in vivo. NSG mice were implanted with firefly luciferase-labeled THP-1 cells (THP1-FFluc) with high CD70 expression. Mice were injected with 100,000 THP1 cells / mouse and 5M CBNK cells / mouse. Tumor bioluminescence imaging showed that CB-NK cells transduced with trCD27 CAR could reduce tumor burden caused by CD70-high THP-1 cells (Figure 9A). In addition, survival curves showed a significant survival benefit for THP-1 bearing NSG mice when a single dose of CB-NK cells transduced with various trCD27 CARs was injected when compared to non-transduced (NT) cells and cells transduced with IL15 CAR or CD70-IL15 CAR (Figure 9B). The trCD27 #15 and trCD27 #23 CAR constructs showed the most significant survival benefit. p=0.0027 (THP-1+NT vs. THP-1+CD27 #15 or #23); p=0.001 (THP-1+IL15 vs. THP-1+CD27 #15 or #23); p=0.001 (THP-1+CD70-IL15 vs. THP-1+CD27 #15 or #23). Mantel-Cox test was used to estimate p-values.

[0330] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made therein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, apparatus, manufacture, compositions, means, methods and steps described herein. As one skilled in the art would readily appreciate from this disclosure, any process, apparatus, manufacture, composition, means, method or step, whether currently existing or later developed, that performs substantially the same function or achieves substantially the same result as the corresponding embodiments described herein may be utilized in accordance with the present disclosure. Thus, the appended claims are intended to include such processes, apparatus, manufacture, compositions, means, methods or steps within their scope.

Claims

1. 1. A polynucleotide encoding an anti-CD70 chimeric antigen receptor (CAR) comprising a signal peptide, an anti-CD70 antigen binding domain, a transmembrane domain, and at least one intracellular domain, wherein the anti-CD70 antigen binding domain does not comprise an antibody.

2. The polynucleotide of claim 1, wherein the anti-CD70 antigen binding domain is comprised in the extracellular domain of CD27, full-length CD27, a codon-optimized CD27 extracellular domain, a codon-optimized full-length CD27, and / or comprises, consists of, or essentially consists of the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:

5.

3. the transmembrane domain is derived from CD28, the T cell receptor alpha chain, the T cell receptor beta chain, the T cell receptor zeta chain, CD3zeta, CD3epsilon, CD3gamma, CD3delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, or DAP12; and / or 3. A polynucleotide according to claim 1 or 2, comprising, consisting of or consisting essentially of the sequence of SEQ ID NO:3 or SEQ ID NO:

7.

4. said at least one intracellular domain comprising: CD3 zeta ITAM-containing signaling domain, and / or an intracellular costimulatory domain selected from the group consisting of CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, NKp80, and combinations thereof; and / or 4. The polynucleotide according to any one of claims 1 to 3, comprising, consisting of or consisting essentially of one or more of the sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

5. the signal peptide is derived from CD27 or granulocyte macrophage colony stimulating factor receptor (GMSCF-R); and / or 5. The polynucleotide according to any one of claims 1 to 4, comprising, consisting of or consisting essentially of the sequence of SEQ ID NO:

6.

6. said signal peptide comprises, consists of or consists essentially of the sequence of SEQ ID NO:6, the antigen-binding domain comprises, consists of or consists essentially of the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5; the transmembrane domain comprises, consists of, or consists essentially of the sequence of SEQ ID NO:3 or SEQ ID NO:7; and The polynucleotide of any one of claims 1 to 5, wherein the intracellular domain comprises one or more of SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

12.

7. The polynucleotide of any one of claims 1 to 6, wherein the CAR comprises one or more of the following: (a) signal peptide (SP) of CD27, extracellular domain (EC) of CD27, transmembrane domain (TMD) of CD27, intracellular domain (ICD) of DAP12, and CD3 zeta; (b) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, DAP12 ICD; (c) CD27 SP, CD27 EC, CD28 TMD, DAP12 ICD, and CD3 zeta; (d) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, DAP12 ICD, and CD3 zeta; (e) CD27 SP, CD27 EC, CD27 TMD, natural killer group 2 member D (NKG2D) ICD, and CD3 zeta; (f) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, NKG2D ICD, and CD3 zeta; (g) CD27 SP, CD27 EC, CD28 TMD, natural killer group 2 member D (NKG2D) ICD, and CD3 zeta; (h) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, NKG2D ICD, and CD3 zeta; (i) CD27 SP, CD27 EC, CD27 TMD, 4-1BB ICD, and CD3 zeta; (j) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, 4-1BB ICD, and CD3 zeta; (k) CD27 SP, CD27 EC, CD28 TMD, 4-1BB ICD, and CD3 zeta; (l) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, 4-1BB ICD, and CD3 zeta; (m) CD27 SP, CD27 EC, CD27 TMD, DAP10 ICD, and CD3 zeta; (n) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, DAP10 ICD, and CD3 zeta; (o) CD27 SP, CD27 EC, CD28 TMD, DAP10 ICD, and CD3 zeta; (p) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, DAP10 ICD, and CD3 zeta; (q) CD27 SP, CD27 full length (FL), CD27 TMD, CD27 ICD, and CD3 zeta; (r) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, and CD3 zeta; (s) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, CD28 ICD, and CD3 zeta; (t) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, CD28 ICD, and CD3 zeta; (u) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, 4-1BB ICD, and CD3 zeta; (v) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, 4-1BB ICD, and CD3 zeta; (w) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD, and CD3 zeta; (x) GMSCF-R SP, CD27 FL, CD27 TMD, CD27 ICD, DAP10 ICD, and CD3 zeta; (y) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, DAP12 ICD, and CD3 zeta; (z) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, DAP12 ICD, and CD3 zeta; (aa) CD27 SP, CD27 FL, CD27 TMD, CD27 ICD, NKG2D ICD, and CD3 zeta; (bb) GMSCF-R SP, codon-optimized CD27 FL, codon-optimized CD27 TMD, codon-optimized CD27 ICD, NKG2D ICD, and CD3 zeta; (cc) CD27 SP, CD27 EC, CD27 TMD, and CD3 zeta; (dd) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, and CD3 zeta; (ee) CD27 SP, CD27 EC, CD28 TMD, and CD3 zeta; (ff) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD28 TMD, and CD3 zeta; (gg) CD27 SP, CD27 EC, CD27 TMD, CD28 ICD, and CD3 zeta; (hh) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, CD28 ICD, and CD3 zeta; (ii) CD27 SP, CD27 EC, CD28 TMD, CD28 ICD, and CD3 zeta; or (jj) GMSCF-R SP, codon-optimized CD27 EC, CD28 TMD, and CD3 zeta.

8. The polynucleotide according to any one of claims 1 to 7, wherein the polynucleotide comprises any one of the sequences of SEQ ID NO: 1 or SEQ ID NO: 15 to 37.

9. The polynucleotide of any one of claims 1 to 8, wherein the polynucleotide comprises SEQ ID NO:

30.

10. An immune cell comprising the polynucleotide according to any one of claims 1 to 9.

11. The immune cell of claim 10, wherein the immune cell is a natural killer (NK) cell, a T cell, a gamma-delta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, or a dendritic cell.

12. The immune cell of claim 10, wherein the NK cell is derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, a cell line, or a mixture thereof, or is a CD56+ NK cell.

13. The NK cells are 13. The immune cell of claim 11 or 12, which expresses one or more exogenously provided cytokines, including IL-15, IL-2, IL-12, IL-18, IL-21, IL-7, or a combination thereof.

14. A composition comprising a cell having a polynucleotide according to any one of claims 1 to 8, the composition being used in a method for killing CD70-positive cells in an individual, the method comprising the step of administering a therapeutically effective amount of the cell to the individual.

15. The composition of claim 14, wherein the CD70 positive cells are T regulatory cells or CD70-expressing cancer cells.

16. the cell carrying the polynucleotide is a NK cell, a T cell, a gamma-delta T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, a gamma-delta T cell, or a dendritic cell; The NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, a cell line, or a mixture thereof; and / or Allogeneic or autologous to the individual; 16. The composition according to claim 14 or 15.

17. the composition is administered to the individual one or more times, and where, if cells carrying the polynucleotide are administered one or more times, the duration of time between administrations of the cells to the individual is 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, or 1 year or more; and 17. The composition of any one of claims 14 to 16, administered by injection, intravenously, intraarterially, intraperitoneally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, topically, by perfusion, into the tumor microenvironment, or a combination thereof.

18. The composition of any one of claims 14 to 17, wherein the composition is administered in combination with an effective amount of a further treatment, the further treatment comprising an antibody, surgery, gene therapy, immunotherapy or hormone therapy.

19. the individual is a human, CD70-expressing cancer; and / or have acute myeloid leukemia, lymphoma, lung cancer, kidney cancer, bladder cancer, melanoma, glioblastoma, breast cancer, head and neck cancer, mesothelioma, multiple myeloma, pancreatic cancer, or a combination thereof; The composition according to any one of claims 14 to 18.

20. The composition of any one of claims 14 to 19, wherein CD70 positive cells are identified in the individual.

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