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 of U.S. Provisional Application No. 63 / 141,016, filed on January 25, 2021, and U.S. Provisional Application No. 63 / 270,414, filed on October 21, 2021, the entire disclosures of both of which are incorporated herein by reference.

[0002] (Sequence Listing) This application includes a sequence listing submitted in ASCII format, the entire disclosure of which is incorporated herein by reference. 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 fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine.

Background Art

[0004] Adoptive cancer immunotherapy by gene reprogramming of natural killer (NK) cells has clinically relevant applications and advantages such as 1) innate antitumor surveillance that does not require prior sensitization, 2) an allogeneic transplantation effect without graft-versus-host reactivity, and 3) direct cell-mediated cytotoxicity and cytolysis of target tumors. The development and self-tolerance, alloreactivity, and acquisition of effector functions of human NK cells are adaptive processes of licensing, calibration, and targeting. At the molecular level, specific activating and inhibitory receptors aggregate extracellular signals, balance them, and integrate them into different effector functions to direct the functions of NK cells. The functional activity and responsiveness of NK cells to external stimuli follow a "rheostat" model of continuous education and are thus reprogrammable. Genetic modification to direct the effector functions of NK cells is an effective way to utilize the cytotoxic ability to kill tumor cells.

[0005] CD70, the ligand of cytokine receptor CD27, is expressed not only in hematological malignancies such as acute myeloid leukemia (AML) and lymphoma, but also in many solid cancers such as kidney cancer, bladder cancer, lung cancer, triple-negative breast cancer, renal cell carcinoma, pancreatic cancer, and melanoma. Since it is only transiently observed in activated T lymphocytes, B lymphocytes, and dendritic cells, it is an attractive "pan-cancer antigen". Unlike other AML targets, CD70 is not expressed in normal hematopoietic stem cells. Therefore, in contrast to most current clinical CAR-T therapies for AML, cytopenia persists after CAR therapy, and the likelihood of the recipient requiring hematopoietic stem cell transplantation is low. Thus, it is particularly advantageous as a target for AML immunotherapy. This disclosure provides solutions to long-felt needs in the technical field of cancer treatment for CD70-positive cancers.

Summary of the Invention

[0006] This disclosure is directed to methods and compositions for the treatment or prevention of cancers having CD70-expressing cells. This disclosure relates to specific anti-CD70 chimeric antigen receptors (CARs), and their use for targeting all types of CD70-positive cancers. In certain embodiments, the antigen-binding domain targeting CD70, including upon specific binding of CD70, is not an antibody. In certain embodiments, the anti-CD70 CAR lacks all types of antibodies and includes an scFv that binds to CD70. In certain embodiments, the extracellular domain of the anti-CD70 CAR does not include an scFv or other type of antibody.

[0007] In certain embodiments, the antigen-binding domain targeting CD70 is a native receptor of CD70, such as the receptor CD27. In certain embodiments, some or all of CD27 is incorporated into the CAR molecule. In embodiments of this disclosure, the antigen-binding domain present in the anti-CD70 CAR molecule consists of some or all of the extracellular domain of CD27, and in certain embodiments, the CAR molecule may or may not utilize the transmembrane domain of CD27. In some cases, the CD28 transmembrane domain is incorporated instead of the CD27 transmembrane domain, even though the extracellular domain of CD27 is used in the CAR. Certain intracellular domains, such as CD3zeta alone or a combination of CD3zeta and DAP10 or DAP12, may be utilized in the CAR, and in some cases, the intracellular domain does not include the 4-1BB domain.

[0008] Embodiments of this disclosure include any polynucleotide encoding part or all of an anti-CD70 CAR. Specific embodiments include a polynucleotide encoding an anti-CD70 CAR comprising an antigen-binding domain, a transmembrane domain, and at least one intracellular domain (including one or more costimulatory domains), wherein the CAR comprises an antigen-binding domain that does not contain an antibody. A vector is envisioned that contains the anti-CD70 CAR and optionally an expression construct encoding another gene. The vector may or may not be viral. Cells containing the vector are also envisioned and may include any type of immune cell, such as at least NK cells and T cells. In some cases, the cells may be 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 for treating or preventing any type of cancer are included herein, including those by administering cells expressing a specific anti-CD70 CAR in a therapeutically effective dose, for the purpose of improving or preventing cancer, reducing the risk of cancer, reducing the severity of cancer, preventing metastasis or its risk, or delaying the onset of cancer.

[0010] Embodiments of this disclosure include methods for targeting CD70-expressing cells, in which case the cells are cancerous, but in other cases the CD70-expressing cells are immunomodulatory cells such as Tregs.

[0011] This disclosure includes methods for genetically engineering human NK cells (including those derived from umbilical cord blood (CB)) to target CD70+ve tumors using CD27-ectodomain CARs. Included herein are various novel CAR constructs that support NK cell survival and proliferation by fusing the extracellular binding portion of CD27 (CD27eCAR, or CD27 EC) with CD3ζ alone, or with an activation signaling endodomain combined with a co-stimulatory or adapter signaling domain such as 4-1BB, CD28, DAP10, or DAP12, or with a cytokine gene such as IL15, IL-12, IL-18, or IL-21. A series of in vitro studies have confirmed the activity of CD27eCAR / IL-15-transformed CB-NK cells against AML and lymphoma targets.

[0012] Embodiments of this disclosure include polynucleotides 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 contain 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 is composed of an extracellular domain of CD27 and / or can be codon-optimized. In certain embodiments, the antigen-binding domain includes, consists of, or is essentially composed of SEQ ID NO: 2. In some embodiments, the antigen-binding domain and the transmembrane domain include, consist of, or is essentially composed of SEQ ID NO: 1. The sequence encoding the transmembrane domain may be codon-optimized, and / or the transmembrane domain may be any of the following: CD28, the α chain of the T cell receptor, the β chain of the T cell receptor, the zeta chain of the T cell receptor, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, or DAP12. In certain embodiments, the CD28 transmembrane domain may include, consist of, or essentially consist of SEQ ID NO: 3. The CAR may contain two or more intracellular domains, or three or more intracellular domains. The intracellular domains may contain any ITAM-containing signaling domain. The intracellular domains may contain CD3 zeta. The intracellular domain may be an intracellular co-stimulatory 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.

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

[0014] In certain embodiments, a polynucleotide encodes a CAR, and 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 CD3 zeta; (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 CD3 zeta; (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 CD3 zeta; (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) GMSCF-R SP, codon-optimized CD27 EC, codon-optimized CD27 TMD, CD28 ICD, and CD3zeta; (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] Embodiments of this disclosure include cells containing any polynucleotides incorporated 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, immutable NKT (iNKT) cells, B cells, macrophages, MSCs, or dendritic cells. If the cells are NK cells, they may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic stem cells, cell lines, or mixtures thereof. The NK cell line may be the NK-92 cell line, other NK cell lines derived from tumor 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] Embodiments of the present disclosure include a population of immune cells of the present disclosure, the cells present in a suitable culture medium. Specifically, the immune cells are NK cells. The immune cells may or may not be obtained from cryopreservation.

[0017] In certain embodiments, there are methods for killing CD70-positive cells in an individual, comprising the step of administering to the individual a therapeutically effective amount of cells possessing any polynucleotides as incorporated herein. The cells may be NK cells, T cells, gamma delta T cells, immutable 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 may not be cancer cells but T regulatory cells. The individual has or is suspected of having a cancer that expresses CD70. For example, the individual has or is suspected of having 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 cells may be homogeneous or autologous with respect to the individual. The individual may be human. In some embodiments, the cells are administered to the individual once or more times. The interval between administrations of cells to the individual may be 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or more than 1 year.

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

[0019] In certain embodiments, cells are administered to an individual by injection, intravenous, intra-arterial, intraperitoneal, intratracheal, intratumoral, intramuscular, endoscopic, intravesical, intracranial, percutaneous, subcutaneous, local, perfusion, within the tumor microenvironment, or a combination thereof. In certain embodiments, the method further includes the step of identifying CD70-positive cells within the individual.

[0020] The features and technical advantages of this disclosure have been outlined rather broadly above, in order to better facilitate understanding of the detailed description below. The following describes additional features and advantages that form the subject matter of the claims herein. Those skilled in the art will understand that the disclosed ideas and specific embodiments can readily be used as a basis for modifying or designing other structures to accomplish the same purpose as the present design. They will also understand that such equivalent structures will not deviate from the spirit and scope set forth in the appended claims. Novel features of the designs disclosed herein, along with further purposes and advantages, will be better understood from the following description when considered in conjunction with the accompanying figures, both in terms of organization and operation. However, it should be explicitly understood that each figure is provided for illustrative and explanatory purposes only and is not intended to define the limits of this disclosure.

[0021] For a more complete understanding of this disclosure, please refer here to the following description taken with the attached drawings. [Brief explanation of the drawing]

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

[0023] [Figure 2] Figures 2A-2E show various anti-CD70 CAR structures.

[0024] The following table provides information regarding the section (tr) and full-length (fl) CD27 constructs included herein, as well as the respective reference numbers used in the data. S.No refers to the serial number. [Table 1] [Table 2]

[0025] Regarding this table, the following information pertains to the tr and fl CD27 structures, as well as the respective identification numbers used in the figures:

[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] [Figure 3] Figure 3 shows the transfection efficiencies of cleaved (tr) and full-length (fl) CD27 constructs in 293T cells.

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

[0038] [Figure 5]Figure 5A reveals CD70 expression on the surface of tumor target, CD70-positive Raji cells. Figure 5B reveals CD70 expression on the surface of tumor target, CD70-positive Karpas cells. Figure 5C reveals the killing activity of umbilical cord blood natural killer (CB-NK) cells transduced with various trCD27 constructs against CD70-positive Raji and CD70-positive Karpas cells using a 4-hour annexin V assay. Figure 5C shows that various trCD70-transduced CB-NK cells exhibit better killing of both Raji cells (black bars) and Karpas cells (gray bars) when compared to NT (non-transduced) CBNK cells, IL15 (construct with only the 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 illustrates CD107a degranulation (a marker of NK cell cytotoxicity) by CB-NK cells transduced with various trCD27 constructs into Raji and Karpas cells. trCD27 CAR-transduced CBNK cells and cancer cells (Raji and Karpas) were co-cultured in a 1:1 ratio in the presence of CD107a antibody for 6 hours. After incubation, CD107a expression was evaluated by flow cytometry. In the three bars forming a group, the left (green) bar represents the control, consisting of CB-NK cells not co-cultured with cancer cells; the center (black) bar represents trCD70-transduced CB-NK cells co-cultured with Raji cells; and the right (gray) bar represents trCD70-transduced CB-NK cells co-cultured with Karpas cells. Most 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.

[0040] [Figure 7] Figure 7A shows the killing activity of CB-NK cells transduced with various trCD27 constructs against Raji cells, using Incucyte live imaging. Figure 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 cells or Karpas cells in a 1:1 ratio, and the real-time cytotoxicity of NK cells against Raji and Karpas cells was measured hourly over a 12-hour period. trCD27-transduced CB-NK cells showed enhanced cytotoxicity 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 antitumor activity of CB-NK cells transduced with various trCD27 constructs against Raji cells with high CD70 expression. Figure 8B shows the in vivo antitumor 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 survival curves 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 survival curves after injection of CB-NK cells transduced with various trCD27 CARs. [Modes for carrying out the invention]

[0043] In accordance with long-standing patent law practice, the words “a” and “an,” when used herein in conjunction with the word “comprising,” including in the claims, mean “one or more.” Some embodiments of the Disclosure may consist of one or more elements, process steps and / or methods of the Disclosure, or may be essentially composed of one or more elements, process steps and / or methods of the Disclosure. Any method or composition described herein is intended to be practiced with respect to any other method or composition described herein, and various embodiments may be combined.

[0044] Throughout this specification, unless otherwise required by context, the words “comprise” (inclusive), “comprises” (inclusive), and “comprising” (inclusive) shall be understood to mean that they include the specified process or element or group of processes or elements, but do not exclude any other process or element or group of processes or elements. “Consisting of” (consisting of) means that whatever follows the phrase “consisting of” is included and limited thereto. Thus, the phrase “consisting of” (consisting of) indicates that the enumerated elements are required or essential, and that other elements are not necessary. “Consisting essentially of” (consisting essentially of) means that any element enumerated after this phrase is included and limited to other elements that do not interfere with or contribute to the activity or effect specified in the disclosure for the enumerated elements. Therefore, the phrase "consisting essentially of" indicates that the enumerated elements are required or essential, but the other elements are optional and may or may not be present depending on whether or not they affect the activity or function of the enumerated elements.

[0045] Throughout this specification, references to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,” or any combination thereof, mean that the specific features, structures, or characteristics described in relation to such embodiment are included in at least one embodiment of the present invention. Therefore, not all occurrences of the aforementioned phrases in various places throughout this specification necessarily refer to the same embodiment. Furthermore, such specific features, structures, or characteristics may be combined in any preferred manner in one or more embodiments.

[0046] Where used herein, the terms “or” and “and / or” are used to describe multiple components in combination or mutually exclusive. For example, “x, y and / or z” can mean “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 particularly intended that x, y or z may be excluded from a particular embodiment.

[0047] Throughout this application, the term “about” is used in accordance with its plain and common sense meaning in the fields of cell biology and molecular biology to indicate that a given value includes the standard deviation of errors in the device or method used each time to obtain that value.

[0048] The term “manipulated” as used herein refers to entities produced by human hands, including cells, nucleic acids, polypeptides, and vectors. In at least some cases, the manipulated entities are synthetic and include elements that do not exist in nature and are not composed in the manner used herein. In specific embodiments, vectors are manipulated by recombinant nucleic acid technology, and cells are manipulated by transfection or transduction with the manipulated vector.

[0049] As used herein, “prevent” and similar terms (e.g., “prevented,” “preventing,” etc.) refer to approaches to prevent a disease or condition (e.g., cancer), to suppress a disease or condition (e.g., cancer), or to reduce the likelihood of a disease or condition (e.g., cancer) occurring or recurring. 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 severity, impact, symptoms, and / or burden of a disease or condition prior to its onset or recurrence.

[0050] The term “sample” (or “specimen”), as used herein, generally refers to a biological sample. A sample may be obtained from tissue or cells from an individual. In some cases, a sample may include, or be derived from, tissue biopsies, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, or waste tissue. A sample may be isolated from its source before collection. Examples, but not limited to, include blood (including, but not limited to, umbilical cord blood), serum, plasma, cerebrospinal fluid, pleural fluid, amniotic fluid, lymph, saliva, urine, feces, tears, sweat, bone marrow or mucosal exudate, and other bodily fluids isolated from their primary source before collection. In some cases, a sample is isolated from its primary source (cells, tissues, bodily fluids (e.g., blood), environmental samples, etc.) during the sample preparation period. A sample may or may not be purified from its primary source, or may or may not be concentrated. In some cases, the primary source is homogenized prior to further processing. Samples may be filtered or centrifuged to remove pia membrane, lipids, or particulate matter. Samples may also be purified or concentrated for nucleic acids or treated with RNases. Samples may contain intact, fragmented, or partially degraded tissue or cells.

[0051] Where used herein, the term “subject” generally refers to an individual having a biological sample being processed or analyzed, and in specific cases having or being suspected of having cancer. A subject can be any biological or animal subject that is the subject of a method or material, including mammals, e.g., humans; laboratory animals (e.g., primates, rats, mice, rabbits); livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens); domestic pets (e.g., dogs, cats, and rodents); horses; and genetically modified non-human animals. A subject can be a patient, e.g., a patient having or being suspected of having a disease (e.g., a benign or malignant neoplasm, i.e., cancer) (which may be indicated as a medical condition). A subject may be currently receiving or may have received treatment. A subject may be asymptomatic. A subject may be a healthy individual, but one who desires cancer prevention. The term “individual” may be used interchangeably in at least some cases. Where used herein, “subject” or “individual” may or may not be housed in a medical facility, and may be treated as an outpatient of a medical facility. An individual may receive one or more medical compositions via the internet. An individual may include human or non-human animals of any age, and therefore individuals may include both adults, juveniles (i.e., children), and infants, and individuals in utero. The term is not intended to imply the need for medical treatment, and therefore individuals may voluntarily or involuntarily become part of an experiment, whether clinical or in support of basic scientific research.

[0052] As used herein, “treatment” or “treating” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment may, where necessary, involve either alleviation or improvement of the symptoms of the disease or condition, or delaying the progression of the disease or condition. “Treatment” does not necessarily imply the complete eradication or cure of the disease or condition or its associated symptoms.

[0053] This disclosure includes targeting CD70-expressing cancers using anti-CD70 CARs, in some cases where the CARs are expressed on the surface of NK cells derived from umbilical cord blood. Numerous novel CAR molecules are described herein, which are constructed by fusing an extracellular binding portion of CD27 (CD27eCAR) with a signaling end domain for activation that incorporates CD3ζ either alone or in combination with a co-stimulatory signaling domain or an adapter signaling domain (e.g., 4-1BB, CD28, DAP10, or DAP12), and in at least some cases with a cytokine gene (e.g., IL-15, IL-12, IL-18, or IL-21). In some embodiments, the CAR is a fusion of the extracellular domain of CD27 (a native ligand for CD70) that is specific to the human CD70 antigen. In addition, in some cases, codon optimization has been used to improve the surface expression of CD27 on the surface of transduced cells. In some cases, vectors containing expression constructs encoding anti-CD70 CARs also carry cytokine genes, such as human interleukin-15 (IL-15), IL-12, IL-18, or IL-21, to help the survival and maintenance of the vector-receiving cells, including NK cells, for example.

[0054] I. Chimeric antigen receptor (CAR) The immune cells of this disclosure may be genetically engineered to express one or more antigen-binding receptors (e.g., engineered CARs) that target one or more antigens (e.g., CD70). For example, immune cells may be modified to express a CAR that is antigen-specific to CD70. Other CARs may be expressed by the same cells as the CD70 antigen receptor-expressing cells, and they may be directed to different antigens. In some cases, immune cells may be engineered to express CD70-specific CARs, for example, by knock-in of CARs using CRISPR / Cas technology.

[0055] Various suitable methods for modifying cells are known in this art. See, for example, Sambrook and Ausubel (see above). For example, cells may be transduced to express CARs that are antigen-specific for cancer antigens using transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0056] In some embodiments, the cells include one or more nucleic acids introduced by genetic engineering that encode one or more antigen-targeting receptors (at least one of which may be directed toward CD70), and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterogeneous, i.e., not normally present in the cells or samples obtained from the cells, and are, for example, nucleic acids obtained from another organism or cell, such as nucleic acids not normally found in the cells being engineered and / or in the organism from which such cells originate. In some embodiments, the nucleic acids are not naturally occurring, for example, nucleic acids not found in nature (e.g., chimeras).

[0057] This disclosure encompasses CD70-specific CARs comprising a) an extracellular domain including an antigen-binding region targeting 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, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR, which includes an antigen-binding region or antigen-binding domain. In one embodiment, a transmembrane domain inherently associated with one of the domains in the CAR is used, but in other embodiments, no such transmembrane domain is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to minimize interaction with other components of the receptor complex by avoiding binding of such domain to transmembrane domains of the same or different surface membrane proteins.

[0058] This disclosure relates to an anti-CD70 CAR that includes an antigen-binding domain without the use of an antibody or antibody fragment. In certain embodiments, instead of an anti-CD70 CAR that utilizes an antibody or antibody fragment as the antigen-binding domain, the CAR utilizes part or all of CD27 in the CAR, including in some cases using the extracellular domain of CD27 as the antigen-binding domain of the CAR.

[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 number NG_031995.1, and is incorporated herein by reference in its entirety. The nucleotide sequence encoding the entire CD27 protein includes the following sequence:

[0060] (Sequence ID 9)

[0061] Any polynucleotide included in this disclosure may utilize sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 9.

[0062] An example of a complete wild-type CD27 protein sequence is NCBI GenBank® accession number P26842 (which is also identical to the amino acid sequence in accession number 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] (Sequence No. 4)

[0064] In certain embodiments, part or all of SEQ ID NO: 4 is utilized in the CAR. In certain embodiments, the full-length wild-type (or codon-optimized) CD27 protein is utilized in the CAR, while in other embodiments, a truncated form of the wild-type CD27 protein is utilized as the antigen-binding domain in the CAR. In specific examples, part or all of the extracellular domain of CD27 is used, so that the truncated CD27 used as the antigen-binding domain is the truncated form 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 includes the following sequence:

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

[0067] Any polynucleotide included by this disclosure may utilize sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 63.

[0068] The polypeptide sequence encoding the truncated form of the wild-type CD27 protein includes the following sequence:

[0069] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR(Sequence ID 5)

[0070] Any polypeptide included by this disclosure may utilize sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to Sequence ID No. 5.

[0071] In specific cases where the full-length CD27 protein is used in a CAR molecule, additional transmembrane domains are not utilized. In specific cases where the full-length CD27 protein is used in a CAR molecule, additional intracellular signaling domains may or may not be utilized in the CAR, because the full-length CD27 protein contains its own intracellular signaling domain. In specific cases where the full-length CD27 protein is used in a CAR molecule, additional signaling peptides may or may not be utilized, even though the full-length CD27 protein contains its own signaling peptide. In certain embodiments, CD3 zeta (and / or DAP10, DAP12, 2B4, CD28, NKG2D, 41BB, or any ITAM-containing signaling domain) is utilized in the CAR.

[0072] In one example, the CD27 region used as the antigen-binding domain in the CAR molecule contains amino acids 1-211 of SEQ ID NO: 4 (including the TMD of CD27), or consists of amino acids 1-211 of SEQ ID NO: 4 (including the TMD of CD27), or essentially consists of amino acids 1-211 of SEQ ID NO: 4 (including the TMD of CD27). In another example, the CD27 region used as the antigen-binding domain in the CAR molecule contains amino acids 1-191 of SEQ ID NO: 4 (without the TMD of CD27), or consists of amino acids 1-191 of SEQ ID NO: 4 (without the TMD of CD27), or essentially consists of amino acids 1-191 of SEQ ID NO: 4 (without the TMD of CD27). In a specific example, the CD27 region used in the CAR molecule contains, consists of, or is essentially composed of the following amino acids: 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 ~241, 1~242, 1~243, 1~244, 1~245, 1~246, 1~247, 1~248, 1~249, 1~250, 1~251, 1~252, 1~253, 1~254, 1~255, 1~256, 1~57, 1~258, 1~259, or the whole (1~260); in specific embodiments, such amino acids in these ranges are consecutive. In some embodiments, regions of SEQ ID NO: 4 having N-terminal shortenings, for example, shortenings 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 are utilized. In certain cases, the N-terminus may contain shortenings 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, as well as shortenings at the C-terminus, such as the shortening endpoints listed above.

[0073] In certain embodiments, any region of the CAR molecule is codon-optimized. In some embodiments, the antigen-binding domain includes any of the examples of CD27 sequences contained herein, but is codon-optimized. In specific cases, the antigen-binding domain includes a shortened C-terminal CD27 (e.g., the extracellular domain) and is also codon-optimized. In certain aspects, the antigen-binding domain includes amino acids 1-191 of CD27, or consists of amino acids 1-191 of CD27, or is essentially composed of amino acids 1-191 of CD27, and is also codon-optimized. In other aspects, the antigen-binding domain and transmembrane domain include amino acids 1-211 of CD27, or consists of amino acids 1-211 of CD27, or is essentially composed of amino acids 1-211 of CD27, and is also codon-optimized.

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

[0075] In some cases, the signal peptide of CD27 is used in the CAR (MARPHPWWLCVLGTLVGLS(SEQ ID NO: 6);ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCA(SEQ ID NO: 61)), or a sequence is used that has 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. In some cases, the signal peptide of GMCSF-R is used in the CAR (MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 14);ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA (SEQ ID NO: 62)), or a sequence is used that has 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.

[0076] CAR may or may not contain the TM of CD27 (ILVIFSGMFLVFTLAGALFLH (Sequence ID 7); ATCCTTGTGATCTTCTCTGGAATGTTCCTTGTTTTCACCCTGGCCGGGGCCCTGTTCCTCCAT (Sequence ID 66)), or may or may not contain sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to Sequence ID 7 or Sequence ID 66.

[0077] CAR may or may not contain the ICD of CD27 which is QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (Sequence ID 8), or a sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to Sequence ID 8.

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

[0079] In some embodiments, the ICD of CD28 (RSKRSRLLHSD YMNMTPRRPGPTRKHYQPYA PPRDFAAYRS; SEQ ID NO: 11) 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: 11 is used.

[0080] In some embodiments, the ICD(KRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEEGGCEL(SEQ ID NO: 12);AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO: 68)) of 4-1BB 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: 12 or SEQ ID NO: 68 is used.

[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 is used which has 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.

[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 is used that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 71 or SEQ ID NO: 74.

[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 is used that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 70 or SEQ ID NO: 73.

[0084] In certain embodiments, codon-optimized CD27 is used, one example being:

[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 included by this disclosure may utilize sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 13.

[0087] In certain embodiments, codon-optimized extracellular domains of CD27 are utilized, one example being:

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

[0089] Any polynucleotide included by this disclosure may utilize sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 64.

[0090] Figures 2A to 2E provide specific examples of CAR constructs containing particular components. These are specific examples in which the CAR construct may contain full-length CD27 (FL; all 260 amino acids). When full-length CD27 is not available as an alternative, the extracellular domain of CD27 (CD27 EC) may be used, which is residues 20 to 191 of CD27. In certain embodiments, when a shortened CD27 is used in the CAR, the intracellular costimulatory domain (ICD) of CD27 may not be available in the CAR, and / or the transmembrane domain (TMD) of CD27 may not be available in the CAR.

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

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

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

[0094] Example (21) in Figure 2D includes the SP of CD27, the EC of CD27, the TMD of CD27, the ICD of 4-1BB, and the CD3 zeta. Example (22) in Figure 2D includes the SP of GMSCF-R, the EC of codon-optimized CD27, the TMD of codon-optimized CD27, the ICD of 4-1BB, and the CD3 zeta. Example (23) in Figure 2D includes the SP of CD27, the EC of CD27, the TMD of CD28, the ICD of 4-1BB, and the CD3 zeta. Example (24) in Figure 2D includes the SP of GMSCF-R, the EC of codon-optimized CD27, the TMD of CD28, the ICD of 4-1BB, and the CD3 zeta. Example (25) in Figure 2D includes the SP of CD27, the EC of CD27, the TMD of CD27, the ICD of DAP10, and the CD3 zeta. Example (26) in Figure 2D includes the SP of GMSCF-R, the EC of codon-optimized CD27, the TMD of codon-optimized CD27, the ICD of DAP10, and the 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 the CD3 zeta. Example (28) in Figure 2D includes the SP of GMSCF-R, the EC of codon-optimized CD27, the TMD of CD28, the ICD of DAP10, and the 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 the 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 the 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 the 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 the 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 the CD3 zeta. Example (34) in Figure 2E includes the SP of GMSCF-R, the codon-optimized EC of CD27, the TMD of CD28, the ICD of NKG2D, and the CD3 zeta. Example (35) in Figure 2E includes the SP of CD27, the EC of CD27, the TMD of CD28, the ICD of NKG2D, and the CD3 zeta. Example (36) in Figure 2E includes the SP of GMSCF-R, the codon-optimized EC of CD27, the TMD of CD28, the ICD of NKG2D, and the CD3 zeta.

[0096] An example of an anti-CD70 CAR of this disclosure includes the following Sequence ID No. 1, which contains the transmembrane domain of CD28 (underlined in Sequence ID No. 1 below) and the extracellular domain of CD27 (ununderlined in Sequence ID No. 1 below), functionally linked to the signal peptide of CD27 (bold in Sequence ID No. 1 below):

[0097] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV(Sequence ID 1)

[0098] The extracellular domain of CD27 in Sequence ID No. 1 contains the following sequence as Sequence ID No. 2: MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR

[0099] The CD28 transmembrane domain of Sequence ID 1 contains the following sequence as Sequence ID 3:FWVLVVVGGVLACYSLLVTVAFIIFWV(TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(Sequence ID 65)).

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

[0101] This disclosure provides cells that accept a vector encoding at least one CAR (including immune cells such as NK cells), where the CAR may be, for example, a first-generation, second-generation, third-generation, or later generation. The CAR may be bispecific or non-bispecific for two or more different antigens, one of which is CD70. In certain embodiments, the CAR is not a bivalent tandem CAR. The CAR may contain one or more co-stimulatory domains. For example, each co-stimulatory domain may contain one or more co-stimulatory domains, for example, members 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 a combination thereof. In specific embodiments, the CAR contains a CD3 zeta. In certain embodiments, the CAR lacks one or more specific co-stimulatory domains; for example, the CAR may lack 4-1BB and / or CD28.

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

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

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

[0105] In some embodiments, the transmembrane domains can be obtained from either natural or synthetic sources. When the source is natural, the domains in some aspects are derived from any membrane-bound or transmembrane protein. Transmembrane regions include transmembrane regions derived from the alpha, beta, or zeta chains of T cell receptors, 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 (e.g., DAP10 or DAP12, etc.) (i.e., membrane The transmembrane domain includes at least one or more transmembrane domains of cell receptors (alpha, beta, or zeta chains), 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 (e.g., DAP10 or DAP12). Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain mainly consists of hydrophobic residues (e.g., leucine and valine). In some aspects, a triplet of phenylalanine, tryptophan, and valine may be found at the respective ends of the synthetic transmembrane domain.

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

[0107] In certain embodiments, the anti-CD70 CAR may have a nucleotide sequence less than or equal to:

[0108] CD27tr28tdmCD3zIL15:

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

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

[0111] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV (Sequence ID: 38)

[0112] CD27Tr28tmd41BBicd3zIL15:

[0113]

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

[0115] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 39)

[0116] GSPco27Tr28tmd41BBicCD3zIL15:

[0117]

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

[0119] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 40)

[0120] CD27Tr28tmdDAP10icd3z15:

[0121]

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

[0123] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKD CDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVGGVLACYSLLVTVAFI IFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 41)

[0124] GSPco27Tr28tmdDAP10IL15:

[0125]

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

[0127] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLV KDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAF IIFWVLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 42)

[0128] CD27Tr28tmdDAP12icd3z15:

[0129]

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

[0131] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKA AQCDPCIPGVFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPRG RGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 43)

[0132] GSPco27Tr28tmddap12icd15:

[0133]

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

[0135] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVYFLGRLVPR GRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 44)

[0136] CD27Tr28tmdNKG2Dic3z15:

[0137]

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

[0139] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKA AQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKSKV VPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 45)

[0140] GSPco27Tr28tmdNKG2Dicd3z15:

[0141]

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

[0143] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVSANERCKS KVVPCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 46)

[0144] CD27Tr41BBicd3z15:

[0145]

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

[0147] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 47)

[0148] GSPco27Tr41BBicd3z15:

[0149]

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

[0151] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDC DQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 48)

[0152] CD27TrCD3ZIL15:

[0153]

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

[0155] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCE PGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGM FLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 49)

[0156] GSPco27Tr3z15:

[0157]

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

[0159] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQM CEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSG MFLVFTLAGALFLHKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 50)

[0160] CD27TrCD28icd3z15:

[0161]

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

[0163] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 51)

[0164] GSPco27Tr28CD3z15:

[0165]

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

[0167] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKD CDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHRSKRSR LLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 52)

[0168] CD27TrCD28tmdicd3z15:

[0169]

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

[0171] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQH RKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 53)

[0172] GSPco27Tr28tmdicCD3z15:

[0173]

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

[0175] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCD QHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 54)

[0176] CD27TrDAP10icd3z15:

[0177]

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

[0179] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLV KDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFL HLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 55)

[0180] GSPco27FLdap10icd3z15:

[0181]

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

[0183] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTF LVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALF LHLCARPRRSPAQEDGKVYINMPGRGKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 56)

[0184] CD27TrDAP12icd3z15:

[0185]

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

[0187] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRGA AEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 57)

[0188] GSPco27FLdap12icd3z15:

[0189]

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

[0191] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQ HRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHYFLGRLVPRGRG AAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 58)

[0192] CD27TrNKG2Dic3z15:

[0193]

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

[0195] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVVPC RQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 59)

[0196] GSPco27TrNKG2Dicd3z15:

[0197]

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

[0199] MTRVTNSPSLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWKGPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLPCSRMLLLVTSLLLCELPHPAFLLIPATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQ HRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHSANERCKSKVV PCRQKQWRTSFDSKKLDLNYNHFESMEWSHRSRRGRIWGMKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (Sequence ID: 60)

[0200] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source or a cDNA source, or it 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 preferable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA. Furthermore, it may be even more advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0201] The chimeric construct is intended to be introduced into any type of immune cell, either as naked DNA or in a suitable vector. Various methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in an appropriate orientation for expression.

[0202] Alternatively, viral vectors (e.g., retroviral vectors, adenovirus vectors, adeno-associated virus vectors, or lentiviral vectors) can be used to introduce chimeric constructs into immune cells. Suitable vectors for use according to the methods of this disclosure are non-replicating in immune cells. Many virus-based vectors are known in which the number of viral copies maintained in the cell is sufficiently low to maintain cell viability: for example, vectors based on HIV, SV40, EBV, HSV, or BPV. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

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

[0204] One or more cytokines may be co-expressed from a vector as polypeptides distinct from the antigen receptor. For example, interleukin-15 (IL-15) is tissue-limited and is found in serum or systemically at any level only under pathological conditions. IL-15 possesses several desirable attributes for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and proliferation of natural killer cells, promotes the eradication of established tumors by mitigating the functional suppression of tumor resident cells, and inhibits activation-induced cell death (AICD). In addition to IL-15, other cytokines are conceivable. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used for human application. NK cells expressing IL-15 are capable of uninterrupted supportive cytokine signaling useful for their survival after injection.

[0205] In specific embodiments, cells express one or more exogenously provided cytokines. For example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, GMCSF, or a combination thereof. Another example is the cytokine IL-21. Another example is the cytokine IL-12. Since cytokines are expressed from intracellular expression vectors, they may be exogenously provided to NK cells. Alternatively, endogenous cytokines in cells may be upregulated by manipulating the regulation of endogenous cytokine expression, for example, by genetic recombination at the promoter site(s) of the cytokine. When cytokines are provided to cells in expression constructs, the cytokines may be encoded from the same vector as the suicide gene and / or anti-CD70 CAR. In some embodiments, this disclosure relates to the co-utilization of the CAR with IL-15. In some embodiments, this disclosure relates to the co-utilization of the CAR with IL-21. In some embodiments, this disclosure relates to the co-utilization of the CAR with IL-12.

[0206] III. Suicide Genes In certain embodiments, suicide genes are used in conjunction with anti-CD70 cell therapy to suppress the use of anti-CD70 cell therapy and to prepare for terminating cell therapy under desired circumstances and / or at desired time. Suicide genes are used in transdextrins for the purpose of inducing death in transdextrins when necessary. Cells of this disclosure that have been modified to accept vectors encompassed by this disclosure may contain one or more suicide genes. In some embodiments, the term “suicide gene” is defined, as used herein, as a gene that, when administered with a prodrug or other agent, results in the transfer of its gene product to a compound that kills its host cell. In other embodiments, the suicide gene encodes a gene product that, when desired, is targeted by an agent that targets the suicide gene product (e.g., an antibody).

[0207] In some cases, when an individual receiving and / or previously receiving cell therapy exhibits one or more symptoms of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or toxicity to the target / extratumor toxicity, or when there is a risk of having one or more symptoms, including imminent ones, cell therapy may be contingent on the use of one or more suicide genes of any kind. The use of suicide genes may be part of a planned protocol for treatment, or may be used only when the need for such use is recognized. In some cases, cell therapy is no longer needed, and therefore cell therapy is terminated by the use of one or more agents targeting suicide genes or gene products derived therefrom.

[0208] The use of suicide genes may be initiated when at least one adverse event has begun in an individual, which may be recognized by any means, including during routine monitoring, which may or may not have been ongoing since the initiation of cell therapy. Adverse events(s) may be detected during examinations and / or tests. If an individual has cytokine release syndrome (which may also be referred to as a cytokine storm), the individual may have, for example, elevated inflammatory cytokines(s) (simply 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 / hepatic dysfunction; or a combination thereof. If an individual is neurotoxic, the individual may have confusion, delirium, dysplasia, and / or epileptic seizures. In some cases, individuals are tested for markers associated with the development and / or severity of cytokine release syndrome (e.g., C-reactive protein, IL-6, TNF-alpha, and / or ferritin).

[0209] Examples of suicide genes include engineered non-secretory (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009; the whole of which is incorporated herein by reference), which may be affected by the 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; as well as deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase (a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine) may also be utilized. Other suicide genes include, for example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzyme, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

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

[0211] IV. Pharmaceutical Compositions The pharmaceutical compositions of this disclosure contain an effective amount of cells expressing anti-CD70 CAR dissolved or dispersed in a pharmaceutically acceptable carrier. The expression “pharmaceutically or pharmacologically acceptable” indicates molecular entities and compositions that, when administered appropriately to animals (e.g., humans), do not produce adverse reactions, allergic reactions, or other undesirable reactions. The preparation of pharmaceutical compositions containing cells expressing anti-CD70 CAR will be known to those skilled in the art in light of this disclosure, as exemplified by Remington: The Science and Practice of Pharmacy (21st edition, Lippincott Williams and Wilkins, 2005; incorporated herein by reference). Furthermore, it will be understood that for administration to animals (e.g., humans), the preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0212] As used herein, “pharmaceutically acceptable carrier” includes all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobials, antifungals), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, colorants, substances of such kinds, and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Printing Company, 1990, pp. 1289-1329); this is incorporated herein by reference). Any carrier is intended for use in a pharmaceutical composition unless a conventional carrier is contraindicated with the active ingredient.

[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 a route of administration such as injection. The compositions disclosed at this time may be administered intravenously, intradermally, percutaneously, subarachnoidally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucous membraneally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion directly immersing target cells, via catheter, by lavage, in cream, in lipid compositions (e.g., liposomes), or by any combination of the foregoing (see, for example, Remington's Pharmaceutical Sciences, 18th edition (Mack Printing Company, 1990); which is incorporated herein by reference).

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

[0215] Furthermore, in accordance with this disclosure, compositions of this disclosure suitable for administration are provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier must be assimilated and encompass liquid carriers, semi-solid carriers (i.e., pastes), or solid carriers. Unless any conventional medium, drug, diluent, or carrier is detrimental to the recipient or to the therapeutic efficacy of the composition contained therein, its use in an administerable composition for use in carrying out the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, and fillers, or combinations thereof. The composition may also contain various antioxidants to slow the oxidation of one or more components. In addition, prevention of microbial action can be provided by preservatives, such as various antimicrobial 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 this disclosure, compositions are combined with carriers in any convenient and practical manner, namely by dissolution, suspension, emulsification, mixing, encapsulation, and absorption. Such procedures are routine for those skilled in the art.

[0217] In specific embodiments of this disclosure, the composition is combined with or completely mixed with a semi-solid carrier or a solid carrier. The mixing can be carried out in any convenient manner, for example, 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 compositions include buffers, amino acids (e.g., glycine and lysine), and carbohydrates (e.g., dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol).

[0218] In further embodiments, the disclosure may relate to the use of a pharmaceutically acceptable lipid vehicle composition comprising cells expressing anti-CD70 CAR and, if necessary, an aqueous solvent. As used herein, the term “lipid” will be defined to include any of the broad range of substances that are characteristically insoluble in water and extractable by organic solvents. A wide variety of compounds in this broad class are well known to those skilled in the art, and as used herein, lipids are not limited to any particular structure, whatever that structure may be. Examples include a wide range of compounds containing various long-chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., designed or manufactured by humans). However, lipids are usually biological substances. A wide variety of biological lipids are well known in the art, and these include, for example, triglycerides, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glycolipids, sulfatides, lipids with ether-linked and ester-linked fatty acids, and polymerizable lipids, as well as combinations thereof. Naturally, compounds other than those specifically described herein and understood by those skilled in the art as lipids are also included in 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 lipid-containing solution, soluble with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained as a suspension in lipids, contained with micelles or liposomes, or complexed with micelles or liposomes, or otherwise associated with lipids 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 compositions of this disclosure administered to animal patients can be determined based on various physical and physiological factors, such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. Depending on the dosage and route of administration, the preferred dosage and / or effective dose may vary depending on the subject's response. In any case, the administering officer will determine the concentration of the active ingredient(s) in the composition and the appropriate dosage(s) for the individual subject.

[0221] In certain embodiments, a pharmaceutical composition may contain, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may constitute, for example, between about 2% and about 75% of the weight of the unit, or between about 25% and about 60%, and any range that can be derived therein. Naturally, the amount of one or more active compounds in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit of the compound. Various factors, such as solubility, bioavailability, biological half-life, route of administration, and shelf life of the product, as well as other pharmacological considerations, will be intended by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment plans may be desirable.

[0222] In other, but not limited, doses may also include, per administration, approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body weight, approximately 100 micrograms / kg / body weight, approximately 200 micrograms / kg / body weight, approximately 350 micrograms / kg / body weight, approximately 500 micrograms / kg / body weight, approximately 1 milligram / kg / body weight, approximately 5 milligrams / kg / body weight, approximately 10 milligrams / kg / body weight, approximately 50 milligrams / kg / body weight, approximately 100 milligrams / kg / body weight, approximately 200 milligrams / kg / body weight, approximately 350 milligrams / kg / body weight, approximately 500 milligrams / kg / body weight, up to approximately 1000 mg / kg / body weight or more, and any range that can be derived therefrom. In the ranges that can be derived from the figures listed herein, in any case not limited to these, doses such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, and approximately 5 micrograms / kg / body weight to approximately 500 milligrams / kg / body weight can be administered based on the above figures.

[0223] A. Compositions and formulations for the digestive tract In certain embodiments of this disclosure, 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, orally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilated food carrier, or encapsulated in a rigid or soft gelatin capsule, or compressed into a tablet, or taken directly with food in a meal.

[0224] In certain embodiments, the active compound may be incorporated with an excipient and used in the form of ingestible tablets, buccal tables, lozenges, capsules, elixirs, suspensions, syrups, and wafers (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Patents No. 5,641,515, 5,580,579, and 5,792,451, each of which is incorporated herein by reference in whole, in particular). Tablets, lozenges, pills, and capsules may also contain the following: binders (e.g., tragacanth gum, 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), flavorings, and odorants (e.g., peppermint, wintergreen oil, cherry flavoring, orange flavoring, etc.). When the drug unit is in capsule form, the capsule may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or, in other cases, to alter the physical form of the drug unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the drug form is a capsule, the capsule may contain various carriers (e.g., liquid carriers) in addition to the above types of materials. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coating prevents the denaturation of the composition in the acidic pH of the stomach or upper intestine. See, for example, U.S. Patent No. 5,629,001. When it reaches the small intestine, the basic pH there 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).Elixir syrups may contain active compounds, sucrose as a sweetener, methylparaben and propylparaben as preservatives, colorants, and flavoring agents (e.g., cherry or orange flavoring). Naturally, all materials used in the preparation of any dosage unit must be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, active compounds may be incorporated into sustained-release preparations and formulations.

[0225] For oral administration, the compositions of this disclosure may, alternatively, be incorporated with one or more excipients in the form of mouthwashes, toothpastes, oral tablets, oral sprays, or sublingual oral formulations. For example, a mouthwash may be prepared incorporating the required amount of the active ingredient in a suitable solvent (e.g., sodium borate solution (Dober's solution)). Alternatively, the active ingredient may be incorporated in an oral solution (e.g., an oral solution containing sodium borate, glycerin, and potassium bicarbonate), or dispersed in a toothpaste, or added in a therapeutically effective amount to a composition which may contain water, binders, abrasives, flavoring agents, foaming agents, and wetting agents. Alternatively, the compositions may be in the form of tablets or solutions which may be placed under the tongue, or otherwise disintegrate in the mouth.

[0226] Further formulations suitable for other forms of gastrointestinal administration include suppositories. Suppositories are solid drug formulations of various weights and shapes, usually with added drugs, for insertion into the rectum. After insertion, suppositories soften, melt, or dissolve in the lumen. Conventional carriers for suppositories may generally include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from a mixture containing, for example, an active ingredient in the range of about 0.5% to about 10%, preferably in the range of about 1% to about 2%.

[0227] B. Compositions and formulations for parenteral use In further embodiments, the composition may be administered via parenteral routes. As used herein, the term “parenteral” includes routes that bypass the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, subarachnoidally, subcutaneously, or intraperitoneally. U.S. Patents 6,613,308, 5,466,468, 5,543,158, 5,641,515, and 5,399,363 (each of which is incorporated herein by reference in whole, in particular).

[0228] Solutions of active compounds, as free bases or pharmacologically acceptable salts, may be prepared in water, which is suitably mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, which is incorporated herein by reference in its entirety). In all cases, the forms must be sterile and fluid enough to be readily injectable. The forms must be stable under manufacturing and storage conditions and kept against contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (i.e., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Adequate fluidity may be maintained, for example, by the use of coatings (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it would be preferable to include an isotonic agent (e.g., sugar or sodium chloride). Long-term absorption of the injectable composition can be achieved by using absorption-delaying agents (e.g., aluminum monostearate and gelatin) in the composition.

[0229] For parenteral administration in aqueous solutions, for example, the solution must be suitably buffered if necessary, and the liquid diluent must first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, a variety of sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one drug may be dissolved in an isotonic NaCl solution and added to a subcutaneous injection solution, or it may be injected at the proposed injection site, or both (see, e.g., Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Some variation in the dosage will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine an appropriate dose for the individual subject. Furthermore, for administration to humans, the preparation must meet sterility standards, pyrogenicity standards, general safety standards, and purity standards as required by the FDA Office of Biologics.

[0230] Sterile injectable solutions are prepared by incorporating the required amount of active compound, along with various other components listed above, into a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a base dispersion medium and other required components derived from the components listed above. For sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredients, plus any additional desired components, from their pre-sterile filtered solutions. The powdered compositions are combined with or without stabilizers in a liquid carrier (e.g., water or saline solution).

[0231] C. Other pharmaceutical compositions and formulations In other specific embodiments of the present invention, active compound cells expressing anti-CD70 CAR may be formulated for administration via a wide variety of routes, such as topical administration (i.e., transdermal administration), mucosal administration (intranasal administration, vaginal administration, etc.) and / or inhalation.

[0232] Pharmaceutical compositions for topical administration may contain active compounds formulated for medicinal applications (e.g., ointments, pastes, creams, or powders). Ointments include all oily compositions for topical application based on adsorption, emulsification, and water solubility, while creams and lotions are such compositions containing only an emulsifying base. Topically administered medicinal drugs may contain penetration enhancers to facilitate the adsorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones, and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as other suitable absorbent ointment bases, emulsifying ointment bases, or water-soluble ointment bases, whatever type of ointment base may be. Topical preparations may also contain emulsifiers, gelling agents, and antimicrobial preservatives, as needed, to retain 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 certain 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 directly delivering compositions 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 whole). Similarly, in drug delivery, delivery using intranasal microparticle resins (Takenaga et al., 1998) and delivery using lysophosphatidylglycerol compounds (U.S. Patent No. 5,725,871, which is specifically incorporated herein by reference in whole) are also widely known in the pharmaceutical art. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045 (which is specifically incorporated herein by reference in whole).

[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 would consist of a suspension of an 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. The suitable container 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.

[0235] V. Combination Therapy In certain embodiments, the compositions and methods of this embodiment are associated with cancer treatment that is additive to compositions comprising cells expressing anti-CD70 CAR. Further treatment may include radiotherapy, surgery (e.g., mammography 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 foregoing. Further treatment may take the form of adjuvant therapy or neoadjuvant therapy.

[0236] In some embodiments, further treatment is the administration of one or more small molecule enzyme inhibitors or one or more metastasis inhibitors. In some embodiments, further treatment is the administration of side effect limiting agents (e.g., drugs intended to reduce the occurrence and / or severity of side effects of the procedure, e.g., antiemetics). In some embodiments, further treatment is radiotherapy. In some embodiments, further treatment is surgery. In some embodiments, further treatment is a combination of radiotherapy and surgery. In some embodiments, further treatment is gamma irradiation. In some embodiments, further treatment is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent (one or more). Further treatment may be one or more chemotherapeutic agents known in the art.

[0237] Immunotherapy (in addition to the cell therapy of the Disclosure) may be administered before, during, after, or in various combinations with further cancer treatment (e.g., immune checkpoint therapy). These administrations may occur at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments in which immunotherapy is administered to a patient separately from one or more of the compositions of the Disclosure, it will generally be ensured that a meaningful period does not end between the respective delivery times, and as a result, the two compounds will still be able to provide a favorable synergistic effect to the patient. In such cases, it is intended that the immunotherapy and the disclosed compositions may be administered to the patient within a range of approximately 12 to 24 hours or 72 hours from each other, more specifically within a range of approximately 6 to 12 hours from each other. In some circumstances, it may be desirable to significantly extend the time between treatments, in which case days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) may elapse between each administration.

[0238] Administering any of the compounds or cell therapies of this embodiment to a patient would follow general protocols for the administration of such compounds, taking into account the toxicity of the action source, if any. Therefore, in some embodiments, there is a step to monitor for toxicity that may result from the combination therapy.

[0239] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, 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, neck, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, cancer is recurrent cancer. In some embodiments, cancer is stage I cancer. In some embodiments, cancer is stage II cancer. In some embodiments, cancer is stage III cancer. In some embodiments, cancer is stage IV cancer.

[0240] Cancer can be, but is not limited to, the following histological types: neoplasm, malignant; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix Carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed type of hepatocellular carcinoma and cholangiocarcinoma; columnar adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid cancer; carcinoid tumor, malignant; branchio-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; oxyphilic carcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma Carcinoma; skin adnexal cancer; apocrine gland cancer; sebaceous gland cancer; ceruminous gland cancer; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous cell 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; chromaffin cell tumor; glomus angiosarcoma; malignant melanocytoma Melanoma; Apigmented melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevi; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Fetal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymal tumor, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Undifferentiated germ cell tumor; Fetal carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Perivascular cell tumor, malignant; Lymphangiosarcoma; Osteosarcoma; Paraosteal osteosarcoma; Chondrosarcoma;Chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastoma; ameloblastoma, malignant; ameloblastoma; pineal glandoma, malignant; chordoma; glioma, malignant; ependymal cell tumor; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astrocytoblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; undifferentiated neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granular Cellular tumors, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell type, diffuse; malignant lymphoma, follicular; mycosis fungoides; other designated non-Hodgkin lymphomas; malignant histiocytoma; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma 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 range of chemotherapeutic agents may be used according to this embodiment. The term “chemotherapy” refers to the use of drugs to treat cancer. “Chemotherapeutic agent” is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within cells, for example, whether and at what stage of the cell cycle they affect. 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, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and biposulfan; aziridine compounds, e.g., benzodopa, carbocon, meturedopa and uredopa; ethyleneimine compounds and methylamelamine compounds, e.g., altoretamine, triethylenemelamine, etc. Ethylene phosphoramides, triethiylenethiophosphoramides, and trimethylolomelamine; acetogenins (especially bratacin and bratacinone); camptothecins (including their synthetic analog, topotecan); bryostatins; callistatins; CC-1065 (including its synthetic analogs, adzeresin, karzeresin, and bizeresin); cryptophycins (especially cryptophycin 1 and cryptophycin 8) ;Drastatin;Duocalmycin (including synthetic analogs KW-2189 and CB1-TM1);Erytherobin;Pancratistatin;Sarcodictyins;Spongistatin;Nitrogen mustards, e.g., chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, Phenesterine, prednimustine, trophosphamide, and uracil mustard, etc.; nitrosourea compounds, e.g., carmustine, chlorozotosine, photemustine, lomustine, nimustine, and ranimustine, etc.; antibiotics, e.g., engine antibiotics (e.g., calichemycin, especially calichemycin gamma I and calichemycin omega I1), etc.; dinemisins, e.g., dinemisin A; bisphosphonate compounds, e.g., clodronate, etc.; esperamicins;Similarly, neocardinostatin chromophore and related pigment protein enediin antibiotics include chromophore, acrasinomycin, actinomycin, authrarnnycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, and 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, olibomycins, peplomycin, potophyllomycin Phytolic acid analogs include folic acid (potfiromycin), puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, pteropterin, and trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamipri ¹ and thioguanine, etc.; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and phloxuridine, etc.; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone, etc.; antiadrenal agents, e.g., mitotane and trilostane, etc.; folic acid supplements, e.g., frolinic acid, etc.; acegraton; aldofsphamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabucil; bisantren;Edatraxate; defofamine; demecoltin; diaziquan; elformithine; elliptinium acetate; epotilones; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and anthamitosines; mitogwazone; mitoxantrone; mopidanmol; nitraerine; pe Intostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, loridine A, and anguidine); urethane; vindesine; dacarbazine; mannomustine ; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-Thiogunine; Mercaptopurine; Platinum-coordinated complexes, e.g., cisplatin, oxaliplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxanthrone; Vincristine; Vinorelbine; Novanthrone; Teniposide; Edatrexate; Daunomycin; A Minopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids, e.g., retinoic acid; Capecitabine; Carboplatin, Procarbazine, Plicomycin, Gemcitabien, Navelbine, Farnesyl protein transferase inhibitors, Transplatinum, and any pharmaceutically 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 those commonly known as gamma rays, X-rays, and / or the induced delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also intended: e.g., microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. It is likely that all of these elements affect a wide range of damage to DNA, to DNA precursors, to DNA replication and repair, and to chromosome assembly and maintenance. The application range for X-rays ranges from a daily dose of 50 to 200 roentgens over long periods (3 to 4 weeks) to a single dose of 2,000 to 6,000 roentgens. The application range for radioisotopes varies widely and depends on the half-life of the radioisotope, the intensity 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 therapies) can be used in combination with or in conjunction with the methods of the present embodiment. In the context of cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may serve as an effector of treatment alone. Alternatively, the antibody may mobilize 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. In an alternative, the effector may be a lymphocyte that bears surface molecules that interact either directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells that are different from those having a knockdown or knockout of TGF-beta R2.

[0245] Antibody-drug conjugates have emerged as a groundbreaking approach to cancer treatment development. Antibody-drug conjugates (ADCs) include monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs. In this approach, the high specificity of the MAb to its antigen target is combined with highly potent cytotoxic drugs, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells with elevated levels of antigen. Targeted drug delivery also minimizes exposure to normal tissue, leading to 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 demonstrated the effectiveness of this approach. Currently, more than 30 ADC drug candidates are 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 depend on identifying and validating novel targets suitable for this effort, as well as creating targeting MAbs. 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 susceptible to targeting, i.e., some marker that is not present on most other cells. There are many tumor markers, any of which may be suitable for targeting in the context of the present embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosine kinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. One alternative aspect of immunotherapy is to combine the anti-cancer effect with an immunostimulatory effect. Immunostimulatory molecules also exist, including 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 immunoadjuvants 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 therapy such as any type of interferon, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. 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. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapy described herein.

[0248] In some embodiments, immunotherapy may involve immune checkpoint inhibitors. Immune checkpoints can either enhance (e.g., co-stimulatory molecules) or desensitize (weaken) signals. Suppressive 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 activator gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and the 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 prophylactic, diagnostic, or staging, therapeutic, and palliative surgeries. Therapeutic surgery includes excision, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in combination with other treatments, such as the procedures of this embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision refers to the physical removal of at least part of the tumor. In addition to tumor excision, surgical procedures include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0250] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment may be achieved by perfusion, direct injection, or local application of further anti-cancer treatment to the area. Such treatment may be repeated, for example, every day, every two days, every three days, every four days, every five days, every six days or every seven days, or every week, every two weeks, every three weeks, every four weeks and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months or every twelve months. These treatments may also be in varying doses.

[0251] E. Other medications Other agents may be used in combination with certain aspects of this embodiment to improve the therapeutic efficacy of the treatment. These further agents include agents that affect the upregulation and GAP binding of cell surface receptors, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, 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 bindings will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is further intended that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of this embodiment to improve the efficacy of the treatment.

[0252] VI. Vectors CD70-targeted CARs may be delivered to recipient cells, including NK cells, by any suitable vector, including viral or nonviral vectors. Examples of viral vectors include, at a minimum, retroviral vectors, lentiviral vectors, adenovirus vectors, or adeno-associated virus vectors. Examples of nonviral vectors include, at a minimum, plasmids, transposons, lipids, nanoparticles, liposomes, and combinations thereof.

[0253] When NK cells are transduced by a vector encoding a CD70-targeted CAR, and transduction into the cells of one or more other genes, such as a suicide gene and / or cytokines and / or voluntary therapeutic gene products, the CD70-targeted CAR, the voluntary suicide gene, and the voluntary 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 cytokine(s) are expressed from the same vector molecule (e.g., the same viral vector molecule). In such cases, the expression of the CD70-targeted CAR, the suicide gene, and / or cytokine(s) may or may not be regulated by the same regulatory element(s). When the CD70-targeted CAR, the suicide gene, and / or cytokine(s) 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 once or more times in the same vector).

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

[0255] A. Adjustment element The expression cassettes contained in the vectors useful in this disclosure particularly include a eukaryotic transcription promoter functionally linked to a protein-coding sequence, a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence (in the 5'-3' direction). Promoter and enhancer that regulate the transcription of protein-coding genes in eukaryotic cells may consist of numerous gene elements. Cellular mechanisms collect and integrate the regulatory information conveyed by each element, thereby enabling different genes to develop distinct, often complex, patterns of transcriptional regulation. Promoter used in connection with this disclosure includes, for example, constitutive promoters, inductive promoters, and tissue-specific promoters. When vectors are used to create cancer therapies, promoters may be effective under hypoxic conditions.

[0256] B. Promoter / Enhancer The expression constructs provided herein include promoters for expressing antigen receptors and other cistron gene products. Promoters generally contain sequences that function to locate the start site for RNA synthesis. The best-known example of this sequence is the TATA box; however, in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the late SV40 gene, discrete elements overlapping the start site itself help to locate the start. Further promoter elements regulate the transcription initiation frequency. Typically, these are located in the upstream region of the start site, but many promoters have been shown to also contain functional elements downstream of the start site. To bring the coding sequence "under the control" of the promoter, the 5' end of the transcription start site in the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription and promotes the expression of the encoded RNA.

[0257] The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are reversed or moved relative to one another. For example, in the tk promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decrease. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which are cis-acting regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0258] Promoters may be naturally associated with a nucleic acid sequence, such as those obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters may be indicated as “endogenous.” Similarly, enhancers may be 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 the coding nucleic acid segment under the control of a recombinant or heterologous promoter (which indicates a promoter not normally associated with the nucleic acid sequence in its natural environment). Recombinant or heterologous enhancers also indicate enhancers not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not “naturally present,” i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthesizing promoter and enhancer nucleic acid sequences, various sequences may be prepared using recombinant cloning and / or nucleic acid amplification techniques, including PCR®, in relation to the compositions disclosed herein. Furthermore, it is intended that regulatory sequences that guide the transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria and chloroplasts) may also be used.

[0259] Naturally, it will be important to use promoters and / or enhancers that effectively guide the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the field of molecular biology generally understand that various promoters, various enhancers, and various cell type combinations are used for protein expression (see, e.g., Sambrook et al. (1989), which is incorporated herein by reference). The promoters used may be useful under appropriate conditions for guiding high levels of expression of the introduced DNA segment, such as being constitutive, tissue-specific, inducible, and / or favorable in the large-scale production of recombinant proteins and / or recombinant peptides. Promoters may be heterogeneous or endogenous.

[0260] In addition, any promoter / enhancer combination (for example, combinations from the Eukaryotic Promoter Database EPDB via the World Wide Web at epd.isb-sib.ch / ) could also be used to induce expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, provided that a suitable bacterial polymerase is provided either as part of a delivery complex or as a further gene expression construct.

[0261] Examples of promoters, though not limited to them, include early or late viral promoters, e.g., the early or late promoter of SV40, the pre-early promoter of cytomegalovirus (CMV), the early promoter of Roussarcoma virus (RSV), etc.; promoters of eukaryotic cells, e.g., the beta-actin promoter, the GADPH promoter, the metallothionein promoter, etc.; and chained response element promoters, e.g., the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter near the minimal TATA box (tre). It is also possible to use the promoter sequence of human growth hormone (e.g., the human growth hormone minimal promoter described in GenBank®, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (which is available from ATCC; catalog number ATCC45007). In certain embodiments, the promoter is 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 expressing therapeutic genes is applicable to the implementation of this disclosure.

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

[0263] C. Initiation signal and chain expression Specific start signals may also be used in the expression constructs provided herein for efficient translation of coding sequences. These signals may include ATG start codons or adjacent sequences. Exogenous translational control signals, including ATG start codons, 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 start codons 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 start codons can be either native or synthetic. Expression efficiency may be enhanced by including appropriate transcriptional enhancer elements.

[0264] In certain embodiments, the use of intra-sequence ribosome entry site (IRES) elements is employed to generate multiple gene or polycistron messages. IRES elements bypass the 5' methylation cap-dependent ribosome scanning model of translation, allowing translation to be initiated at an internal site. Various IRES elements derived from two members of the Picornaviridae family (polio and encephalomyocarditis) are described, as are IRES derived from mammalian messages. IRES elements can be ligated to heterologous open reading frames. Multiple open reading frames can be transcribed together, but each separated by an IRES, thereby resulting in a polycistron message. The IRES element allows each open reading frame to contact a ribosome for efficient translation. Multiple genes can be efficiently expressed using only one promoter / enhancer to transcribe just one message.

[0265] As detailed elsewhere herein, certain 2A sequence elements may be used to effect linked or co-expression of genes in the constructs provided in this disclosure. For example, cleavage sequences may be used to co-express multiple genes by linking multiple open reading frames to form a single cistron. One exemplary cleavage 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, but in an alternative embodiment, the same vector utilizes two or more of the same 2A sequences. Various examples of 2A sequences are provided in U.S. Patent Application Publication No. 2011 / 0065779, which is incorporated herein by reference in its entirety.

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

[0267] E. Selection markers and screening markers In some embodiments, cells such as NK cells containing the anti-CD70 CAR expression construct of this disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would impart a identifiable change to the cell, thereby enabling the easy identification of cells containing the expression vector. Generally, a selection marker provides a property that enables selection. A positive selection marker is one in which the presence of the marker enables selection, while a negative selection marker is one in which the presence of the marker hinders selection. An example of a positive selection marker is a drug resistance marker.

[0268] Typically, the inclusion of drug selection markers aids in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeosin, and histidinol are useful selection markers. In addition to markers that provide phenotypes enabling the identification of transformants based on the implementation of conditions, other types of markers are also intended, including screening markers (e.g., GFP) on which colorimetric analysis is the basis. Alternatively, screening markers as negative selection markers, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), may be used. Those skilled in the art will also know how immunological markers may be used in conjunction with FACS analysis. The markers used are considered irrelevant as long as they can be expressed concurrently with the nucleic acid encoding the gene product. Further examples of selection and screening markers are widely known to those skilled in the art.

[0269] VII.Cells This disclosure encompasses any type of immune cell that accepts a vector encoding a CD70-targeted CAR and which may also encode at least one cytokine and at least one suicide gene. In some cases, various vectors encode a CAR, while others encode a suicide gene and / or cytokine. 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, in certain cases the cell is an NK cell. NK cells may be derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or bone marrow. NK cells may be derived from cell lines, for example, NK-92 cells, for example, but are not limited to these. NK cells may be umbilical cord blood mononuclear cells, such as CD56+ NK cells.

[0270] In some cases, the cells are not immortalized cell lines but cells obtained from an individual (e.g., primary cells). For example, in some cases, the cells are immune cells obtained from an individual. As one example, the cells are T lymphocytes obtained from an individual. As another example, the cells are cytotoxic cells obtained from an individual. As yet another example, the cells are stem cells (e.g., peripheral blood stem cells) or primordial cells obtained from an individual.

[0271] In certain embodiments, the cells of the Disclosure may be specifically formulated and / or cultured in a specific medium. The cells may be formulated in a manner suitable for delivery to a recipient without adverse effects.

[0272] In a particular context, the culture medium can be prepared using any of the following media used for culturing animal cells, as well as any combination thereof, such as AIM V medium, X-VIVO-15 medium, NeuroBasal medium, EGM2 medium, TeSR medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Modified MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham medium, RPMI-1640 medium, and Fischer medium, or any combination thereof, however, the medium may not be particularly limited to them as long as it can be used to culture animal cells. In particular, the medium may be heterogeneous or chemically defined.

[0273] The culture medium may be serum-containing, serum-free, or free of heterogeneous components. To prevent contamination by components from other animal species, the serum may be derived from the same animal as the stem cells. Serum-free media refer to media that do not contain untreated or unpurified serum, and therefore, serum-free media may include media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0274] The culture medium may or may not contain serum substitutes. Examples of serum substitutes include those appropriately containing albumin (e.g., lipid-rich albumin, bovine albumin, albumin substitutes (e.g., recombinant albumin or humanized albumin), vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes can be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679 (which is incorporated in its entirety herein). Any commercially available substitute may be more conveniently used. Commercially available substitutes include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0275] In certain embodiments, the culture medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, twelve, thirteenth, fif In specific embodiments, one or more of these may be explicitly excluded.

[0276] In some embodiments, the medium further contains vitamins. In some embodiments, the medium contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelf, ten, thirteen, twelve, and thirteen (and any range that can be derived therefrom): 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 contains a combination thereof or salts thereof. In some embodiments, the medium contains, or essentially consists 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 vitamins include biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamin A, or a combination or salt thereof, or essentially derived from them. In some embodiments, the medium further includes proteins. In some embodiments, the proteins include albumin or bovine serum albumin, BSA fraction, catalase, insulin, transferrin, superoxide dismutase, or a combination thereof. In some embodiments, the medium further includes 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 includes one or more of the following: B-27® supplement, heterogeneous B-27® supplement, GS21® supplement, or a combination thereof. In some embodiments, the culture medium comprises amino acids, monosaccharides, inorganic ions, or further comprises these.In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations thereof or salts thereof. In some embodiments, the culture medium further includes one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the culture medium includes one or more vitamins discussed herein and / or one or more proteins discussed herein and / or one or more of the following, or essentially consists of these: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement Fillers, non-containing B-27® supplements, GS21® supplements, amino acids (e.g., arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (e.g., sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or their salts, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be expressly excluded.

[0277] The culture medium may also contain one or more externally added fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins (one or more), growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, and / or inorganic salts. In specific embodiments, one or more of these may be explicitly excluded.

[0278] One or more components of the culture medium may be added at concentrations 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 from these.

[0279] In specific embodiments, the cells of this disclosure are formulated in particular. The cells may or may not be formulated as a cell suspension. In specific cases, the cells are formulated in single-dose form. The cells may be formulated for systemic or topical administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may contain one or more cryopreservatives, e.g., DMSO (e.g., in 5% DMSO). The cell formulation may contain albumin, including human albumin, and specific formulations contain 2.5% human albumin. The cells may be specifically formulated for intravenous administration; for example, the cells are formulated for intravenous administration over a period of less than one hour. In certain embodiments, the cells are in the form of a formulated cell suspension that is stable at room temperature for one, two, three, or four hours or longer from thawing.

[0280] In some cases, immune cells are expanded before use and / or before manufacture. In some cases, immune cells are NK cells expanded in the presence of an effective amount of antigen-presenting cells, including universal antigen-presenting cells (UAPCs), in any preferred ratio. Cells may be cultured with UAPCs in ratios of 10:1-1:10, 9:1-1:9, 8:1-1:8, 7:1-1:7, 6:1-1:6, 5:1-1:5, 4:1-1:4, 3:1-1:3, 2:1-1:2, or 1:1, including, for example, a 1:2 ratio. In some cases, NK cells are enlarged in the presence of IL-2 at concentrations such as 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] Following genetic modification with one or more vectors, NK cells may be injected immediately or stored. In certain scenarios, after genetic modification, cells may be augmented ex vivo as a bulk population over several days, weeks, or months, approximately 1, 2, 3, 4, 5 days, or more, after the genes have been transferred into the cells. In further scenarios, transfectants are cloned, and clones that clearly demonstrate the presence of a single integrated or episome-maintained expression cassette or plasmid, and the expression of a CD70-targeted CAR, are augmented ex vivo. Clones selected for augmentation clearly demonstrate the ability to specifically recognize and lyse CD70-expressing target cells. Recombinant immune cells may be augmented by stimulation with IL-2 or other cytokines that bind to a common gamma chain (e.g., IL-7, IL-12, IL-15, and IL-21). Recombinant immune cells may be augmented by stimulation with artificial antigen-presenting cells. In further developments, genetically modified cells may be cryopreserved.

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

[0283] Cells may be obtained directly from an organism or from a storage facility or other storage facility. Cells used therapeutically may be autologous or homogeneous with respect to the organism to which they are given therapeutically.

[0284] Cells may be obtained from individuals in need of treatment for medical conditions, and after cell manipulation to express CD70-targeted CARs, the cells may be returned to the individual from which they were originally obtained. In some cases, cells are stored for subsequent use in the same individual or another. In such cases, cells may or may not be further modified prior to use.

[0285] Among NK cells that accept CD70-targeted CARs, the NK cells that may be included in a population of cells, and that population, may have a majority that can be transduced with one or more CD70-targeted CARs. The cell population may contain 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the 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 these cells to express one or more other target genes (including therapeutic genes) depending on the intended purpose (one or more). For example, a party interested in treating CD70-positive cancer may modify these cells to express a second CAR for the same or a different antigen. Other modifications to cells may include modifications that tailor the cells to the needs of the individual receiving the cells, including specific needs for a particular type of cancer the individual suffers from.

[0287] VIII. Cellular gene editing In certain embodiments, cells containing an anti-CD70 CAR are gene-edited to alter the expression of one or more endogenous genes in those cells. In specific cases, cells are modified to have reduced expression levels of one or more endogenous genes, including inhibition of the expression of one or more endogenous genes (which may be indicated as knockout). Such cells may or may not be enlarged.

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

[0289] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as through RNA-guided endonucleases (RGENs). For example, the modification may be performed using clustered and regularly arranged short palindromic sequence repeats (CRISPR) and CRISPR-related (Cas) proteins. In some embodiments, CpF1 may be used instead of Cas9. Generally, the “CRISPR system” refers collectively to CRISPR-related (“Cas”) genes (including sequences encoding Cas genes), tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracrmate sequences (in the context of the endogenous CRISPR system, “serial repeat sequences” and partial serial repeat sequences processed by tracrRNA), guide sequences (also referred to as “spacers” in the context of the endogenous CRISPR system), and / or other sequences and transcripts and other elements derived from the CRISPR locus that are involved in or direct the expression of such sequences and transcripts.

[0290] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide) RNA that sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9) that has nuclease function (e.g., two nuclease domains). One or more elements of the CRISPR system may originate from a type I, type II, or type III CRISPR system, for example, from a specific organism (e.g., Streptococcus pyogenes) that contains an endogenous CRISPR system.

[0291] In some embodiments, a Cas nuclease and gRNA (including a fusion of a target sequence-specific crRNA and a default tracrRNA) are introduced into a cell. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to its target site, e.g., a gene, through complementary base pairing. The target site may be selected based on the position immediately 5' of a protospacer adjacent motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, the CRISPR system features elements that facilitate the formation of the CRISPR complex at the site of the target sequence. Typically, the “target sequence” refers to a sequence designed so that the guide sequence is complementary, and the hybridization of the target sequence and the guide sequence facilitates the formation of the CRISPR complex. If there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex, complete complementarity is not necessarily required.

[0292] The CRISPR system can induce disruption or modification, as discussed herein, following a double-strand break (DSB) at a target site. In other embodiments, a Cas9 variant, considered a “nickase,” is used to introduce a nick into the single strand at the target site. For example, to improve specificity, a pair of nickases may be used, each led by a pair of different gRNAs targeting the sequence, and when the nicks are introduced simultaneously, a 5' overhang is introduced. In other embodiments, a catalytically inactive Cas9 is fused to a heterogeneous effector domain, such as a transcription repressor or transcription activator, to affect gene expression.

[0293] The target sequence may contain any polynucleotide, such as DNA polynucleotides or RNA polynucleotides. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within a cell organelle. Generally, a sequence or template that can be used for recombination to a targeted gene locus containing the target sequence is referred to as an “edit template,” “edited polynucleotide,” or “edited sequence.” In some embodiments, an exogenous template polynucleotide may be referred to as the edit template. In some embodiments, the recombination is homologous recombination.

[0294] Typically, in the context of endogenous CRISPR systems, the formation of a CRISPR complex (which includes a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins) results in a 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 from the target sequence). Tracr sequences that may contain or consist of all or part of a wild-type tracr sequence (for example, approximately 20 nucleotides, 26 nucleotides, 32 nucleotides, 45 nucleotides, 48 ​​nucleotides, 54 nucleotides, 63 nucleotides, 67 nucleotides, 85 nucleotides or more, or more than approximately 20 nucleotides, more than approximately 26 nucleotides, more than approximately 32 nucleotides, more than approximately 45 nucleotides, more than approximately 48 nucleotides, more than approximately 54 nucleotides, more than approximately 63 nucleotides, more than approximately 67 nucleotides, more than approximately 85 nucleotides or more) may also form part of a CRISPR complex, such as by hybridization along at least part of the tracr sequence to all or part of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of the CRISPR complex (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).

[0295] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into a cell such that the expression of one or more elements of the CRISPR system directs the formation of a CRISPR complex at one or more target sites. Alternatively, the components can be delivered to the cell as proteins and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more further vectors providing any components of the CRISPR system not included in the first vector. These vectors may contain 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 in one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0296] The vector may contain regulatory elements 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, and Csm4. Examples include Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, Cpf1 (Cas12a), their homologs or modified versions thereof. These enzymes are publicly known; for example, the amino acid sequence of the Cas9 protein of S. pyogenes can be found in the SwissProt database as accession number Q99ZW2.

[0297] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). In some embodiments, Cpf1 (Cas12a) may be used instead of Cas9. The CRISPR enzyme may direct the cleavage of one or both strands at a target sequence location, such as within the target sequence and / or within the complementary strand of the target sequence. A vector may encode a mutated CRISPR enzyme compared to the corresponding wild-type enzyme, and the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartate to alanine in the RuvC I catalytic domain of Cas9 from S. pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (single-strand cleavage). In some embodiments, the Cas9 nickase may be used in combination with guide sequences, e.g., two guide sequences that target the sense and antisense strands of its DNA target, respectively. This combination can be used to introduce nicks into both strands and to induce NHEJ or HDR.

[0298] In some embodiments, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells of a particular organism (e.g., mammals including, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates) or cells derived from such organisms. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to increase expression in a target host cell by maintaining the native amino acid sequence while replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the host cell's gene. Different species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon usage frequency between organisms) often correlates with messenger RNA (mRNA) translation efficiency, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of a selected tRNA in a cell usually reflects that it is the codon most frequently used in peptide synthesis. Therefore, based on codon optimization, genes can be adapted to optimal gene expression in a given organism.

[0299] Generally, the guide sequence is any polynucleotide sequence that has sufficient complementarity to the target sequence to hybridize with the target sequence and to direct the sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and the 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 greater 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 preferred alignment algorithm.

[0300] The optimal alignment can be determined using any suitable algorithm for aligning sequences. Non-restrictive examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0301] A CRISPR enzyme can be part of a fusion protein containing one or more heterologous protein domains. A CRISPR enzyme fusion protein may contain any further protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. 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, and autofluorescent proteins including green fluorescent protein (GFP), 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). Further domains that may form part of a fusion protein containing a CRISPR enzyme are described in U.S. Patent Application Publication No. 20110059502, incorporated herein by reference.

[0302] IX. Treatment Method In various embodiments, CD70-targeted CAR constructs, nucleic acid sequences, vectors, and host cells, as intended herein, and / or pharmaceutical compositions containing them, are used to prevent, treat, or improve cancerous diseases, such as neoplastic diseases. In certain embodiments, the pharmaceutical compositions of the Disclosure may be particularly useful in preventing, improving, and / or treating cancer, including, for example, cancers that express CD70 and may or may not be solid tumors. An individual may use the treatment methods of the Disclosure, for example, as a first treatment or after (or in conjunction with) another treatment, for example, after HSCT. The immunotherapy methods may be tailored to the needs of an individual with cancer based on the type and / or stage of cancer, and in at least some cases, the immunotherapy may be modified for the individual during the course of the treatment process.

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

[0304] Tumors for which this treatment method is useful include malignant cell types of any type, such as malignant cell types found in solid tumors or hematological malignancies. 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 this disclosure, immune cells expressing anti-CD70 CAR are delivered to an individual in need, for example, an individual with cancer. The cells then enhance 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 immune cells in two or more doses, the interval between doses must be sufficient to allow time for propagation in the individual, and in specific embodiments, the interval between doses is one, two, three, four, five, six, seven or more days.

[0306] The NK cells on which CD70-targeted CARs are utilized may, in certain embodiments, be NK, T, or invariant NKT cells engineered for cell therapy for mammals. In such cases where the cells are NK cells, the NK cell therapy may be of any kind, and the NK cells may be of any kind. In a specific embodiment, the cells are NK cells engineered to express one or more CD70-targeted CARs and are given to an individual in a therapeutically effective amount (in the range of 10³ to 10¹⁰) that improves in the individual at least one symptom associated with CD70-expressing cells. In a specific embodiment, the cells are NK cells into which the CD70-targeted CAR is transduced. The therapeutically effective amount is 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 pieces to 10 6 pieces, 10 3 pieces to 10 5 pieces, 10 3 pieces to 10 4 pieces, 10 4 pieces to 10 10 pieces, 10 4 pieces to 10 9 pieces, 10 4 pieces to 10 8 pieces, 10 4 pieces to 10 7 pieces, 10 4 pieces to 10 6 pieces, 10 4 pieces to 10 5 pieces, 10 5 pieces to 10 10 pieces, 10 5 pieces to 10 9 pieces, 10 5 pieces to 10 8 pieces, 10 5 pieces to 10 7 pieces, 10 5 pieces to 10 6 pieces, 10 6 pieces to 10 10 pieces, 10 6 pieces to 10 9 pieces, 10 6 pieces to 10 8 pieces, 10 6 pieces to 10 7 pieces, 10 7 pieces to 10 10 pieces, or 10 7 pieces to 10 9 pieces, 10 7 pieces to 1...... 8 pieces, 10 8 pieces to 10 10 pieces, 10 8 pieces to 10 9 pieces, or 10 9 pieces to 10 10 It may be cells of pieces. Therefore, in a specific embodiment, an individual having CD70-positive cancer is given a therapeutically effective amount of cells expressing one or more CD70-targeted CARs, including NK cells, once or multiple times.

[0307] In certain embodiments, the disclosure intends, in part, cells expressing CD70 CARs, CD70-targeted CAR constructs, CD70-targeted CAR nucleic acid molecules, and CD70-targeted CAR vectors that can be administered either alone or in any combination using standard vectors and / or gene delivery systems, and in at least in some aspects with pharmaceutically acceptable carriers or excipients. In certain embodiments, the nucleic acid molecule or vector may be stably integrated into the target genome after administration.

[0308] In specific embodiments, viral vectors that are specific to certain cells or tissues and persist in, for example, NK cells may be used. Suitable pharmaceutical carriers and excipients are widely known in the art. Compositions prepared in accordance with this disclosure can be used for the prevention, treatment, or delay of the diseases identified above.

[0309] Furthermore, this disclosure relates to a method for the prevention, treatment, or improvement of neoplastic diseases, comprising the step of administering to a subject in need an effective amount of cells expressing a CD70-targeted CAR, nucleic acid sequence, or vector, as intended herein and / or produced by a process as intended herein.

[0310] Possible indications for administration of one or more exemplary CD70-targeted CAR cell compositions include cancerous diseases, including neoplastic diseases, such as B-cell malignancies, multiple myeloma, breast cancer, or lung cancer. Exemplary indications for administration of one or more CD70-targeted CAR cell compositions include cancerous diseases, including any malignant tumor that expresses CD70. Administration of one or more compositions of the present disclosure is useful for all stages and types of cancer, including, for example, minimal residual disease, early-stage cancer, advanced cancer, and / or metastatic and / or refractory cancer.

[0311] A therapeutically effective amount of the produced cells can be administered by parenteral administration via many routes, including intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intratumoral injection, subarachnoid injection, intraventricular injection, intra-articular injection, or infusion via a reservoir.

[0312] The therapeutically effective amount of cells produced for use in adoptive cell therapy is the amount that achieves the desired effect in the subject being treated. For example, this could be the amount of immune cells needed to inhibit cancer development or induce cancer regression.

[0313] The produced cell population can be administered in a treatment plan consistent with the disease, for example, in one or several doses over one to several days to improve the disease state, or in regular doses over a long period to suppress disease progression and prevent disease recurrence. The exact dose to be used in formulation will also depend on the route of administration and the severity of the disease or disorder, and must be determined according to the physician's judgment and the individual patient's condition. The therapeutically effective amount of cells will depend on the subject being treated, the severity and type of pain, and the mode of administration. In some embodiments, the dose that may be used in the treatment of a human subject is 1 m 2 There are at least 1 × 10 T cells per unit. 3 each, at least 1 × 10 4 pieces, 3.8×10 4 each, at least 3.8 × 10 5 each, at least 3.8 × 10 6 each, at least 3.8 × 10 7 each, at least 3.8 × 10 8 each, at least 3.8 × 10 9 pieces, or at least 3.8 × 10 10 It ranges from one unit. In a particular embodiment, the dose used in the treatment of a human subject is 1 m 2 The number of T cells per area is approximately 3.8 × 10⁻⁶ 9 Approximately 3.8 x 10 10The range is up to 10. In a further embodiment, the therapeutically effective amount of T cells is about 5 × 10 6 Approximately 7.5 × 10⁻⁶ cells / kg body weight 8 It can change down to the cell / kg body weight level, for example, approximately 2 × 10⁻¹⁶ cells per kg of body weight. 7 Approximately 5 x 10 from a single cell 8 Down to individual cells, or approximately 5 x 10⁶ cells per kg of body weight. 7 Approximately 2 × 10⁶ cells 8 It is possible to alter cells down to the individual cell level. The precise amount of T cells can be easily determined by those skilled in the art based on the subject's age, weight, sex, and physiological state. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0314] This disclosure further encompasses co-administration protocols with other compounds that act via immune cells (e.g., bispecific antibody constructs, targeted toxins, or other compounds). Clinical treatment plans for co-administration of one or more of the compounds of the present invention may include simultaneous co-administration, co-administration before or after administration of other components. 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 this disclosure are also intended to include the pharmaceutical compositions described above herein, either alone or in combination with further pharmaceuticals to be administered to an individual requiring medical treatment or intervention.

[0316] X. Kit of this disclosure Any of the compositions described herein may be included in a kit. In examples that are not limited thereto, a kit may include an anti-CD70 CAR molecule, cells containing the same, and / or reagents for generating the same, any of which may be included in a preferred container means in a kit of this disclosure. A kit may include immune cells, including NK cells, a vector, an expression construct polynucleotide (whether viral or not) for insertion into the vector, and any kind of CD27 sequence, as incorporated herein. Primers for amplifying any polynucleotide may be included. In some cases, a kit may include cryopreserved cells, including NK cells. Reagents for transfection or transduction into cells may be included.

[0317] The kit composition may be packaged in an aqueous medium or in a lyophilized form. The kit container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means that may contain, preferably suitably, one or more components in small portions. If two or more components are present in the kit, the kit will also generally include a second, third or other further container that may separately contain the additional components. However, various combinations of components may be included in the vial. The kits of this disclosure will also typically include means for containing an anti-CD70 CAR molecule, cells containing it, and / or reagents for producing it in tight containment for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vial.

[0318] When the components of the kit are provided in one and / or more liquid solutions, the liquid solutions are aqueous solutions, in which case sterile aqueous solutions are particularly assumed. The composition may also be formulated into a syringe-injectable composition. In such cases, the container means may be a syringe, pipette and / or other similar device from which the composition may be applied to an infected area of ​​the body, injected into an animal body, and / or 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 (one or more). When reagents and / or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. It is also assumed that the solvent may be provided in a separate container.

[0320] Regardless of the number and / or type of containers, the kits of this disclosure may also include, and / or be packaged with, instruments for assisting the injection / administration and / or placement of the final composition into the body of an animal. Such instruments may be syringes, pipettes, forceps, and / or any such medically approved delivery vehicle. In some embodiments, a variety of reagents or devices or containers may be included in the kit for ex vivo use. [Examples]

[0321] The following embodiments are included to illustrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following embodiments represent techniques found by the inventors to function well in the implementation of the present invention and may therefore be considered to constitute preferred embodiments for its implementation. However, it should be understood that many variations can be made in light of this disclosure in the specific embodiments disclosed, and that many variations may still produce similar or analogous results without departing from the spirit and scope of the present invention.

[0322] Example 1 CD27-containing CARs for targeting CD70-positive tumors The inventors first confirmed CD70 expression in AML patient samples. Tsne plots from mass cytometry data showed high CD70 expression in primary AML samples (n=54), but not in healthy CB CD34+ cells (n=10) (Figure 1A). As shown in Figure 1B, the Tsne plots also showed various bone marrow markers in lineage-negative cells from CB samples (upper CB set from Figure 1A) and AML samples (lower AML population from Figure 1A). CD70 expression in AML samples was similar to that of other well-characterized bone marrow markers.

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

[0324] Figure 4 shows that the TrCD27 construct exhibited higher transduction efficiency compared to the flCD27 construct. Retroviral supernatant collected from transfection experiments (Figure 3) was used for transduction into umbilical cord blood-derived NK (CBNK) cells, and RetroNectin was used to enhance transduction efficiency. Transduction efficiency was measured 48 hours post-transduction by examining CD27 surface expression in 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, IgG surface expression in CBNK cells was used to determine transduction efficiency. Under these conditions, the flCD27 construct showed insufficient transfection and transduction efficiency, highlighting the importance of the trCD27 construct.

[0325] In Figures 5A-5C, transduced trCD27 constructs induced apoptosis in cancer cells. The Annexin V-LIVE / DEAD® Fixable Aqua staining assay was performed to investigate whether transduced trCD27 constructs CBNK cells could induce death in 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). Transduced trCD27 constructs NK cells and cancer cells were co-cultured in a 1:1 ratio for 4 hours and stained with Annexin V-LIVE / DEAD® Fixable Aqua. When various trCD70-transduced CBNK cells (e.g., #3, #13, #15, #17, #19, #20, etc.) were compared to NT CBNK cells, IL15 CAR-transduced CBNK cells, or CD70-IL15 CAR-transduced CBNK cells (Figure 5C), they exhibited superior killing power against both Raji cells (black bars) and Karpas cells (gray bars).

[0326] In Figure 6, CB-NK cells transduced with the trCD27 construct 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) in a 1:1 ratio for 6 hours in the presence of CD107a antibody. After incubation, CD107a expression was evaluated by flow cytometry. Most trCD70-transduced CB-NK cells showed enhanced cytotoxic activity against Raji cells (center black bar) and Karpas cells (right gray bar) when compared to NT CB-NK cells, IL15 CAR-transduced CB-NK cells, or CD70-IL15 CAR-transduced CB-NK cells. CB-NK cells not co-cultured with cancer cells showed minimal basal levels of CD107a expression (left green bar).

[0327] Figures 7A and 7B demonstrate that transduced CB-NK cells with the trCD27 construct 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) in a 1:1 ratio, and the real-time cytotoxicity of NK cells against Raji and Karpas cells was measured hourly over a 12-hour period. All trCD70 CAR-transduced CB-NK cells showed 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] Figures 8A and 8B demonstrate that transduced trCD27 CAR CB-NK cells possess effective antitumor activity against CD70-positive Raji cells in vivo. NSG mice were transplanted with firefly luciferase-labeled Raji cells (Raji-FFluc) exhibiting high CD70 expression. Mice were injected with 20,000 Raji cells / mouse and 5M CBNK cells / mouse. Bioluminescence imaging of tumors showed that transduced trCD27 CAR CB-NK cells could reduce tumor burden caused by high-CD70 Raji cells (Figure 8A). In addition, survival curves compared non-transduced (NT) cells and cells transduced with IL15 CAR or CD70-IL15 CAR showed a significant survival benefit to NSG mice with Raji cells when various trCD27 CAR transduced CB-NK cells were injected (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 the p-values.

[0329] Figures 9A and 9B demonstrate that CB-NK cells transduced with the trCD27 CAR possess effective antitumor activity against CD70-positive AML cells in vivo. NSG mice were transplanted with firefly luciferase-labeled THP-1 cells (THP1-FFluc) that expressed high CD70. Mice were injected with 100,000 THP1 cells per mouse and 5 million CBNK cells per mouse. Bioluminescence imaging of tumors showed that CB-NK cells transduced with the trCD27 CAR could reduce the tumor burden caused by THP-1 cells with high CD70 expression (Figure 9A). In addition, survival curves showed a significant survival benefit for NSG mice with THP-1 when a single dose of various trCD27 CARs was injected into CB-NK cells, 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). The Mantel-Cox test was used to estimate p-values.

[0330] While the Disclosure and its merits are described in detail, it should be understood that various changes, substitutions, and modifications may be made herein without departing from the spirit and scope of the design as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, apparatus, manufactures, compositions, means, methods, and steps described herein. As will be readily apparent to those skilled in the art, processes, apparatus, manufactures, compositions, means, methods, or steps that perform substantially the same function as or achieve substantially the same results as the corresponding embodiments described herein, whether currently existing or to be developed in the future, may be utilized in accordance with this Disclosure. Accordingly, the appended claims are intended to include within their scope such processes, apparatus, manufactures, compositions, means, methods, or steps.

Claims

1. A polynucleotide encoding an anti-CD70 chimeric antigen receptor (CAR), comprising a signal peptide, an anti-CD70 antigen-binding domain including the extracellular domain of CD27, a transmembrane domain not comprising the transmembrane domain of CD27, and at least one intracellular domain, wherein the anti-CD70 antigen-binding domain is antibody-free.

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

5.

3. The polynucleotide according to claim 1 or 2, wherein the transmembrane domain comprises, or essentially comprises, a transmembrane domain derived from CD28, the α chain of a T cell receptor, the β chain of a T cell receptor, the ζ chain of a T cell receptor, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, or DAP12, is codon-optimized, and / or contains the sequence of SEQ ID NO: 3 or SEQ ID NO:

7.

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

12.

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

6.

6. The signal peptide contains the sequence of SEQ ID NO: 6, consists of the sequence of SEQ ID NO: 6, or is essentially composed of the sequence of SEQ ID NO:

6. The antigen-binding domain comprises or essentially comprises the sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO:

5. The transmembrane domain comprises, or essentially comprises, the sequence of SEQ ID NO: 3 or SEQ ID NO: 7; and The polynucleotide according to any one of claims 1 to 5, wherein the intracellular domain includes one or more of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

7. The polynucleotide according to any one of claims 1 to 6, wherein the CAR comprises one or more of the following: (c) SP of CD27, EC of CD27, TMD of CD28, ICD of DAP12, and zeta of CD3; (d) SP of GMSCF-R, EC of codon-optimized CD27, TMD of CD28, ICD of DAP12, and CD3 zeta; (g) SP of CD27, EC of CD27, TMD of CD28, ICD of natural killer group 2 member D (NKG2D), and CD3 zeta; (h) SP of GMSCF-R, EC of codon-optimized CD27, TMD of CD28, ICD of NKG2D, and CD3 zeta; (k) SP of CD27, EC of CD27, TMD of CD28, ICD of 4-1BB, and zeta of CD3; (l) SP of GMSCF-R, codon-optimized EC of CD27, TMD of CD28, ICD of 4-1BB, and CD3 zeta; (o) SP of CD27, EC of CD27, TMD of CD28, ICD of DAP10, and zeta of CD3; (p) SP of GMSCF-R, EC of codon-optimized CD27, TMD of CD28, ICD of DAP10, and CD3 zeta; (ee) CD27 SP, CD27 EC, CD28 TMD, and CD3 zeta; (ff) SP of GMSCF-R, EC of codon-optimized CD27, TMD of codon-optimized CD28, and CD3 zeta; (ii) SP of CD27, EC of CD27, TMD of CD28, ICD of CD28, and zeta of CD3; 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 a polynucleotide encoding the polypeptide described in SEQ ID NO: 1 or one of the sequences of SEQ ID NOs: 15 to 37.

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

30.

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

11. The immune cells according to claim 10, wherein the immune cells are natural killer (NK) cells, T cells, gamma-delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, MSCs, or dendritic cells.

12. The immune cells according to claim 10, wherein the immune cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, cell lines, or mixtures thereof, or are CD56+ NK cells.

13. The immune cells according to claim 11 or 12, wherein the immune cells express 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 cells having polynucleotides as described in any one of claims 1 to 8, wherein the composition is used in a method for killing CD70-positive cells in an organism, the method comprising the step of administering a therapeutically effective amount of cells to the organism.

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

16. The cells having the polynucleotides are NK cells, T cells, gamma-delta T cells, invariant NKT (iNKT) cells, B cells, macrophages, MSCs, or dendritic cells; The NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, bone marrow, cell lines, or mixtures thereof; and / or The composition according to claim 14 or 15, which is of the same species or self-derived from the individual.

17. The composition according to any one of claims 14 to 16, wherein the composition is administered to the individual once or more times, wherein, in the case of one or more administrations of cells containing polynucleotides, the duration of time between administrations of cells to the individual is 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or more than 1 year; and is administered by injection, intravenously, intraarterially, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, intracranially, percutaneously, subcutaneously, locally, by perfusion, into the tumor microenvironment, or a combination thereof.

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

19. The aforementioned individual is a human, A composition according to any one of claims 14 to 18, having a cancer that expresses CD70; and / or 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.

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

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