Compositions and methods for cellular immunotherapy

Abstract

Provided are methods for treating a subject of a solid tumor expressing GPC3 comprising administering to the subject an anti-GPC3 chimeric antigen receptor immunoresponsive cell wherein the administering is performed after or concurrently with subjecting the subject to a lymphopenia treatment. A kit for use in the method is also provided.

Description

Cross-referencing

[0001] This application claims the benefit of Chinese Patent Application No. CN201610256568.9, filed on April 22, 2016, the contents of which are incorporated herein by reference in their entirety. This application is a divisional application of the invention patent application filed on April 21, 2017, with application number CN201780038997.1 and entitled "Composition and Method for Cell Immunotherapy". Background Technology

[0002] Cancer has a significant social impact worldwide. In 2016 alone, an estimated 1,685,210 new cancer diagnoses were recorded in the United States, and 595,690 people were projected to die from the disease. According to... Journal of Oncology Practice （ Erikson 2007 ） By 2020, approximately one in 19 of the 18.2 million Americans will have either been diagnosed with or survived cancer, up from one in 26 in 2005 among the 11.7 million Americans.

[0003] Chimeric antigen receptors (CARs) are recombinant receptors for antigens that redirect the specificity and function of T cells and other immune cells to a single molecule. The use of CARs in cancer immunotherapy can bypass the barriers of active immunization, rapidly generating tumor-targeting T cells. Once expressed in cells, CAR-modified cells can exert immediate and long-term effects in subjects.

[0004] Chimeric antigen receptor (CAR) T-cell therapy, which edits a cancer patient's T cells to recognize their tumors, has shown efficacy in treating blood cancers. In recent clinical trials, CAR T-cell therapy significantly improved outcomes for patients with advanced, otherwise incurable blood cancers such as leukemia and lymphoma. In contrast, CAR T-cell therapy for solid tumors faces a number of unique challenges. These challenges include identifying the antigens expressed that clearly distinguish tumors from normal tissue, and establishing effective tumor-killing capabilities within the tumor to reduce tumor size. Summary of the Invention

[0005] There is an urgent need for alternative and effective treatments for various solid tumors. This invention addresses this need and also provides related advantages. Therefore, this invention discloses a method for treating a subject with a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3). In some cases, the method may include administering anti-GPC3 chimeric antigen receptor immune response cells to the subject. In some cases, administration may be performed after or concurrently with lymphopenic therapy in the subject. In some cases, the immune response cells may be NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells). In some cases, administration of anti-GPC3-CAR T cells to the subject may be performed after subjecting the subject to lymphopenic therapy. In some cases, the amount of GPC3-CAR T cells that may be administered to the subject is at least about 5 × 10⁻⁶. 4 Cells / kg. In some cases, the amount of GPC3-CAR T cells administered to the subject was approximately 5 × 10⁻⁶. 4 To approximately 1 × 10 12Applications per kg. Administration can effectively reduce tumor size by at least 30%, as measured by computed tomography (CT) scans. In some cases, administration can effectively stabilize tumor size, defined as a change of less than 10% from the baseline measurement of the tumor lesion diameter as measured by CT scans. In some cases, administration of anti-GPC3-CAR T cells in combination with lymphopenia treatment synergistically extends median survival by at least approximately 6 months compared to administration of anti-GPC3-CAR T cells alone. In some cases, solid tumors may be liver cancer, gastric cancer, thyroid cancer (e.g., thyroid tumors), lung cancer, breast cancer, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, noneminomatous germ cell carcinoma of the testis, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer. In some cases, the anti-GPC3 chimeric antigen receptor may include an antigen-binding unit that can specifically bind to the C-terminus of GPC3. In some cases, the antigen-binding unit may contain a sequence that exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some cases, anti-GPC3-CAR may contain a sequence that exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.Anti-GPC3-CAR may contain a sequence exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with any of SEQ ID NOs 28 to 30. In some cases, anti-GPC3-CAR T cells may contain at least two intracellular signaling domains. In some cases, anti-GPC3-CAR T cells may contain at least three intracellular signaling domains. The intracellular signaling domains may be selected from signaling domains derived from CD3, CD28, 4-1BB, OX40, DAP10, or ICOS. In some cases, lymphopenic therapy may include reducing the number of regulatory T cells in the subject. Reducing the number of regulatory T cells may include reducing the number of regulatory T cells by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, based on the amount of regulatory T cells in the subject’s circulating CD4. + and CD25 + Cells are analyzed by flow cytometry. In some cases, lymphopenia treatment may include administration of radiation or biological agents to the subject. Lymphopenia treatment may also include administration of chemotherapy to the subject. Administration of chemotherapy to the subject may include administration of chemotherapeutic agents selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof. In some cases, at least one chemotherapy agent may be administered to the subject before administration of anti-GPC3-CAR T cells. In some cases, lymphopenia treatment may reduce the number of lymphocytes by at least about 20%, which is measured by complete blood count (CBC) analysis.

[0006] This article may also disclose a method that further includes administering anti-GPC3-CAR T cells to the subject a second time. In some cases, the subject may have a refractory, persistent, or progressive disease. The anti-GPC3-CAR T cells may be autologous or allogeneic relative to the subject.

[0007] This article may also disclose a method that further includes administering at least one immunostimulant to a subject concurrently with or after administration of anti-GPC3 chimeric antigen receptor immune response cells. The immunostimulant may be selected from aldehyde interleukin (IL-2), IL-3, IL-6, IL-11, GM-CSF, and any combination thereof. The biological agent may be an antibody against an antigen expressed on lymphocytes.

[0008] This article discloses a kit for administering anti-GPC3-CAR immune response cells to subjects exhibiting solid tumors, comprising: an effective amount of anti-GPC3-CAR immune response cells; a chemotherapeutic agent effective in reducing lymphocytes present in the subject; and instructions for administering the anti-GPC3-CAR immune response cells to the subject after or concurrently with the chemotherapeutic agent. In some cases, the anti-GPC3-CAR immune response cells may be NK cells or T cells (anti-GPC3-CAR T cells). The chemotherapeutic agent may be selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof. The kit may further comprise approximately 60 mg / kg to approximately 80 mg / kg of cyclophosphamide or approximately 25 mg / kg of cyclophosphamide formulated for administration to subjects in need. 2 Approximately 35 mg / m 2 Fludarabine. In some cases, the kit may contain approximately 1 x 10 4 One cell to approximately 1 x 10 12 Anti-GPC3-CAR T cells. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells at least 12 hours after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells at least 24 hours after chemotherapy. Anti-GPC3-CAR T cells can be formulated for intravenous injection. Anti-GPC3-CAR T cells can be formulated for intra-arterial injection into the liver of a subject who may contain a solid tumor.

[0009] Specifically, this application provides the following technical solutions:

[0010] 1. A method of treating a subject with a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3), the method comprising administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or concurrently with lymphopenia treatment of the subject.

[0011] 2. The method according to item 1, wherein the immune response cells are NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells).

[0012] 3. The method according to item 2, wherein the administration of the anti-GPC3-CAR T cells to the subject is performed after the subject has received lymphopenia treatment.

[0013] 4. The method according to item 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is at least about 5 × 10⁻⁶.4 per kg.

[0014] 5. The method according to item 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is approximately 5 × 10⁻⁶. 4 From approximately 1 × 10 12 per kg.

[0015] 6. The method according to claim 1, wherein the application effectively reduces the tumor size by at least 30%, the tumor size being measured by computed tomography (CT) scan.

[0016] 7. The method according to item 1, wherein the application effectively stabilizes tumor size, and the stabilized tumor size is a change of less than 10% in the baseline measurement of the diameter of the tumor lesion as measured by computed tomography (CT) scan.

[0017] 8. The method according to item 2, wherein the combination of administration of anti-GPC3-CAR T cells and lymphopenia treatment synergistically extends the median survival of the subject by at least about 6 months compared to administration of the anti-GPC3-CAR T cells alone.

[0018] 9. The method according to item 1, wherein the solid tumor is liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell cancer, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer.

[0019] 10. The method of claim 1, wherein the anti-GPC3 chimeric antigen receptor comprises an antigen-binding unit that specifically binds to the C-terminus of GPC3.

[0020] 11. The method according to claim 10, wherein the antigen-binding unit comprises a sequence exhibiting at least 90% sequence homology to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0021] 12. The method according to claim 1, wherein the anti-GPC3-CAR comprises a sequence exhibiting at least 90% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30.

[0022] 13. The method according to claim 1, wherein the anti-GPC3-CAR comprises a sequence that exhibits at least 90% sequence identity with any one of SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30.

[0023] 14. The method according to item 2, wherein the anti-GPC3-CAR T cell comprises at least two intracellular signal transduction domains.

[0024] 15. The method according to item 2, wherein the anti-GPC3-CAR T cell comprises at least three intracellular signal transduction domains.

[0025] 16. The method according to item 14 or 15, wherein the intracellular signal transduction domain is selected from the signal transduction domains of CD3, CD28, 4-1BB, OX40, DAP10 or ICOS.

[0026] 17. The method according to claim 1, wherein the lymphopenia treatment comprises reducing the amount of regulatory T cells in the subject.

[0027] 18. The method of claim 17, wherein reducing the amount of regulatory T cells comprises reducing the number of said regulatory T cells by at least about 30%, the amount of said regulatory T cells being determined by the amount of circulating CD4+ in the subject. + and CD25 + Flow cytometry analysis of cells.

[0028] 19. The method of claim 1, wherein the lymphopenia treatment further comprises administering radiation or a biological agent to the subject.

[0029] 20. The method of claim 1, wherein the lymphopenia treatment further comprises administering other chemotherapy to the subject.

[0030] 21. The method of claim 20, wherein administering other chemotherapy to the subject comprises administering a chemotherapeutic agent selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof.

[0031] 22. The method according to item 21, wherein the chemotherapeutic agent is administered to the subject at least once prior to the administration of the anti-GPC3-CAR T cells.

[0032] 23. The method according to item 1, wherein the lymphopenia treatment reduces the number of lymphocytes by at least about 20%, the number of lymphocytes being measured by complete blood cell count (CBC) analysis.

[0033] 24. The method according to item 2 further includes administering the anti-GPC3-CAR T cells to the subject a second time.

[0034] 25. The method according to item 1, wherein the subject suffers from a refractory, persistent, or progressive disease.

[0035] 26. The method according to item 2, wherein the anti-GPC3-CAR T cells are autologous or allogeneic relative to the subject.

[0036] 27. The method according to claim 1 further includes administering at least one immunostimulant to the subject concurrently with or after the administration of the anti-GPC3 chimeric antigen receptor immune response cells.

[0037] 28. The method according to item 27, wherein the immunostimulant is selected from interleukin (IL-2), IL-3, IL-6, IL-11, GM-CSF and any combination thereof.

[0038] 29. The method according to claim 19, wherein the biological agent is an antibody that targets an antigen expressed on lymphocytes.

[0039] 30. A kit for administering anti-GPC3-CAR immune response cells to a subject exhibiting a solid tumor, comprising:

[0040] (a) An effective amount of anti-GPC3-CAR immune response cells;

[0041] (b) Chemotherapy agents that effectively reduce the presence of lymphocytes in the subjects; and

[0042] (c) Instructions for administration of anti-GPC3-CAR immune response cells to the subject after or simultaneously with the chemotherapy agent.

[0043] 31. The kit according to item 30, wherein the anti-GPC3-CAR immune response cells are NK cells or T cells (anti-GPC3-CAR T cells).

[0044] 32. The chemotherapeutic agent according to claim 30, wherein the chemotherapeutic agent is selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof.

[0045] 33. The kit according to claim 30 further comprises about 60 mg / kg to about 80 mg / kg of cyclophosphamide or about 25 mg / kg of cyclophosphamide formulated for administration to a subject in need. 2 Approximately 35 mg / m 2 Fludarabine.

[0046] 34. The medicine box according to item 31, comprising about 1 x 10 8 To approximately 1 x 10 11 One anti-GPC3-CAR T cell.

[0047] 35. The kit according to item 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells after administration of the chemotherapeutic agent.

[0048] 36. The kit according to item 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells at least 12 hours after administration of the chemotherapeutic agent.

[0049] 37. The kit according to item 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells at least 24 hours after administration of the chemotherapeutic agent.

[0050] 38. The kit according to item 31, wherein the anti-GPC3-CAR T cells are formulated for intravenous injection.

[0051] 39. The kit according to claim 31, wherein the anti-GPC3-CAR T cells are formulated for intra-arterial injection into the liver of the subject containing the solid tumor. Incorporation

[0052] All publications, patents, and patent applications mentioned herein are incorporated by reference as if each individual publication, patent, or patent application were specifically and individually incorporated by reference. In the event of any conflict between the terminology used herein and that used in the incorporated references, the terminology used herein shall prevail. Attached Figure Description

[0053] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be gained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments in which the principles of this disclosure may be utilized; in the drawings:

[0054] Figure 1 This demonstrates a second-generation CAR-T vector encoding a chimeric antigen receptor targeting GPC3.

[0055] Figure 2 The ratio of lymphocytes to baseline levels in individuals H01, H02, or H03 treated with cyclophosphamide and / or fludarabine is shown.

[0056] Figure 3 The images show magnetic resonance imaging (MRI) scans of liver sections before and after treatment.

[0057] Figure 4A The non-radioactive cytotoxicity assays of CytoTox 96® on 33-28 BBZ, 92-28 BBZ, and 4-28 BBZ cells co-cultured with control SK-HEP-1 cells are shown. Figure 4B The non-radioactive cytotoxicity assays of CytoTox 96® on 33-28Z, 92-28Z, 92-BBZ and 92-28BBZ co-cultured with the second control cell line CHO-K1 are shown, with effector:target ratios of 3:1, 1:1 or 1:3.

[0058] Figure 5A The non-radioactive cytotoxicity assay of CytoTox 96® on 33-28 BBZ, 92-28 BBZ, and 4-28 BBZ co-cultured with GPC3-positive Huh-7 liver cancer cells is shown. Figure 5B The assay is shown in 33-28BBZ, 92-28BBZ, and 4-28BBZ cells co-cultured with control cells transduced to express GPC3, CHO-K1-GPC3+, with effector:target ratios of 3:1, 1:1, or 1:3.

[0059] Figure 6A The CytoTox 96® non-radioactive cytotoxicity assay is shown on anti-GPC3 CAR-T cells co-cultured with HepG2 (HCC) in 92-28Z, 92-BBZ, 92-28BBZ or empty vector. Figure 6B The CytoTox 96® non-radioactive cytotoxicity assay is shown on anti-GPC3 CAR-T cells co-cultured with Hep3B (HCC) in 92-28Z, 92-BBZ, 92-28BBZ, or empty vector. Figure 6CThe CytoTox 96® non-radioactive cytotoxicity assay is shown on anti-GPC3 CAR-T cells co-cultured with PLC / PRF / 5 (hepatocellular carcinoma) in 92-28Z, 92-BBZ, 92-28BBZ or empty vector.

[0060] Figure 7A The CytoTox 96® non-radioactive cytotoxicity assay performed on anti-GPC3 CAR-T cells 33-28Z or 33-28BBZ co-cultured with Huh-7 cells is shown (effectant:target ratio of 3:1, 1:1 or 1:3). Figure 7B The non-radioactive cytotoxicity assay of CytoTox 96® on anti-GPC3 CAR-T cells 92-28Z or 92-28BBZ co-cultured with the gastric cancer cell line KATO-III is shown (effectant:target ratio of 3:1, 1:1 or 1:3).

[0061] Figure 8A Mice treated with second-generation CAR-T92-28Z showed significantly reduced tumor size compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8B Tumor images are shown, demonstrating that mice treated with second-generation CAR-T 92-28Z and transplanted with Huh-7 cells had significantly smaller tumors compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8C Mice treated with second-generation CAR-T 92-28Z also showed a significant reduction in tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8D Mice treated with second-generation CAR-T 92-28Z also showed significantly reduced inhibition compared to mice treated with third-generation CAR-T 92-28BBZ or saline.

[0062] Figure 9 The specific dissolution results of various second- and third-generation anti-GPC3 CAR constructs 4-28Z, 4-28BBZ, 4-4-28BBZ, 4-14-28BBZ, 4-20-28BBZ, 4-35-28BBZ, and 4-42-28BBZ co-cultured with Huh7 (HCC) cells in a cytoTox 96® non-radioactive cytotoxicity assay are shown. The effector:target ratio is 3:1 or 1:1.

[0063] Figure 10AThe CytoTox 96® non-radioactive cytotoxicity assay is shown on anti-GPC3 CAR-T cells 33-28Z, 92-28Z, 92-BBZ, 92-28BBZ or empty vector co-cultured with A431 (GPC3 negative). Figure 10B The non-radioactive cytotoxicity assays of CytoTox 96® performed on anti-GPC3 CAR-T cells co-cultured with Huh-7 (GPC3-positive) CAR-T cells in 33-28Z, 92-28Z, 92-BBZ, 92-28BBZ, or empty vector are shown. CytoTox assays were performed at effector:target ratios of 3:1, 1:1, or 1:3.

[0064] Figure 11A Results from a xenograft mouse model are shown, in which mice treated with second-generation CAR-T 92-28Z had significantly reduced tumor volume (mm²) compared to mice treated with third-generation CAR-T 92-28BBZ or empty vector. 3 ). Figure 11B The results showed that mice treated with second-generation CAR-T 92-28Z had significantly reduced tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 11C Images of tumors from mice treated with empty vector, 92-28BBZ, or 92-28Z CAR-T are shown. Detailed Implementation

[0065] The following description and examples illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein and is therefore subject to variation. Those skilled in the art will recognize that many variations and modifications are possible within the scope of this disclosure. Unless otherwise stated, any embodiment may be combined with any other embodiment.

[0066] As used herein, unless otherwise stated, some embodiments of the invention take into account numerical ranges. Various aspects of the invention may be presented in range format. It should be understood that the range format description is for convenience and brevity only and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of ranges should be considered as explicitly disclosing all possible subranges and individual numerical values ​​within those ranges. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. When a range exists, it includes the range endpoints. definition

[0067] As used herein, unless otherwise specified, the article “a” means one or more, unless otherwise expressly stated.

[0068] As used herein, unless otherwise stated, terms such as “comprising,” “containing,” and “including” mean “including.”

[0069] As used herein, the term “activation” and its grammatical equivalents can refer to the process by which a cell transitions from a quiescent state to an active state. This process may include responses to antigens, migration, and / or phenotypic or genetic alterations to a functionally active state. For example, the term “activation” can refer to the stepwise process of T cell activation. For instance, a T cell may require at least two signals to become fully activated. The first signal may occur after the TCR is conjugated by the antigen-MHC complex, and the second signal may occur through the conjugation of co-stimulatory molecules (Table 3). In vitro, anti-CD3 can mimic the first signal and anti-CD28 can mimic the second signal. For example, engineered T cells can be activated by expressed CARs. As used herein, “T cell activation” or “T cell triggering” can refer to the state of a T cell that has been adequately stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0070] As used herein, the term "antigen-binding unit" refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules (i.e., molecules containing antigen-binding sites that specifically bind to antigens ("immune response"). The term "antigen-binding unit" also includes immunoglobulin molecules from various species, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibody (e.g., IgG) consists of four polypeptide chains: two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Immunoglobulins represent a large family of molecules, including several types such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or modified antibodies and their fragments. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen-binding units." The term "antigen-binding unit" also includes any molecular structure containing polypeptide chains with a specific shape suitable for recognizing epitopes, wherein one or more non-covalently binding interactions stabilize the complex between the molecular structure and the epitope.

[0071] If an antigen-binding unit binds to an antigen with greater affinity or affinity than other reference antigens, including peptides or other substances, then it is considered to have a “specific binding” or “immune response” to the antigen.

[0072] As used herein, “antigen” refers to a substance that is specifically recognized and bound by an antigen-binding unit. Antigens may include peptides, proteins, glycoproteins, polysaccharides, and lipids; portions thereof and combinations thereof. Non-limiting exemplary antigens include human and mouse GPC3 and other homologs. “Antigen” may also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can act as an antigen.

[0073] As used herein, the term "immunoglobulin" or "Ig" can refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called chimeric antigen receptors or antigen receptors. This class of proteins includes five members: IgA, IgG, IgM, IgD, and IgE, with IgG being the most common circulating antibody. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is crucial in the defense against bacteria and viruses. For example, CARs can recognize tumor cell antigens.

[0074] The term "anti-GPC3 antibody" can refer to an antibody or antibody-binding site that is capable of binding to GPC3 with sufficient affinity to distinguish GPC3 from other antigens expressed by the cell. In one embodiment, the anti-GPC3 antibody binds to less than about 10% of the antibody binding to GPC3, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody binding to GPC3 may have <1 μM, <100 nM, <10 nM, <5 nM, <4 nM, <3 nM, <2 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 μM). -8 M or smaller, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M). In some embodiments, the anti-GPC3 antibody binds to an epitope of GPC3 that is conserved among GPC3 from different species.

[0075] As used herein, the term "autologous" and its grammatical equivalents can refer to substances originating from the same organism. For example, a sample (e.g., cells) may be removed, processed, and returned to the same subject (e.g., a patient) at a later time. Autologous processes are distinguished from allogeneic processes, in which the donor and recipient are different subjects.

[0076] As used herein, “xenotransplantation” and its grammatical equivalents can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, where the recipient and donor are of different species. The transplantation of cells, organs, and / or tissues described herein can be used for xenotransplantation into humans. Xenotransplantation includes, but is not limited to, vascularized xenotransplantation, partially vascularized xenotransplantation, non-vascularized xenotransplantation, xenodressing, xenobandage, and xenostructures.

[0077] As used herein, “allogeneic transplantation” and its grammatical equivalents (e.g., allogeneic transplantation) can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, wherein the recipient and the donor are different individuals of the same species. The transplantation of cells, organs, and / or tissues described herein can be used for allogeneic transplantation into humans. Allogeneic transplantation includes, but is not limited to, vascularized allogeneic transplantation, partially vascularized allogeneic transplantation, non-vascularized allogeneic transplantation, allogeneic dressings, allobandages, and allogeneic structures.

[0078] As used herein, “autologous transplantation” and its grammatical equivalents (e.g., autologous transplantation) can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, wherein the recipient and the donor are the same individual. The transplantation of cells, organs, and / or tissues described herein can be used for autologous transplantation into humans. Autologous transplantation includes, but is not limited to, vascularized autologous transplantation, partially vascularized autologous transplantation, non-vascularized autologous transplantation, autologous dressings, autologous bandages, and autologous structures.

[0079] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered molecule that can be expressed by immune cells, including but not limited to T cells. When expressed in T cells, a CAR can redirect T cells to induce the killing of target cells with artificial receptor-directed specificity. The extracellular binding domain of a CAR can be derived from mouse, humanized, or fully human monoclonal antibodies. "Anti-GPC3-CAR" is a CAR that can bind to GPC3.

[0080] As used herein, the term "epitope" and its grammatical equivalents can refer to a portion of an antigen that can be recognized by antibodies, B cells, T cells, or engineered cells. For example, an epitope can be a cancer epitope recognized by TCRs. Multiple epitopes within an antigen can also be recognized. Epitopes can also be mutated.

[0081] As used herein, the term "engineering" and its grammatical equivalents can refer to one or more alterations to nucleic acids (e.g., nucleic acids within an organism's genome). The term "engineering" can also refer to alterations, additions, and / or deletions of genes. Engineered cells can also refer to cells with added, deleted, and / or altered genes.

[0082] As used herein, the terms “cell” or “engineered cell” and their grammatical equivalents can refer to cells of human or non-human animal origin. Engineered cells can also refer to cells that express CAR.

[0083] As used herein, the term "Good Manufacturing Practice" (GMP) and its grammatical equivalents can refer to products that are safe, effective, or pure according to FDA regulations. GMP is sometimes also referred to as "cGMP," where "c" stands for "current." Product manufacturers may employ the latest technologies and systems to comply with GMP regulations for their products. GMP-compatible products are typically used in clinical settings, which are the opposite of research environments.

[0084] As used herein, the term "transfection" refers to the introduction of foreign nucleic acids into eukaryotic cells. Transfection can be accomplished by a variety of methods known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0085] The term "stable transfection" or "stable transfection" refers to the introduction and integration of foreign nucleic acids, DNA, or RNA into the genome of a transfected cell. The term "stable transfectant" refers to a cell in which foreign DNA has been stably integrated into the genomic DNA.

[0086] As used herein, the terms “encoding nucleic acid molecule,” “encoding DNA sequence,” and “encoding DNA” refer to the sequence or order of deoxyribonucleotides along a chain of deoxyribonucleic acid (DNA). The sequence of these deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0087] As used herein, the term "subject" refers to any animal, such as a mammal or marsupial. Subjects of this invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0088] The term “recipient” and its grammatical equivalents used in this article can refer to a person or non-human animal receiving therapy or treatment.

[0089] As used herein, the term "peripheral blood lymphocytes" (PBL) and its grammatical equivalents can refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes can also refer to lymphocytes not located in organs. Peripheral blood lymphocytes can include T cells, NK cells, B cells, or any combination thereof.

[0090] The term "immune response cells" can refer to cells capable of initiating an immune response, including but not limited to T cells, B cells, and NK T cells, their respective precursor cells, and their progeny. Immune response cells can also refer to lymphoid or bone marrow cells.

[0091] As used in this article, the term "T cell" and its grammatical equivalents can refer to T cells of any origin. For example, T cells can be primary T cells, such as autologous T cells, cell lines, etc. T cells can also be human or non-human.

[0092] As used herein, the terms “T cell activation” or “T cell triggering” and their grammatical equivalents can refer to the state of T cells that have been adequately stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. In some cases, “complete T cell activation” can be analogous to triggering T cell cytotoxicity. T cell activation can be measured using a variety of assays known in the art. Assays can include ELISA, ELISPOT for measuring cytokine secretion, flow cytometry for measuring intracellular cytokine expression (CD107), flow cytometry for measuring proliferation, and cytotoxicity assays (51Cr release assays) for determining target cell elimination. These assays typically use controls (unengineered cells) to compare with engineered cells (CAR T cells) to determine the relative activation of engineered cells compared to controls. Additionally, these assays can compare engineered cells incubated or in contact with target cells that do not express the target antigen. For example, CD19-CAR T cells incubated with target cells that do not express CD19 can be compared.

[0093] The term "sequence" and its grammatical equivalents, when used to refer to a nucleotide sequence, may include DNA or RNA; and may be single-stranded or double-stranded. Nucleic acid sequences can be mutated. Nucleic acid sequences can have any length, for example, from 2 to 1,000,000 or more nucleotides (or any integer value between or above), such as about 100 to about 10,000 nucleotides or about 200 to about 500 nucleotides. How to use

[0094] In one aspect, this article discloses a method for treating subjects exhibiting solid tumors expressing phosphatidylinositol proteoglycan-3 (GPC3). The subject method generally includes the step of administering anti-GPC3 chimeric antigen receptor immune-response cells to the subject, wherein the administration is performed after or concurrently with lymphopenic therapy.

[0095] Subjects treated with the methods disclosed herein may exhibit cancer or solid tumors. Typically, cancer cells or solid tumor cells express one or more tumor antigens. Commonly, the tumor antigen is phosphatidylinositol proteoglycan-3 (GPC3). Subjects exhibiting cancer or solid tumors expressing GPC-3 may be termed GPC3-positive cancer.

[0096] Cancers expressing GPC3 include, but are not limited to, liver cancer, stomach cancer, esophageal cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell carcinoma, melanoma, adenoma, adrenal carcinoma, schwannoma, malignant fibrous histiocytoma, or any combination thereof. The subject method is applicable to the treatment of squamous cell carcinoma or adenocarcinoma, gastrointestinal neuroendocrine carcinoma, or any other cancer disclosed in "Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms. (2013) 44, 542-550 Human Pathology". Furthermore, the immune-response cells disclosed herein, such as the anti-GPC3-CAR-T cells, can be used to target ovarian cancer, cholangiocarcinoma, mesothelioma, breast cancer, lung squamous cell carcinoma, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, intrahepatic and extrahepatic cancer, gallbladder cancer, invasive ductal carcinoma, clear cell carcinoma, large eosinophilic tumor, papillary carcinoma, adenocarcinoma, and lobular and medullary carcinoma of the breast. Other targets for anti-GPC3 therapy may include those found in "Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues. (2008) 129:899-906 Am J Clin Pathol".

[0097] In some cases, GPC3 expression in cancer cells or tumor cells can be assessed by flow cytometry or immunohistochemistry. GPC3 levels on cancer cells or tumor cells can be classified as low, intermediate, or high.

[0098] In some cases, cancer cells or tumor cells may express GPC3 on their cell surface. For example, GPC3 expression on the cell surface can be determined using antibodies against GPC3 using methods such as immunohistochemistry or flow cytometry. Alternatively, GPC3 mRNA expression can be considered to be associated with GPC3 expression on the cell surface and can be determined using methods selected from in situ hybridization and RT-PCR.

[0099] In some cases, GPC3 is encoded by a gene containing a nucleic acid sequence that exhibits at least 50% sequence identity with the reference gene in Table 2. GPC3 may be encoded by a gene containing a nucleic acid sequence that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with the reference gene in Table 2.

[0100] In some implementations, anti-GPC3 chimeric antigen receptor immune response cells are administered to subjects who exhibit solid tumors expressing GPC3, either after or simultaneously with lymphopenia treatment.

[0101] Anti-GPC3 chimeric antigen receptors (CARs) typically comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that controls cellular activation of the immune response. In some cases, anti-GPC3 CARs further include a hinge or spacer region. In some cases, anti-GPC3 CARs further include one or more co-stimulatory domains.

[0102] Chimeric antigen receptors typically include an extracellular antigen-binding region. In one embodiment, the extracellular antigen-binding region may be fully human. In other cases, the extracellular antigen-binding region may be humanized. In still other cases, the extracellular antigen-binding region may be mouse or chimeric, wherein the extracellular antigen-binding region consists of amino acid sequences from at least two different animal species. In some cases, the extracellular antigen-binding region may be non-human. Various antigen-binding regions can be engineered to target GPC3. Non-limiting examples include single-chain variable fragments (scFv) from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments, or natural ligands that bind to their homologous receptors. Extracellular antigen-binding regions may include scFv, Fab, or natural ligands and any derivatives thereof. Extracellular antigen-binding regions may refer to molecules other than intact antibodies, which may include portions of intact antibodies and bind to antigens that can bind to intact antibodies. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0103] Extracellular antigen-binding regions (e.g., scFv, Fab, or natural ligands) can be a portion of the CAR that determines antigen specificity. Extracellular antigen-binding regions can bind to any complementary target. They can be derived from antibodies with known variable region sequences. They can also be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, they can be obtained from whole-exome sequencing of tumor cells or primary cells such as tumor-infiltrating lymphocytes (TILs).

[0104] In some cases, the binding specificity of the extracellular antigen-binding region can be determined by the complementarity-determining region, or CDR (such as the light chain CDR or heavy chain CDR). In many cases, binding specificity can be determined by both the light chain CDR and the heavy chain CDR. A given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket compared to other reference antigens, which can confer greater affinity and / or specificity to antigens such as GPC3. For example, a CDR specific for phosphatidylinositol proteoglycan-3 can be expressed in the extracellular binding region of a CAR, so that a CAR targeting GPC3 can enable immune-responding cells to target tumor cells expressing GPC3.

[0105] In some aspects of any of the embodiments disclosed herein, the extracellular antigen-binding region, such as the scFv, may include a light chain CDR that is specific for GPC3. The light chain CDR may be a complementarity-determining region of the light chain of an antigen-binding unit (such as the scFv of a CAR). The light chain CDR may comprise a sequence of consecutive amino acid residues, or two or more consecutive amino acid residue sequences separated by non-complementarity-determining regions such as framework regions and optionally side-attached. In some cases, the light chain CDR may comprise two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. In some cases, the light chain CDR may comprise three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In some instances, a group of CDRs present on a common light chain may be collectively referred to as light chain CDRs.

[0106] In some aspects of any of the embodiments disclosed herein, the extracellular antigen-binding region, such as scFv, may include a heavy chain CDR that is specific for GPC3. The heavy chain CDR may be a complementarity-determining region of the heavy chain of the antigen-binding unit (such as scFv). The heavy chain CDR may comprise a sequence of consecutive amino acid residues, or two or more consecutive amino acid residue sequences separated by non-complementarity-determining regions such as frames and optionally side-attached. In some cases, the heavy chain CDR may comprise two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. In some cases, the heavy chain CDR may comprise three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In some cases, a group of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.

[0107] In some cases, the extracellular antigen-binding domain targeting GPC3 can be expressed by anti-GPC3 CAR immune response cells. In some cases, the CDR, light chain, and / or heavy chain binding to the GPC3 antigen can be contained within the extracellular antigen-binding domain of CAR immune response cells, such as CAR T cells. In some cases, modified anti-GPC3 CDRs can be expressed on the extracellular antigen-binding domain of CAR immune response cells and have approximately 50% to approximately 100% homology with the original anti-GPC3 CDR. In some cases, modified anti-GPC3 CDRs may contain approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% homology with unmodified anti-GPC3 CDRs.

[0108] In some cases, GPC3-targeting scFvs can be expressed by anti-GPC3 CAR immune response cells. In some cases, GPC3 antigen-binding CDRs, light chains, and / or heavy chains can be expressed on scFvs of CAR immune response cells, such as CAR T cells. In some cases, modified anti-GPC3 CDRs can be expressed on scFvs of CAR immune response cells, such as CAR T cells, and have approximately 50% to approximately 100% homology with the original anti-GPC3 CDR. In some cases, modified anti-GPC3 CDRs may contain approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% homology with unmodified anti-GPC3 CDRs. Table 1: Exemplary anti-GPC3 extracellular antigen binding regions:

[0109] In a preferred embodiment, the extracellular antigen-binding region specifically recognizes GPC3. GPC3 may contain a sequence that is at least 80% identical to the GPC3 gene referenced in Table 2. In some cases, the extracellular antigen-binding region may target the N-terminus, C-terminus, or any portion from the N-terminus to the C-terminus of GPC3. The C-terminus of GPC3 may be covalently linked to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. In some cases, the C-terminus may contain about 1 to about 800 bases. The C-terminus may contain about 1, 50, 100, 150, 300, 500, 600, up to about 800 bases from the C-terminal tip. In some cases, the extracellular antigen-binding region may target the GPI anchor of GPC3. Table 2: GPC3

[0110] Extracellular antigen-binding domains (AGBs) can be modified in various ways using genetic engineering. In some cases, the ADB can be mutated to select for ADBs with higher affinity for their targets. In others, the affinity of the ADB for its target can be optimized for targets that are expressed at low levels in normal tissues. This optimization can be performed to minimize potential toxicity. In still others, clones of ADBs with higher affinity for the membrane-bound form of the target may be superior to their soluble counterparts. This modification is possible because some targets can also be detected at varying levels of soluble forms, and targeting them can cause unintended toxicity.

[0111] In some cases, the extracellular antigen-binding region may share approximately 50% to approximately 100% homology with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some cases, the extracellular antigen-binding region may contain approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% homology with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some cases, the extracellular antigen-binding region may be mouse, humanized, or fully human. The extracellular antigen-binding region may be approximately 1% to approximately 100% human. In some cases, the extracellular antigen-binding region can be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to approximately 100% of the human body.

[0112] In some cases, the extracellular antigen-binding region (CAR) contains a hinge or spacer region. The terms hinge and spacer region are used interchangeably. A hinge can be considered the CAR portion that provides flexibility to the CAR. In some cases, the hinge can be used to detect CARs on the cell surface, particularly when antibodies to detect the CAR are ineffective or unavailable. For example, the length of the hinge derived from immunoglobulins may need to be optimized depending on the location of the epitope on the target target of the CAR-binding region.

[0113] In some cases, the hinge may not belong to an immunoglobulin, but rather to the natural hinge of another molecule, such as the CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues known to play a role in the interaction between the CD8 co-receptor and MHC molecules. These cysteine ​​and proline residues can affect the performance of the CAR.

[0114] CAR hinges can be adjustable in size and can be compensated to some extent to normalize the orthogonal synaptic distance between CAR-responding cells and target cells. This morphology of the immune synapse between immune-responding cells and target cells also defines the distances that cannot be functionally bridged by CARs due to distal membrane epitopes on cell surface target molecules; even with short-hinge CARs, the synaptic distance cannot be brought close enough for signal transduction. Similarly, proximal membrane CAR target antigen epitopes have been described, where signal transduction output was observed only against the background of long-hinge CARs. The hinge can be adjusted according to the extracellular antigen-binding region used. The hinge can have any length.

[0115] Transmembrane domains anchor CARs to the cell's plasma membrane. The native transmembrane portion of CD28 can be used with CARs. In other cases, the native transmembrane portion of CD8α can also be used with CARs. "CD8" may refer to a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with NCBI reference number NP_001759 or its stimulatory fragment thereof. "CD8 nucleic acid molecule" may refer to a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region may be the native transmembrane portion of CD28. "CD28" may refer to a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with NCBI reference number NP_006130 or its stimulatory fragment thereof. "CD28 nucleic acid molecule" may refer to a polynucleotide encoding a CD28 polypeptide. In some cases, the transmembrane portion may include the CD8α region.

[0116] The intracellular signaling domain of a CAR can be responsible for activating at least one of the effector functions of an immune response cell in which the CAR has been placed. CARs can induce effector functions of T cells, such as cytotoxic activity or co-activities including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire chain of the signaling domain. In some cases, a truncated portion of the intracellular signaling domain is used. In some cases, the term "intracellular signaling domain" therefore means any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals.

[0117] Preferred examples of signal transduction domains for CAR may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that work together to initiate signal transduction upon target-receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0118] In some cases, the intracellular signal transduction domain may contain a signal transduction motif, referred to as an immune receptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signal transduction sequences include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. However, in a preferred embodiment, the intracellular signal transduction domain originates from the CD3ζ chain.

[0119] An example of a T-cell signaling domain containing one or more ITAM motifs is the CD3ζ domain, also known as the T-cell receptor T3ζ chain or CD247. This domain is part of the T-cell receptor-CD3 complex and plays a crucial role in coupling antigen recognition with several intracellular signal transduction pathways involving primary effector activation of T cells. As used herein, CD3ζ is primarily targeted at human CD3ζ and its isotypes (as known from Swissprot entry P20963), including proteins with substantially identical sequences. Similarly, the complete T-cell receptor T3ζ chain is not required as part of a chimeric antigen receptor, and any derivative containing the signaling domain of the T-cell receptor T3ζ chain is suitable, including any functional equivalents thereof.

[0120] Intracellular signal transduction domains can be selected from any of the domains in Table 3. In some cases, domains can be modified such that homology with any reference domain can be from about 50% to about 100%. Any of the domains in Table 3 can be modified such that the modified version can contain about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% homology.

[0121] The intracellular signaling domain of a CAR may further include one or more co-stimulatory domains. The intracellular signaling domain may contain a single co-stimulatory domain, such as the ζ-chain (generation 1 CAR) or CD28 or 4-1BB (generation 2 CAR). In other instances, the intracellular signaling domain may contain two co-stimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (generation 3).

[0122] Along with intracellular signaling domains such as CD8, these co-stimulatory domains can generate downstream activation of kinase pathways that support gene transcription and functional cellular responses. The co-stimulatory domains of CARs can activate proximal signaling proteins associated with the CD28 (phosphatidylinositol-4,5-bisphosphate-3-kinase) or 4-1BB / OX40 (TNF-α receptor-associated factor adaptor) pathways, as well as MAPK and Akt activation.

[0123] In some cases, signals generated by CAR can be combined with secondary or co-stimulatory signals. Regarding co-stimulatory domains, chimeric antigen receptor-like complexes can be designed to include several possible co-stimulatory domains. As is known in the art, in naive T cells, the involvement of T cell receptors alone is insufficient to induce complete activation of T cells into cytotoxic T cells. Complete and effective T cell activation requires a second co-stimulatory signal. Several receptors that have been reported to provide co-stimulation for T cell activation include, but are not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, and 4-1BB. These co-stimulatory molecules utilize signaling pathways that share the common characteristic of synergizing with primary T cell receptor activation signals. These co-stimulatory signaling domains provide signals that can synergize with primary effector activation signals derived from one or more ITAM motifs (e.g., the CD3ζ signaling domain) and fulfill the needs for T cell activation.

[0124] In some cases, adding a co-stimulatory domain to the chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells. In another embodiment, the T cell signaling domain and the co-stimulatory domain are fused together to form a signaling region. Table 3. Co-stimulatory domains

[0125] In some cases, the subject CAR may contain a sequence or a portion thereof that exhibits approximately 50% to approximately 100% sequence identity with any one of SEQ ID NO: 9 to SEQ ID NO: 13 in Table 4. In some cases, the subject CAR may contain a sequence that exhibits approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% sequence identity with any one of SEQ ID NO: 9 to SEQ ID NO: 13. Table 4: CAR-T domains containing exemplary hinges, transmembrane domains, and intracellular domains:

[0126] In some cases, the subject CAR may contain a sequence that exhibits approximately 50% to approximately 100% sequence identity with any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 (Table 5). In some cases, the subject CAR may contain a sequence that exhibits approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% sequence identity with any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. Table 5: Exemplary Anti-GPC3 CAR

[0127] In some cases, anti-GPC3 CARs may possess at least one or more of the following properties in any combination: a) binding to recombinant human GPC3; b) binding to recombinant cynomolgus monkey GPC3; c) binding to endogenous GPC3 on the surface of HepG2 cells; d) binding to cynomolgus monkey GPC3 expressed on the surface of 293 cells; e) binding to endogenous GPC3 on the surface of cancer cells; f) binding to endogenous GPC3 on the surface of hepatocellular carcinoma cells; g) binding to endogenous GPC3 on the surface of cell lines selected from HepG2, Hep3B, Huh7, and JHH-7; h) binding to epitopes of human GPC3 from amino acids 25 to 137; i) binding to trans-human GPC3. Epitope binding at the furin cleavage site at amino acid R358 / S359 of GPC3; j) binding to full-length mature human GPC3, but not to the N-terminal or C-terminal fragment of human GPC3; k) binding to epitopes in amino acids 420 to 470 of human GPC3; l) binding to epitopes in amino acids 470 to 509 of human GPC3; m) competing with antibody 7H1 for binding to human GPC3; n) competing with antibody 4G7 for binding to human GPC3; o) competing with antibody 15G1 for binding to human GPC3; binding to the C-terminal fragment of human GPC3; and / or p) competing with antibody 4A11 for binding to human GPC3.

[0128] Transgenic genes encoding GPC3 CAR can be incorporated into cells. For example, they can be incorporated into immune-responding cells such as T cells. When inserted into a cell, the transgene can be a complementary DNA (cDNA) segment, a copy of messenger RNA (mRNA), or the gene itself residing in its original genomic DNA region (with or without introns).

[0129] Nucleic acid (e.g., DNA) encoding a transgenic sequence can be randomly inserted into the chromosome of a cell. Random integration can be achieved by any method of introducing nucleic acid (e.g., DNA) into the cell. For example, such methods can be, but are not limited to, electroporation, acoustic pore effect, use of a gene gun, lipid transfection, calcium phosphate transfection, use of dendritic cells, microinjection, and the use of viral vectors, including adenovirus, AAV, and retroviral vectors, and / or group II ribonucleases.

[0130] DNA encoding transgenes can be introduced into cells via electroporation. DNA can also be introduced into cells via lipid transfection, infection, or transformation. Electroporation and / or lipid transfection can be used to transfect primary cells. Electroporation and / or lipid transfection can be used to transfect primary hematopoietic cells. DNA can also be introduced into the cellular genome without the use of homologous recombination. In some cases, the flanking sites of the DNA can be engineered sites complementary to targeted double-strand breaks in the genome. In some cases, DNA can be excised from polynucleotides, allowing it to insert into double-strand breaks without homologous recombination.

[0131] The transgene to be inserted can be flanked by engineered sites similar to targeted double-strand break sites in the genome to excise the transgene from multiple nucleic acids, thereby allowing it to be inserted into the double-strand break region.

[0132] The DNA encoding the transgene can also be designed to include a reporter gene, allowing the presence of the transgene or its expression product to be detected by activating the reporter gene. Any reporter gene (such as those disclosed above) can be used. Cells containing the transgene can be selected by choosing cells in a cell culture where the reporter gene has been activated.

[0133] CAR expression can be verified by expression assays (e.g., qPCR) or by measuring RNA levels. Expression levels can also indicate copy number. For example, very high expression levels may indicate that more than one copy of the CAR has been integrated into the genome. Alternatively, high expression may indicate that the transgene is integrated into a highly transcribed region, such as near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as via Western blotting.

[0134] The subject-specific anti-GPC3 CAR immune response cells may contain one or more transgenes. One or more transgenes may express a CAR protein that recognizes and binds to at least one epitope (e.g., GPC3) on an antigen, or bind to a mutated epitope on the antigen. The CAR may be a functional CAR. The subject-specific anti-GPC3 CAR immune response cells may also contain one or more CARs, or may contain a single CAR and a secondary engineered receptor.

[0135] Transgenic cells can encode suicide genes. Targeted tumor regression in response to CAR immune response cells can be accompanied by toxicity, as demonstrated in many effective treatments for cancer patients. In some cases, when the target antigen is shared between tumor and normal tissue, CAR immune response cells may be unable to distinguish between tumor and normal tissue (“target-on / off-target” toxicity). In other cases, a systemic perturbation of the immune system, known as cytokine release syndrome (CRS), can occur. CRS can include systemic inflammatory response syndrome or a cytokine storm, which may result from the rapid in vivo expansion of CAR immune response cells. CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure in severe cases. In most cases, this toxicity is associated with the in vivo expansion of infused CAR immune response cells, which can cause a general perturbation of the immune system and release high levels of pro-inflammatory cytokines such as TNFα and IL-6.

[0136] In some cases, CAR immune response cells targeting antigens shared with normal tissues can be generated, such that they transiently express CAR, for example, after electroporation of the mRNA encoding the receptor. Furthermore, significant efforts have been made to further engineer CAR immune response cells by including a safety switch that allows for significant elimination of CAR immune response cells in cases of severe target toxicity. The CAR-encoding vector can be combined with a safety switch such as an inducible caspase-9 gene (activated by a dimerizing chemical inducer) or a truncated form of the EGF receptor R (activated by the monoclonal antibody cetuximab) or RQR8.

[0137] The subject matter of anti-GPC3 CAR immune response cells may encode a suicide gene transgene. The transgene may also include a CAR receptor or another similar receptor. The suicide gene can induce the elimination of CAR immune response cells. The suicide gene can be any gene that induces apoptosis in the CAR immune response cells. The suicide gene may be encoded within a viral vector along with the anti-GPC3 CAR.

[0138] One or more transgenic genes may originate from different species. For example, one or more transgenic genes may include human genes, mouse genes, rat genes, pig genes, bovine genes, dog genes, cat genes, monkey genes, chimpanzee genes, or any combination thereof. For example, a transgenic gene may originate from humans and have a human gene sequence. One or more transgenic genes may include human genes. In some cases, one or more transgenic genes are not adenovirus genes.

[0139] As described above, transgenes can be inserted into the genome of immune-responding cells in a random or site-specific manner. For example, a transgene can be inserted into a random locus in the genome of an immune-responding cell. These transgenes can be functional, for example, fully functional if inserted at any location in the genome. For example, a transgene can encode its own promoter or can be inserted at a location controlled by an endogenous promoter. Alternatively, a transgene can be inserted into a gene, such as an intron or exon, promoter, or non-coding region. Insertable transgenes can cause the insertion to interrupt the gene, such as an endogenous immune checkpoint.

[0140] Sometimes, more than one copy of a transgene can insert into more than one random locus in the genome. For example, multiple copies can insert into random loci in the genome. This can lead to an increase in overall expression compared to a single random insertion of the transgene. Alternatively, one copy of the transgene can insert into a gene, and another copy can insert into a different gene. Transgenes can be targeted so that they can insert into specific loci in the genome of immune-responding cells.

[0141] In some cases, polynucleotides containing sequences encoding the anti-GPC3 CAR can be in the form of plasmid vectors. Plasmid vectors may contain promoters. In some cases, the promoters may be constitutive. In some cases, the promoters may be inducible. The promoters may be derived from CMV, U6, MND, or EF1a. In some cases, the promoter may be adjacent to the CAR sequence. In some cases, the plasmid vector further contains a splice acceptor. In some cases, the splice acceptor may be adjacent to the CAR sequence. The promoter sequence may be a PKG or MND promoter. The MND promoter may be a synthetic promoter containing the U3 region of a modified MoMuLV LTR and a myeloproliferative sarcoma virus enhancer.

[0142] In some cases, polynucleotides encoding anti-GPC3 CAR themes can be engineered for delivery to cells via non-viral technologies. In other cases, polynucleotides can be Good Manufacturing Practice (GMP) compatible reagents.

[0143] The expression of polynucleotides encoding the anti-GPC3 CAR can be controlled by one or more promoters. Promoters can be ubiquitous, constitutive (unregulated promoters that allow for continuous transcription of the associated gene), tissue-specific, or inducible promoters. The expression of transgenes inserted near or around the promoter can be regulated. For example, transgenes can be inserted around or adjacent to ubiquitous promoters. Some ubiquitous promoters can be the CAGGS promoter, hCMV promoter, PGK promoter, SV40 promoter, or ROSA26 promoter.

[0144] Promoters can be endogenous or exogenous. For example, one or more transgenes can be inserted near or around an endogenous or exogenous ROSA26 promoter. Furthermore, promoters can be specific to immune-responding cells. For example, one or more transgenes can be inserted near or around a porcine ROSA26 promoter.

[0145] Tissue-specific or cell-specific promoters can be used to control the location of expression. For example, one or more transgenes can be inserted near or around a tissue-specific promoter. Tissue-specific promoters can be FABP promoters, Lck promoters, CamKII promoters, CD19 promoters, keratin promoters, albumin promoters, aP2 promoters, insulin promoters, MCK promoters, MyHC promoters, WAP promoters, or Col2A promoters.

[0146] Tissue-specific or cell-specific promoters can be used to control the location of expression. For example, one or more transgenes can be inserted near or around a tissue-specific promoter. Tissue-specific promoters can be FABP promoters, Lck promoters, CamKII promoters, CD19 promoters, keratin promoters, albumin promoters, aP2 promoters, insulin promoters, MCK promoters, MyHC promoters, WAP promoters, or Col2A promoters.

[0147] Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing the inducer as needed. Expected inducible promoters can be, but are not limited to, Lac, Tac, Trc, Trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and / or Trex.

[0148] In addition, although not required for expression, transgenic sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and / or polyadenylation signals.

[0149] In some cases, the transgene encodes a GPC3-resistant CAR, where the transgene inserts into a safe harbor, resulting in GPC3-resistant CAR expression. In some cases, the transgene inserts into the PD1 and / or CTLA-4 loci. In other cases, the transgene is delivered to cells via lentivirus for random insertion, with PD1 or CTLA-4 specific nucleases provided as mRNA. In some cases, the transgene is delivered via viral vector systems such as retroviruses, AAVs, or adenoviruses along with mRNA encoding nucleases specific to the safe harbor (e.g., AAVS1, CCR5, albumin, or HPRT). Cells may also be treated with mRNA encoding PD1 and / or CTLA-4 specific nucleases. In some cases, the CAR-encoding polynucleotide is provided via a viral delivery system along with mRNA encoding HPRT-specific nucleases and PD1 or CTLA-4 specific nucleases. CARs that can be used with the methods and compositions disclosed herein may comprise all types of these chimeric proteins, including first-, second-, and third-generation designs. Other reagents such as CCR2 or siRNA can be used to reduce PD-1 expression.

[0150] In some cases, retroviral vectors (gamma-retroviruses or lentiviruses) can be used to introduce transgenes into immune-responding cells. For example, a transgene encoding a CAR (e.g., an anti-GPC3 CAR) or any receptor that binds to the GPC3 antigen, or a variant or fragment thereof, can be cloned into a retroviral vector and its expression can be driven by its endogenous promoter, retroviral long terminal repeats, or promoters specific to the target cell type of interest. Non-viral vectors can also be used. Non-viral vector delivery systems may include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vectors such as liposomes or poloxamers.

[0151] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors offer additional advantages over vectors derived from oncogenetic retroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells. They also have the added advantage of low immunogenicity. Adenoviral vectors have the advantage of not integrating into the genome of target cells, thus avoiding negative integration-related events.

[0152] Cells can be transfected with CAR-encoded transgenes. Transgene concentrations can range from approximately 100 picograms to approximately 50 micrograms. In some cases, the amount of nucleic acids (e.g., ssDNA, dsDNA, RNA) that can be introduced into the cells can be varied to optimize transfection efficiency and / or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample to power pores. In some cases, the amount of nucleic acids (e.g., dsDNA) required for optimal transfection efficiency and / or cell viability can be cell type specific. In some cases, the amount of nucleic acids (e.g., dsDNA) used for each sample can directly correspond to transfection efficiency and / or cell viability. For example, a range of transfection concentrations. The vector-encoded transgene can be integrated into the cell genome. In some cases, the integration of the vector-encoded transgene is positive. In other cases, the integration of the vector-encoded transgene is negative.

[0153] In some cases, the starting cell density for viral delivery used for cell modifications such as CAR can be altered to optimize transfection efficiency and / or cell viability. In some cases, the starting cell density used for transfecting or transducing cells with a viral vector can be less than approximately 1 x 10⁻⁶ cells / year. 5 1 x 10⁻⁶ cells. In some cases, the starting cell density for cell modification using a viral vector can be at least about 1 x 10⁻⁶ cells. 5 From one cell to at least approximately 5 x 10 7 Cell density. In some cases, the starting cell density for optimal transfection efficiency and / or cell viability can be cell type specific. For example, for macrophages, 1.5 x 10⁻⁶ cells. 6 The initial cell density can be optimal (e.g., to provide the highest viability and / or transfection efficiency). In another example, for human cells, 5 x 102 6 The initial cell density for each cell type can be optimal (e.g., to provide the highest viability and / or transfection efficiency). In some cases, a certain range of initial cell densities may be optimal for a given cell type. For example, for human cells such as T cells, 5.6 x 10⁶ cells / mL is optimal. 6 5 x 10 7 The initial cell density can be optimal (e.g., to provide the highest viability and / or transfection efficiency).

[0154] The efficiency with which a nucleic acid sequence encoding a CAR can be integrated into the cell genome using, for example, a viral system, can be approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher than 99.9%. In some cases, the detection rate of CAR on the cell membrane of engineered cells can be approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher than 99.9%, as measured by flow cytometry.

[0155] In some cases, immune response cells can be stem memory T cells composed of CD45RO (-), CCR7 (+), CD45RA (+), CD62L+ (L-selectin), CD27+, CD28+ and / or IL-7Rα+. SCM The stem memory cells may also express CD95, IL-2Rβ, CXCR3, and / or LFA-1, and exhibit many functional properties different from those of the stem memory cells. Alternatively, the immune response cells may also be central memory T cells containing L-selectin and CCR7. CM Cells, including central memory cells, may secrete IL-2, but not IFNγ or IL-4. Immune response cells can also be effector memory T cells containing L-selectin or CCR7. EM Cells produce effector cytokines such as IFNγ and IL-4.

[0156] The carrier can be delivered in vivo by administration to an individual patient, typically via systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or local administration, as described below. Alternatively, the carrier can be delivered ex vivo to cells, such as cells removed from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then typically re-implanted into the patient after selection of cells in which the carrier has been incorporated. The cells can be expanded before or after selection.

[0157] Suitable immune response cells for expressing anti-GPC3-CAR can be autologous or non-autologous cells for the subject in need.

[0158] A suitable source of immune response cells can be obtained from the subject. In some cases, T cells can be obtained. These T cells can be obtained from many sources, including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissues from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain circumstances, any number of techniques known to those skilled in the art, such as Ficoll, can be used. TM T cells are obtained from blood units collected from a subject. In one embodiment, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes (including T cells, monocytes, granulocytes, and B cells), other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected via apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps.

[0159] Alternatively, the cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells may be part of a mixed cell population exhibiting different phenotypic characteristics. Cell lines may also be obtained from transformed T cells according to the methods described above. Cells may also be obtained from a cell therapy library. Modified cells resistant to immunosuppressive therapy may be obtained by any of the methods described herein. A desired cell population may also be selected prior to modification. An engineered cell population may also be selected after modification. Engineered cells may be used for autologous transplantation. Alternatively, the cells may be used for allogeneic transplantation. In some cases, cells are administered to the same patient whose sample was used to identify cancer-related target sequences. In other cases, cells are administered to a different patient than the patient whose sample was used to identify cancer-related target sequences.

[0160] In some cases, the immune response cells can be primary cells, including primary T cells, stem cells, or progenitor cells. Progenitor cells can be hematopoietic progenitor cells. The cells used in this invention can be human cells. Suitable cells can be expanded in vitro. Suitable cells can also be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), IL-7Rα(+), or combinations thereof.

[0161] In some cases, suitable primary cells include peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subpopulations, such as, but not limited to, T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cell, including any T cell (TIL), such as tumor-infiltrating cells, such as CD3+ T cells, CD4+ T cells, CD8+ T cells, or any other type of T cell. T cells may also include memory T cells, memory stem T cells, or effector T cells. T cells may also be selected from a bulk population, for example, T cells selected from whole blood. T cells may also be expanded from a bulk population. T cells may also be biased towards a specific population and phenotype. For example, T cells may be phenotyped as including CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+), and / or IL-7Rα (+). Suitable cells may be selected that contain one or more markers selected from the following: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+), and / or IL-7Rα (+). Suitable cells also include stem cells, for example, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. Suitable cells may include any number of primary cells, such as human cells, non-human cells, and / or mouse cells. Suitable cells may be progenitor cells. Suitable cells may be derived from the subject to be treated (e.g., a patient). Suitable cells may be derived from a human donor. Suitable cells may be stem memory T cells composed of CD45RO (-), CCR7 (+), CD45RA (+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+. SCM The stem memory cells may also express CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit many functional properties different from those of the stem memory cells. Suitable cells may be central memory T cells containing L-selectin and CCR7. CM The central memory cells may secrete, for example, IL-2, but not IFNγ or IL-4. Suitable cells may also be effector memory T cells containing L-selectin or CCR7. EM Cells produce effector cytokines such as IFNγ and IL-4.

[0162] In some cases, the method may include enriching the CD8+ of cultured T cells prior to rapid cell expansion. +T cells. After culturing T cells in IL-2, they can be isolated using, for example, CD8 microbeads (e.g., using CliniMACS). plus The CD8 microbead system (Miltenyi Biotec) causes T cells to consume CD4. + Cells and enriched CD8 + Cells. Not bound by any specific theory, CD4 can be considered... + CD25 + Regulatory T cells can impair antitumor responses. Therefore, it can be considered that enriching CD8+ in cultured T cells... + T cells and reduce or eliminate CD4 + Cells can improve the anti-tumor CD4 response in adoptive transfer + Effects on cells, improving response rates in patients and / or reducing the effects of CD4+ + The toxicity observed is due to the production of cytokines. Additionally, CD8+ can be considered enriched. + “Young” T cells have proven more reliable and predictable than hybrid T cells in rapid clinical-scale expansion.

[0163] Cells can be Good Manufacturing Practice (GMP) compatible agents. Cells can be part of combination therapies to treat cancer, infection, autoimmune disorders, or graft-versus-host disease (GVHD) in subjects in need. In some cases, the cells of the present invention can be administered as a monotherapy to subjects in need.

[0164] In some cases, the cells expressing the thematic CAR comprise a heterogeneous population of T cells. In other cases, the cells used may consist primarily of CD4 and CD8 T cells in varying proportions. The CD4 and CD8 cells may possess the phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells may also possess the phenotypic characteristics of effector-memory cells. In another embodiment, the cells may be central-memory cells.

[0165] Suitable cells that can be isolated from a donor can be at any developmental stage, including but not limited to fetuses, newborns, young adults, and adults. For example, donor immune response cells can be isolated from adults. Donor human immune response cells can be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old. For example, immune response cells can be isolated from individuals under 6 years of age. Immune response cells can also be isolated from individuals under 3 years of age. Donors can be over 10 years old.

[0166] Methods for obtaining suitable cells may include selection based on given markers. Such markers may include GFP, resistance genes, cell surface markers, or endogenous tags. Cells can be selected using any endogenous marker. Applicable cell selection techniques include flow cytometry and / or magnetic column chromatography. The selected cells can then be infused into the subject. The selected cells can also be expanded to a large number. The selected cells can be expanded prior to infusion.

[0167] The number of cells necessary for therapeutic efficacy in a patient can vary depending on cell viability and the efficiency of genetic modification of cells (e.g., the efficiency of transgene integration into one or more cells, or the expression level of proteins encoded by the transgene). In some cases, the products of genetically modified cell viability (e.g., proliferation) and the efficiency of transgene integration may correspond to a therapeutic aliquot of cells available for administration to a subject. In some cases, an increase in genetically modified cell viability may correspond to a decrease in the number of cells necessary for therapeutic efficacy in a patient. In some cases, an increase in the efficiency of transgene integration into one or more cells may correspond to a decrease in the number of cells necessary for therapeutic efficacy in a patient. In some cases, determining the number of cells necessary for therapeutic efficacy may include determining a function corresponding to changes in cell viability over time. In some cases, determining the number of cells necessary for therapeutic efficacy may include determining a function corresponding to changes in the efficiency at which the transgene can be integrated into one or more cells relative to time-related variables (e.g., cell culture time, electroporation time, cell stimulation time). In some cases, therapeutically effective cells may be a cell population comprising approximately 30% to approximately 100% anti-GPC3 CAR expression on the cell surface. In some cases, therapeutically effective cells may express approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% to greater than approximately 99.9% of anti-GPC3 CAR on the cell surface, as measured by flow cytometry.

[0168] As mentioned above, various cells can be used to express the subject CAR. Anti-GPC3 CARs can be present in the plasma membrane of eukaryotic cells such as mammalian cells, among which suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, stem cell progeny, progeny cells, and NK cells.

[0169] When present in the plasma membrane of eukaryotic cells, CARs can be active in the presence of their binding targets. For example, anti-GPC3 CARs can be active in the presence of GPC3. Targets, such as GPC3, can be expressed on the membrane. Targets can also be soluble (e.g., not cell-bound). Targets can be present on the surface of cells, such as target cells. Targets can be present on solid surfaces, such as lipid bilayers; and so on. Targets can be soluble, such as soluble antigens. Targets can be antigens. Antigens can be present on the surface of cells, such as target cells. Antigens can be present on solid surfaces, such as lipid bilayers; and so on. In some cases, the target can be an epitope of an antigen. In the methods disclosed herein, the antigen is typically GPC3, and the target cells expressing GPC3 are cancer cells or tumor cells.

[0170] In some cases, when a CAR is present on the cell's plasma membrane and is activated by binding to its target, it can lead to cytotoxic activity of the cell against the target, which expresses an antigen on its cell surface that binds to the CAR-binding domain. For example, in some cases, the cell may be a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte). The CAR of this disclosure, when present on the cell's plasma membrane and activated by binding to its target, can increase the cytotoxic activity of the cytotoxic cell against the target cell, which expresses an antigen on its cell surface that binds to the CAR-binding domain. For example, in some cases, the cell may be an NK cell or a T lymphocyte. The CAR of this disclosure, when present on the cell's plasma membrane and activated by binding to its target, can increase the cytotoxic activity of the cell by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold compared to the cytotoxic activity of the cell when the target is not bound.

[0171] In some cases, when activated by binding to its target, CARs can lead to other CAR activation-related events, such as proliferation and expansion (due to increased cell division or anti-apoptotic responses). Cell proliferation can be visually measured by observing cell clusters under a microscope. Cell expansion can be measured using a hematology counter. In some cases, CAR-T cells may exhibit increased cell proliferation and expansion compared to equivalent T cells (non-CAR T cells). The increased cell expansion compared to equivalent cells can be approximately 1 to approximately 20-fold. The increased cell expansion compared to equivalent cells can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20-fold. Cell expansion can be measured over a period of time. For example, cell expansion can occur between cell collection and infusion into the subject. In other cases, cell expansion can be performed from 1 day to approximately 30 days after collection. Cell expansion can be performed approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 days after collection. In some cases, cells can be expanded using a rapid expansion protocol (REP) prior to infusion into the subject. In some cases, REP can occur within approximately 14 days.

[0172] In some cases, when activated by binding to its targets, CARs can lead to other CAR activation-related events, such as regulation of intracellular signaling, cell differentiation, or cell death. In some cases, CAR expression on cells can alter intracellular signaling. In other cases, CAR expression can alter cell differentiation.

[0173] The subject-specific anti-GPC3 CAR immune response cells can be administered to cancer or tumor subjects expressing GPC3 during or after lymphopenia treatment.

[0174] In some cases, anti-GPC3 CAR immune response cells are administered following lymphopenic therapy. This treatment reduces circulating lymphocytes in the treated subject, or essentially depletes circulating lymphocytes (i.e., lymphopenia). For example, anti-GPC3 CAR immune response cells can be administered from at least 1 hour to at least 1 week after lymphopenic therapy. For example, anti-GPC3 CAR immune response cells can be administered at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or 7 days or longer after lymphopenic therapy. In some cases, CAR-T cells can be administered approximately 1, 2, 3, 4, 5, 6 weeks or longer after lymphopenic therapy.

[0175] In some cases, host lymphopenia can promote the expansion of anti-GPC3 CAR immune response cells, such as CAR T cells. On the one hand, lymphopenia can create "space" for upcoming adoptive transfer cells, and on the other hand, it can induce the homeostatic expansion of these cells. The latter effect may be mediated by chemoablation of endogenous regulatory T cells, which typically secrete inhibitory cytokines (such as TGF-β and IL-10) that limit the expansion of effector cells, such as CAR T cells. In some cases, lymphopenia treatment can increase the in vivo expansion of anti-GPC3 CAR immune response cells by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 times compared to treatment with anti-GPC3 CAR immune response cells without lymphopenia treatment.

[0176] Furthermore, due to less competition and increased production of lymphosplenic cells, T-cell growth homeostatic cytokines such as IL-7 and IL-15, which are typically present in limited quantities, become readily available. Therefore, lymphopenia can be induced prior to the infusion of anti-GPC3 CAR immune-response cells to increase the efficacy of these cells in treated subjects. In some cases, lymphopenia treatment, measured by tumor reduction or control, can improve antitumor efficacy by approximately 10% to approximately 100% compared to treatment without lymphopenia treatment. For example, lymphopenia treatment can improve antitumor efficacy by approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100% compared to treatment without lymphopenia treatment.

[0177] Lymphopenia can be achieved using various methods. Total body irradiation (TBI) or cytotoxic drugs can be used to induce lymphopenia prior to the administration of anti-GPC3 CAR immune response cells. While these methods may be intended to deplete the recipient's lymphatic compartments, they can also promote the presentation of tumor antigens by triggering tumor cell death and antigen release. These antigens can then be received and presented by antigen-presenting cells (APCs) to enhance the activation of anti-GPC3 CAR immune response cells.

[0178] Radiation and / or chemotherapy can induce host cell activation, leading to the release of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-4, and the upregulation of co-stimulatory molecules such as CD80. In some cases, lymphopenic therapy can improve anti-GPC3 CAR immune response cell therapy by activating host cells. In other cases, lymphopenic therapy can improve anti-GPC3 CAR immune response cell therapy by upregulating co-stimulatory molecules. In addition to enhanced APC function and availability, premodulation protocols can also disrupt the integrity of the mucosal barrier through radiation-induced apoptosis of these organ lining cells. Damage to the gut can allow bacterial products such as LPS to translocate into the systemic circulation. LPS, in turn, can... in vivo Activating anti-GPC3 CAR immune response cells can enhance anti-tumor responses. Therefore, pro-inflammatory cytokines and microbial products provide key "danger signals" for the activation and maturation of dendritic cells (DCs), thereby enhancing anti-GPC3 CAR immune response cell-mediated tumor therapy.

[0179] In some cases, lymphopenic therapy can selectively deplete cells in the body that express CD25, including humanized anti-Tac (anti-CD25) and ONTAK™ (IL-2 conjugated with diphtheria toxin).

[0180] In some cases, chemotherapy agents may be administered to achieve lymphopenia in the subject. In some cases, the subject may receive nonmyeloablative lymphopenic chemotherapy. Nonmyeloablative lymphopenic chemotherapy can be any suitable such therapy and can be administered via any appropriate route. Chemotherapy regimens may include the use of a single alkylating agent, such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine, and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone, and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin, and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, and leucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and leucovorin), and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed-dose prednisone, bleomycin, and leucovorin).

[0181] Non-myeloablative lymphopenic chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is GPC3 positive, as it may be metastatic. The preferred route of administration for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. Preferably, about 60 mg / kg of cyclophosphamide may be administered over two days, followed by about 25 mg / kg of [unspecified drug]. 2 Fludarabine was administered for approximately five days.

[0182] In some cases, lymphopenia may be performed to minimize cellular rejection in the immune-mediated anti-GPC3 CAR immune response. For example, a subject may be treated with cyclophosphamide (Cy) followed by fludarabine (Flu) lymphopenia. The dosage of chemotherapeutic agents such as Cy can be from about 1 mg / kg to about 200 mg / kg. The dosage of Cy for the subject can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 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, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or up to approximately 200 mg / kg.

[0183] Alternatively, an exemplary dosage and regimen for Cy treatment could be approximately 0.5–5 g / m². 2 / day, preferably 0.6~3 g / m 2 / day, preferably 1~2 g / m 2 / day; lasts 1-3 days, preferably 1-2 days;

[0184] The dose of Flu for the subjects can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 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, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or up to approximately 200 mg / m² 2 .

[0185] Alternatively, an exemplary dose and regimen for fludarabine treatment could be approximately 20–80 mg / m². 2 / day, preferably 25~70mg / m² 2 / day or 25~30 mg / m 2 / day, lasting 2-10 days, 3-8 days, or 4, 5, 6 or 7 days.

[0186] In some cases, Cy and Flu can be used in combination. For example, the initial dose of Flu is approximately 10–60 mg / m². 2 / day or 15~50 mg / m 2 / day or 20~30 mg / m 2 Within the range of 0.2-1 mg / m², continue for 2-8 days or 3-6 days, followed by Cy administration at a dose of approximately 0.2-1 mg / m². 2 / day or 0.3~0.8 mg / m 2 / day or 0.4~0.6 g / m 2 / day, lasting 1-5 days or 2-3 days.

[0187] In some cases, subjects may receive Cy and Flu treatment prior to anti-GPC3 CAR immune response cell infusion, with Cy administered once at a dose of 60 mg / kg and Flu at a dose of 25 mg / m². 2Flu is administered 3 to 5 times. Prior to anti-GPC3 CAR immune response cell infusion, subjects may receive approximately 0 to 20 doses of lymphodepletant. Prior to anti-GPC3 CAR immune response cell infusion, subjects may receive approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 doses of lymphodepletant such as Cy or Flu. In other cases, subjects may receive lymphodepletant such as Cy and / or Flu concurrently with anti-GPC3 CAR immune response cell infusion. In other cases, lymphodepletant such as Cy and / or Flu may be administered to subjects after anti-GPC3 CAR immune response cell infusion. In other cases, lymphodepletant may be administered to subjects before, concurrently with, and / or after anti-GPC3 CAR immune response cell infusion. In some cases, subjects may not receive lymphopenic therapy. In some cases, different doses of lymphocyte-depleting agents may be used during the course of the regimen. For example, a subject may receive 60 mg / kg of Cy before receiving anti-GPC3 CAR immune response cells, and then receive 40 mg / kg of Cy concurrently with anti-GPC3 CAR immune response cells. A complete blood count (CBC) may be performed to determine the extent of lymphopenia and whether additional administration may be necessary. In some cases, Cy may be administered alone. In other cases, Flu may be administered alone. In some cases, Cy and Flu may be used alternately during the regimen.

[0188] Lymphopenia can improve the expansion of anti-GPC3 CAR immune response cells. Lymphopenia can also improve the persistence of anti-GPC3 CAR immune response cells in the blood. The expansion and persistence of anti-GPC3 CAR immune response cells can be detected by flow cytometry using anti-CAR antibodies. The persistence of anti-GPC3 CAR immune response cells can also be measured by evaluating the copy number of anti-GPC3 CAR immune response cells using qPCR.

[0189] In some cases, the acceptable dose of anti-GPC3 CAR immune response cells was reduced if lymphopenia treatment was administered, compared to subjects who may not have received lymphopenia treatment.

[0190] In some cases, lymphopenic therapy can be administered over a period of time. For example, lymphopenic therapy can be administered over a course of treatment from 1 minute to approximately 24 hours. Lymphopenic therapy can be administered for 1 minute, 15 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or up to approximately 24 hours. Lymphopenic agents can be administered in combination with other agents such as diluents, uroprotectants, excipients, or combinations thereof. For example, sodium 2-mercaptoethanesulfonate (mesna) can be included in lymphopenic therapy. In some cases, subjects may receive cyclophosphamide intravenously at 60 mg / kg / day in 250 ml D5W for approximately 2 days, or mesna at 15 mg / kg / day over 1 hour for 2 days. Mesna can be administered in combination with various doses of lymphocyte-depleting agents. For example, mesna can be administered at doses from approximately 1 mg / kg / day up to approximately 50 mg / kg / day. Mesna can be administered at doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or up to approximately 50 mg / kg / day. In some cases, for obese or pediatric subjects, the dosage can be calculated using actual weight. Actual weight can be the average of actual weight and ideal weight. For example, for men, ideal weight can be calculated as 50 kg + 2.3 (the number of inches over 60 inches). For women, ideal weight can be calculated as 45.5 kg + 2.3 (the number of inches over 60 inches). In some cases, Flu administration may be 25 mg / m² daily. 2 Flu is administered via intravenous infusion (IVPB) over 30 minutes daily for 5 days. In some cases, Flu can be administered before Cy. In some cases, Flu can be administered concurrently with Cy. In some cases, Flu can be administered after Cy. In some cases, Flu can be started approximately 1 to 2 hours after Cy and mesna.

[0191] Total body radiation therapy (TBI) can take the form of radiation therapy. As the name suggests, TBI can involve radiation to the entire body, but in modern practice, the lungs may be partially shielded to reduce the risk of radiation-induced lung injury. TBI can be administered in various doses. For example, TBI can be administered at doses of approximately 10 to approximately 12 Gy. In some cases, TBI may be administered in fractions, delivering smaller doses in several smaller doses rather than delivering the entire dose at once. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 different doses of radiation may be administered.

[0192] The reported D0 value (the amount of ionizing radiation necessary to eradicate a specific cell type) for hematopoietic stem cells can be from about 0.5 to about 1.4 Gy, while the D0 value for human leukemia cell lines can be from about 0.8 to about 1.5 Gy, indicating that both cell types are radiosensitive. The ideal dosing regimen may depend on the patient's age, disease, and intended type of treatment. Myeloablative TBI can be administered at a dose of about 12 to about 15 Gy, in about 8 to about 12 fractions over about 4 days, about 2 to 3 times daily. In some cases, myeloablative TBI can be administered at a dose of about 5 to about 20 Gy. Myeloablative TBI can be administered at doses of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20 Gy. In some cases, myeloablative TBI can be administered in about 5 to about 20 fractions. Myeloablative TBI can be administered in approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to approximately 20 sessions. Myeloablative therapy can be administered over approximately 1 to approximately 10 days. Myeloablative therapy can be administered over approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to approximately 10 days.

[0193] In some cases, low-dose TBI may comprise a dose of about 2 to about 8 Gy administered in about 1 to about 4 fractions. In some cases, the low dose may comprise about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or up to 8 Gy. Low-dose TBI may be administered to subjects who cannot tolerate bone marrow removal due to age or comorbidities.

[0194] TBI can be applied using parallel, opposing pairs of high-energy photon beams, with the photon beam intensity ranging from about 4 to about 18 MV for TBI. In some cases, the photon beam intensity for TBI can be from about 1 to about 25 MV. The photon beam intensity can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or up to about 25 MV. TBI can be performed using devices such as the VarianClinac iX or the Siemens Artiste.

[0195] In some cases, the application rate can be from about 5 cGy / min to about 100 cGy / min. The application rate can be approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or up to 100 cGy / min.

[0196] In some cases, certain organs may be shielded during radiation. For example, during whole-body radiation, the liver, lungs, brain, heart, or a combination thereof may be shielded. Biological reagents

[0197] In some cases, biological agents such as antibodies can be used to deplete immune cells. Monoclonal antibodies (mAbs) that are lysogenic to lymphocytes can be another means of inducing lymphopenia. In some cases, antibodies for T-cell lymphopenia that can be administered prior to the infusion of anti-GPC3 CAR immune response cells should be effective but transiently present in vivo to allow for rapid infusion and proliferation of the infused anti-GPC3 CAR immune response cells. The antibodies used can target markers expressed on the surface of cells such as lymphocytes. In some cases, lymphopenic antibodies can be anti-CD3, anti-CD4, anti-CD8, anti-CD45, anti-CD25, anti-CD52, and any combination thereof.

[0198] Antibodies such as alenzusab (Campath-1H), bortezomib, thymoglobulin (rabbit ATG, Genzyme), ATGAM (horse ATG, Pfizer), and alenzusab (Campath-1H) can be used. Rituximab (IDEC-C2B8), GA101, humanized anti-CD20 IgG1, XmAb5574, Fc-engineered anti-CD19 antibody, afasicept (LFA3-Ig), fingolimod (FTY720), anti-thymocyte globulin (ATG), anti-CD4 antibody, anti-CD3 antibody, and anti-CD8 antibody can also be used as lymphopenic agents. In some cases, cladribine (2-CdA, Leustatin®), a purine analogue similar to fludarabine, can be used. In some cases, antibody lymphopenic therapy may include a single antibody (such as alenzusab (anti-CD52)). In other cases, antibody-based lymphopenia treatment may include at least two antibodies, such as alenzumab and anti-CD45. In some cases, lymphopenia treatment may include multiple lymphopenia modalities, such as radiation combined with antibody therapy or chemotherapy combined with radiation. Chemotherapy and antibody therapy may also be used in combination.

[0199] Antibiotics, antifungals, and antivirals may be administered to subjects as prophylaxis during lymphopenia treatment. For example, prophylaxis may include 500 mg Altrex daily, one q MWF tablet of ceftriaxone DS, and 200 mg fluconazole daily. Drug treatment continues until the absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) return to their pre-treatment baseline. Biological agents may also be doxorubicin.

[0200] One or more cytokines may be introduced together with the cells. Cytokines can be used to promote the expansion of metastatic cells (including adoptive tumor-specific cells) within the tumor microenvironment. In some cases, IL-2 can be used to promote the expansion of the cells described herein. Cytokines such as IL-1 may also be used. Other relevant cytokines in the field of immunotherapy, such as IL-2, IL-7, IL-12, and L-21, or any combination thereof, may also be used. In some cases, recombinant cytokines are used.

[0201] In some cases, T-cell growth factors may be administered. Growth factors can be administered via any suitable route. If more than one T-cell growth factor is administered, they may be administered simultaneously or sequentially in any order, and via the same or different routes. T-cell growth factors such as interleukin-2 (IL-2) may be administered as an intravenous bolus. The dose of T-cell growth factors such as IL-2 is considered high by those skilled in the art. Preferably, about 720,000 IU / kg of IL-2 may be administered three times daily until tolerated. In some cases, about 5 to about 15 doses of IL-2 may be administered, with an average of about 9 doses. The dose of T-cell growth factors may be from about 0 to about 20. T-cell growth factors may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about 20 times.

[0202] IL-2 can be administered as an intravenous bolus at a dose of 720,000 IU / kg (based on total body weight). In some cases, IL-2 can be administered over 15 minutes. In some cases, IL-2 can be administered over 20 minutes. IL-2 can also be administered via immediate injection. In some cases, IL-2 can be administered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 minutes. 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or up to 6 hours.

[0203] In some cases, IL-2 can be initiated within 24 hours of cell infusion and continued for up to approximately 4 days (up to 12 doses). In some cases, IL-2 can be administered for up to approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after the initial administration.

[0204] IL-2 can be administered every 8 hours. In some cases, IL-2 can be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after the initial administration. In some cases, IL-2 administration may be discontinued if toxicity is detected. In some cases, if a patient experiences grade 3 or 4 toxicity due to adeleukin, the dose may be delayed or discontinued, unless it is a common reversible grade 3 toxicity of adeleukin, such as diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or systemic symptoms and laboratory changes. In some cases, an additional dose may be given if these toxicities can be easily reversed within 24 hours with supportive measures. Additionally, the decision to continue or discontinue administration may be made at the discretion of the treating physician.

[0205] In some cases, immune response cells expressing anti-GPC3CAR may have increased anti-tumor efficacy compared to comparable immune response cells that do not express anti-GPC3CAR.

[0206] In some cases, antitumor efficacy may refer to cytotoxic activity. In others, it may refer to persistence. Antitumor efficacy can also refer to the ability of cells to target tumors. Various in vitro assays can be used to measure antitumor efficacy. For example, cytotoxic capacity can be measured by ELISA measuring the release of interleukin-2 (IL-2) or interferon-γ (IFNγ). Cytotoxic activity can be measured by killing assays such as the chromium-51 release assay or co-culture assay. In some cases, anti-GPC3 CAR immune-response cells may exhibit increased antitumor efficacy and capacity compared to comparable cells.

[0207] The efficacy of anti-GPC3 CAR therapy can be evaluated in various ways. Efficacy can refer to the degree of anti-tumor efficacy in controlling, reducing, or eliminating tumors, such as GPC3-positive tumors. Therapeutic efficacy can also refer to the expansion, persistence, tumor targeting, and any combination thereof of CAR immune response cells.

[0208] Subjects eligible for anti-GPC3 CAR immune response cell therapy, such as anti-GPC3 CAR T-cell therapy, can be evaluated during infusion, immediately after infusion, or years after infusion. For example, treated subjects may return to the clinic for evaluation from approximately one day to a lifetime. Treated subjects can be evaluated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, or up to 90 years after initial administration of the subject CAR immune response cell therapy. In some cases, the evaluation plan may include daily, weekly, monthly, or annual monitoring. In other cases, subjects may be observed more frequently as clinically indicated. Evaluation may include physical examination, chemical evaluation, complete blood count, thyroid group assessment, toxicity assessment, computed tomography (CT) scans of body regions, apheresis, and any combination thereof.

[0209] In some cases, apheresis can be performed approximately 1 to 10 weeks before and after administration of the subject-specific CAR immune response cell infusion. At other time points, peripheral blood lymphocytes (PBLs) can be obtained from whole blood using purification via Ficoll gradient centrifugation. Aliquots of peripheral blood mononuclear cells (PBMCs) can be cryopreserved for immunomonitoring of cell function. In some cases, various assays, including those evaluating specific lysis and cytokine release, metabolomics and bioenergetics studies (using the hippocampus), intracellular FACS of cytokine production, ELISA spot assays, and lymphocyte subset analysis, can be used to evaluate the immune relevance of subject-specific CAR immune response cell therapy. Typically, a difference of approximately 2 to 3-fold in these assays indicates a real biological difference. In some cases, differences of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and up to approximately 5-fold, as measured in vitro after anti-GPC3 CAR immune response cell therapy, may indicate therapeutic efficacy.

[0210] In some cases, topical CAR immune response cell therapy can reduce tumor size by at least 30%, as measured by computed tomography (CT) scans or MRI. Anti-GPC3 CAR immune response cell therapy can reduce tumor size by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to approximately 100%. CAR-T therapy can eliminate tumors, as measured by CT scans. In some cases, anti-GPC3 CAR immune response cell therapy can effectively stabilize tumor size, where the change in tumor size from a baseline measurement of the tumor lesion diameter as measured by computed tomography (CT) scan is less than 10%. For example, the tumor size may not expand after administration of anti-GPC3 CAR immune response cells. In some cases, stability can be considered as a change in tumor size of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to pre-treatment measurements.

[0211] In some cases, the efficacy of anti-GPC3 CAR immune response cells can be considered based on subject survival time. For example, subjects treated with anti-GPC3 CAR immune response cells, such as anti-GPC3 CAR T cells, may survive longer than untreated subjects or subjects treated with different therapies.

[0212] In some cases, the efficacy of anti-GPC3 CAR immune response cells can be improved by adding secondary therapies such as lymphopenic therapy. Secondary therapies can act synergistically with anti-GPC3 CAR immune response cell therapy. In some cases, secondary therapies can produce an additive effect with anti-GPC3 CAR immune response cell therapy. Secondary therapies can include lymphopenia and other forms of cell therapy, antibody therapy, chemotherapy, radiotherapy, surgery, anti-angiogenic therapy, and any combination thereof.

[0213] Treatment response can be evaluated using the international criteria outlined in the Revised Criteria for Evaluation of Treatment Efficacy in Solid Tumors (RECIST) guidelines (version 1.1). In cases of malignant lymph nodes, the RECIST criteria can be used to assess changes in the maximum diameter (one-dimensional measurement) and minimum diameter of the tumor lesion. For example, a measurable lesion can be defined as one that can be precisely measured in at least one dimension (the longest diameter to be recorded) as >20 mm (via chest X-ray), >10 mm (via CT scan), or >10 mm (via clinical examination using calipers). To be considered pathologically enlarged and measurable, a lymph node can be >15 mm on the short axis when assessed via CT scan. All other lesions (or disease sites), including small lesions (pathological lymph nodes with a maximum diameter <10 mm or a short axis ≥ 10 to <15 mm), are considered non-measurable. Bone lesions, meningeal diseases, ascites, pleural / pericardial effusions, cutaneous lymphangitis / pneumonia, and inflammatory breast diseases are considered non-measurable.

[0214] All measurable lesions, representing up to two per organ and up to five per systemic organs, can be identified as target lesions and recorded and measured at baseline. Target lesions can be selected based on their size (the lesion with the longest diameter), representing all relevant organs, but should otherwise be those suitable for repeatable measurements. It is possible that sometimes the largest lesion is not suitable for repeatable measurement; in such cases, the second largest repeatable lesion should be selected. The sum of the diameters of all target lesions (longest diameter for non-nodular lesions, short axis for nodular lesions) can be calculated and reported as the baseline sum diameter. If lymph nodes are included in the sum, only the short axis should be added to the sum. The baseline sum diameter will be used as a reference to further characterize any objective tumor regression in the measurable dimensions of the disease.

[0215] In some cases, clinical lesions can be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and have a diameter of approximately 10 mm as assessed using calipers (e.g., skin nodules). In cases where calipers cannot be used to measure the lesion, CT scans or MRI may also be used. In some cases, CT scans may produce tissue sections of approximately 5 mm or smaller. In some cases, CT scans may have scan thicknesses of 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, or 0.5 mm. If a CT scan has a section thickness greater than 5 mm, the minimum measurable lesion can be twice the section thickness. In some cases, MRI may also be performed to evaluate the subject. Ideally, the same type of scanner should be used, and the image acquisition protocol should be as close as possible to that of the previous scan when determining treatment efficacy. Where possible, breath-hold scanning techniques should be used for body scans. In some cases, fluorodeoxyglucose (FDG)-positron emission tomography (PET) may be used to measure treatment efficacy.

[0216] Once a subject is evaluated, target lesions are categorized as stable disease (SD), progressive disease (PD), partial response (PR), and / or complete response (CR). SD can be considered as a minimum sum of diameters with no reduction sufficient to qualify for PR or an increase sufficient to qualify for PD. PD can be considered as a minimum sum of diameters with a target lesion diameter that increases by at least 20%, with the minimum sum of diameters as a reference (or the baseline sum if possible). In some cases, in addition to a relative increase of approximately 20%, the sum must also demonstrate an absolute increase of at least approximately 5 mm. PR can be a reduction of at least approximately 30% in the diameter of the target lesion, with the baseline sum of diameters as a reference. CR can be the elimination of the target lesion.

[0217] In some cases, the combination of anti-GPC3-CAR immune response cells (such as anti-GPC3 CAR T cells) and lymphopenia treatment synergistically increased median survival by at least approximately 6 months compared to administration of anti-GPC3 immune response cells alone (Table 6). In some cases, the combination of anti-GPC3-CAR immune response cell therapy and lymphopenia treatment synergistically increased survival by at least approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or up to approximately 25 years compared to administration of anti-GPC3 immune response cells alone. Table 6: Comparison of Constructs

[0218] The subject matter of anti-GPC3 CAR immune response cells can be formulated into drugs and used to treat people or mammals diagnosed with diseases such as cancer. These drugs can be administered to people or mammals in combination with one or more chemotherapeutic agents or compounds.

[0219] Populations of CAR immune-response cells, such as CAR T cells, can be formulated for administration to subjects using techniques known to those skilled in the art. Formulations containing CAR immune-response cell populations may contain pharmaceutically acceptable excipients. Excipients included in the formulation will serve different purposes, depending on, for example, the subset of T cells used and the administration method. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tensioning agents, fillers, and lubricants. Formulations containing CAR immune-response cell populations are typically prepared and cultured in the absence of any non-human components such as animal serum. A formulation may contain one CAR immune-response cell population, or more, such as two, three, four, five, six, or more CAR immune-response cell populations. For example, a formulation may contain one CAR T cell population, or more, such as two, three, four, five, six, or more CAR T cell populations.

[0220] Formulations comprising anti-GPC3 CAR immune response cell populations can be administered to subjects using modalities and techniques known to those skilled in the art. Exemplary modalities include, but are not limited to, intravenous injection. Other modalities include, but are not limited to, intratumoral, intradermal, subcutaneous (SC, sq, sub-Q, Hypo), intramuscular (IM), intraperitoneal (IP), intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid region), intracranial, intraspinal, and intrathecal (spinal fluid). Any known device suitable for parenteral injection or infusion of the formulation can be used to achieve such administration.

[0221] The formulation containing a CAR immune response cell population administered to a subject contains a number of CAR immune response cells that are effective in treating and / or preventing a specific indication or disease. Therefore, a therapeutically effective CAR immune response cell population can be administered to the subject. Typically, an administration of approximately 1 x 102 CAR ... 4 To approximately 1 x 10 10 A formulation containing approximately 1 x 10 CAR immune response cells. In most cases, the formulation will contain approximately 1 x 10 CAR immune response cells. 5 To approximately 1 x 10 9 CAR immune response cells, approximately 5 x 10 5 To approximately 5 x 10 8 One CAR immune response cell or approximately 1 x 10 6To approximately 1 x 10 7 The number of CAR immune response cells administered to a subject will vary widely, depending on the location, origin, identity, extent, and severity of the cancer, as well as the age and condition of the individual to be treated. The physician will ultimately determine the appropriate dose to be used.

[0222] Tumor-targeting molecules are administered to subjects before, simultaneously with, or after the administration of CAR immune response cells. The tumor-targeting molecules bind to target cells in the subject by associating with tumor-associated antigens or tumor-specific antigens. Tumor-targeting molecules can be formulated for administration to subjects using techniques known to those skilled in the art. Formulations of tumor-targeting molecules may contain pharmaceutically acceptable excipients. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tensioning agents, fillers, and lubricants.

[0223] Tumor-targeting molecules can be administered to subjects using modalities and techniques known to those skilled in the art. Exemplary modalities include, but are not limited to, intravenous, intraperitoneal, and intratumoral injection. Other modalities include, but are not limited to, intradermal, subcutaneous (SC, sq, sub-Q, Hypo), intramuscular (IM), intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid region), intracranial, intraspinal, and intrathecal (spinal fluid). In some cases, CAR-T can be administered locally at the site of a tumor lesion, such as a liver lesion. Any known device suitable for parenteral injection or infusion of the formulation can be used to achieve such administration.

[0224] A formulation containing a tumor-targeting molecule is administered to the subject in an amount effective in treating and / or preventing a specific indication or disease. Typically, a formulation containing at least about 0.1 mg / kg body weight to about 100 mg / kg body weight of a tumor-targeting molecule is administered to the subject requiring treatment. In most cases, the dose is about 1 mg / kg to about 100 mg / kg body weight of labeled protein daily, taking into account the route of administration, symptoms, etc. The physician will determine the appropriate dose to be used.

[0225] In one embodiment, the chimeric antigen receptor is used to stimulate an immune response mediated by immune cells. For example, a T-cell-mediated immune response is an immune response involving T-cell activation. Activated antigen-specific cytotoxic T cells are able to induce apoptosis in target cells that display epitopes of foreign antigens on their surface, such as cancer cells displaying tumor antigens. In another embodiment, the chimeric antigen receptor is used to deliver anti-tumor immunity in mammals. Due to the T-cell-mediated immune response, the subject will develop anti-tumor immunity.

[0226] In some cases, methods of treating a subject with cancer may include administering to the subject a formulation of one or more tumor-targeting molecules, wherein these molecules bind to cancer cells, and administering one or more therapeutically effective subject-specific CAR immune-response cell populations, wherein the CAR immune-response cells bind to the tumor-targeting molecules and induce cancer cell death. Another embodiment may relate to a method of treating a subject with cancer, comprising administering to the subject a therapeutically effective subject-specific anti-GPC3 CAR immune-response cell population, wherein the CAR immune-response cells bind to cancer cells, thereby inducing cancer cell death.

[0227] The frequency of administration for the two formulations, one containing anti-GPC3 CAR immune response cells and the other containing anti-GPC3 CAR immune response cells in combination with tumor-targeting molecules, will vary depending on factors including the disease being treated, the components containing CAR immune response cells and tumor-targeting molecules, and the administration modality. Each formulation can be administered independently four, three, two, or once daily; every other day, every three days, every four days, every five days, every six days, once a week, every eight days, every nine days, every ten days, once every two weeks, once a month, and once every two months.

[0228] As used herein, the terms "chemotherapeutic agent" or "chemotherapeutic compound" and their grammatical equivalents can refer to chemical compounds useful in cancer treatment. Chemotherapy cancer agents that can be used in combination with the disclosed CAR immune-response cells include, but are not limited to, mitotic inhibitors (vinblastine alkaloids). These include vincristine, vinblastine, vindesin, and Navelbine™ (vinorelbine, 5'-nordehydrovinblastine). In other embodiments, cancer chemotherapeutic agents include topoisomerase I inhibitors, such as camptothecin compounds. As used herein, "camptothecin compounds" include Camptosar™ (irinotecan hydrochloride), Hycamtin™ (topotecan hydrochloride), and other compounds derived from camptothecin and its analogues. Another class of chemotherapeutic cancer agents that can be used in the methods and compositions disclosed herein are podophyllotoxin derivatives, such as etoposide, teniposide, and mitoxatophodin. This disclosure also includes other chemotherapeutic cancer agents called alkylating agents, which alkylate genetic material in tumor cells. These include, but are not limited to, cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphamide, carmustine, busulfan, chlorambucil, beluguine, uramustine, chlomaphazin, and dacarbazine. This disclosure includes antimetabolites as chemotherapeutic agents. Examples of these types of agents include cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Another class of cancer chemotherapeutic agents that can be used in the methods and compositions disclosed herein include antibiotics. Examples include, but are not limited to, doxorubicin, bleomycin, actinomycin D, daunorubicin, styromycin, mitomycin C, and donomycin. Many commercially available liposomal formulations of these compounds are present. This disclosure also includes other cancer chemotherapeutic agents, including but not limited to antitumor antibodies, dacarbazine, azacytidine, acridine, melphalan, ifosfamide, and mitoxantrone.

[0229] The subject of this study, anti-GPC CAR immune response cells, can be administered in combination with other antitumor agents, including cytotoxic / antitumor agents and anti-angiogenic agents. Cytotoxic / antitumor agents can be defined as agents that attack and kill cancer cells. Some cytotoxic / antitumor agents can be alkylating agents, which alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphamide, carmustine, busulfan, chlorambucil, belugutine, uramustine, chlomaphazin, and dacarbazine. Other cytotoxic / antitumor agents can be tumor cell antimetabolites, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Other cytotoxic / antitumor agents can be antibiotics, such as doxorubicin, bleomycin, actinomycin D, daunorubicin, styromycin, mitomycin C, and donomycin. Many commercially available liposomal formulations of these compounds are also available. Other cytotoxic / antitumor agents can be mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, acridine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0230] Anti-angiogenic agents may also be used. Suitable anti-angiogenic agents for the disclosed methods and compositions include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin-1 (including α and β), interleukin-12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and-2 (TIMP-1 and TIMP-2). Small molecules may also be used, including topoisomerases such as raszosen and topoisomerase II inhibitors with anti-angiogenic activity.

[0231] Other anticancer agents that can be used in combination with the target anti-GPC3 CAR immune response cells include, but are not limited to: acivitine; doxorubicin; acodazole hydrochloride; acroline; adorine; interleukin; hexamethylmelamine; ampicillin; ametrine acetate; aminoglutethimide; acridine; anastrozole; atrazomycin; asparaginase; triamcinolone; avastin; azacitidine; azatiprine; azotocin; palmastat; benzoxetine; bicalutamide; and bisaminoglycan hydrochloride. ; Dermaclofen; Bleomycin sulfate; Buquina sodium; Brompirimidine; Busulfan; Actinomycin C; Calotestosterone; Carbetamide; Carbetin; Carboplatin; Carmustine; Carrubicin hydrochloride; Carzelazoline; Sildenafil; Chlorisac; Siroxam; Cisplatin; Cladribine; Clinapordine mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin D; Daunorubicin hydrochloride; Decitabine; Dextromethorphan; Dezadazidine Ning; Dezaguanine Mesylate; Diaconazole; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifen; Droloxifen Citrate; Drotadazole Propionate; Dazomycin; Edatraxa; Eflunomide Hydrochloride; Exalucin; Enloplatin; Enpromethazine; Epilapidil; Epirubicin Hydrochloride; Ibuproazole; Exorubicin Hydrochloride; Estradiol; Estradiol Sodium Phosphate; Ethamidazole; Etoposide; Etoposide Phosphate; Etoposide; Fatamicin Hydrochloride Trazodazole; Fazarabine; Fenivel Aamine; Fluorouracil; Fludarabine Phosphate; Fluorouracil; Flucitabine; Phosphorione; Fostracin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofocin; Interleukin II (including recombinant interleukin II or rIL2); Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-I; Interferon γ-I b; Isopropylplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Riazol hydrochloride; Lometraxo sodium; Lomustine; Loxoanthraquinone hydrochloride; Masrophenone; Metformin; Nitrogen mustard hydrochloride; Medroxyprogesterone acetate; Meropenem; Minoril; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorpheniramine; Metotepazole; Mitolidomide; Mitocarcin; Mitocin; Mitocelin; Mitocin C; Mitospec; Mitotan; Mitoanthraquinone hydrochloride; Mycophenolic acid; Nocodazole; Nogamycin; Omar Platinum; Oxysulex; Paclitaxel; Pegaspargase; Peribacin; Pendimethalin; Pelosinomycin Sulfate; Pephosphonamide; Piperabromide; Piperabromide; Pirroanthraquinone Hydrochloride; Procainamide; Promethene; Porphyromycin Sodium; Pofibromycin; Prednisone; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazosulfan; Lipoadenosine; Rogulam; Safungo; Safungo; Semustine; Citricazone; Sodium Phosphatidylcholine; Sparmycin; Spiromustine; Spiroplatin; Streptomycin; Streptozotocin; Sulfonamide; Tadalafil; Ticogallan Sodium; Tegafur; Tiloanthraquinone Hydrochloride; Temopofen;Teniposide; Tiroxicon; Testrolide; Thiomipurine; Thioguanine; Thiotepa; Tiazofurin; Tirazamine; Toremifene Citrate; Tritoprolone Acetate; Tricerebin Phosphate; Trimethotraxa; Trimethotraxa Glucuronate; Triptorelin; Tobaccochloride Hydrochloride; Uramustine; Uretepa; Vapotetide; Vertepofen; Vincristine Sulfate; Vincristine Sulfate; Vincristine Sulfate; Vinpicidin Sulfate; Vincristine Sulfate; Vincristine Sulfate; Vinrocin Sulfate; Vinorelbine Tartrate; Vinrodine Sulfate; Vorticillin Sulfate; Zorniplatin; Netostatin; Zorubicin Hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; ararubicin; acylfulren; adenocyclopentol; adorexin; aldehyde-interleukin; ALL-TK antagonists; hexamethylmelamine; amimastatin; amidox; amifostine; aminolevulinic acid; amrarubicin; acridine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antraloxetine; anti-dorsalizing morphogenetic protein-1; antiandrogens; prostate cancer; antiestrogens; antitumor ketones; antisense oligonucleotides; glycine afedipine; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atametastan; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azamycin; diazotyrosine; berry gibberellin III derivative; balanol; balanol; CAR / ABL antagonist; benzodihydropyrrolidone; benzoyl astrosaponin; β-lactam derivative; β-alethine; β-clamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisaccharin; diazidopropyltriazine; diazidopropyltriazine; bistratene A; breflate; brompirimidine; bdolotentan; sulfoxide imine; calcipotriol; calcium phosphate protein C; camptothecin derivative; canarypox IL-2; capecitabine; formamide-amino-triazole; carboxyamine-triazole; CaRest M3; CARN 700; Cartilage-derived inhibitors; Carvacrol; Casein kinase inhibitors (ICOS); Scutellaria baicalensis extract; Bombyx mori antimicrobial peptide B; Cetrolec; Dihydroporphyrin; Chloroquinoxaline sulfonamide; Cicalprost; Cisporphyrin; Cladribine; Clomiphene analogues; Clotrimazole; Collosimycin A; Collosimycin B; Compressorin A4; Compressorin analogues; Conagenin; Crambescidin 816; Krestorin; Nostocin 8; Nostocin A derivatives; Curacin A;Cyclopentaquinone; Cycloplatin; Cypemycin; Cytarabine octadecyl phosphate sodium; Cytolysin; Hexestrol phosphate; Dacizumab; Decitabine; Dehydroepiandrosterone B; Deserelin; Dexamethasone; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan; Dextromethorphan B; Didox; Diethylnorspermide; Dihydro-5-azacytidine; 9-Dihydrotamethasone; Dioxamycin; Diphenylspirostine; Docetaxel; Doxorubicin; Dolasin Agar; Deoxyfluorouridine; Droloxifen; Drocannabinol; Ducamycin SA; Ebuseline; Ecomustine; Edifosine; Edrecolomab; Eflunomide; Elimite; Ethiributine; Epirubicin; Eprepitant; Estrogen-mustine analogs; Estrogen agonists; Estrogen antagonists; Ethanol; Etoposide phosphate; Exemestane; Fatrazol; Fazalabin; Fenivel Aamine; Filgrass; Finasteride; Vrapin; Flucalostem; Fluasterone; Fludarabine; Fludorunicin hydrochloride hydrochloride); phenomenox; formestan; fostracin; formustin; gadolinium texaphyrin; gallium nitrate; gallotaline; ganirilac; gelatinase inhibitor; gemcitabine; glutathione inhibitor; hepsulfam; thiazolinone; hexamethylene diacetamide; hypericin; ibandronic acid; idarubicin; edoxifene; icormenone; imofoxetine; ilomastastat; imidazopiclone; imiquimod; immunostimulatory peptide; insulin-like growth factor-1 receptor inhibitor; interferon agonist; interferon; interleukin; iodobenzylguanidine; iododoxorubicin; 4-sweet potato picrol; iloprapra; isolaridine; isobengazole; ishomohalicondrin B; isitasetron; jasplakinolide; kahalalide F F); spirotin triacetate; Lanreitide; Ranamidine; Levofloxacin; Lentinan sulfate; Leptostatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprorelin + estrogen + progesterone; Leuprorelin; Levamisole; Riazole; Linear polyamine analogs; Lipophilic disaccharide peptides; Lipophilic platinum compounds; Lissoclinamide 7; Lobaplatin; Earthworm phospholipids; Lometrozole; Clonidamine; Loxoanthraquinone; Lovastatin; Loxoribin; Letopecan; Lutetium texaphyrin; Lysofylline; Dissolving peptide; Metformin; Manostatin A; Marimastatin; Masoroxyfen; Mammary filament inhibitor; Matrix metalloproteinase inhibitor; Minoliquilizer;Merbarone; Avorelin; Methionase; Metoclopramide; MIF inhibitor; Mifepristone; Mitefocin; Mililastine; Mimatched double-stranded RNA; Mitoguanidine hydrazone; Dibromoceramide; Mitomycin analogue; Mitonaphthylamine; Mitotoxin fibroblast growth factor-saponin; Mitoxantrone; Mofarotin; Moraxillamine; Human chorionic gonadotropin monoclonal antibody; Monophospholipid A+ Mycobacterium cell wall SK; Mopidamine; Multidrug resistance gene inhibitor; Therapy based on multiple tumor suppressor gene 1; Nitrogen mustard anticancer agent; Indian Ocean sponge B (mycaperoxide) B) Mycobacterium cell wall extract; myriaporone; N-acetyldinarin; N-substituted benzamide; nafarilin; nagrestip; naloxone + pentazocine; napavin; napterpin; natosine; nedaplatin; nemorubicin; neridonic acid; neutral endopeptidase; nilumethicone; nisamycin; nitric oxide regulator; nitrooxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; ondansetron; ondansetron; oracin; oral cytokine inducer; omaliplatin; ozaratron ; Oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoyl raffinate; pamidronate; ginsenoside triol; panomiphen; parabactin; pozoniprid; pegasparginase; peldesine; sodium pentosan polysulfate; pentostatin; pentrazodazole; perfluorobromide; pefosamide; phenazinomycin; benzoylpyridinium; phenylacetate; phosphatase inhibitor; picibanil; pilocarpine hydrochloride; pirarubicin; pirarubicin; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compound; platinum-triamine complex; porphyrin sodium; pofibromycin; prednisone; propyl diacidone; prostaglandin J2; proteasome inhibitor; protein A-based immunomodulator; protein kinase C inhibitor; microalgal protein kinase C inhibitor; protein tyrosine phosphatase inhibitor; purine nucleoside phosphorylase inhibitor; rubigin; pyrazoline acridine; pyridoxine-modified hemoglobin polyethylene oxide conjugate; RAF antagonist; raltitrexed; ramosetron; ras farnesyltransferase inhibitor; ras inhibitor; ras-GAP inhibitor; demethylated retepritin; rhenium etradicate Re 186; rhizomycin; ribozyme; RII isotretinoin; rogulimine; roxithoreline; romotide; roquimetac; rubiginone B1; ruboxyl; safingo; saintopin; SarCNU; inositol A; saxaglastine; Sdi 1. Analog; Smustine;Aging-derived inhibitors 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding proteins; cizonan; sobuzosen; boraxane; sodium phenylacetate; solverol; somatostatin-binding protein; sonaringin; phosphatidylcholine; spicamycin D; spiromostatin; sennapandine; spongistatin 1; squalamine; stem cell inhibitors; stem cell division inhibitors; stipiamide; matrix lysozyme inhibitors; sulfinosine; potent vasoactive intestinal peptide antagonists; suradista; suramin; sorghum extract; synthetic glycosaminoglycans; tamustine; tamoxifen methyl iodide; tauromustine; tazarotene; tecogallan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temopofol; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomin e; bacterial embryonic stem cell; thiazolinone; thrombopoietin; thrombopoietin mimics; thymosin; thymopoietin receptor agonists; thymotrehnem; thyroid-stimulating hormone; ethyl cinnamate; teirazamine; timolocinolone dichloride; topsentin; toremifene; pluripotent stem cell factor; translation inhibitors; retinoic acid; triacetyluridine; tricerebroside; trimethoprim; triptorelin; tropisetron; tolostaniel; tyrosine kinase inhibitors; tyrosine phosphorylation inhibitors; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitors; urokinase receptor antagonists; valproic acid; variolin B; erythrocyte gene therapy vector systems; verarate; veratramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; voroxetine; zanoteron; zebuline; benzyl-Vitamin C; and fenestrated succinate. In one implementation, the anticancer drug is 5-fluorouracil, paclitaxel, or leucovorin.

[0232] In some cases, the subject anti-GPC3 CAR immune response cells can be introduced via injection, catheter, etc. In some cases, immunostimulants may also be included, including but not limited to interleukins (e.g., IL-2, IL-3, IL-6, and IL-11, and others), colony-stimulating factors (such as G-CSF, M-CSF, and GM-CSF), interferons (e.g., gamma interferon), and erythropoietin. In some cases, subjects may be treated with CAR-T, immune-consuming agents, and immunostimulants. IL-2 may be used to treat subjects to enhance the performance of the CAR-T cell product. In some cases, the immunostimulant may be a recombinant protein. The immunostimulant may also include the active portion of the protein. In some cases, the immunostimulant may include only a portion of the protein. This portion of the protein may be approximately 50%, 60%, 70%, 80%, 90%, or up to approximately 100% of the protein.

[0233] Compositions containing subject-specific anti-GPC3 CAR immune response cells, such as CAR T cells, can be readily provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are slightly easier to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within suitable viscosity ranges to provide longer contact time with specific tissues. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. If desired, sterile injectable solutions can be prepared by incorporating genetically modified CAR immune response cells for practicing the invention with various amounts of other desired ingredients into a suitable solvent in the desired amount. Such compositions can be mixed with suitable carriers, diluents, or excipients such as sterile water, physiological saline, glucose, dextrose, etc. The compositions can also be lyophilized. The composition may contain excipients such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or viscosity-enhancing additives, preservatives, flavoring agents, colors, etc., depending on the route of administration and desired preparation. Suitable formulations can be prepared by referring to standard texts such as “REMINGTON'S PHARMACEUTICAL SCIENCE,” 17th edition, 1985, which is incorporated herein by reference. Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Extended absorption of injectable drug forms can be achieved by using delayed-absorption agents such as aluminum monostearate and gelatin. However, according to the invention, any carrier, diluent, or additive used must be compatible with genetically modified CAR immune-response cells or their progenitor cells.

[0234] In some cases, the compositions can be isotonic, meaning they can have the same osmotic pressure as blood and tears. The required isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextran, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred, particularly for buffers containing sodium ions. If desired, a pharmaceutically acceptable thickener can be used to maintain the viscosity of the composition at a selected level. Methylcellulose is preferred because it is readily available, economical, and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener will depend on the agent chosen. It is important to use the amount that achieves the selected viscosity. Clearly, the selection of a suitable carrier and other additives will depend on the exact route of administration and the nature of the specific dosage form, such as a liquid dosage form (e.g., whether the composition will be formulated as a solution, suspension, gel, or another liquid form, such as a delayed-release or liquid-filled form).

[0235] In some cases, such as in compositions, formulations, and methods for treating cancer, the unit dose of the applied composition or formulation may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg. In some cases, the total amount of the composition or formulation applied may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 g.

[0236] In some cases, pharmaceutical compositions containing subject-specific anti-GPC3 CAR immune response cells, such as CAR T cells, can be administered alone or together with pharmaceutically acceptable carriers or excipients via any route of administration, and such administration can be in single or multiple doses. More specifically, the pharmaceutical composition can be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, sugar tablets, handcandy, powders, sprays, aqueous suspensions, injectable solutions, elixirs, syrups, etc. Such carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents. Furthermore, such oral pharmaceutical formulations can be appropriately sweetened and / or flavored using various types of reagents commonly used for such purposes.

[0237] For example, cells can be administered to patients in conjunction with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, or cytarabine (also known as ARA-C). In some cases, engineered cells can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immunoablation agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines, and radiation. Engineered cell compositions can also be administered to patients in conjunction with bone marrow transplantation and T-cell ablation therapies using chemotherapeutic agents such as fludaribine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (e.g., before, simultaneously with, or after). In some cases, engineered cell compositions may be administered following B-cell ablation therapy, which uses agents such as rituximab (Rituxan) that react with CD20. For example, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject may receive an infusion of engineered cells, such as expanded engineered cells. Additionally, expanded engineered cells may be administered before or after the procedure. Engineered cells obtained by any of the methods described herein can be used in patients in need of treatment for host-vG rejection and graft-versus-host disease (GvHD). Therefore, methods for treating patients in need of treatment for host-vG rejection and graft-versus-host disease (GvHD) may be considered, including treating patients by administering an effective amount of genetically engineered cells containing inactivated TCRα and / or TCRβ. medicine box

[0238] This document may disclose kits containing compositions. It may also disclose kits for the treatment or prevention of cancer, pathogen infection, immune disorders, or allogeneic transplantation. In one embodiment, the kit may contain a unit dose of a therapeutic or prophylactic composition containing an effective amount of cells, including one or more anti-GPC3 CARs. In some embodiments, the kit includes a sterile container that may contain a therapeutic or prophylactic vaccine; such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug. In some cases, the subject matter anti-GPC3 CAR immune response cells, such as CAR T cells, may be provided with instructions regarding the administration of CAR immune response cells to subjects suffering from or at risk of cancer, pathogen infection, immune disorders, or allogeneic transplantation. The instructions typically contain information relating to the use of the composition for the treatment or prevention of cancer, pathogen infection, immune disorders, or allogeneic transplantation. In some cases, the medicine box may contain approximately 1 x 10 4 One cell to approximately 1 x 10 12 Cells. In some cases, the kit may contain at least approximately 1 x 103 cells. 5 1 cell, at least about 1 x 10 6 1 cell, at least about 1 x 10 7 1 cell, at least approximately 4 x 10 7 Each cell, at least approximately 5 x 10 7 1 cell, at least approximately 6 x 10 7 1 cell, at least approximately 6 x 10 7 1 cell, at least approximately 8 x 10 7 1 cell, at least approximately 9 x 10 7 1 cell, at least about 1 x 10 8 1 cell, at least about 2 x 10 8 1 cell, at least approximately 3 x 10 8 1 cell, at least approximately 4 x 10 8 Each cell, at least approximately 5 x 10 8 1 cell, at least approximately 6 x 10 8 1 cell, at least approximately 6 x 10 8 1 cell, at least approximately 8 x 10 8 1 cell, at least approximately 9 x 10 8 1 cell, at least about 1 x 10 9 1 cell, at least about 2 x 10 9 1 cell, at least approximately 3 x 10 9 1 cell, at least approximately 4 x 10 9 Each cell, at least approximately 5 x 109 1 cell, at least approximately 6 x 10 9 1 cell, at least approximately 6 x 10 9 1 cell, at least approximately 8 x 10 9 1 cell, at least approximately 9 x 10 9 1 cell, at least about 1 x 10 10 1 cell, at least about 2 x 10 10 1 cell, at least approximately 3 x 10 10 1 cell, at least approximately 4 x 10 10 Each cell, at least approximately 5 x 10 10 1 cell, at least approximately 6 x 10 10 1 cell, at least approximately 6 x 10 10 1 cell, at least approximately 8 x 10 10 1 cell, at least approximately 9 x 10 10 1 cell, at least about 1 x 10 11 1 cell, at least about 2 x 10 11 1 cell, at least approximately 3 x 10 11 1 cell, at least approximately 4 x 10 11 Each cell, at least approximately 5 x 10 11 1 cell, at least approximately 6 x 10 11 1 cell, at least approximately 6 x 10 11 1 cell, at least approximately 8 x 10 11 1 cell, at least approximately 9 x 10 11 One cell or at least about 1 x 10 12 Cells. For example, the kit may contain approximately 5 x 10 cells. 10 1 cell. In another example, the kit may contain 3x10 cells. 6 10 cells; cells can be expanded to approximately 5 x 102 10 One cell was administered to the subject.

[0239] In some cases, the kit may contain allogeneic cells. In some cases, the kit may contain cells that can have genomic modifications. In some cases, the kit may contain "off-the-shelf" cells. In some cases, the kit may contain cells that can be expanded for clinical use. In some cases, the kit may contain contents for research purposes.

[0240] In some cases, the package insert contains at least one of the following: a description of the therapeutic agent; a dosage regimen and administration for the treatment or prevention of tumor formation, pathogen infection, immune disorders, or allogeneic transplantation or their symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The package insert may be printed directly on the container (if present), or as a label applied to the container, or as a separate sheet, brochure, card, or leaflet provided in or with the container. In some cases, the package insert provides a procedure for administering anti-GPC3 CAR immune response cells, such as anti-GPC3 CAR T cells, after administration of the chemotherapy agent. In some cases, the package insert provides a procedure for administering anti-GPC3 CAR immune response cells before administration of the chemotherapy agent. In some cases, the package insert provides a procedure for administering anti-GPC3 CAR immune response cells concurrently with administration of the chemotherapy agent. In some cases, the package insert provides a procedure for administering anti-GPC3 CAR immune response cells at least 12 hours after administration of the chemotherapy agent. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells at least approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, or up to 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells at least 24 hours after chemotherapy. Anti-GPC3 CAR immune response cells can be formulated for intravenous injection. Anti-GPC3 CAR immune response cells can be formulated for intra-arterial injection into the liver of subjects who may contain solid tumors.

[0241] In some cases, the kit may contain cyclophosphamide and / or fludarabine, which are formulated for use at doses of approximately 60 mg / kg to approximately 80 mg / kg and approximately 25 mg / m², respectively. 2 Approximately 35 mg / m 2 Administer to subjects who require it. In some cases, the kit may contain pediatric doses.

[0242] Recombination methods are well known in the art. Unless otherwise stated, the practice of this invention employs conventional techniques of molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are well explained in the following literature, such as "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (Gait, ed., 1984); "Animal Cell Culture" (Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (Wei and Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, eds., 1987); "Current Protocols in Molecular Biology" (Ausubel et al., ed., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (Coligan et al., ed., 1991). These techniques are applicable to the production of polynucleotides and peptides and can therefore be considered in the preparation and practice of this invention. Particularly useful techniques are discussed in the following sections. Other applications

[0243] Effective adoptive cell transfer-based immunotherapy (ACT) can be used to treat patients with cancer (e.g., metastatic cancer). For example, autologous peripheral blood lymphocytes (PBLs) can be modified using the non-viral or viral methods disclosed herein to express an anti-GPC3 chimeric antigen receptor (CAR) that recognizes GPC3 on cancer cells or tumor cells, and can be used in the disclosed methods and kits. This invention relates to compositions and methods for use in immunotherapy (including, but not limited to, cancer therapy) that utilize human or humanized chimeric antigen receptors after or concurrently with lymphopenic therapy in a subject. The chimeric antigen receptor utilizes a human or humanized chimeric antigen receptor construct after or concurrently with lymphopenic therapy in a subject.

[0244] These compositions and methods can provide cancer therapies with numerous advantages. In some cases, the methods may include modifying immune-responding cells so that the immune-responding cells are substantially independent of the presence or activity of the major histocompatibility complex (MHC). In some cases, the polynucleotides described herein may encode chimeric antigen receptors. Methods for preparing immune-responding cells expressing chimeric antigen receptors such as anti-GPC3 CAR are also disclosed. Therapeutic methods in which anti-GPC3 CAR immune-responding cells can be administered to a patient with cancer or disease may also be disclosed.

[0245] This article describes a method for treating diseases (e.g., cancer) in recipients, including transplanting one or more subject-specific anti-GPC3 CAR immune response cells into the recipient after or simultaneously with lymphopenia treatment.

[0246] Autologous lymphocyte infusion can be used for treatment. Autologous peripheral blood mononuclear cells (PBMCs) can be collected from patients in need of treatment, and T cells can be activated and expanded using methods described herein and known in the art, and then infused back into the patient. In other cases, allogeneic cells can be used to treat the patient. Subject CAR immune response cell populations can be formulated for administration; and administered to the subject using techniques known to those skilled in the art. The expanded cells can then be grown under conditions similar to those of unmodified cells, thereby allowing the modified cells to be expanded and used for various purposes.

[0247] The methods disclosed herein may include transplantation. Transplantation can refer to adoptive transplantation of cell products. Transplantation can be autologous transplantation, allogeneic transplantation, xenotransplantation, or any other type of transplantation. For example, transplantation can be xenotransplantation. Transplantation can also be allogeneic transplantation.

[0248] In some cases, approximately 5x10 10 The subject administers anti-GPC3 CAR immune response cells to the subjects. In some implementations, approximately 5 x 10 10 Each cell represents the median number of cells administered to the subject. In some implementations, approximately 5 x 10-1 10 Each cell is essential to influence the treatment response in a subject. In some implementations, at least approximately 1 x 10-1 cells are required. 7 1 cell, at least about 2 x 10 7 1 cell, at least approximately 3 x 10 7 1 cell, at least approximately 4 x 10 7 Each cell, at least approximately 5 x 10 7 1 cell, at least approximately 6 x 10 7 1 cell, at least about 7 x 10 7 1 cell, at least approximately 8 x 10 7 1 cell, at least approximately 9 x 10 71 cell, at least about 1 x 10 8 1 cell, at least about 2 x 10 8 1 cell, at least approximately 3 x 10 8 1 cell, at least approximately 4 x 10 8 Each cell, at least approximately 5 x 10 8 1 cell, at least approximately 6 x 10 8 1 cell, at least about 7 x 10 8 1 cell, at least approximately 8 x 10 8 1 cell, at least approximately 9 x 10 8 1 cell, at least about 1 x 10 9 1 cell, at least about 2 x 10 9 1 cell, at least approximately 3 x 10 9 1 cell, at least approximately 4 x 10 9 Each cell, at least approximately 5 x 10 9 1 cell, at least approximately 6 x 10 9 1 cell, at least about 7 x 10 9 1 cell, at least approximately 8 x 10 9 1 cell, at least approximately 9 x 10 9 1 cell, at least about 1 x 10 10 1 cell, at least about 2 x 10 10 1 cell, at least approximately 3 x 10 10 1 cell, at least approximately 4 x 10 10 Each cell, at least approximately 5 x 10 10 1 cell, at least approximately 6 x 10 10 1 cell, at least about 7 x 10 10 1 cell, at least approximately 8 x 10 10 1 cell, at least approximately 9 x 10 10 1 cell, at least about 1 x 10 11 1 cell, at least about 2 x 10 11 1 cell, at least approximately 3 x 10 11 1 cell, at least approximately 4 x 10 11 Each cell, at least approximately 5 x 10 11 1 cell, at least approximately 6 x 10 11 1 cell, at least about 7 x 10 11 1 cell, at least approximately 8 x 10 11 1 cell, at least approximately 9 x 10 11 One cell or at least about 1 x 10 12 Cells. For example, approximately 5 x 10 10 One cell is administered to the subject. In another instance, cells can be obtained from 3x10 cells. 6 Each cell was expanded to approximately 5 x 102 10Individual cells are then administered to the subject. In some cases, the cells are expanded to a sufficient number for treatment. For example, 5 x 102 7 Individual cells can undergo rapid expansion to produce a sufficient number for therapeutic use. In some cases, a sufficient number for therapeutic use may be 5 x 10-1. 10 Any number of cells can be infused for therapeutic purposes. For example, a subject can be infused with 1x10 cells. 6 Up to 5x10 12 The number of cells (including end values). Patients may be infused with as many cells as can be generated for them. In some cases, not all cells infused into a patient are engineered. For example, at least 90% of the cells infused into a patient may be engineered. In other cases, at least 40% of the cells infused into a patient may be engineered. In some embodiments, the method may include calculating the amount of engineered cells necessary to influence a treatment response in a subject and / or administering them to the subject. In some embodiments, calculating the amount of engineered cells necessary to influence a treatment response includes cell viability and / or the efficiency with which the anti-GPC3 CAR transgene has been integrated into the cell's genome. In some embodiments, the cells administered to the subject to influence a treatment response may be live cells. In some embodiments, to achieve a therapeutic response in a subject, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, and at least about 10% of the cells are live cells. In some embodiments, to influence a therapeutic response in a subject, the cells applied to the subject may be cells having one or more transgenes successfully integrated into the cell's genome. In some implementations, in order to achieve a therapeutic response in subjects, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, and at least about 10% of the cells have one or more CAR transgenes successfully integrated into the cell genome.

[0249] In some cases, subject-specific anti-GPC3 CAR immune response cells may be administered to the subject, wherein the administerable CAR immune response cells can be approximately 1 day to approximately 35 days old. For example, the administered cells may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or up to 40 days old. The age of the CAR immune response cells can be considered from the time of stimulation. The age of the CAR immune response cells can be considered from the time of blood component apheresis. The age of the CAR immune response cells can be considered from the time of transduction. In some implementations, the CAR immune response cells that can be administered to the subject are approximately 10 days to approximately 14 days or approximately 20 days old. In some cases, the “age” of the CAR immune response cells can be determined by the length of the telomeres. For example, "young" CAR immune response cells may have longer telomere lengths than "consumed" or "old" CAR immune response cells. Without being bound by any specific theory, it can be assumed that immune response cells lose approximately 0.8 kb of telomere length per week in culture, and that young CAR immune response cell cultures may have telomeres approximately 1.4 kb longer than those of immune response cells approximately 44 days old. Without being bound by any specific theory, it is assumed that longer telomere lengths may be correlated with a patient's positive objective clinical response and cell persistence in vivo.

[0250] In some cases, cells are isolated from the subject organism, transfected with nucleic acids (e.g., genes or cDNA), and re-infused back into the subject organism (e.g., a patient).

[0251] Cells (e.g., engineered cells or engineered primary T cells) can function before, after, and / or during transplantation. For example, transplanted cells can function for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days after transplantation. Transplanted cells can function for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation. Transplanted cells can function for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation. In some cases, transplanted cells can function for up to the recipient's lifetime.

[0252] Furthermore, the transplanted cells performed 100% of their expected normal function. The transplanted cells also performed 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, and 49% of their expected normal function. 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or up to approximately 100%.

[0253] The transplanted cells can also perform more than 100% of their normal expected function. For example, the transplanted cells can perform 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or up to about 5000% of their normal expected function.

[0254] Transplantation can be performed using any type of graft. Sites may include, but are not limited to, the subcapsular space of the liver, spleen, kidney, omentum, submucosa of the stomach or intestine, segments of small intestinal vessels, venous sacs, testis, brain, spleen, or cornea. For example, a subcapsular graft can be performed. Intramuscular grafting is also possible. Portal vein grafting is also an option.

[0255] Following treatment (e.g., any treatment disclosed herein), transplant rejection can be improved compared to transplanting one or more wild-type cells into the recipient. For example, transplant rejection can be hyperacute rejection. Transplant rejection can also be acute rejection. Other types of rejection can include chronic rejection. Transplant rejection can also be cell-mediated or T-cell-mediated rejection. Transplant rejection can also be natural killer cell-mediated rejection.

[0256] Improving transplantation can refer to mitigating hyperacute rejection, which may include reducing, alleviating, or weakening adverse effects or symptoms. Transplantation can also refer to adoptive transplantation of cell products.

[0257] Another indicator of a successful transplant can be the number of days the recipient does not require immunosuppressive therapy. For example, following the treatments (e.g., transplantation) described herein, the recipient may not require immunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. This can indicate a successful transplant. It can also indicate that the transplanted cells, tissues, and / or organs have not been rejected.

[0258] In some cases, recipients may not need immunosuppressive therapy for at least 1 day. Recipients may not need immunosuppressive therapy for at least 7 days. Recipients may not need immunosuppressive therapy for at least 14 days. Recipients may not need immunosuppressive therapy for at least 21 days. Recipients may not need immunosuppressive therapy for at least 28 days. Recipients may not need immunosuppressive therapy for at least 60 days. Furthermore, recipients may not need immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0259] Another indicator of a successful transplant can be the number of days the recipient needs to reduce immunosuppressive therapy. For example, following the treatments described herein, the recipient may need reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. This can indicate a successful transplant. It can also indicate that the transplanted cells, tissues, and / or organs have not been rejected or have minimal rejection.

[0260] In some cases, the recipient may require at least one day of reduced immunosuppressive therapy. The recipient may require at least seven days of reduced immunosuppressive therapy. The recipient may require at least 14 days of reduced immunosuppressive therapy. The recipient may require at least 21 days of reduced immunosuppressive therapy. The recipient may require at least 28 days of reduced immunosuppressive therapy. The recipient may require at least 60 days of reduced immunosuppressive therapy. Furthermore, the recipient may require reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0261] Reduced immunosuppressive therapy can refer to less immunosuppressive therapy compared to the immunosuppressive therapy required when transplanting one or more wild-type cells into a recipient.

[0262] Immunosuppressive therapy can include any treatment that suppresses the immune system. Immunosuppressive therapy can help alleviate, minimize, or eliminate transplant rejection in recipients. For example, immunosuppressive therapy can include immunosuppressive drugs. Immunosuppressive drugs that can be used before, during, and / or after transplantation are not limited to MMF (mycophenolate mofetil (Cellcc)), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alenzusab (Campath), anti-CD20 (rituximab), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (abatacept / Orencia), and beraccept (LEA29Y). The following are listed as immunosuppressants / antibiotics: Sirolimus (Rapimune), everolimus, tacrolimus (Prograf), dalizumab (Ze-napax), baliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyguanidin, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody. In addition, one or more immunosuppressants / antibiotics may be used together or sequentially. One or more immunosuppressants / antibiotics may be used for induction therapy or maintenance therapy. The same or different drugs may be used during the induction and maintenance phases. In some cases, dalizumab (Zenapax) can be used for induction therapy, while tacrolimus (Prograf) and sirolimus (Rapimune) can be used for maintenance therapy. Dalizumab (Zenapax) can also be used for induction therapy, while low-dose tacrolimus (Prograf) and low-dose sirolimus (Rapimune) can be used for maintenance therapy. Immunosuppression can also be achieved using non-pharmacological regimens, including but not limited to whole-body irradiation, thymic irradiation, and total and / or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs. Example

[0263] The invention is described in further detail with reference to the following experimental embodiments. These embodiments are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should not in any way be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent from the teachings provided herein. Example 1: Construction of GPC3-resistant CAR vector

[0264] An exemplary lentiviral plasmid vector was constructed using a third-generation self-inactivated lentiviral vector system. This system comprises a packaging plasmid pMDLg RRE (Addgene) encoding Gag / Pol, a packaging plasmid pRSV-REV (Addgene) encoding Rev, an envelope plasmid pCMV-VSV-G (Addgene) encoding VSV-G, and a recombinant expression vector encoding the CAR gene based on the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE (Addgene). This system effectively reduces the risk of forming replicative lentiviral (RCL) particles.

[0265] The empty vector pRRLSIN-cPPT.PGK-GFP.WPRE contains a promoter for elongation factor-1α (EF-1α), and an Mlu is inserted between this promoter and the CD8αsp signal peptide. I Cleavage site. Specifically, the pWPT-EGFP vector (Addgene) was double-digested with ClaI / SalI (NEB) to re-obtain a 1.1 kb DNA fragment, which was then ligated with T4 DNA ligase to pRRLSIN-cPPT.PGK-GFP.WPRE, which had been double-digested with ClaI / SalI (NEB). The ligation mixture was then transformed into host cells TOP10. Positive clones were identified by colony PCR and confirmed by sequencing, yielding the recombinant plasmid pRRLSIN-cPPT.EF-1α-EGFP.WPRE. The EF-1α promoter (SEQ ID NO:33, containing Mlu) of the CD8α signaling domain (also known as fragment 1, 442 bp) was amplified using upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 31), downstream primer 5'-CGGCCTGGCGGCGTGGAG-3' (SEQ ID NO: 32), and pRRLSIN-cPPT.EF-1α-EGFP.WPRE as a template. I The cleavage site was identified, and amplification was performed under the following conditions: initial denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 68°C for 30 s; and after 25 cycles, extension at 68°C for 10 min. Agarose gel electrophoresis confirmed that the amplified bands had the expected fragment size.

[0266] Humanized antibody 35 has been described in Chinese patent application number CN201510481235.1, which is capable of specifically recognizing humanized GPC3 protein. To construct the 35-CAR lentiviral plasmid, the following conditions were used during amplification to provide the heavy chain variable domain fragment: a fragment containing the heavy chain variable domain 35 (SEQ ID NO: 80 in CN201510481235.1) was used as a template, with upstream primer 5'-ctccacgccgccaggccggaggtgcagctggtgcag-3' (SEQ ID NO: 34) and downstream primer 5'-GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGGTG-3' (SEQ ID NO: 35); the following conditions were used to provide the light chain variable domain fragment: a fragment containing the light chain variable domain 35 (SEQ ID NO: 79, CN201510481235.1) was used as a template, with upstream primer 5'-GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTTCTACAGCTAC-3' (SEQ ID NO: 80) 36), and downstream primer 5'-CGGCGCTGGCGTCGTGGTACGTTTGATCTCCAGCTTGGTG-3' (SEQ ID NO: 37). Bridged PCR amplification of the heavy and light chain variable domain primers provided a 35 scFv fragment (SEQ ID NO: 38, also known as fragment 2, 765 bp) containing sequences repeating the upstream CD8α signal peptide and the downstream hinge region. PCR amplification was performed under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s, annealing at 58 °C for 40 s; extension at 68 °C for 40 s; and after 25 cycles, extension at 68 °C for 10 min. The amplified bands were confirmed to be of the expected fragment size by agarose gel electrophoresis.

[0267] Using upstream primer 5'-accacgacgccagcgccg-3' (SEQ ID NO: 39) and downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 40), pWPT-eGFP-F2A-GPC3-28Z as a template (see Chinese patent application CN 104140974 A), Hinge-28Z (SEQ ID NO: 41, fragment 3, 703 bp) (with internal Sal) was amplified. I(Cutting site). PCR amplification was performed under the following conditions: initial denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s; extension at 68°C for 30 s; and a final extension at 68°C for 10 min after 25 cycles. The amplified bands were confirmed to be of the expected fragment size by agarose gel electrophoresis. Equimolar amounts (approximately 50 ng) of fragments 1, 2, and 3 were amplified by overlap PCR under the following conditions: initial denaturation at 94°C for 4 min; denaturation at 94°C for 40 s; annealing at 60°C for 40 s; extension at 68°C for 140 s; and a final extension at 68°C for 10 min after 5 cycles. The mixture was then supplemented with DNA polymerase, upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 31) and downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 40), and amplified for 25 cycles under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s; extension at 68 °C for 140 s; and final extension at 68 °C for 10 min. The theoretical size of the resulting 35-28Z (SEQ ID NO: 41) was 1874 bp. The amplified product was confirmed by agarose gel electrophoresis to have the expected fragment size, and its sequence is shown in SEQ ID NO: 23.

[0268] Using Mlu I and Sal I The pRRLSIN-cPPT.EF-1α-EGFP.WPRE vector and 35-28Z were digested, then ligated using T4 ligase and transformed into TOP10. Positive clones were identified by colony PCR and confirmed by Invitrogen sequencing, confirming the acquisition of pRRL-EF-1α-35-28Z. Example 2: Packaging of GPC3-CAR lentivirus

[0269] Anti-GPC3-CAR lentiviral stock solution (0.75 L) was prepared using embryonic kidney cells 293 T (ATCC: CRL-11268). Specifically, 293 T cells were cultured at 1.17 × 10⁻⁶ cells per cell line. 4 / cm 2The culture was inoculated into 5 layers of CellStack (Corning). On the day of transfection, solution A was prepared containing 133.2 μg of packaging plasmid pRSV-Rev, 133.2 μg of pMDLg / pRRE, 51.56 μg of pCMV-VSV-G, and 111.7 μg of pRRL-EF-1α-35-28Z. This was achieved by dissolving 966 μg of all plasmids in 17 ml of DMEM basal medium and gently mixing. Solution B was prepared by dissolving 2.9 mg of PEI (Polysciences) in 17 ml of DMEM basal medium, gently mixing the solutions, and incubating at room temperature for 5 min. Solution A was then added to solution B, mixed thoroughly, and incubated at room temperature for 20-25 min. The 293 T culture was then transferred from the CellStack to a 1 L flask. The combined solution containing the plasmids and PEI was then added to the culture solution and gently mixed. The resulting solution was then transferred to a Cellstack and incubated at 37°C and 5% CO2 for 5 h. The medium was then replaced with fresh DMEM supplemented with 10% FBS (Life Technology). The culture was then incubated at 37°C and 5% CO2 for 48 h. The lentivirus stock solution was subsequently obtained again, filtered through a 0.45 μm (Millipore) filter, concentrated, purified using a KrosFlo® IIi tangential flow filtration system (Spectrum), washed with AIM-V (Life Technology), and stored. Viral titration was performed according to the method described in CN104140974A. The viral titer was calculated to be approximately 1 × 10⁻⁶. 8 / ml. The virus was distributed at a rate of 1×10⁻⁶. 8 / Packaged in small vials and stored at -80°C for later use. Example 3: Preparation of anti-GPC3-CAR T cells and in vitro antitumor assay against liver cancer cells

[0270] CAR T cells were prepared in an immune cell preparation laboratory according to cGMP standards. Specifically, human peripheral blood mononuclear cells (PBMCs) were obtained by separating peripheral blood or leukocyte-enriched blood from subjects using a COBE Spectra blood component separator via density gradient centrifugation. PBMCs were mixed with magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Life Technology) at a 1:3 PMBC to magnetic bead ratio, stimulated with recombinant human IL-2 (ShanghaiHua Xin High Biotechnology Co., Ltd.) at a final concentration of 300 U / mL, and cultured for 48 h. Subsequently, activated T cells were transduced using the aforementioned lentivirus at approximately 5 MOI, with transduction efficiency enhanced by RetroNectin (Takara). Free virus was removed by medium replacement via low-speed centrifugation (100 g × 10 min) at 24 h and 48 h post-transduction. Magnetic beads were removed at 72 h post-transduction. The above centrifugation was repeated to obtain a density of 5 x 10⁻⁶ cells / mL. 5 Subcultured at / mL. Subsequently, at approximately 3 x 10⁹ / mL every other day. 5 Cells were passaged at a density of 300 U / mL, and recombinant human IL-2 was added to the lymphocyte culture medium at a final concentration of 300 U / mL.

[0271] After 10 to 12 days of in vitro culture, CAR expression in transduced CAR T cells was determined by flow cytometry. In other words, the positive transduction rate of CAR T cells was measured, and it is shown in Table 7. Furthermore, in vitro antitumor activity was assessed by co-incubating with high GPC3-expressing hepatocellular carcinoma cells (Huh-7), low GPC3-expressing PLC / PRF / 5, and GPC3-negative SK-HEP-1 cells for 18 h, with effector-to-target ratios of 3:1, 1:1, and 1:3. Detailed procedures are described in CN104140974A. Table 8 presents the in vitro antitumor activity results at an effector-to-target ratio of 1:1.

[0272] The results showed that approximately 40% to 80% of the anti-GPC3-CAR T cells expressed CAR. Furthermore, the anti-GPC3-CAR T cells exhibited specific killing activity against GPC3-positive Huh-3 and PLC / PRF / 5 cells in a dose-dependent manner based on the effector:target ratio, while they showed no killing activity against GPC3-negative SK-HEP-1 (Table 8), indicating that the anti-GPC3-CAR T cells prepared by subjects in this disclosure have excellent targeted killing activity.

[0273] Table 7: Overview of positive transduction rate and administration regimen of anti-GPC3-CAR T cells from subjects

[0274] Table 8: In vitro killing effect of anti-GPC3-CAR T cells from subjects (effectant:target ratio of 3:1, 1:1 and 1:3, cultured in vitro for 18 h) Example 4: Changes in lymphocytes after lymphopenia

[0275] After confirming that no abnormalities occurred during the preparation of anti-GPC3-CAR T cells in the subjects, a comprehensive assessment of the subjects' various physical conditions was conducted to determine their suitability for in vivo lymphopenia treatment. A primary lymphopenia regimen was designed for appropriate subjects.

[0276] The following two lymphopenia regimens were designed: Regimen 1: Cyclophosphamide (CTX) single-drug lymphopenia regimen, at a dose of 1 g / m². 2 / day x 1 day. Option 2: Lymphopenia regimen combining fludarabine (FLU) and cyclophosphamide, dose 20-30 mg fludarabine / m 2 / day x 4 days + 500 mg cyclophosphamide / m 2 / day x 2days.

[0277] Implementing this scheme involves adjustments to the following condition: calculating the human body surface area according to Stevenson's formula (Chinese Journal of Physiology, 12:327, 1937). That is, surface area (m²) 2 = 0.0061 x height (cm) + 0.0128 x weight (kg) - 0.1529. For example, an adult who is 170 cm tall and weighs 60 kg has a height of 1.6521 m. 2 The surface area. Subjects who were unsuitable for lymphopenia treatment due to their existing physical condition or their own choice were assigned to the second cohort. A total of three subjects (H01, H02, and H03) received different lymphopenia treatments. One subject (H04) was not ablated due to his physical condition and his own choice. The protocols are shown in Table 9.

[0278] Table 9: Overview of Lymphopenia Protocols Used in Subjects

[0279] H01 1 This indicates that subject H01 was initially treated with anti-GPC3-CAR T-cell therapy; H01 2This indicates that subject H01 received a second dose of anti-GPC3-CAR T-cell therapy; and D0 is defined as the day of the anti-GPC3-CAR T-cell administration. Following the above lymphopenia regimen, the absolute lymphocyte counts in the subjects varied. Lymphopenia treatment with cyclophosphamide monotherapy resulted in a lymphopenia reduction of approximately 40% to approximately 72%. Lymphopenia treatment with the combination of fludarabine and cyclophosphamide produced a more significant difference in lymphopenia reduction compared to cyclophosphamide monotherapy, ranging from approximately 82% to approximately 96.5%. Specifically, Figure 2 The changes in the ratio of absolute lymphocyte count to baseline at each monitoring time point before and after lymphopenia treatment are shown. These results demonstrate that the combination therapy of fludarabine and cyclophosphamide results in superior lymphopenia compared to cyclophosphamide monotherapy. Example 5: Clinical response of subjects after ablation

[0280] Approximately 2 days after lymphopenia treatment, subjects were administered anti-GPC3-CAR T-cell therapy intravenously. The dosage of anti-GPC3-CAR T-cells administered is presented in Table 7. A summary of the subjects' clinical responses after treatment is shown in Table 10. Results demonstrate that, to date, the three subjects who received lymphopenia treatment have stable disease (SD) or partial disease (PR) following anti-GPC3-CAR T-cell therapy. Conversely, subject H04, who did not receive lymphopenia treatment, showed progressive disease on MRI 4 weeks after anti-GPC3-CAR T-cell therapy.

[0281] Regarding AFP results, due to the insensitivity of AFP, the prognosis of subject H02 could not be assessed from tumor markers. For the other two subjects, their AFP levels were slightly decreased, as shown in Table 10. It should be noted that subject H03 showed an 87% reduction in AFP compared to baseline before treatment after lymphopenia treatment with combined fludarabine and cyclophosphamide. Simultaneously, imaging showed partial clinical response at the target site. Figure 3 The MRI results of subject H03 before and 10 weeks after treatment are shown.

[0282] Table 10: Overview of clinical responses in subjects after anti-GPC3-CAR T-cell therapy

[0283] Note: ↓ indicates a decrease in AFP levels; ↑ indicates an increase in AFP levels; PD indicates progressive disease; CR indicates a complete response; and PR indicates a partial response. Safety data and clinical observations showed that no subjects experienced intolerable toxicities or side effects after treatment with anti-GPC3-CAR T cells. All subjects experienced some degree of fever and increased GRP. Subjects H01 and H04 experienced shivering after administration. Subject H03 had a decrease in albumin due to fever. By ingesting exogenous nutrients and albumin, the subject had normal albumin levels after the fever subsided (Table 11).

[0284] Table 11: Overview of toxicities or side effects in subjects following administration of anti-GPC3-CAR T cells after lymphopenia treatment.

[0285] Furthermore, no cytokine release syndrome, which frequently occurs during CAR T-cell therapy for hematologic malignancies, was observed in this clinical study, indicating that anti-GPC3-CAR T cells are safe.

[0286] In summary, clinical studies using CAR-GPC T-cell therapy for GPC3-positive hepatocellular carcinoma demonstrate that subjects receiving effective lymphopenic therapy benefited from anti-GPC3-CAR T-cell therapy, while those not receiving such therapy did not. Furthermore, neither subjects receiving nor not receiving lymphopenic therapy exhibited intolerable toxicities or side effects; both groups experienced some adverse reactions such as fever and increased CRP, suggesting that lymphopenic therapy may not have exacerbated these adverse reactions. Moreover, in terms of the effect of lymphopenic therapy and the benefit to subjects, the combined fludarabine and cyclophosphamide lymphopenic therapy regimen may be superior to the cyclophosphamide monotherapy regimen. In conclusion, anti-GPC3-CAR T-cell therapy following effective lymphopenic therapy can provide an effective treatment solution for the clinical management of GPC3-positive hepatocellular carcinoma. Example 6: Phenotypic Analysis

[0287] The expression of cell surface molecules was determined by flow cytometry using standard methodologies. Monoclonal antibodies conjugated to the following proteins—phycoerythrin, fluorescein isothiocyanate, and / or dinophyll chlorophyll—were used: CD3, CD4, CD8, CD30, CCR4, CD45RA, CD45RO, CCR7, CD62L, CD56, and αβT cell receptor (BD Biosciences PharMingen). CAR expression on T cells was detected using anti-CAR antibodies. Samples were analyzed using FACSCalibur (BD Biosciences PharMingen), and data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA). At least 10,000 positive events were measured for each sample. Example 7: Cytotoxicity assay: Liver cancer

[0288] To assess the potential cytotoxicity of second- or third-generation CAR-T transduced T cells, a CytoTox96® non-radioactive cytotoxicity assay (Promega) was performed. In short, anti-GPC3 CAR-T cells at concentrations of 33-28 BBZ, 92-28 BBZ, and 4-28 BBZ were compared with control cells SK-HEP-1 (… Figure 4A ) and CHO-K1 ( Figure 4B Co-culture was performed with an effector-to-target ratio of 3:1, 1:1, or 1:3, and target cells at a density of 10,000 per well. The cells were incubated at 37°C for 18 hours. After incubation, visible wavelength absorbance data were collected using a standard 96-well plate reader.

[0289] To evaluate antitumor cytotoxicity, CAR-T cells (33-28BBZ, 92-28BBZ, and 4-28BBZ) were co-cultured with GPC3-positive hepatocellular carcinoma cells (Huh-7) or control cells transduced to express GPC3 and CHO-K1-GPC3+. The effector-to-target ratio was 3:1, 1:1, or 1:3, with 10,000 target cells per well. Cells were incubated at 37°C for 18 hours. Figure 5A and Figure 5B After incubation, absorbance data at visible wavelengths were collected using a standard 96-well plate reader.

[0290] For all effectors: target ratio, cytotoxicity is the average of 5 replicate wells. result

[0291] Cytotoxicity data showed that the 33-28BBZ, 92-28BBZ, and 4-28BBZ CAR-T constructs were cytotoxic to tumor cell lines expressing GPC3 at ratios of 3:1, 1:1, and 1:3, but not to control cell lines that did not express GPC3. Example 8: Cytotoxicity assay: Liver cancer group

[0292] To assess the potential cytotoxicity of second- or third-generation CAR-T transduced T cells, the CytoTox96® non-radioactive cytotoxicity assay (Promega) was performed. In short, anti-GPC3 CAR-T cells were transduced with HepG2 (HCC) at 92-28Z, 92-BBZ, 92-28BBZ, or an empty vector. Figure 6A ), Hep3B (HCC) Figure 6B ) or PLC / PRF / 5 (liver cancer) ( Figure 6C Co-culture was performed with an effector-to-target ratio of 3:1, 1:1, or 1:3, at a target cell density of 10,000 cells / well, and incubated at 37°C for 18 hours. To assess the cytotoxicity of second- and third-generation CAR-T cells, similar CytoTox 96® assays were performed using 92-28Z, 92-BBZ, 92-28BBZ, or an empty vector co-cultured with Huh-7 hepatocellular carcinoma cells exhibiting high GPC3 expression. Figure 7A After incubation, absorbance data at visible wavelengths were collected using a standard 96-well plate reader. result

[0293] Cytotoxicity data showed that the second- and third-generation constructs exhibited comparable cytotoxicity across all hepatocellular carcinoma cell lines, including HCC and liver cancer cell lines, at ratios of 3:1, 1:1, and 1:3. The second- and third-generation constructs were able to target tumor cells expressing different levels of GPC3, revealing broad tumor-targeting potential. Example 9: Cytotoxicity assay: Gastric cancer

[0294] To evaluate the potential cytotoxicity of second- or third-generation CAR-T transduced T cells in the gastric cancer tumor environment, a non-radioactive CytoTox 96® Promega assay was performed. In short, anti-GPC3 CAR-T cells (92-28Z, 92-28BBZ, or empty vector) were co-cultured with KATO-III at effector:target ratios of 1:3, 1:1, and 3:1, at 10,000 cells / well, and incubated at 37°C for 18 hours. result

[0295] Cytotoxicity data showed that the second- and third-generation constructs were effective against the gastric cancer tumor line KATO-III ( Figure 7B It exhibits considerable cytotoxicity, with cells transduced with empty vectors showing no activity. Data indicate that CAR-T cells are specific to their tumor targets and do not show reactivity in tissues that do not express the GPC3 antigen. Example 10: Comparison of scFv 33 and scFv 92

[0296] To compare CAR-T cells with different scFv targeting GPC3, second- or third-generation CAR-T transduced T cells were used in the CytoTox 96® non-radioactive cytotoxicity assay (Promega). In short, anti-GPC3 CAR-T cells (33-28Z, 92-28Z, 92-BBZ, 92-28BBZ, or empty vector) were co-cultured with Huh-7 cells or control A431 cells (GPC3 negative) at an effector:target ratio of 3:1, 1:1, or 1:3, with 10,000 target cells per well, and incubated at 37°C for 18 hours. After incubation, absorbance data at visible wavelengths were collected using a standard 96-well plate reader. result

[0297] Cytotoxicity data showed that, at effector-to-target ratios of 3:1, 1:1, and 1:3, the construct with scFv 92 exhibited increased cytotoxicity compared to the construct with scFv 33. Figure 10B Cytotoxicity is antigen-specific, as no cytotoxicity was detected when co-cultured with the GPC3-negative tumor cell line A431. Figure 10A . Example 11: Comparison of Second-Generation and Third-Generation CAR-T

[0298] To compare second- and third-generation CAR-T cells, CAR-T-transduced T cells were used in the CytoTox 96® non-radioactive cytotoxicity assay (Promega). In short, anti-GPC3 CAR-T cells transduced in 4-28Z, 4-28BBZ, 4-4-28BBZ, 4-14-28BBZ, 4-20-28BBZ, 4-35-28BBZ, 4-42-28BBZ, or empty vector were co-cultured with Huh7 (HCC) cells at an effector:target ratio of 3:1 or 1:1, with 10,000 target cells per well, and incubated at 37°C for 18 hours. After incubation, absorbance data at visible wavelengths were collected using a standard 96-well plate reader. result

[0299] Cytotoxicity data showed that the second and third generation constructs were cytotoxic, with 4-20-28BBZ, 4-35-28BBZ, and 4-42-28BBZ exhibiting higher cytotoxicity, as measured by lysis. Figure 9 . Example 12: Proliferation Assay

[0300] The proliferation of anti-GPC3 CAR-T cells after exposure to target cells (Huh-7, GPC3+) or control cells (SK-HEP-1, GPC3-) was determined by carboxyfluorescein succinimide dilution assay.

[0301] One week after transduction, control T lymphocytes and anti-GPC3 CAR-T cells were labeled with 1.5 μmol / L fluorescein diacetate succinimide (CFSE; Invitrogen) and plated with irradiated tumor targets (GPC3-positive and GPC3-negative lines) at an effector:target (E:T) ratio of 5:1. CD4 counts were measured by flow cytometry on day 4 of co-culture. + and CD8 + CFSE dilution on T cells. Example 13: Patient-derived tumor graft mouse model and lymphopenia

[0302] NOD / SCID mice were each implanted with a 2x2x2 mm lung cancer tumor. The tumors were implanted approximately 27 days post-implantation, or when the tumor size reached 220 mm. 3 At that time, cyclophosphamide was administered intravenously to mice, and 1x10 mg / L was administered intraperitoneally. 7 Empty vector, hu92-28Z, or hu92-28BBZ CAR-T cells. A second administration is given on day 34. Tumor volume is measured using calipers approximately every 3–7 days until day 52 post-inoculation. result

[0303] On day 51 post-tumor inoculation, mice treated with second-generation CAR-T 92-28Z showed significantly smaller tumor size compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8A and Figure 8B Mice treated with second-generation CAR-T 92-28Z also showed significantly reduced tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8C Mice treated with second-generation CAR-T 92-28Z also showed significantly reduced inhibition compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8D . Example 14: Xenograft HCC Mouse Model

[0304] NOD.Cg-Prkdc in infants aged 8 to 12 weeks scid Il2rg tm1Wjl 0.5x10 smears were implanted in the left flank of / SzJ (NSG) mice (Bar Harbor, ME). 6 One Huh-7 tumor cell. Seven days after tumor inoculation, 1x10-1 doses were administered via tail vein injection. 7 Mice were treated with empty vector-transduced T cells, hu92-28Z, or hu92-28BBZ. Following treatment, mice were monitored at least weekly using calipers for 35 days after tumor cell inoculation. result

[0305] Compared with mice treated with T cells transduced with empty vector, treatment with hu92-28Z and hu92-28BBZ showed significant antitumor activity. Figure 11A The results showed that mice treated with hu92-28Z or hu92-28BBZ had significantly reduced tumor volume on day 35 after tumor cell inoculation. Figure 11B The results showed that mice treated with hu92-28Z or hu92-28BBZ had significantly smaller tumors compared to mice treated with cells transduced with an empty vector, as demonstrated by the reduction in their weight. Figure 11C Images of xenografted tumors are shown. Example 15: Clinical Expansion of Anti-GPC3 CAR T Cells

[0306] To generate a large number of transduced T cells, a rapid expansion protocol (REP) was used to induce cell proliferation. Prior to REP, T cells were cultured with anti-CD3, anti-CD28, and IL-2, and transduced the day after culture initiation as described above. Cells were grown at 75 cm⁻¹ 2 The cells were cultured in flasks at 37°C and 5% CO2. Cell counts were performed every 2 days at a concentration of 0.5 × 10⁻⁶ cells / mL. 6 Cells were suspended at a concentration of 100 cells / mL in fresh T cell culture medium containing 300 IU / mL IL-2, and kept in culture for the remainder of the time. Example 16: Clinical Trial

[0307] Patients with evaluable hepatocellular carcinoma underwent apheresis to isolate peripheral blood mononuclear cells. Lymphocytes were isolated, transduced with anti-GPC3 CAR virus, amplified, and aliquoted for immunological testing. On days -7 and -6 prior to CAR-T administration, patients received intravenous cyclophosphamide pretreatment at 60 mg / kg / day for 2 days over 1 hour. On days -7 and -3 prior to CAR-T administration, patients received 25 mg / m² / day for 5 days over 30 minutes. 2 A pretreatment regimen of fludarabine administered via intravenous infusion (IVPB) per day. During the pretreatment regimen, patients underwent a complete blood count (CBC) test daily.

[0308] In the first part of the Phase I study, one patient in each group began dose escalation, with each patient starting at a dose of 10. 9 Individual anti-GPC3 CAR-T therapy. Individual patients are treated with a half-logarithmic increment. Therefore, the following dosage will be used: 10 9 1 cell, 3x10 9 10 cells, 10 10 1 cell, 3 x 10 10 1 cell and at most 1 x 10 11 Cells. Autologous anti-GPC3 CAR-T cells were administered intravenously over 20 to 30 minutes via a non-filtering tube.

[0309] All patients returned to the clinic for evaluation 6 weeks after administration of the CAR-T cell product.

[0310] Table 12: Sequences

Claims

1. The use of GPC3-CAR-expressing immune response cells, cyclophosphamide, and fludarabine in the preparation of drugs for the treatment of tumors.

2. The use according to claim 1, wherein the subject has undergone lymphopenia treatment prior to administration of the immune response cells, wherein the lymphopenia treatment comprises administering a chemotherapy agent to the subject, wherein administering the chemotherapy agent to the subject comprises administering cyclophosphamide and fludarabine.

3. The use according to claim 1 or 2, wherein the immune response cells are autologous or allogeneic relative to the subject.

4. The use according to any one of claims 1-3, wherein the intracellular signal transduction region of the CAR is selected from the intracellular signal transduction domains of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, 4-1BB, or CD66d.

5. Use of immune effector cells in the preparation of medicaments for treating GPC3-positive tumors, wherein the immune effector cells express chimeric antigen receptors (CARs) that recognize GPC3.

6. The use of immune effector cells expressing a chimeric antigen receptor (CAR) that recognizes GPC3 in the preparation of a medicament containing the cells and cyclophosphamide and fludarabine, wherein the immune effector cells and cyclophosphamide and fludarabine are formulated to provide a greater therapeutic effect than the sum of the effects of the reagents alone.

7. A composition comprising immune effector cells expressing a chimeric antigen receptor (CAR) that recognizes GPC3 and a chemotherapeutic agent.

8. A method for treating GPC3-positive tumors, the method comprising administering at least one course of immune effector cells to a subject, the immune effector cells expressing a chimeric antigen receptor (CAR) that recognizes GPC3, wherein the dose of the immune effector cells in each course is from about 5 x 10⁴ cells / kg to 1 x 10¹² cells / kg; preferably, lymphopenia treatment is performed before or concurrently with the administration of the immune effector cells to the subject.

9. A medicine box, comprising: (a) Effective amounts of immune response cells expressing GPC3-CAR; (b) a chemotherapeutic agent, wherein the chemotherapeutic agent comprises cyclophosphamide and fludarabine; and (c) Instructions for administering the immune response cells to the subject after the chemotherapy agent.

10. Use of anti-GPC3 chimeric antigen receptor (CAR) immune response cells in the preparation of a medicament for treating solid tumors in a subject, wherein the subject has undergone lymphopenia treatment prior to or concurrently with administration of the medicament, wherein the lymphopenia treatment comprises administration of a chemotherapeutic agent to the subject, wherein administration of the chemotherapeutic agent to the subject comprises administration of cyclophosphamide and fludarabine, wherein the extracellular domain of the CAR comprises an antigen-binding unit that exhibits specific binding to the C-terminus of GPC3.

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