Compositions and methods for treating cancer using chimeric antigen receptors targeting glypican 3

Abstract

The present disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor T cells that target glypican 3.

Description

[Technical Field]

[0001] The present disclosure relates to the treatment of cancer using chimeric antigen receptor T cells. [Background technology]

[0002] 1. Chimeric antigen receptor T cell therapy Chimeric antigen receptor (CAR) T-cell therapy is a specific form of cell-based immunotherapy that uses genetically engineered T cells to fight cancer. In CAR T-cell therapy, T cells are collected from a patient's blood, engineered ex vivo to express a CAR containing both an antigen-binding domain and a T-cell activation domain, expanded into a larger population, and administered to the patient. CAR T cells act as a living drug, binding to cancer cells and causing their destruction. When successful, the effects of CAR T-cell therapy tend to be long-lasting, as evidenced by the persistence and expansion of CAR T cells detected in patients long after clinical remission.

[0003] 2. Structure and function of CAR The antigen-binding domain of a CAR is an extracellular region that targets a surface antigen on tumor cells. Suitable target antigens can be proteins, phosphorylated proteins, peptide-MHC, carbohydrates, or glycolipid molecules. Ideal target antigens are widely expressed on tumor cells to enable targeting of a high percentage of cancer cells. Ideal candidate target antigens also typically have minimal expression in normal tissues, limiting off-tumor and on-target toxicity. The antigen-binding domain of a CAR contains a targeting moiety, such as an antibody single-chain variable fragment (scFv), directed against the target antigen.

[0004] The T cell activation domain of the CAR is intracellular and activates T cells in response to the antigen binding domain interacting with its target antigen. The T cell activation domain can include one or more costimulatory domains, which are the intracellular domains of known activating T cell receptors. The choice and positioning of the costimulatory domain within the CAR construct influences the function and fate of the CAR T cell, as costimulatory domains have different effects on the kinetics, cytotoxic function, and safety profile of CAR T cells.

[0005] The extracellular antigen-binding domain and intracellular T cell activation domain of the CAR are connected by a transmembrane domain, a hinge, and an optional spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on tumor cells. It can be used alone or with a spacer domain that allows the scFv to protrude from the T cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface.

[0006] CAR T therapy directed against the B-lymphocyte antigen CD19 (Kymriah®, Novartis) has shown promise for pediatric acute lymphoblastic leukemia, and CAR T therapy directed against a B-cell maturation antigen (bb2121, a collaboration between Celgene® and Bluebirdbio®) has shown promise for relapsed / refractory multiple myeloma. More recent data suggest that CAR approaches may be effective against solid tumors. GD2 CAR natural killer T cell (NKT) therapy has shown activity in neuroblastoma (Heczey A, et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood; 124(18):2824-33, 2014), and mesothelin CAR T with pembrolizumab has demonstrated antitumor activity against mesothelioma. However, additional targets for treating solid tumors are needed.

[0007] 3. Challenges of CAR T cell therapy Unfortunately, the complexity of CAR T cell-based therapy can lead to undesirable and potentially dangerous consequences. Off-tumor effects, such as neurotoxicity and acute respiratory distress syndrome, are potential adverse effects of CAR T cell therapy and can be fatal. Cytokine release syndrome (CRS) is the most common acute toxicity associated with CAR T cells. CRS occurs when lymphocytes become highly activated and release excessive amounts of inflammatory cytokines. Patients with CRS sometimes exhibit elevated serum levels of interleukin-2, interleukin-6, interleukin-1 beta, GM-CSF, and / or C-reactive protein when these factors are measured. CRS is classified by severity and diagnosed as grade 1–4 (mild to severe), with more severe cases clinically characterized by hyperthermia, hypotension, hypoxia, and / or multiorgan toxicity. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR T-cell therapy experienced CRS, with 50% of these patients developing grade 3–4 symptoms.

[0008] Therefore, additional CAR T cell-based therapies are needed to expand the armamentarium of effective cancer treatments. However, new CAR T cell therapies must be designed to effectively treat cancer while minimizing the risk of developing dangerous inflammatory responses such as CRS. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure describes compositions and methods for treating cancer using CAR T cells.

[0010] As described below, in a first aspect, the disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D), and the CAR construct provides an isolated nucleic acid sequence that does not induce cytokine production in GPC3-expressing cells.

[0011] In some embodiments of the first aspect, the antigen-binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof.

[0012] In some embodiments of the first aspect, the antigen-binding domain is a Fab or a single chain variable fragment (scFv).

[0013] In some embodiments of the first aspect, the antigen-binding domain is an scFv comprising the nucleic acid sequence of SEQ ID NO:33 or SEQ ID NO:34.

[0014] In some embodiments of the first aspect, the isolated nucleic acid further encodes a transmembrane domain, a costimulatory domain, and a signaling domain.

[0015] In some embodiments of the first aspect, the transmembrane domain comprises a CD28 transmembrane domain.

[0016] In some embodiments of the first aspect, the costimulatory domain comprises one or more of CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains.

[0017] In some embodiments of the first aspect, the costimulatory domain comprises one or more of a CD28, 4-1BB, and CD3 zeta costimulatory domain.

[0018] In some embodiments of the first aspect, the signal domain comprises a sequence encoding a CSFR2 signal peptide.

[0019] In some embodiments of the first aspect, the anti-GPC3 CAR further comprises a hinge / spacer domain.

[0020] In some embodiments of the first aspect, the hinge / spacer domain is an IgG4P hinge / spacer.

[0021] In some embodiments of the first aspect, the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:26.

[0022] In a second aspect, the present disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0023] In some embodiments of the second aspect, the VH comprises the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29.

[0024] In some embodiments of the second aspect, the VL comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30.

[0025] In some embodiments of the second aspect, the anti-GPC3 CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain.

[0026] In some embodiments of the second aspect, the anti-GPC3 CAR comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:25.

[0027] In a third aspect, the present disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0028] In some embodiments, the present disclosure provides a cell comprising the vector of the third aspect.

[0029] In a fourth aspect, the present disclosure provides a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), and the CAR construct provides cells that do not induce cytokine production in GPC3 cells.

[0030] In some embodiments of the fourth aspect, the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0031] In a fifth aspect, the present disclosure provides a cell comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0032] In some embodiments of the fifth aspect, the VH comprises the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29.

[0033] In some embodiments of the fifth aspect, the VL comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30.

[0034] In some embodiments of the fifth aspect, the CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain.

[0035] In some embodiments of the fifth aspect, the CAR comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:25.

[0036] In some embodiments of the fifth aspect, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0037] In some embodiments of the fifth aspect, the cells exhibit anti-tumor immunity upon contact with tumor cells that express GPC3.

[0038] In a sixth aspect, the present disclosure provides a method for treating cancer, comprising administering to a subject in need of cancer treatment an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. The method provides a method.

[0039] In some embodiments of the sixth aspect, the method further comprises inhibiting tumor growth, inducing tumor regression, and / or prolonging survival in the subject.

[0040] In some embodiments of the sixth aspect, the cells are autologous cells.

[0041] In some embodiments of the sixth aspect, the autologous cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0042] In some embodiments of the sixth aspect, the cancer is a solid tumor.

[0043] In some embodiments of the sixth aspect, the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

[0044] In some embodiments of the sixth aspect, the cancer is hepatocellular carcinoma.

[0045] In some embodiments of the sixth aspect, the method further comprises administering to the subject an effective amount of an anti-TNFα antibody.

[0046] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken in conjunction with the appended claims, which should be noted as being defined by the references thereto, and not by any specific discussion of the features and advantages set forth in this description.

[0047] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles and operation of the present disclosure. The present invention also relates to the following: [Item 1] An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), said CAR comprising an antigen-binding domain specific for glypican 3 (GPC3), said antigen-binding domain having an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), wherein the CAR construct does not induce cytokine production in GPC3 cells. [Item 2] 2. The isolated nucleic acid sequence of item 1, wherein the antigen-binding domain of the encoded CAR comprises an antibody or an antigen-binding fragment thereof. [Item 3] 3. The isolated nucleic acid sequence of item 2, wherein the antigen-binding domain of the encoded CAR is a Fab or a single-chain variable fragment (scFv). [Item 4] 4. The isolated nucleic acid sequence of item 3, wherein the antigen-binding domain is an scFv comprising the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34. [Item 5] 5. The isolated nucleic acid sequence of any one of items 1 to 4, further encoding a transmembrane domain, a costimulatory domain, and a signaling domain. [Item 6] 6. The isolated nucleic acid sequence of item 5, wherein the encoded transmembrane domain comprises a CD28 transmembrane domain. [Item 7] 6. The isolated nucleic acid sequence of item 5, wherein the encoded costimulatory domain comprises one or more of the CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains. [Item 8] 6. The isolated nucleic acid sequence of item 5, wherein the encoded costimulatory domain comprises one or more of a CD28, 4-1BB, and CD3 zeta costimulatory domain. [Item 9] 6. The isolated nucleic acid sequence of item 5, wherein the encoded signal domain comprises a sequence encoding a CSFR2 signal peptide. [Item 10] 10. The isolated nucleic acid sequence of any one of items 1 to 9, further encoding a hinge / spacer domain. [Item 11] 11. The isolated nucleic acid sequence of item 10, wherein the encoded hinge / spacer domain is an IgG4P hinge / spacer. [Item 12] 2. The isolated nucleic acid sequence of claim 1, wherein the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:26. [Item 13] An anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and The VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. An anti-GPC3 CAR. [Item 14] 14. The anti-GPC3 CAR of item 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. [Item 15] 14. The anti-GPC3 CAR of item 13, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. [Item 16] 16. The anti-GPC3 CAR according to any one of items 13 to 15, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain. [Item 17] 17. The anti-GPC3 CAR of item 16, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. [Item 18] 1. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34. [Item 19] A cell containing the vector according to item 18. [Item 20] A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ) and the CAR construct does not induce cytokine production in GPC3 cells. [Item 21] 21. The cell of item 20, wherein the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34. [Item 22] A cell comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and A cell wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. [Item 23] 23. The cell of item 22, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. [Item 24] 23. The cell of item 22, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. [Item 25] 25. The cell of any one of items 22 to 24, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain. [Item 26] 26. The cell of any one of Items 22 to 25, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. [Item 27] 27. The cell according to any one of items 19 to 26, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell. [Item 28] 28. The cell of item 27, wherein the cell exhibits anti-tumor immunity when contacted with tumor cells expressing GPC3. [Item 29] 1. A method of treating cancer, comprising: The method comprises administering to a subject in need of cancer treatment an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and A method wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. [Item 30] 30. The method of claim 29, further comprising inhibiting tumor growth, inducing tumor regression, and / or prolonging survival of the subject. [Item 31] 30. The method of claim 29, wherein the cells are autologous cells. [Item 32] 32. The method of claim 31, wherein the autologous cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. [Item 33] 33. The method of any one of items 29 to 32, wherein the cancer is a solid tumor. [Item 34] 34. The method of claim 33, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. [Item 35] 35. The method of claim 34, wherein the cancer is hepatocellular carcinoma. [Item 36] 36. The method according to any one of items 29 to 35, further comprising administering to the subject an effective amount of an anti-TNFα antibody. [Brief explanation of the drawings]

[0048] [Figure 1A]GPC3 expression in cancer and normal tissues. 1A. Anti-GPC3 antibody staining in hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and ovarian cancer. 1B. Immunohistochemical staining (IHC) results for human colonic ganglion tissue. [Figure 1B] Continuation of Figure 1A. [Figure 2] Comparison of the heavy and light chain variable regions of single-chain variable fragments (scFv), GPC3-1 and GPC3-2. [Figure 3A] Binding of cell surface GPC3 CAR to soluble GPC3 protein. KD values ​​are shown (fits are shown as solid lines). [Figure 3B] Surface plasmon resonance binding of anti-GPC3 scFv-Fc to soluble GPC3 protein. Average values ​​of k, kd, and KD are reported for both interactions (fits shown as solid lines). [Figure 3C] This is a continuation of Figure 3B. [Figure 4A] Cytokine production upon in vitro administration of chimeric antigen receptor (CAR) constructs to cells with or without target antigen. GPC3 CAR T results in antigen-specific cytokine production. Cell lines are listed from left to right for each construct in the order listed in the legend. 4A. Results for three cytokines are shown. UT: Untransduced T cells (donor T cells that are activated and proliferating but not carrying the CAR transgene). 4B. Results for interferon gamma (IFN-γ) for a subset of constructs are shown. Cell types (all negative except HEPG2) are indicated in the right legend. [Figure 4B] This is a continuation of Figure 4A. [Figure 5] Cytotoxicity of CAR in HCC cell lines. Constructs used are indicated in the right legend. E:T ratio: effector:target ratio. UT: untransduced T cells. [Figure 6-1]Cytotoxicity of GPC3-1 in HCC cell lines expressing low levels of GPC3. 6A. GPC3 expression was assessed by flow cytometry for the indicated cell lines. 6B. Receptor density on the indicated cell lines. 6C. Cytotoxicity of GPC3-1 against the cell lines indicated in the legend, with effector:target ratios of 3:1 (upper graph) and 0.3:1 (lower graph). 6D. KT50 (time to kill 50% of targets) of GPC3-1 against the indicated cell lines at two different effector:target ratios. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7] Multifunctionality testing of GPC3-2 and GPC3-1 CAR T constructs. The scFv is shown to the left of each row and the costimulatory domain is shown at the top of each chart. [Figure 8A] 8A. Effect of chimeric antigen receptor T cell (CAR T) transplantation on body weight. Constructs used are indicated in the right legend. BW: body weight. ACT: adoptive T cell therapy. UT: untransduced T cells. PBS: phosphate-buffered saline. 7B. IHC showing CAR-T accumulation in lung tissue of GPC3 CAR-T-treated mice. [Figure 8B] This is a continuation of Figure 8A. [Figure 9] Effect of CAR T administration on tumor volume. Constructs used are indicated in the right legend. ACT: adoptive T cell therapy. UT: untransduced T cells. PBS: phosphate buffered saline. [Figure 10] Effect of CAR T administration on survival. Constructs used are indicated in the right legend. ACT: adoptive T cell therapy. UT: untransduced T cells. PBS: phosphate buffered saline. [Figure 11A]Fluorescence-activated cell sorting (FACs) studies of GPC3-1 CAR T cell differentiation and exhaustion using different costimulatory domains. Results are shown for spleen (11A and 11B) and tumor cells (11C and 11D). Dot plots show the frequency of CD3+ T cells infiltrating each organ for each construct (11A and 11C). GPC3 CAR-T with the 4-1BB / CD3 zeta (BZ) signaling domain exhibited more central memory and less exhaustion than CD28 / CD3 zeta (28Z) in vivo. The costimulatory domains used are indicated at the top of each panel. The markers assayed are indicated on the x- and y-axes. FSC: forward scatter. EM: effector memory. CM: central memory. TN: naive T cells. [Figure 11B] This is a continuation of Figure 11A. [Figure 11C] This is a continuation of Figure 11B. [Figure 11D] This is a continuation of Figure 11C. [Figure 12] Persistence of GPC3-1 CAR T in Hep3B and HepG2 tumors. Constructs used are indicated in the right legend. CD3 percentages are indicated. ACT: adoptive T cell therapy. UT: untransduced T cells. [Figure 13] Effect of GPC3-1BZ treatment on body weight of non-tumor-bearing and tumor-bearing mice. The constructs used are indicated in the legend on the right. BW: body weight. ACT: adoptive T cell therapy. TZ is GPC3-1 TZ. UT: untransduced T cells. PBS: phosphate-buffered saline. [Figure 14] Tumor volume and blood collection time points for cytokine analysis of GPC3-1 BZ and GPC3-1 TZ. The time points for blood collection for subsequent cytokine response testing are indicated by arrows. The constructs used are shown in the legend on the right. ACT: adoptive T cell therapy. UT: untransduced T cells. PBS: phosphate-buffered saline. [Figure 15] Maximum systemic cytokine response (IFN-γ) with GPC3-1 CAR T treatment. Data shown is from the blood draw on day 8, when the maximum cytokine response was observed. UT: untransduced T cells. PBS: phosphate-buffered saline. [Figure 16]Histology of Hep3B tumor tissue in NOD scid gamma (NSG) immunodeficient mice. Top: untreated control. Bottom: animal treated with GPC3-1 BZ CAR T cells. Images shown at 20x. [Figure 17] Histology of enteric neural tissue in NOD scid gamma (NSG) immunodeficient mice. Left: untreated control. Right: animals treated with GPC3-1 BZ CAR T cells. [Figure 18] Cell surface GPC3 quantification. From top to bottom: A375 cells (GPC3 negative), HepG2 cells (high GPC3), Hep3B cells (intermediate / low GPC3), and Huh7 cells (low GPC3). The area under the peak indicates the population of cells expressing the protein at the level indicated on the x-axis. APC: allophycocyanin. [Figure 19] Cytokine enzyme-linked immunosorbent assay (ELISA) results after 24 hours of exposure to GPC3-1 BZ T cells. Cell lines are listed from left to right in the order listed top to bottom in the legend. [Figure 20] Immunohistochemical staining for GPC3 of representative tumor xenografts from two HCC cell lines. Both xenografts were scored as intensity = 2. Data show that tumors with at least 25% positive GPC3 expression at moderate intensity respond to GPC3-1 BZ CAR-T. [Figure 21-1] Determination of relative surface GPC3 expression. FSC: forward scatter. APC: allophycocyanin. MFI: mean fluorescence intensity. The frequency of GPC3 in each gate is shown in the dot plot on the left (12.7%, 24%, and 9.34%, respectively). The histogram on the right shows the expression of GPC3 in the sorted population, confirming purity and homogeneity. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22]Cytokine ELISA after 24 hours of exposure to GPC3-1 BZ. Results are shown for T cells only, A375 cells (GPC3 negative), and low, moderate, and high GPC3 expressers. Results are shown from left to right in each panel for each cell type in the order listed top to bottom in the legend. UT: untransduced T cells. TZ and BZ: GPC3-1 TZ and GPC3-1 BZ. [Figure 23] Interferon gamma (IFNγ) levels in different cell types after CAR T treatment. The constructs used are indicated on the x-axis. Results are shown from left to right for each construct in each cell type in the order listed top to bottom in the legend. TZ: GPC3-1 TZ. UT: Untransduced T cells. Moderate: Treatment with Moderate cells only. [Figure 24] Cytokine levels in neuronal tissue cell types after treatment with GPC3-1 CAR T. Constructs used are indicated on the x-axis. Results are shown from left to right for each construct in each cell type in the order listed top to bottom in the legend. UT: untransduced T cells. Moderate: treated with moderate cells only. [Figure 25] Tumor volume following treatment with CAR T and anti-CRS-associated cytokine antibodies. CRS = cytokine release syndrome. Top: Tumor volume when treated with different CAR and antibody schemes. Bottom: Studies of individual subjects treated with GPC3-1 BZ+PBS, GPC3-1 BZ+anti-IL-6, and GPC3-1 BZ+anti-TNF-α. MEDI7028 is GPC3-1 BZ. ACT: adoptive T cell therapy. UT: untransduced T cells. PBS: phosphate buffered saline. [Figure 26A] Testing treatment with higher doses of CAR T and anti-TNFα in a resistant HCC model (Huh7). Anti-TNFα is used to attenuate the toxicity of high doses of CAR-T and promote anti-tumor activity. The constructs used are listed in the right legend. 26A. Test scheme. BW: body weight. 26B. Tumor growth. iv: intravenous administration. 26C. Body weight change. [Figure 26B] This is a continuation of Figure 26A. [Figure 26C] This is a continuation of Figure 26B. DETAILED DESCRIPTION OF THE INVENTION

[0049] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton, et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0050] As used herein, the terms "comprise" and "include," and variations thereof (e.g., "comprises," "comprising," "include," and "including") will be understood to indicate the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms while retaining their ordinary meaning.

[0051] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The percentages disclosed herein can vary in amounts of ±10, 20, or 30% from the disclosed values ​​and remain within the intended scope of the disclosure.

[0053] Unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed herein as ranges can be considered to be any specific value or subrange within the range set forth in various embodiments of the present disclosure, down to one-tenth of the unit of the lower limit of the range, unless the content clearly dictates otherwise.

[0054] As used herein, ranges and amounts can be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%," and the term "about," which also means "5%," can also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5% to 5.5%. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0055] As used herein, the terms "or" and "and / or" can describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0056] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used instead of or interchangeably with any of these terms.

[0057] As used herein, "protein" can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to form a multimeric protein.

[0058] An "isolated" material, e.g., an isolated nucleic acid, is a material that is not in its natural environment, although not necessarily purified. For example, an isolated nucleic acid is a nucleic acid that is not produced or located in its native or natural environment (e.g., a cell). An isolated material can be separated, fractionated, or at least partially purified by any suitable technique.

[0059] As used herein, the terms "antibody" and "antigen-binding fragment thereof" refer to at least the minimum portion of an antibody capable of binding to a particular antigen to which the antibody is targeted, e.g., at least some of the complementarity-determining regions (CDRs) of the heavy chain (VH) variable domain and the light chain (VL) variable domain in the context of a typical antibody produced by a B cell. Antibodies or antigen-binding fragments thereof can be or be derived from polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab'), Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising a VL or VH domain alone or in combination with a portion of the opposing domain (e.g., an entire VL domain and a partial VH domain having one, two, or three CDRs), and fragments produced by a Fab expression library. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892, 019. Antibody molecules encompassed by the present disclosure can be of or derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0060] As used herein, the term "polynucleotide" includes single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA.

[0061] As used herein, the term "vector" may refer to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that enables it to replicate in that host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. Certain types of vectors contemplated herein may be combined with or incorporated into viruses to facilitate cell transformation.

[0062] A "transformed" cell, or "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. Nucleic acid molecules may be introduced into such cells by various techniques, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0063] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (e.g., an epitope) to its cognate binding domain (e.g., a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a population of epotopes and paratopes (i.e., antigens and antigenic domains).

[0064] As used herein, the terms "treat," "treatment," or "treatment of," when used in the context of cancer treatment, refer to alleviating disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy to alleviate symptoms, signs, or causes of a disease when administered to a subject. Treatment also refers to the alleviation or reduction of at least one clinical symptom and / or inhibiting or slowing the progression of a disease state and / or preventing or delaying the onset of a disease or condition.

[0065] As used herein, the terms "subject," "individual," or "patient" refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0066] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, e.g., CAR T cells, is an amount sufficient to carry out a particularly stated or intended purpose, such as treating cancer. An "effective amount" can be determined based on routine experimentation for the stated purpose.

[0067] 2. Overview The present disclosure is directed to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More particularly, the present disclosure relates to CAR cell therapy in which transformed cells, such as T cells, express a CAR that targets glypican 3 (GPC3). Still further, the CAR constructs, transformed cells expressing the constructs, and therapies utilizing the transformed cells disclosed herein can provide robust cancer treatments with minimal risk of cytokine release syndrome (CRS) or indiscriminate cytokine release in non-GPC3-expressing cells.

[0068] Without wishing to be bound by theory, GPC3 is believed to be a viable cancer target across multiple modalities, including bispecific T cell engagers, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). The carcinoembryonic antigen GPC3 is a GPI-linked heparin sulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction and stimulates Wnt signaling. GPC3 competes with patched for Hh binding, alleviating smoothened inhibition and inducing GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) growth. Furthermore, GPC3 expression levels have been shown to correlate with the stage and grade of HCC.

[0069] Furthermore, GPC3 is considered a promising target for CAR cell therapy, and therefore, antibodies and CAR constructs derived from these antibodies have been developed, as described herein.

[0070] 3. Design of CAR Constructs The CAR constructs of the present disclosure can have several components, many of which can be selected based on the desired or sophisticated function of the resulting CAR construct. In addition to the antigen-binding domain, the CAR construct can have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. The selection of one component over another (i.e., the selection of a particular costimulatory domain of one receptor over a costimulatory domain of a different receptor) can affect clinical efficacy and safety profiles.

[0071] 4. Antigen-binding domain Antigen-binding domains contemplated herein can comprise antibodies or one or more antigen-binding fragments thereof. One contemplated CAR construct targeting GPC3 comprises a single-chain variable fragment (scFv) comprising light and heavy chain variable regions from one or more antibodies specific for GPC3, linked either directly or via a flexible linker (e.g., GGGS repeats having 1, 2, 3, or more repeats).

[0072] The antigen-binding domain of a CAR targeting GPC3 as disclosed herein can vary in its binding affinity to the GPC3 protein. The relationship between binding affinity and efficacy may be more nuanced in the context of CARs compared to antibodies, where higher affinity is generally desirable. For example, preclinical studies on receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CARs derived from high-affinity scFvs (dissociation constant 0.56 nM) have shown an increased therapeutic index compared to low-affinity variants. Conversely, other examples have reported that engineering scFvs to lower affinity improves discrimination between cells with altered antigen density. This may be useful for improving therapeutic specificity for antigens differentially expressed in tumor versus normal tissues.

[0073] Various methods can be used to confirm the binding affinity of an antigen-binding domain. In some embodiments, a methodology that excludes avidity effects can be used. Avidity effects often involve multiple antigen-binding sites simultaneously interacting with multiple target epitopes in a multimerized structure. Thus, avidity functionally represents the cumulative strength of multiple interactions. An example of a methodology that excludes avidity effects is one in which one or both interacting proteins are monomeric / monovalent, since multiple simultaneous interactions are not possible if one or both partners contain only a single interaction site.

[0074] 5. Spacer domain The CAR constructs of the present disclosure may have a spacer domain that provides conformational freedom to facilitate binding to the target antigen on the target cell. The optimal length of the spacer domain may depend on the proximity of the binding epitope to the target cell surface. For example, proximal epitopes may require longer spacers, while distal epitopes may require shorter ones. In addition to promoting CAR binding to the target antigen, achieving an optimal distance between the CAR cell and the cancer cell may also help sterically block large inhibitory molecules from the immunological synapse formed between the CAR cell and the target cancer cell. CARs targeting GPC3 may have long, intermediate, or shorter spacers. Long spacers can include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural or with modifications common to therapeutic antibodies, such as the S228P mutation), while the CH3 region itself can be used to construct an intermediate spacer (approximately 120 amino acids). Shorter spacers can be derived from segments (less than 60 amino acids) of CD28, CD8α, CD3, or CD4. Short spacers can also be derived from the hinge region of an IgG molecule. These hinge regions can be derived from any IgG isotype and may or may not contain mutations common in therapeutic antibodies, such as the S228P mutation described above.

[0075] 6. Hinge domain CARs targeting GPC3 may also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to target antigens on tumor cells. It can be used alone or together with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably; for example, an IgG4 sequence can be considered both a "hinge" and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0076] A CAR targeting GPC3 may further comprise a sequence containing a signal peptide. The signal peptide acts to prompt the cell to translocate the CAR to the cell membrane. Examples include an IgG1 heavy chain signal polypeptide, an Ig kappa or lambda light chain signal peptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, a CD8a signal polypeptide, or a CD33 signal peptide.

[0077] 7. Transmembrane domain The CAR targeting GPC3 may further comprise a sequence containing a transmembrane domain. The transmembrane domain may comprise a hydrophobic α-helix spanning the cell membrane. Although the characteristics of the transmembrane domain have not been studied in as much detail as other aspects of the CAR construct, they may potentially affect CAR expression and the association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0078] 8. Costimulatory Domain CARs targeting GPC3 may further comprise one or more sequences forming a costimulatory domain. A costimulatory domain is a domain that can enhance or modulate immune effector cell responses. Costimulatory domains can include sequences from one or more of CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The choice of costimulatory domain influences the phenotype and metabolic signature of CAR cells. For example, costimulation of CD28 results in a potent but short-lived effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells engineered with CARs containing a 4-1BB costimulatory domain tend to expand and persist longer in vivo, have increased oxidative metabolism, are less susceptible to exhaustion, and have an increased ability to generate central memory T cells.

[0079] 9.Cells CAR-based cell therapy can be used with various cell types, such as lymphocytes. Specific types of cells that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, alpha beta T cells, gamma delta T cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells (Tregs). In one embodiment, CAR cells for treating a subject are autologous. In other embodiments, CAR cells can be derived from a genetically similar, but not identical, donor (allogeneic).

[0080] 10. CAR cell production The CAR constructs of the present disclosure can include several combinations of the modular components described herein. For example, in some embodiments of the present disclosure, the CAR construct includes a GPC3-1 scFv antigen-binding domain. In some embodiments, the CAR includes a GPC3-2 scFv antigen-binding domain. In some embodiments of the present disclosure, the CAR construct includes a CSFR2 signal peptide. In some embodiments, the CAR construct includes an IgG4P hinge / spacer domain with an S228P mutation. In some embodiments, the CAR construct includes a CD28 transmembrane domain.

[0081] Different costimulatory domains can be utilized in the CAR constructs of the present disclosure. In some embodiments, the CAR construct comprises a costimulatory domain derived from the intracellular domain of CD3z. In some embodiments, the CAR construct comprises a CD28 costimulatory domain. In some embodiments, the CAR construct comprises a 4-1BB costimulatory domain. In some embodiments, the CAR construct comprises costimulatory domains derived from CD3z and CD28. In some embodiments, the CAR construct comprises costimulatory domains derived from CD3z and 4-1BB. In some embodiments, the CAR construct comprises costimulatory domains derived from all of CD3z, CD28, and 4-1BB. In some embodiments, the CAR construct comprises costimulatory domains derived from ICOS, OX-40, and / or GITR.

[0082] 11. Evaluation of CAR Constructs Constructs of the present disclosure were compared and evaluated based on safety and the persistence and establishment of central memory. The low-affinity (high off-rate) scFv GPC3-1 was well evaluated due to its favorable safety. The 4-1BB and CD3z costimulatory domains (both in the same construct) were well evaluated based on their favorable persistence and contribution to a favorable in vivo phenotype (more central memory). The GPC3-1 CAR and GPC3-2 CAR of the present disclosure were comparable to constructs based on published GPC3-targeting CARs. Details of the evaluation can be found in the Examples.

[0083] 12. Implementation In some embodiments, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an antigen-binding domain specific for glypican 3 (GPC3). The antigen-binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), and the CAR construct does not induce cytokine production in GPC3 cells. In some embodiments, the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof. The antigen-binding domain can be a Fab or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain is an scFv comprising the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0084] In some embodiments, the CAR further comprises a transmembrane domain, a costimulatory domain, and a signal domain. The transmembrane domain can be a CD28 transmembrane domain. The costimulatory domain can be one or more of CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains. In one specific embodiment, the costimulatory domain is one or more of CD28, 4-1BB, and CD3 zeta costimulatory domains. The signal domain can be a sequence encoding a CSFR2 signal peptide.

[0085] In some embodiments, the isolated nucleic acid sequence can comprise a hinge / spacer domain. The hinge / spacer domain can be an IgG4P hinge / spacer.

[0086] In some particular embodiments, the isolated nucleic acid sequence encoding the chimeric antigen receptor (CAR) may have the sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:26.

[0087] In other embodiments, the present disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain. The antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH may have a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VL may have a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0088] In some embodiments, the VH can be the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and the VL can be the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the CAR can further have a transmembrane domain, a costimulatory domain, and a signaling domain.

[0089] In some specific embodiments, the anti-GPC3 CAR may have the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:25.

[0090] In other embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence can be SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0091] In other embodiments, the disclosure provides a cell comprising a vector having the nucleic acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0092] In other embodiments, the disclosure provides a cell having a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), and the CAR construct provides a cell that does not induce cytokine production in GPC3 cells. For example, the nucleic acid sequence can be SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0093] In another embodiment, the present disclosure provides a cell expressing an anti-GPC3 chimeric antigen receptor (CAR) on its extracellular surface. The CAR may have an antigen-binding domain that may be an antibody, Fab, or scFv, each having a heavy chain variable region (VH) and a light chain variable region (VL). The VH may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0094] In some embodiments, the VH can have the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29. In some embodiments, the VL can have the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30. The CAR can further comprise a transmembrane domain, a costimulatory domain, and a signaling domain. The cells express a CAR having the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:25.

[0095] In some embodiments, the present disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells that express a CAR on their extracellular surface, where the CAR may have the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. Such cells can exhibit anti-tumor immunity when contacted with tumor cells that express GPC3.

[0096] 13.Treatment of cancer with CARs In some embodiments, the present disclosure provides CAR cells for the treatment of cancer. The compositions (e.g., antibodies, CAR constructs, and CAR cells) and methods of using them described herein are particularly useful for inhibiting the growth or spread of tumor cells, particularly the growth of tumor cells in which GPC3 plays a role.

[0097] Tumors treatable by the compositions of the present disclosure include solid tumors, such as liver, lung, or ovarian tumors, although the cancers listed herein are not intended to be limiting. For example, cancer types contemplated for treatment herein include, for example, NSCLC, advanced solid malignancies, bile duct tumors, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal tumors, esophageal squamous cell carcinoma, extensive stage small cell lung cancer, gastric adenocarcinoma, gastric cancer, gastroesophageal junction cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin's lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal carcinoma, metastatic melanoma, metastatic non-cutaneous melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, fallopian tube cancer, peritoneal tumors, pleural mesothelioma, prostate tumors, recurrent or metastatic PD-L1 positive or negative SCCHN, recurrent squamous cell lung cancer, renal cell carcinoma ... carcinoma), SCCHN, pharynx-pituitary squamous cell carcinoma, laryngeal squamous cell carcinoma, small cell lung cancer, squamous cell carcinoma of the head and neck, squamous cell lung cancer, TNBC, transitional cell carcinoma, unresectable or metastatic melanoma, urothelial cancer, and urothelial carcinoma.

[0098] In one embodiment, the cancers contemplated for treatment herein include any cancer that expresses GPC3 on the cell surface of cancer cells. In one particular example, the cancers contemplated for treatment herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0099] 14. Treatment The CAR-modified cells of the present invention, such as CAR T cells, can be administered alone or as a pharmaceutical composition containing a diluent and / or cytokines or other components related to the cell population. Briefly, the pharmaceutical composition of the present invention can include, for example, CAR T cells as described herein, together with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, such as neutral buffered saline or buffered saline; sulfate; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins, polypeptides, or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical composition of the present invention can be adapted for treatment (or prevention).

[0100] CAR modified cells can also be administered in combination with one or more additional therapeutic agents. In one embodiment, the additional treatment can include an anti-cytokine antibody. For example, one or more anti-TNFα antibodies can be used to attenuate the toxicity of high CAR T doses, which can be associated with CRS-like symptoms and weight loss, and promote anti-tumor activity.

[0101] In certain embodiments, the contemplated therapeutic regimen may include one or more biological and / or chemotherapeutic components, such as CAR T cells and anti-cancer antibodies. For example, the therapeutic regimen could further include other immuno-oncology (IO) therapies, such as immune checkpoint inhibitors (ICIs) and immune system agonists, such as those targeting the PD-1 / PD-L1 axis (PDX).

[0102] Contemplated antibodies include anti-PD-L1 antibodies such as durvalumab (MEDI4736), avelumab, atezolizumab, KNO35, anti-PD-1 antibodies such as nivolumab, pembrolizumab, REGN2810, SHR1210, IBI308, PDR001, anti-PD-1, BGB-A317, BCD-100, and JS001, and anti-CTLA4 antibodies such as tremelimumab or ipilimumab. Additional antibodies are also contemplated herein. Therapeutically effective antibody subparts are also contemplated herein.

[0103] Information regarding durvalumab (or fragments thereof) for use in the methods provided herein can be found in U.S. Patent Nos. 8,779,108, 9,493,565, and 10,400,039, the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the CDR sequences of the variable heavy and variable light chains of the 2.14H9OPT antibody disclosed in the above U.S. patents.

[0104] Information regarding tremelimumab (or an antigen-binding fragment thereof) for use in the methods provided herein can be found in U.S. Pat. No. 6,682,736 (wherein tremelimumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0105] Additional therapeutic agents (chemotherapeutic or biologic) contemplated herein include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, ADRIAMYCIN™ (doxorubicin), cisplatin (MVAC), carboplatin-based regimens, or single-agent taxanes or gemcitabine, temozolomide, or dacarbazine, vinflunine, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Further examples include drugs that target the DNA damage repair system, such as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors, and therapeutic agents that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora B protein kinase activity, and DNA-PK activity.

[0106] Any therapeutic composition or method contemplated herein may be combined with any one or more of the other therapeutic compositions and methods provided herein.

[0107] In some embodiments, the present disclosure provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain. The antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). The VH may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the method further inhibits tumor growth, induces tumor regression, and / or prolongs the survival of the subject.

[0108] In some embodiments, the cells are autologous cells. For example, the autologous cells may be selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0109] In some embodiments, the cancer treated by the method is a solid tumor. For example, the cancer may be hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. In certain embodiments, the cancer is hepatocellular carcinoma.

[0110] The present disclosure provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) and an effective amount of an anti-TNFα antibody.

[0111] It is to be understood that the specific aspects described herein are not limited to the specific embodiments presented and may vary. It is also to be understood that the terminology used herein is for the purpose of describing the specific aspects only and is not intended to be limiting unless specifically defined herein. Furthermore, the specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation, as will be recognized by one of ordinary skill in the art. [Example]

[0112] The following examples illustrate specific embodiments of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the present disclosure in any way. Explanations of terms are provided in Table 1.

[0113] [Table 1]

[0114] Example 1: Expression of GPC3 method GPC3 IHC utilized the mouse monoclonal anti-human GPC3 antibody GC33 (Ventana). Secondary staining was performed with anti-mouse HRP. Human tissue microarrays (TMA, US Biomax) representing hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and ovarian cancer, or human colonic ganglion tissue, were stained for GPC3 expression, and staining intensity and pattern were determined microscopically.

[0115] result GPC3 overexpression is observed in 80% of HCC, 30% of squamous cell lung cancer, and 47% of ovarian clear cell carcinoma. However, GPC3 is undetectable by immunohistochemistry in normal liver tissue, including cirrhosis and hyperplasia samples, and its expression is low in normal tissues (e.g., lung). See Figures 1A and 1B.

[0116] Example 2: Development and affinity testing of scFvs. overview In this example, anti-GPC3 scFvs were developed and their relative affinities for GPC3 were determined.

[0117] method GPC3-1 (SEQ ID NO: 1) and GPC3-2 (SEQ ID NO: 2) have almost the same V H domain (SEQ ID NOs: 27 and 29), but V L The domains (SEQ ID NOs: 28 and 30; see Figure 2) are different: GPC3-2 has a complete germline framework, whereas GPC3-1 does not.

[0118] The apparent binding affinity was determined by cell surface binding of soluble recombinant GPC3 protein to GPC3-1CAR and GPC3-2CAR expressed on the surface of Jurkat cells. CAR constructs were expressed on the surface of Jurkat cells using a lentiviral vector. Cells were stained with various concentrations of recombinant His-tagged GPC3 protein (R&D systems). Bound GPC3 was visualized by staining with a fluorescently conjugated anti-His tag secondary antibody, and cells were analyzed by flow cytometry. Binding curves were fitted to a simple one-site binding model to obtain the apparent K D It was decided that:

[0119] A surrogate measure of binding affinity was determined using a BIAcore surface plasmon resonance system and GPC3-1 and GPC3-2 scFv-Fc fusion proteins. Purified scFv-Fc fusion molecules of GPC3-1 and GPC3-2 were covalently coupled to an amine-reactive SPR sensor chip (CM5, GE Healthcare). For GPC-1, soluble GPC3 protein (R&D Systems) was flowed over the chip surface at concentrations of 14, 28, 57, 114, and 228 nM at a flow rate of 30 μL / min to monitor interactions. For GPC3-2, concentrations of 4, 7, 14, 28, and 57 nM were flowed at the same flow rate. Data were fitted using BIAevaluation software (GE Healthcare) and a simple 1:1 Langmuir binding model, and the data were analyzed using R. max Overall, the fit is good. a , k d and K. D The fit was done locally.

[0120] result In experiments assessing soluble GPC3 binding to GPC3-1CAR and GPC3-2CAR expressed on the surface of Jurkat cells, dThe values ​​were approximately 15 nM for GPC3-1 and 5 nM for GPC3-2 (see Figure 3A). Surface plasmon resonance experiments using the same GPC3 protein and purified GPC3-1 / GPC3-2 scFv-Fc fusion protein used in cell-based binding experiments revealed that the K D The values ​​were approximately 73 nM and 11 nM, respectively. See Table 1 and Figures 3B and 3C.

[0121] [Table 2]

[0122] The reported Kd values ​​for the four scFvs are shown in Table 2.

[0123] [Table 3]

[0124] Example 3. CAR construct development and in vitro testing. overview In this example, an anti-GPC3 CAR construct was developed and tested for the resulting cytokine activity and multifunctionality.

[0125] method CAR Structure. All CAR constructs used the CSFR2 signal peptide (used in many clinical-stage CAR T constructs). The IgG4P (S228P mutant) hinge domain was used as a "spacer" between the scFv and the membrane, and the CD28 transmembrane domain was used. Different costimulatory domains were tested intracellularly, including various combinations of CD28, 4-1BB, and CD3 zeta costimulatory domains. Constructs using costimulatory domains from inducible T cell costimulator (ICOS), OX40, and glucocorticoid-induced TNFR family-related gene (GITR) were also attempted. The sequences of the GPC3-1 and GPC3-2 CAR constructs are shown in SEQ ID NOs: 3-10, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 11-18.

[0126] Other known CARs against GPC3 (based on the GPC3-3 and GPC3-4 scFvs) were constructed for comparison with the GPC3-1 and GPC3-2 CAR constructs. The GPC3-3 CAR contained a short IgG1 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular domain. Another GPC3-3 CAR construct was also developed, containing both the CD28 and 4-1BB costimulatory domains. The GPC3-4 CAR construct contained an IgG4P hinge, a CD28 transmembrane domain, and a 4-1BB costimulatory domain. The sequences of the GPC3-3 CAR and GPC3-4 CAR are shown in SEQ ID NOs: 19-21, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 22-24.

[0127] Generation of CAR T cells. Purified human T cells were injected into 0.2 × 10 6 T cells were seeded at a concentration of 1000 cells / mL + IL-2 (300 IU / mL) in AIM-V medium containing 5% human serum and 1% penicillin-streptomycin. T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Invitrogen), and 24 hours later, transduction was performed by spinoculation. Lentivirus was added to the wells (MOI 100), and the plate was centrifuged at 2000 rpm at 37°C for 2 hours and placed in a 37°C, 5% CO2 incubator. The cell density was adjusted to approximately 0.5–1 × 10 6 Cells were split as needed to maintain 100 cells / mL. CAR-T cells were first immunophenotyped 7 days after transduction, and CAR-T cells were evaluated in in vitro and in vivo functional assays approximately 11 days after transduction.

[0128] Cell line studies. Various cell types were treated with CAR T cells harboring different CAR constructs. For all cytokine studies, 5x10 4CAR-T cells were co-cultured with target cells at a 1:1 ratio in RPMI 10% FCS. After 24 hours, supernatants were collected. Cytokines were analyzed using the Meso Scale Discovery 4-plex kit to detect IFN-γ, IL-2, TNF-α, and IL-10. Cytokine concentrations (picograms / milliliter) were measured.

[0129] Cytotoxicity tests were performed using cell impedance monitoring technology (xCELLigence). 4 Target cells were plated onto the plate, and 24 hours later, CAR-T cells were added at an effector:target (E:T) ratio of 3, 1, or 0.3. After treatment with various CAR constructs, normalized cell index was determined for Hep3B, Huh7, and SNU-182 cells. Hep3B expresses high GPC3 (14kJ / cell), Huh7 expresses intermediate / low GPC3 (7kJ / cell), and SNU-182 is GPC3-negative (0kJ / cell).

[0130] Multifunctionality tests were also performed on CAR constructs. Here, GPC3-1 BZ or the indicated CAR-T cells were co-cultured with Hep3B or A375 cells for 6 hours in the presence of Golgi Stop and a fluorescently labeled antibody against the degranulation marker CD107a. Target engagement induced CAR-T degranulation, which in turn induced binding of fluorescently labeled anti-CD107 present in the culture medium. CD107 accumulation, detected by flow cytometry, was directly proportional to the degree of degranulation and indicates target cell lysis. Because cells were incubated in the presence of Golgi Stop, production of effector cytokines (IFN-γ, IL-2, and TNF-α) could also be assessed by intracellular staining. Boolean gates combining each function (CD107a, IFN-γ, IL-2, and TNF-α) were generated using Flowtop, and resulting pie charts were generated using Spice analysis software.

[0131] result The GPC3-2 and GPC3-3 constructs were observed to produce higher overall TNFα and IL-2 levels compared to GPC3-1. Treatment with CAR T cells bearing the GPC3-1 and GPC3-2 CAR constructs resulted in antigen-specific cytokine production. Meanwhile, the GPC3-4 BZ construct induced cytokines even in GPC3-negative cell types. The GPC3-1 and GPC3-2 constructs did not produce cytokines in the absence of target. Cytokine production appears to depend on antigen density and antigen affinity. See, for example, Figures 4A and 4B.

[0132] The GPC3-1 and GPC3-2 constructs were cytotoxic only against GPC3-expressing cells, whereas the GPC3-4 construct was cytotoxic against both GPC3-positive cells (Hep3B and Huh7) and GPC3-negative cells (SNU-182). The low-affinity CAR (GPC3-1 BZ) exhibited comparable cytotoxicity to the high-affinity (GPC3-2 BZ) CAR. See Figure 5.

[0133] GPC3-1 BZ was cytotoxic to HCC cell lines expressing low levels of GPC3. All target cells tested were highly sensitive to killing by GPC3-1 at E:T ratios of 3:1 and 0.3:1, with only one cell line with low GPC3 expression showing reduced killing at an E:T ratio of 0.3:1. However, the completely equivalent killing rates observed in our isogenic versus GPC3-high and -low Hep3B cell lines suggests that reduced antigen density is not a significant factor limiting CAR-T-mediated cell lysis. See Figure 6.

[0134] Both GPC3-2 and GPC3-1 CAR T cells are polyfunctional regardless of the intracellular domain used. GPC3-1 BZ CAR T cells were polyfunctional, with a higher proportion of cells exhibiting 2+ function. Also, CD28-bearing CAR T cells were less polyfunctional in vitro than CAR T cells bearing the 4-1BB intracellular domain. See Figure 7.

[0135] conclusion Treatment with GPC3-1 resulted in the lowest overall cytokine production of the CARs tested. Both GPC3-1 and GPC3-2 were multifunctional and specifically cytotoxic to GPC3-expressing cells.

[0136] Example 4: In vivo testing of multiple CAR constructs in hepatocellular carcinoma animal models overview In this example, anti-GPC3 CAR constructs were tested in vivo to compare effects on body weight, tumor, and survival.

[0137] method 5×10 6 Hep3B cells were implanted into the flanks of NSG mice (10 mice / group). The average tumor volume was 150 mm 3 When the mice reached a body weight of 1500 mm, they were administered 4 million GPC3-2 BZ or GPC3-1 BZ. Body weight, tumor volume (2x / week), and survival were monitored. Animals that lost 80-90% of their body weight were given nutritional supplements, and animals that lost less than 80% of their body weight were euthanized. Survival events (death) were recorded at 1500 mm. 3 Each experiment was performed in duplicate.

[0138] result Weight loss was observed with the use of the high-affinity GPC3-2 construct, but not the low-affinity GPC3-1 construct, indicating that the low-affinity binder is less toxic in vivo. GPC3-2-based CAR T cells were not tolerated at comparable in vivo doses as GPC3-1-based CAR T cells. The greater toxicity of GPC3-2 BZ correlated with extensive infiltration of CAR T cells in normal mouse lungs. Only minimal levels of infiltration were observed in the lungs of mice treated with GPC3-1 BZ. See Figures 8A and 8B.

[0139] GPC3 CAR T induced Hep3B tumor regression in NSG mice. GPC3-1 BZ showed superior antitumor activity to GPC3-3 BZ and GPC3-4 BZ. See Figure 9.

[0140] GPC3-1 and GPC3-2 CAR T significantly extended the survival of tumor-bearing NSG mice compared with GPC3-3 or GPC3-4 CAR T (p<0.01 vs. GPC3-3 BZ; Kaplan-Meier and Mantel-Cox log-rank). See Figure 10. Similarly, it was determined that deletion of the WPRE had no negative functional impact on GPC3-1 BZ cells in vitro or in vivo.

[0141] conclusion Among the CARs tested, GPC3-1 BZ and GPC3-2 BZ showed the greatest antitumor activity and provided the greatest survival benefit. GPC3-1 BZ was less toxic and less infiltrated into normal tissues than GPC3-2 BZ.

[0142] Example 5: In vivo comparison of GPC3-1 CAR constructs overview In this example, multiple GPC3-1 CAR constructs containing different signaling domains were tested and compared in vivo.

[0143] method Differentiation and Exhaustion Analysis. Differentiation and exhaustion of multiple GPC3-1 CAR constructs were investigated. Mice bearing Hep3B tumors were treated with CAR-T cells with different signaling domains (TZ = GPC3-1 TZ; BZ = GPC3-1 BZ; 28Z = GPC3-1 28Z), and spleens and tumors were analyzed by flow cytometry 7 days after cell injection. Differentiation and exhaustion were assayed using FACs detecting multiple markers in spleen and tumor cells. The differentiation status of T cells was analyzed by combined expression of CD62L and CD45RO (CD62L+ / CD45RO- = naive; CD62L+ / CD45RO+ = central memory; CD62L- / CD45RO+ = effector memory; CD62L- / CD45RO- = effector memory cells re-expressing CD45RA (EMRA)). CD3% was used as a measure of persistence and expansion.

[0144] 5x10 on the mouse 6 Hep3B cells were injected, resulting in a mean size of 150 mm 3 Tumors were established in 100% of the CAR-T cells. Non-tumor-bearing mice or mice bearing Hep3B tumors were administered 4 million GPC3-1 BZ or GPC3-1 TZ T cells. The effect on body weight in both tumor-bearing and non-tumor-bearing mice was measured up to 35 days after administration. Tumor volume was also assayed twice weekly. Animals were bled periodically after administration for analysis of IFN-γ and TNF-α in the blood. Serum cytokines were analyzed 8 days after CAR-T administration. Each experiment was performed in duplicate.

[0145] To investigate the potential for peripheral neurotoxicity in GPC3+ tumor-bearing (Hep3B HCC line) and non-tumor-bearing NSG mice, we administered human anti-GPC3 CAR-T cells to the animals. Histological examination of tumor and enteric nerve tissue was performed in Hep3B tumor-bearing animals treated with GPC3-1 BZ.

[0146] [Table 4]

[0147] result GPC3 CAR-T with a 4-1BB / CD3 zeta (BZ) signaling domain exhibits greater central memory and less attrition than CD28 / CD3 zeta (28Z) in vivo. Results indicate that splenic GPC3-1 BZ CAR T cells are less differentiated than GPC3-1 28Z CAR T cells, while retaining the ability to fully activate and differentiate in antigen-present tumors. See Figures 11A-11D.

[0148] GPC3-1 BZ demonstrated persistence. Expression of activation / exhaustion markers LAG3 and PD1 confirmed that GPC3-1 BZ CAR T cells maintained fewer activated / exhausted cells in the periphery. See Figure 12.

[0149] GPC3-1 BZ did not induce weight loss in either tumor-bearing or non-tumor-bearing mice at tumor-regressing doses. See Figure 13.

[0150] Complete tumor regression was observed only in GPC3-1 BZ CAR T cell-treated mice. See Figure 14.

[0151] Negligible systemic cytokines were detected (transient elevations of IFN-γ and TNF-α measured 7 days after injection on day 8). At the effective CAR T dose, negligible and transient systemic cytokines were detected, and no weight loss was observed. Human IFN-γ and TNF-α were the only cytokines transiently detected at elevated levels in serum after the regression dose of CAR therapy. Additional human or murine cytokine levels, including hIL-2, mIL-10, mIL-6, mTNFα, and mIFNγ, were below the limit of detection (BDL). See Figure 15.

[0152] As the tumors regressed, extensive T cell infiltration and expansion were observed within the tumors. The tumors became smaller due to the reduction of tumor cells, became necrotic, and were infiltrated with mononuclear cells. The mice used lack lymphocytes, so any infiltrating mononuclear cells are likely human CAR-T cells. See Figure 16. The enteric nervous system, which expresses only low levels of GPC3, was not affected. See Figure 17. Minimal mononuclear cell infiltration was observed in the lungs and liver (data not shown).

[0153] conclusion Compared with constructs containing other signaling domains, the GPC3-1 BZ construct exhibits long-lasting activity, promotes a central memory response, and exhibits increased activity against tumors. Furthermore, treatment induces only a transient increase in some cytokines and does not induce weight loss. Treatment induces T cell infiltration and necrosis of tumors, while sparing normal tissue.

[0154] Example 6: Further characterization of GPC3-1 BZ CAR T cells overview In this example, GPC3-1 BZ CAR T cells were further characterized in the treatment of various tumor types and cells with different levels of GPC3 expression. Cytokine responses were analyzed.

[0155] method Cytokine levels in response to treatment with GPC3-1 BZ CAR T cells were examined in tumor types with varying levels of GPC3 expression, and immunohistochemical staining was performed on representative Hep3B and Huh7 tumor xenografts.

[0156] GPC3 expression analysis was also performed on cells within tumor types. Staining intensity was graded on a scale of 1 to 4, with 1 being the lowest intensity and 4 being the highest intensity. A staining intensity of 2 indicates low / moderate intensity. Relative expression of GPC3 expression was determined by FACs. GPC3 expression on Hep3B cells was determined by surface staining with a fluorescently labeled anti-GPC3 antibody and subsequent flow cytometry analysis. Based on GPC3 expression, Hep3B cells were gated as low, medium, or high expressers, and the frequency of GPC3 in each gate was plotted.

[0157] Cytokine levels were measured by ELISA. Cell lines were co-cultured with GPC3-1 BZ CAR T cells at a 1:1 ratio in RPMI 10% FCS. After 24 hours, supernatants were collected and cytokines were analyzed using the Meso Scale Discovery 4-plex Kit to detect IFN-γ, IL-2, TNF-α, and IL-10. Cells were exposed to GPC3-1 BZ T cells for 24 hours, after which cytokine analysis was performed. Cytokine levels in different cell types were examined after GPC3-1 BZ treatment.

[0158] result GPC3-1 BZ CAR T cell-induced cytokine production in GPC3-expressing cell lines was proportional to surface GPC3 expression. See Figures 18-22.

[0159] GPC3-1 BZ CAR T cells did not elicit a cytokine response in GPC3-negative or normal tissues. See Figure 23 and Figure 24.

[0160] conclusion GPC3-1 BZ CAR T cells induce cytokine production at levels proportional to GPC3 expression in treated cells.

[0161] Example 7: Treatment with GPC3-1 CAR T cell constructs and anti-cytokine antibodies overview In this example, GPC3-1 CAR T cell therapy was attempted in combination with an anti-cytokine antibody.

[0162] method Tumors were treated with different combinations of GPC3-1 CAR T cell therapy and anti-cytokine antibodies. Mice bearing Hep3B tumors (10 per group) were administered 5 million GPC3-1 BZ or GPC3-1 TZ transduced cells (TZ = truncated CD3 zeta, a non-signaling negative control) in the presence or absence of 100 μg of anti-human TNFα (golimumab, Janssen) or anti-mouse IL-6 (Bio X Cell).

[0163] We tested high doses (1e7-3e7) of GPC3-1 CAR T in combination with anti-TNFα treatment at two different time points in a resistant HCC model, Huh7. Huh7 tumor-bearing mice (10 / group) received the indicated doses of GPC3-1 BZ T cells (10 million or 3000 cells) and 100 μg of anti-TNFα either on the same day as CAR T treatment (day 0) or 2 days after treatment initiation (day 2).

[0164] result Blockade of TNF-α, but not IL-6, abolished the efficacy of GPC3-1 BZ. See Figure 25.

[0165] Although higher doses of CAR T cells were required to cause tumor growth inhibition in the resistant HCC model Huh7, higher doses were also associated with CRS-like symptoms and weight loss. Weight loss was reversed to achieve tumor growth inhibition with delayed administration of anti-TNFα. See Figures 26A-26C.

[0166] conclusion The use of anti-TNFα treatment in combination with GPC3-1 BZ therapy can alleviate the effects of weight loss caused by high-dose CAR T-cell therapy.

[0167] The embodiments described herein can be practiced in the absence of any element or elements, limitation, or limitations not specifically disclosed herein. These terms and expressions employed are used as terms of description and not as limitations. The use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, and it is recognized that various modifications are possible within the scope of the claimed embodiments. Thus, while the present description has been specifically disclosed in terms of embodiments, it should be understood that those skilled in the art may employ any features, modifications, and variations of the concepts disclosed herein, and that such modifications and variations are deemed to be within the scope of these embodiments as defined by the description and the appended claims. While certain aspects of the present disclosure may be identified herein as particularly advantageous, it is not intended that the present disclosure be limited to these particular aspects of the disclosure.

[0168] A claim or description including "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless the contrary is stated or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process.

[0169] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as a list, for example, in Markush group format, each subgroup of elements is also disclosed, and any element can be excluded from the group.

[0170] In general, when the present disclosure or aspects of the present disclosure are referred to as including certain elements and / or features, it is to be understood that particular embodiments of the present disclosure or particular aspects of the present disclosure consist of, or consist essentially of, such elements and / or features, and for the sake of brevity, those embodiments are not specifically described in these terms herein.

[0171] All patents and publications mentioned herein are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.

[0172] Table 5

[0173] Table 6

[0174] Table 7

[0175] Table 8

[0176] Table 9

[0177] Table 10

[0178] Table 11

[0179] Table 12

[0180] Table 13

[0181] Table 14

[0182] Table 15

[0183] Table 16

[0184] Table 17

Claims

1. 1. An isolated nucleic acid encoding a chimeric antigen receptor (CAR), comprising: The CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the antigen-binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and An isolated nucleic acid, wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

45.

2. 2. The isolated nucleic acid of claim 1, wherein the antigen-binding domain is an scFv encoded by the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO:

34.

3. The isolated nucleic acid of claim 1 or 2, further encoding a transmembrane domain, a costimulatory domain, and a signaling domain.

4. The isolated nucleic acid of claim 3 , wherein the encoded transmembrane domain comprises a CD28 transmembrane domain.

5. 4. The isolated nucleic acid of claim 3, wherein the encoded costimulatory domain comprises one or more of CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains.

6. 4. The isolated nucleic acid of claim 3, wherein the encoded costimulatory domain comprises one or more of a CD28, 4-1BB, and CD3 zeta costimulatory domain.

7. 4. The isolated nucleic acid of claim 3, wherein the encoded signal domain comprises a sequence encoding a CSFR2 signal peptide.

8. The isolated nucleic acid of any one of claims 1 to 7, further encoding a hinge / spacer domain.

9. 9. The isolated nucleic acid of claim 8, wherein the encoded hinge / spacer domain is an IgG4P hinge / spacer.

10. 2. The isolated nucleic acid of claim 1, wherein the sequence of the isolated nucleic acid comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

26.

11. An anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and an anti-GPC3 CAR, wherein the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO:

45.

12. The anti-GPC3 CAR according to claim 11, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

13. The anti-GPC3 CAR according to claim 11, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

14. The anti-GPC3 CAR according to any one of claims 11 to 13, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and a signal domain.

15. The anti-GPC3 CAR of claim 14, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

25.

16. A vector comprising a nucleic acid sequence encoding an anti-GPC3 chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:

34.

17. A cell comprising the vector of claim 16.

18. A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific for glypican 3 (GPC3), and the nucleic acid sequence comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO:

34.

19. A cell comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and A cell wherein the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40, CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO:

45.

20. The cell of claim 19, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

21. The cell of claim 19, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

22. The cell of any one of claims 19 to 21, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain.

23. The cell of any one of claims 19 to 22, wherein the CAR comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

25.

24. The cell of any one of claims 17 to 23, wherein the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

25. The cell of claim 24, wherein the cell exhibits anti-tumor immunity upon contact with tumor cells that express GPC3.

26. A pharmaceutical composition for treating cancer, comprising: The present invention relates to a cell comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 45; The pharmaceutical composition wherein an effective amount of said cells is administered to a subject in need of treatment for cancer.

27. 27. The pharmaceutical composition of claim 26, further for inhibiting tumor growth, inducing tumor regression, and / or prolonging survival in said subject.

28. 27. The pharmaceutical composition of claim 26, wherein the cells are autologous cells.

29. 29. The pharmaceutical composition of claim 28, wherein the autologous cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

30. The pharmaceutical composition according to any one of claims 26 to 29, wherein the cancer is a solid tumor.

31. 31. The pharmaceutical composition of claim 30, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

32. 32. The pharmaceutical composition of claim 31, wherein the cancer is hepatocellular carcinoma.

33. The pharmaceutical composition of any one of claims 26 to 32, wherein an effective amount of an anti-TNFα antibody is further administered to the subject.

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