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

Abstract

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

Description

Background of the Invention Technical Field

[0002] This disclosure relates to the use of chimeric antigen receptor T cells to treat cancer. Background Technology

[0004] 1. Chimeric antigen receptor T-cell therapy

[0005] Chimeric antigen receptor (CAR) T-cell therapy is a specific form of cell-based immunotherapy that uses engineered T cells to fight cancer. In CAR T-cell therapy, T cells are harvested 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 live drugs that bind to cancer cells and cause their destruction. When successful, the effects of CAR T-cell therapy tend to last a long time, as demonstrated by testing the persistence and expansion of CAR T cells in patients long after clinical remission.

[0006] 2. CAR Structure and Function

[0007] The antigen-binding domain of a CAR is an extracellular region that targets surface antigens on tumor cells. Suitable target antigens can be proteins, phosphorylated proteins, peptide-MHC, carbohydrates, or glycolipid molecules. Ideally, the target antigen is widely expressed on tumor cells to enable targeting of a high percentage of cancer cells. Ideal candidate target antigens are also typically expressed minimally in normal tissues, thus limiting extratumor-mediated target toxicity. The antigen-binding domain of a CAR contains a targeting portion of the target antigen, such as an antibody single-chain variable fragment (scFv).

[0008] The T-cell activation domain of a CAR is intracellular and activates T cells in response to an antigen-binding domain that interacts with a target antigen. The T-cell activation domain may contain one or more co-stimulatory domains, which are known intracellular domains that activate T-cell receptors. Because co-stimulatory domains have varying effects on CAR T-cell dynamics, cytotoxicity, and safety, the selection and location of co-stimulatory domains in the CAR construct in vivo can influence CAR T-cell function and fate.

[0009] The extracellular antigen-binding domain and intracellular T-cell activation domain of the CAR are connected by a transmembrane domain, a hinge, and optionally a spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to target antigens on tumor cells. This hinge domain can be used alone or in combination with a spacer domain designed to be located away from the T-cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface.

[0010] CAR T therapy targeting B lymphocyte antigen CD19 Novartis has shown promise in treating childhood acute lymphoblastic leukemia with its CAR T therapy targeting B-cell maturation antigen (“bb2121”). and (Collaboration) shows promise for relapsed / refractory multiple myeloma. 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 cells with pembrolizumab have demonstrated antitumor activity in mesothelioma. However, additional targets are needed for the treatment of solid tumors.

[0011] 3. Challenges of CAR T-cell therapy

[0012] Unfortunately, the complexities of CAR T-cell-based therapies can lead to undesirable and unsafe effects. Extratumor effects, such as neurotoxicity and acute respiratory distress syndrome (ARDS), 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-cell therapy. CRS occurs when lymphocytes are highly activated and release excessive amounts of inflammatory cytokines. When these factors are measured, serum elevations of interleukin-2, interleukin-6, interleukin-1β, GM-CSF, and / or C-reactive protein are sometimes observed in patients with CRS. CRS is graded by severity and diagnosed as one of grades 1–4 (mild to severe), with more severe cases clinically characterized by high fever, hypotension, hypoxia, and / or multi-organ toxicity. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR T-cell therapy experienced CRS, and 50% of these patients presented with grade 3–4 symptoms.

[0013] Therefore, additional CAR T-cell-based therapies are needed to enhance medical devices for effective cancer treatment. However, new CAR T-cell therapies must be designed to effectively treat cancer while minimizing the risk of dangerous inflammatory responses such as CRS. Summary of the Invention

[0014] This disclosure describes compositions and methods for treating cancer using CAR T cells.

[0015] As described below, in a first aspect, this disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific to phosphatidylinositol proteoglycan 3 (GPC3), wherein the antigen-binding domain has an equilibrium dissociation constant (K0) of about 100 nanomolars (nM) or less. D Furthermore, the CAR construct does not induce the production of cytokines in cells expressing GPC3.

[0016] In some embodiments of the first aspect, the CAR antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

[0017] In some embodiments of the first aspect, the antigen-binding domain is a Fab or a single-stranded variable fragment (scFv).

[0018] In some embodiments of the first aspect, the antigen-binding domain is an scFv containing a nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0019] In some embodiments of the first aspect, the isolated nucleic acid further encodes a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0020] In some embodiments of the first aspect, the transmembrane domain includes the CD28 transmembrane domain.

[0021] In some embodiments of the first aspect, the co-stimulatory domain includes one or more of the following: CD28, 4-1BB, CD3ζ, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 co-stimulatory domains.

[0022] In some embodiments of the first aspect, the co-stimulatory domain includes one or more of the CD28, 4-1BB, and CD3ζ co-stimulatory domains.

[0023] In some embodiments of the first aspect, the signal domain contains a sequence encoding the CSFR2 signal peptide.

[0024] In some embodiments of the first aspect, the anti-GPC3 CAR further includes a hinge / spacer sub-domain.

[0025] In some embodiments of the first aspect, the hinge / spacer domain is an IgG4P hinge / spacer.

[0026] 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.

[0027] In a second aspect, this 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 containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 37, a CDR2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR3 containing the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0028] In some embodiments of the second aspect, VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29.

[0029] In some embodiments of the second aspect, VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

[0030] In some embodiments of the second aspect, the anti-GPC3 CAR further includes a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0031] 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.

[0032] In a third aspect, this 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.

[0033] In some embodiments, this disclosure provides cells comprising a carrier of a third aspect.

[0034] In a fourth aspect, this 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 phosphatidylinositol proteoglycan 3 (GPC3), wherein the antigen-binding domain has an equilibrium dissociation constant (K0) of about 100 nanomolars (nM) or less. D Furthermore, the CAR construct does not induce the production of cytokines in GPC3 cells.

[0035] 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.

[0036] In a fifth aspect, this disclosure provides a cell comprising an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 37, a CDR2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR3 containing the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0037] In some embodiments of the fifth aspect, VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29.

[0038] In some embodiments of the fifth aspect, VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

[0039] In some embodiments of the fifth aspect, the CAR further includes a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0040] 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.

[0041] In some embodiments of the fifth aspect, the cell is 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 fifth aspect, the cell exhibits anti-tumor immunity upon contact with tumor cells expressing GPC3.

[0043] In a sixth aspect, this disclosure provides a method for treating cancer, the method comprising: administering to a subject in need an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 37, a CDR2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR3 containing the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0044] In some embodiments of the sixth aspect, the method further includes inhibiting tumor growth, inducing tumor regression, and / or prolonging the survival of the subject.

[0045] In some embodiments of the sixth aspect, the cell is an autologous cell.

[0046] 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.

[0047] In some embodiments of the sixth aspect, the cancer is a solid tumor.

[0048] 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.

[0049] In some embodiments of the sixth aspect, the cancer is hepatocellular carcinoma.

[0050] In some embodiments of the sixth aspect, the method further includes administering an effective amount of anti-TNFα antibody to the subject.

[0051] These and other features and advantages of the invention will be more fully understood from the following detailed description and the appended claims. It should be noted that the scope of the claims is defined by the statements therein, and not by the specific discussion of the features and advantages set forth in this specification. Attached Figure Description

[0052] The accompanying drawings are included to provide a further understanding of the methods and compositions disclosed herein. The drawings illustrate one or more embodiments of the present disclosure and, together with the specification, serve to explain the principles and operation of the disclosure.

[0053] Figure 1A and 1B GPC3 expression in cancerous and normal tissues. 1A. Anti-GPC3 antibody staining results in hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and ovarian cancer. 1B. Immunohistochemical (IHC) results of human colonic ganglion tissue.

[0054] Figure 2 Comparison of the heavy and light variable regions of a single-stranded variable fragment (scFv). GPC3-1 and GPC3-2 are shown.

[0055] Figure 3A Cell surface GPC3 CARs bind to soluble GPC3 protein. K... D Values ​​(shown as a solid line for the fitted values).

[0056] Figure 3B and 3C Surface plasmon resonances between anti-GPC3 scFv-Fcs and soluble GPC3 protein binding were reported. Two interacting kinematics were also described. a k d and K DThe average value (shown as a solid line for the fit).

[0057] Figure 4A and 4B Chimeric antigen receptor (CAR) constructs and their cytokine production after in vitro administration to cells with and without the target antigen. GPC3 CAR T cells induce antigen-specific cytokine production. Cell lines for each construct listed in the legend from top to bottom are shown from left to right. 4A shows the results for three cytokines. UT, untransduced T cells, are activated and expanded donor T cells that have not been transduced by the CAR transgene. 4B shows the results for interferon-γ (IFN-γ) in a subset of the constructs. The right-hand legend shows the cell types (all negative except HEPG2).

[0058] Figure 5 .CAR cytotoxicity in HCC cell lines. The legend on the right shows the constructs used. E:T ratio, effector:target ratio. UT, untransduced T cells.

[0059] Figure 6. Cytotoxicity of GPC3-1 in HCC cell lines expressing low levels of GPC3. 6A. GPC3 expression in designated cell lines assessed by flow cytometry. 6B. Receptor density in designated cell lines. 6C. Cytotoxicity of GPC3-1 to cell lines designated in the legend at effector:target ratios of 3:1 (top) and 0.3:1 (bottom). 6D. KT50 (time to kill 50% of the target) of GPC3-1 in designated cell lines at two different effector:target ratios.

[0060] Figure 7 .Versatility study of GPC3-2 and GPC3-1 CAR T constructs. scFv is shown on the left side of each row, and co-stimulatory domains are shown at the top of each chart.

[0061] Figure 8A and 8B 8A. Effect of chimeric antigen receptor T-cell (CAR T) transplantation on body weight. The legend on the right shows the constructs used. BW, body weight. ACT, adoptive T-cell therapy. UT, untransduced T cells. PBS, phosphate-buffered saline. 7B. IHC depicts CAR-T accumulation in lung tissue of mice treated with GPC3 CAR-T.

[0062] Figure 9 The effect of CAR T administration on tumor volume. The legend on the right shows the constructs used. ACT, adoptive T-cell therapy. UT, untransduced T cells. PBS, phosphate-buffered saline.

[0063] Figure 10The effect of CAR T administration on survival. The legend on the right shows the constructs used. ACT, adoptive T-cell therapy. UT, untransduced T cells. PBS, phosphate-buffered saline.

[0064] Figure 11A-11D Fluorescence activated cell sorting (FACs) studies of GPC3-1 CAR T cell differentiation and exhaustion using different co-stimulatory domains. Results for spleen (11A and 11B) and tumor cells (11C and 11D) are shown. Dot plots show the frequency of CD3+ T cells infiltrating each organ for each construct (11A and 11C). In vivo, GPC3 CAR-T cells with the 4-1BB / CD3ζ (BZ) signaling domain have more central memory and less exhaustion than those with the CD28 / CD3ζ (28Z) domain. The co-stimulatory domains used are shown at the top of each subplot. Assessed markers are shown on the x and y axes. FSC, forward scattering. EM, effector memory. CM, central memory. TN, naive T cells.

[0065] Figure 12 Durability of GPC3-1 CAR T cells in Hep3B and HepG2 tumors. The legend on the right shows the construct used. Percentage of CD3 is shown. ACT, adoptive T-cell therapy. UT, untransduced T cells.

[0066] Figure 13 Effects of GPC3-1BZ treatment on body weight in non-tumor-bearing and tumor-bearing mice. The bottom legend shows the constructs used. BW, body weight. ACT, adoptive T-cell therapy. TZ is GPC3-1 TZ. UT, untransduced T cells. PBS, phosphate-buffered saline.

[0067] Figure 14 Tumor volume and hemorrhage time as analyzed against GPC3-1 BZ and GPC3-1 TZ cytokines. Arrows indicate hemorrhage points in subsequent cytokine response studies. The legend on the right shows the constructs used. ACT, adoptive T-cell therapy. UT, untransduced T cells. PBS, phosphate-buffered saline.

[0068] Figure 15 Maximum systemic cytokine response (IFN-γ) in GPC3-1 CAR T cells. Data from day 8 hemorrhage are shown, with the maximum cytokine response observed on day 8. UT, untransduced T cells. PBS, phosphate-buffered saline.

[0069] Figure 16Histological examination of Hep3B tumor tissue in NOD scidγ (NSG) immunodeficient mice. Top: Untreated control. Bottom: Animals treated with GPC3-1 BZ CAR T cells. Images shown at 20x magnification.

[0070] Figure 17 Histological examination of enteric nerve tissue in NOD scidγ (NSG) immunodeficient mice. Left: Untreated control. Right: Animal treated with GPC3-1 BZ CAR T cells.

[0071] Figure 18 Quantification of cell surface GPC3. From top to bottom: A375 cells (GPC3 negative), HepG2 cells (high GPC3), Hep3B cells (medium / low GPC3), and Huh7 cells (low GPC3). The area under the peak represents the cell population expressing the protein at a specified level on the x-axis. APC, allophycocyanin.

[0072] Figure 19 Results of enzyme-linked immunosorbent assay (ELISA) of cytokines 24 hours after exposure to GPC3-1 BZ T cells. Cell lines listed in the legend are shown from left to right in order from top to bottom.

[0073] Figure 20 Immunohistochemistry against GPC3 was performed on representative tumor xenografts from two HCC cell lines. Both xenografts were scored as intensity = 2. The data indicate that tumors with at least 25% positive GPC3 expression at intermediate intensity will respond to GPC3-1 BZ CAR-T.

[0074] Figure 21 Determination of GPC3 expression on relative surface. FSC, forward scattering. APC, allophycocyanin. MFI, mean fluorescence intensity. The frequencies of GPC3 in each gating group are shown in the dot plot on the left (12.7%, 24%, and 9.34%, respectively). The histogram on the right depicts the expression of GPC3 in the sorting group, confirming purity and homogeneity.

[0075] Figure 22 Cytokine ELISA 24 hours after exposure to GPC3-1 BZ. Results are shown for T cells (only), A375 cells (GPC3 negative), and low, medium (medium / med), and high expression of GPC3. Results are displayed from left to right in each subplot for each cell type listed in the legend from top to bottom. UT, untransduced T cells. TZ and BZ, GPC3-1 TZ and GPC3-1 BZ.

[0076] Figure 23Interferon-γ (IFNγ) levels in different cell types after CAR T treatment. The x-axis shows the constructs used. Results for each construct are shown from left to right for each cell type listed in the legend from top to bottom. TZ, GPC3-1 TZ. UT, untransduced T cells. Culture medium, treated with cell culture medium only.

[0077] Figure 24 Cytokine levels in neural tissue cell types treated with GPC3-1 CAR T. The x-axis shows the construct used. Results for each construct are shown from left to right for each cell type listed in the legend from top to bottom. UT, untransduced T cells. Culture medium, treated with cell culture medium only.

[0078] Figure 25 Tumor volumes treated with CAR T cells and anti-CRS-related cytokine antibodies. CRS = Cytokine Release Syndrome. Top: Tumor volumes after treatment with different regimens of CAR and antibodies. 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.

[0079] Figures 26A-26C Studies of higher CAR-T doses and anti-TNFα treatment in a resistant HCC model (Huh7). Anti-TNFα can be used to attenuate toxicity and promote antitumor activity at higher CAR-T doses. The legend on the right shows the constructs used. 26A. Study mode. BW, body weight. 26B. Tumor growth. iv, intravenous. 26C. Body weight change. Detailed Implementation

[0080] 1. Definition

[0081] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. The following references provide general definitions for several terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al., Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms as used herein have the meanings assigned to them as follows.

[0082] As used herein, the terms “comprise” and “include”, and their variations (e.g., “comprises / comprising”, “includes / including”) should be understood to mean including the stated components, features, elements, or steps, or a group of components, features, elements, or steps, but not excluding any other components, features, elements, or steps, or a group of components, features, elements, or steps. Any of the terms “comprise,” “substantially constitute,” and “comprise” can be replaced by any of the other two terms while retaining their ordinary meaning.

[0083] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used herein include plural indicators.

[0084] The percentages disclosed herein may differ from the disclosed values ​​by ±10%, 20%, or 30%, and are still within the range of the intended disclosure.

[0085] Unless otherwise stated or otherwise clearly apparent from the context and as understood by one of ordinary skill in the art, values ​​expressed herein as ranges in various embodiments of this disclosure may take any specific value or subrange within the stated range, up to one-tenth of the lower limit of that range, unless the context otherwise expressly indicates otherwise.

[0086] As used herein, ranges and quantities may be expressed as “about” a particular value or range. The term “about” also includes the precise quantity. For example, “about 5%” means “about 5%” and also “5%”. The term “about” can also refer to a given value or a range of values ​​within ±10%. Thus, for example, about 5% also means 4.5%–5.5%. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term “about”.

[0087] As used herein, the terms “or” and “and / or” can describe multiple components that are combined or excluded from each other. 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”.

[0088] 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 one or more chains of two or more amino acids. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains having two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" may be used in place of any of these terms, or used interchangeably with them.

[0089] As used herein, “protein” can refer to a single polypeptide, i.e., a single chain of amino acids as defined above, and can also refer to two or more related polypeptides, for example, multimeric proteins formed by disulfide bonds, hydrogen bonds, or hydrophobic interactions.

[0090] "Isolated" substances, such as isolated nucleic acids, are substances not found in their natural environment, although the isolated substance is not necessarily purified. For example, isolated nucleic acids are nucleic acids that do not originate in or are located in their natural or organic environment (such as cells). Isolated substances can be isolated, fractionated, or at least partially purified using any suitable technique.

[0091] As used herein, the terms “antibody” and “antigen-binding fragment thereof” refer to at least a minimal portion of an antibody capable of binding to a specified antigen targeted by the antibody, such as, in the case of a typical antibody produced by B cells, at least some complementarity-determining regions (CDRs) of the variable domains of the heavy chain (VH) and the variable domains of the light chain (VL). An antibody or its antigen-binding fragment may be or derived from polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab′, and F(ab′)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing a single VL or VH domain or a portion thereof that binds to an opposite domain (e.g., the entire VL domain and a portion of the VH domain having one, two, or three CDRs), and fragments generated from a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The antibody molecules covered by this disclosure can be 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 molecules.

[0092] As used herein, the term "polynucleotide" includes both single and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA.

[0093] As used herein, the term "vector" can refer to a nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate within the host cell, such as an origin of replication. A vector may also include one or more optional marker genes and other genetic elements known in the art. Certain types of vectors contemplated herein may associate with or be incorporated into viruses to facilitate cell transformation.

[0094] "Transformed" cells, or "host" cells, are cells in which nucleic acid molecules have been introduced using molecular biology techniques. This article considers all techniques that can introduce nucleic acid molecules into such cells, including transfection with viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA via electroporation, lipid transfection, and particle guns.

[0095] As used herein, the term “affinity” refers to a measure of the strength of binding between an antigen or target (e.g., an epitope) and its homologous binding domain (e.g., a complementary site). As used herein, the term “avidity” refers to the overall stability of the complex between a population of epitopes and complementary sites (i.e., antigens and antigen-binding domains).

[0096] As used herein, when used in the context of treating cancer, the term "treat" refers to alleviating the pathology of a disease, reducing or eliminating disease symptoms, improving survival rates, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to mitigating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of the disease and / or preventing or delaying the onset of the disease or disorder.

[0097] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject for diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0098] As used herein, the term "effective amount" or "therapeutic effective amount" for an applied therapeutic substance (such as CAR T cells) is sufficient for a specific or intended purpose, such as the treatment of cancer. An "effective amount" may be determined empirically and in a conventional manner based on the stated purpose.

[0099] 2. Overview

[0100] This disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More specifically, this disclosure relates to CAR cell therapy in which transformed cells (such as T cells) express a CAR targeting phosphatidylinositol proteoglycan-3 (GPC3). Furthermore, the CAR constructs disclosed herein, the transformed cells expressing these constructs, and the therapies utilizing these transformed cells can provide robust cancer treatment with minimal risk of cytokine release syndrome (CRS) or unselective cytokine release in non-GPC3-expressing cells.

[0101] Unwilling to be bound by theory, GPC3 is considered a viable cancer target in multiple forms, including bispecific T-cell conjugates, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). GPC3 is a carcinoembryonic antigen, a GPI-linked heparin sulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction, thereby stimulating Wnt signaling. GPC3 competes with patched for binding to Hh, which alleviates Smoothened inhibition and induces GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) growth. Furthermore, GPC3 expression levels have shown correlation with HCC stage and grade.

[0102] Furthermore, GPC3 is believed to be a promising target for CAR cell therapy. Therefore, antibodies and CAR constructs derived from these antibodies have been developed as described in this article.

[0103] 3. CAR Component Design

[0104] The CAR construct disclosed herein may have several components, many of which can be selected based on the desired or precise function of the resulting CAR construct. In addition to the antigen-binding domain, the CAR construct may also have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more co-stimulatory domains. Choosing one component over another (i.e., choosing a specific co-stimulatory domain from one receptor, relative to co-stimulatory domains from different receptors) can affect clinical efficacy and safety.

[0105] 4. Antigen-binding domain

[0106] The antigen-binding domains anticipated in this paper may include antibodies or one or more antigen-binding fragments thereof. An anticipated CAR construct targeting GPC3 comprises a single-chain variable fragment (scFv) containing light and heavy chain variable regions from one or more antibodies specific to GPC3, which are directly linked together or linked together via flexible linkers (e.g., repeat sequences of GGGGS having 1, 2, 3 or more repeat sequences).

[0107] As disclosed in this article, the binding affinity of the antigen-binding domain of a GPC3-targeting CAR to the GPC3 protein can vary. In the case of a CAR, the relationship between binding affinity and efficacy may be more nuanced compared to antibodies, which are typically desired to have higher affinity. For example, preclinical studies of a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CAR derived from a high-affinity scFv (with a dissociation constant of 0.56 nM) resulted in an increased therapeutic index compared to a low-affinity variant. Conversely, other examples have been reported where engineered scFvs targeting lower affinity improved the differentiation between cells with different antigen densities. This could improve the therapeutic specificity of antigens that can be used to enhance differential expression of antigens in tumor tissues compared to normal tissues.

[0108] Various methods can be used to determine the binding affinity of antigen-binding domains. In some embodiments, methods that exclude affinity effects can be used. Affinity effects involve multiple antigen-binding sites that interact simultaneously with multiple target epitopes, and typically involve multimeric structures. Therefore, affinity functionally represents the cumulative strength of multiple interactions. An example of a method that excludes affinity effects is any method in which one or both interacting proteins are monomeric / monovalent, because if one or two partner bodies contain only a single interacting site, multiple simultaneous interactions are impossible.

[0109] 5. Spacer subdomain

[0110] The CAR constructs disclosed herein may have a spacer domain to provide conformational freedom, thereby facilitating binding to target antigens on target cells. 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 spacers. In addition to facilitating CAR binding to target antigens, achieving the optimal distance between CAR cells and cancer cells can also help spatially block the entry of large inhibitory molecules into the immune synapse formed between CAR cells and target cancer cells. CARs targeting GPC3 may have long, medium, and short spacers. Long spacers may include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural or with modifications common in therapeutic antibodies, such as the S228P mutation), while the CH3 region may be used alone to construct medium spacers (approximately 120 amino acids). Short spacers may be derived from segments of CD28, CD8α, CD3, or CD4 (<60 amino acids). Short spacers can also originate from the hinge region of IgG molecules. These hinge regions can originate from any IgG isotype and may or may not contain mutations common in therapeutic antibodies, such as the S228P mutation mentioned above.

[0111] 6. Hinge structural domain

[0112] CARs targeting GPC3 can also have hinge domains. 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 in combination with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably—for example, an IgG4 sequence can be considered both a "hinge" sequence and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0113] CARs targeting GPC3 may further include sequences containing signal peptides. The function of the signal peptide is to facilitate the translocation of the CAR to the cell membrane. Examples include IgG1 heavy chain signal peptides, Igκ or λ light chain signal peptides, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptides, CD8a signal peptides, or CD33 signal peptides.

[0114] 7. Transmembrane domain

[0115] CARs targeting GPC3 may further include sequences containing transmembrane domains. These transmembrane domains may include hydrophobic α-helices that cross the cell membrane. The properties of the transmembrane domain have not been studied as extensively as other aspects of the CAR construct, but they may potentially influence CAR expression and association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0116] 8. Co-stimulatory domains

[0117] CARs targeting GPC3 may further include one or more sequences forming a co-stimulatory domain. A co-stimulatory domain is a domain capable of enhancing or modulating the response of immune effector cells. The co-stimulatory domain may include, for example, sequences from one or more of CD3ζ (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The choice of co-stimulatory domain influences the phenotype and metabolic characteristics of CAR cells. For example, CD28 co-stimulation produces an effective, but transient, effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells modified with CARs carrying a 4-1BB co-stimulatory domain tend to expand and persist longer in vivo, exhibit increased oxidative metabolism, are less prone to exhaustion, and have an increased capacity to generate central memory T cells.

[0118] 9. Cells

[0119] CAR cell-based therapies can be used with a variety of cell types, such as lymphocytes. Specific cell types that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, αβT cells, γδT cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells (Tregs). In one embodiment, the CAR cells used to treat the subject are autologous. In other embodiments, the CAR cells may be derived from genetically similar but not identical donors (allogeneic).

[0120] 10. CAR cell production

[0121] The CAR constructs disclosed herein may include combinations of the modular components described herein. For example, in some embodiments of this 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 this disclosure, the CAR construct includes a CSFR2 signal peptide. In some embodiments, the CAR construct includes an IgG4P hinge / spacer domain carrying an S228P mutation. In some embodiments, the CAR construct includes a CD28 transmembrane domain.

[0122] Different co-stimulatory domains that can be used are the CAR constructs disclosed herein. In some embodiments, the CAR construct includes a co-stimulatory domain from an intracellular domain of CD3z. In some embodiments, the CAR construct includes a CD28 co-stimulatory domain. In some embodiments, the CAR construct includes a 4-1BB co-stimulatory domain. In some embodiments, the CAR construct includes co-stimulatory domains from both CD3z and CD28. In some embodiments, the CAR construct includes co-stimulatory domains from both CD3z and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains from all of CD3z, CD28, and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains from ICOS, OX-40, and / or GITR.

[0123] 11. CAR Component Evaluation

[0124] The constructs disclosed herein were compared and evaluated based on safety, persistence, and the establishment of central memory. The low-affinity (high dissociation rate) scFv GPC3-1 was advantageously evaluated due to its improved safety. The 4-1BB and CD3z co-stimulatory domains (both within the same construct) were advantageously evaluated based on their improved persistence and the contribution of a favorable in vivo phenotype (more central memory). The GPC3-1 and GPC3-2 CARs disclosed herein are advantageous compared to constructs based on disclosed GPC3-targeting CARs. Details of the evaluation can be found in the examples.

[0125] 12. Example

[0126] In some embodiments, this disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an antigen-binding domain specific for phosphatidylinositol proteoglycan 3 (GPC3). The antigen-binding domain has an equilibrium dissociation constant (K0) of about 100 nanomolars (nM) or less. DFurthermore, the CAR construct does not induce the production of cytokines in GPC3 cells. In some embodiments, the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof. The antigen-binding domain may be a Fab or a single-stranded variable fragment (scFv). In some embodiments, the antigen-binding domain is an scFv containing the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0127] In some embodiments, the CAR further includes a transmembrane domain, a co-stimulatory domain, and a signaling domain. The transmembrane domain may be a CD28 transmembrane domain. The co-stimulatory domain may be one or more of the following co-stimulatory domains: CD28, 4-1BB, CD3ζ, OX-40, ICOS, CD27, GITR, and MyD88 / CD40. In one specific embodiment, the co-stimulatory domain is one or more of the following co-stimulatory domains: CD28, 4-1BB, and CD3ζ. The signaling domain may be a sequence encoding a CSFR2 signal peptide.

[0128] In some embodiments, the isolated nucleic acid sequence may include a hinge / spacer domain. The hinge / spacer domain may be an IgG4P hinge / spacer.

[0129] In some specific embodiments, the isolated nucleic acid sequence encoding the chimeric antigen receptor (CAR) may have the sequence 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.

[0130] In other embodiments, this disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) including 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 containing the amino acid sequence of SEQ ID NO: 37, a CDR2 containing the amino acid sequence of SEQ ID NO: 38, and a CDR3 containing the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VL may have a CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0131] In some embodiments, VH may be the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and VL may be the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the CAR may further have a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0132] 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.

[0133] In other embodiments, this disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence may 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.

[0134] In other embodiments, this disclosure provides a cell comprising a vector having a 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.

[0135] In other embodiments, this 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 phosphatidylinositol proteoglycan 3 (GPC3), wherein the antigen-binding domain has an equilibrium dissociation constant (K0) of about 100 nanomolars (nM) or less. DThe CAR construct does not induce the production of cytokines in GPC3 cells. For example, the nucleic acid sequence may 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.

[0136] In other embodiments, this disclosure provides a cell that expresses an anti-GPC3 chimeric antigen receptor (CAR) on its extracellular surface. The CAR may have an antigen-binding domain, which 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 include CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39. The VL may include CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0137] In some embodiments, VH may have the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. In some embodiments, VL may have the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The CAR may further include a transmembrane domain, a co-stimulatory domain, and a signaling domain. The cell expresses 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.

[0138] In some embodiments, this disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells expressing a CAR on their extracellular surface, and 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 may exhibit antitumor immunity upon contact with tumor cells expressing GPC3.

[0139] 13. Using CAR to treat cancer

[0140] In some embodiments, this disclosure provides CAR cells for the treatment of cancer. Compositions (e.g., antibodies, CAR constructs, and CAR cells) and methods having the uses described herein can be used, in particular, to inhibit the growth or spread of neoplastic cells; especially the growth of neoplastic cells in which GPC3 functions.

[0141] Growths that can be treated with the compositions disclosed herein include solid tumors, such as solid tumors of the liver, lung, or ovary. However, the cancers listed herein are not intended to be limiting. For example, cancer types intended for treatment in this article include, for instance, NSCLC, advanced solid malignancies, biliary tract 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 lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal cell carcinoma, metastatic melanoma, metastatic non-cutaneous melanoma, multiple myeloma, nasopharyngeal tumors, non-Hodgkin 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, and renal cell carcinoma. Squamous cell carcinoma (SCCHN), hypopharyngeal 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 / urothelial carcinoma.

[0142] In one embodiment, the cancers intended for treatment herein include any cancers in which GPC3 is expressed on the cell surface of cancer cells. In a specific example, cancers intended for treatment herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0143] 14. Treatment methods

[0144] The CAR-modified cells (such as CAR T cells) of the present invention can be administered alone or as part of a pharmaceutical composition having a diluent and / or other components associated with cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include, for example, CAR-T cells as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers, such as neutral buffered saline, buffered saline, etc.; sulfates; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins, peptides, or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions of the present invention are suitable for treatment (or prevention).

[0145] CAR-modified cells can also be administered in conjunction with one or more other therapies. In one embodiment, the other therapies may include anti-cytokine antibodies. For example, one or more anti-TNFα antibodies may be used to attenuate toxicity and enhance anti-tumor activity at higher CAR T doses (which may be associated with CRS-like symptoms and weight loss).

[0146] In one particular embodiment, the intended treatment regimen may include one or more biological components, such as CAR T cells and anti-cancer antibodies and / or chemotherapy components. For example, the intended treatment regimen may additionally include immune checkpoint inhibitors (ICIs), such as immune checkpoint inhibitors targeting the PD-1 / PD-L1 axis (PDX), and other immuno-oncology (IO) therapies, such as immune system agonists.

[0147] Antibodies anticipated include anti-PD-L1 antibodies (such as durvalumab (MEDI4736), avelumab, atezolizumab, and KNO35), anti-PD-1 antibodies (such as nivolumab, pembrolizumab, REGN2810, SHR1210, IBI308, PDR001, anti-PD-1 antibodies, BGB-A317, BCD-100, and JS001), and anti-CTLA4 antibodies (such as tremelimumab or ipilimumab). Additional antibodies are also anticipated. Any therapeutically effective antibody sub-components are also anticipated.

[0148] Information regarding durvalumab (or fragments thereof) used 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 entirety. In one particular aspect, durvalumab or an antigen-binding fragment thereof used in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H9OPT antibody as disclosed in the aforementioned U.S. patents.

[0149] Information regarding the use of trimemumab (or its antigen-binding fragment) in the methods provided herein can be found in U.S. Patent No. 6,682,736 (in which trimemumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0150] Other therapeutic agents (chemotherapeutic agents or biologics) anticipated in this article include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, and adriamycin. TM (doxorubicin), cisplatin (MVAC), carboplatin-based regimens, or single-dose taxane or gemcitabine, temozolomide, or dacarbazine, vinflunine, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Other examples include drugs that target DNA damage repair systems, such as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors and therapeutics that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora protein kinase B activity, and DNA-PK activity.

[0151] Any therapeutic composition or method contemplated herein may be combined with one or more of any other therapeutic compositions and methods provided herein.

[0152] In some embodiments, this disclosure provides a method for treating cancer, the method comprising administering to a subject in need an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) containing 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 CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39. The VL may comprise CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing 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.

[0153] In some embodiments, the cell is an autologous cell. For example, the autologous cell may be selected from the group consisting of: T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0154] In some embodiments, the cancer treated by this 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 one specific embodiment, the cancer is hepatocellular carcinoma.

[0155] This disclosure provides a method for treating cancer, comprising administering to a subject in need an effective amount of cells containing an anti-GPC3 chimeric antigen receptor (CAR) and an effective amount of an anti-TNFα antibody.

[0156] It should be understood that specific aspects of the specification described herein are not limited to the specific embodiments presented and may vary. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting unless specifically defined herein. Furthermore, as those skilled in the art will recognize, the specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation.

[0157] Example

[0158] The following examples illustrate specific embodiments of this disclosure and their various uses. They are presented for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way. Table 1 provides a description of the terminology.

[0159] Table 1. Description of Terms

[0160]

[0161]

[0162] Example 1: Expression of GPC3

[0163] method

[0164] The GPC3 IHC was performed using the mouse monoclonal anti-human GPC3 antibody GC33 (Ventana). Secondary staining was performed using anti-mouse HRP. Human tissue microarrays (TMA, Biomax, representing hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), ovarian cancer, or human colonic ganglion tissue) were stained for GPC3 expression, and staining intensity and pattern were determined by microscopic examination.

[0165] result

[0166] GPC3 is overexpressed in 80% of HCC, 30% of squamous cell lung cancer, and 47% of ovarian clear cell carcinoma. However, GPC3 cannot be detected by immunohistochemistry in normal liver tissue (including cirrhotic and hyperplastic samples) and is expressed at low levels in normal tissues (e.g., lung). See also Figure 1A and 1B .

[0167] Example 2: Development and affinity research of scFv.

[0168] summary

[0169] In this example, an anti-GPC3 scFv was developed and its relative affinity to GPC3 was determined.

[0170] method

[0171] GPC3-1 (SEQ ID NO: 1) and GPC3-2 (SEQ ID NO: 2) have almost the same V H Domains (SEQ ID NO: 27 and 29), but with different V L Domains (SEQ ID NO: 28 and 30; see also) Figure 2 GPC3-2 has a complete phylogenetic framework, while GPC3-1 does not.

[0172] Epigenetic binding affinity was determined by binding soluble recombinant GPC3 protein to the cell surface of GPC3-1 and GPC3-2 CARs expressed on Jurkat cells. CAR constructs were expressed on the surface of Jurkat cells using a lentiviral vector. Cells were stained with different concentrations of recombinant His-labeled GPC3 protein (R&D Systems). Bound GPC3 was visualized by staining with a fluorescently conjugated anti-His-labeled secondary antibody, and cells were analyzed by flow cytometry. Binding curves were fitted to a simple unit-point binding model to determine epigenetic affinity. D .

[0173] Alternative measures of binding affinity were determined using the BIAcore surface plasmon resonance system and the 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 rate of 30 μL / min, and interactions were monitored. For GPC3-2, the same flow rate was used at concentrations of 4, 7, 14, 28, and 57 nM. Data were fitted using BIA evaluation software (GE Healthcare) and a simple 1:1 Langmuir binding model, where the global fit was R0. max And local fitting k a k d and K D .

[0174] result

[0175] In experiments evaluating the binding of soluble GPC3 to GPC3-1 and GPC3-2 CARs expressed on the surface of Jurkat cells, for GPC3-1, K d The value is approximately 15 nM and for GPC3-2, K d The value is approximately 5 nM (see Figure 3A In surface plasmon resonance experiments using the same GPC3 protein and purified GPC3-1 / GPC3-2 scFv-Fc fusion protein as in cell-based binding experiments, for GPC3-1 and GPC3-2, K D The values ​​are approximately 73 nM and 11 nM, respectively. See Table 1 and... Figure 3B and 3C .

[0176] Table 1. Combined unit price value.

[0177]

[0178] Table 2 shows the Kd values ​​reported for the four scFvs.

[0179] Table 2. Dissociation constant (K) d ).

[0180] scFv <![CDATA[K d (nM)]]> GPC3-1 73 GPC3-2 11 GPC3-3 0.5 GPC3-4 12

[0181] Example 3. Development and in vitro study of CAR constructs.

[0182] summary

[0183] In this example, an anti-GPC3 CAR construct was developed and the resulting cytokine activity and versatility were tested.

[0184] method

[0185] CAR Structure. For all CAR constructs, the CSFR2 signal peptide (used in many clinical-stage CAR T constructs) was used. The IgG4P (S228P mutant) hinge domain served as a "spacer" between the scFv and the membrane, and the CD28 transmembrane domain was used. Intracellularly, different co-stimulatory domains (including different combinations of CD28, 4-1BB, and CD3ζ co-stimulatory domains) were tested. Constructs using co-stimulatory domains from inducible T cell co-stimulators (ICOS), OX40, and glucocorticoid-induced TNFR family-associated genes (GITR) were also explored. Sequences of the GPC3-1 and GPC3-2 CAR constructs are shown in SEQ ID NO: 3-10, and the corresponding nucleic acid sequences are shown in SEQ ID NO: 11-18.

[0186] Other known CARs targeting GPC3 (based on GPC3-3 and GPC3-4 scFv) were constructed for comparison with GPC3-1 and GPC3-2 CAR constructs. The GPC3-3 CAR contains a short IgG1 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular domain. Another GPC3-3 CAR construct containing both the CD28 and 4-1BB costimulatory domains was also developed. The GPC3-4 CAR construct contains an IgG4P hinge, a CD28 transmembrane domain, and a 4-1BB costimulatory domain. The sequences of the GPC3-3 and GPC3-4 CARs are shown in SEQ ID NOs: 19-21, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 22-24.

[0187] CAR T cell production. Purified human T cells were generated at 0.2 x 10⁻⁶. 6T cells were seeded at a concentration of 1 cell / 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 Dynabead (Invitrogen) and transduced by spinoculation after 24 hours. Lentiviral virus was added to wells (MOI 100), and the plates were centrifuged at 2000 rpm and 37°C for 2 hours and then incubated at 37°C with 5% CO2. Cells were separated as needed to maintain a density of approximately 0.5–1 x 10⁻⁶ cells / mL. 6 Cell density of cells / mL. Immunophenotyping of CAR-T cells was performed 7 days after transduction, and CAR-T cells were evaluated in in vitro and in vivo functional assays approximately 11 days after transduction.

[0188] Cell lines tested. Multiple cell types were treated with CAR T cells containing different CAR constructs. For all cytokine studies, 5x10 [cell lines were used]. 4 CAR-T cells and target cells were co-cultured at a 1:1 ratio in RPMI 10% FCS. After 24 hours, the supernatant was 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 / mL) were determined.

[0189] Cytotoxicity studies were conducted using cell impedance monitoring technology (xCELLigence). Plates were laid in 3x10⁻⁶ cells / mL. 4 Target cells were selected, and CAR-T cells were added 24 hours later at an effector-to-target (E:T) ratio of 3, 1, or 0.3. Normalized cell indices were determined for Hep3B, Huh7, and SNU-182 cells treated with various CAR constructs. Hep3B cells expressed high levels of GPC3 (14 kJ / cell), Huh7 cells expressed medium / low levels of GPC3 (7 kJ / cell), and SNU-182 cells were negative for GPC3 (0 kJ / cell).

[0190] Multifunctionality studies were also conducted on the CAR construct. Here, GPC3-1 BZ or designated CAR-T cells were co-cultured with Hep3B or A375 for 6 hours in the presence of Golgi Stop and a fluorophore-labeled antibody against the degranulation marker CD107a. Target binding induced CAR-T degranulation, followed by binding to the fluorescently labeled anti-CD107 present in the culture medium. CD107 accumulation, as detected by flow cytometry, was proportional to the degree of degranulation, and this accumulation indicated target cell lysis. Since the cells were incubated in the presence of Golgi Stop, the production of effector cytokines (IFN-γ, IL-2, TNF-α) was also assessed by intracellular staining. Boolean gating for each function (CD107a, IFN-γ, IL-2, and TNF-α) was generated using Flowtop, and pie charts of the results were generated using Spice analysis software.

[0191] result

[0192] Higher levels of TNFα and IL-2 output were observed overall in the GPC3-2 and GPC3-3 constructs compared to GPC3-1. Treatment of CAR T cells with the GPC3-1 and GPC3-2 CAR constructs produced antigen-specific cytokines. On the other hand, the GPC3-4 BZ construct induced cytokines even in GPC3-negative cell types. In the absence of a target, the GPC3-1 and GPC3-2 constructs did not produce cytokines. Cytokine production appears to be antigen-density and affinity dependent. See also Figure 4A and 4B .

[0193] The GPC3-1 and GPC3-2 constructs were cytotoxic only to cells expressing GPC3, while the GPC3-4 construct was cytotoxic to both GPC3-positive cells (Hep3B and Huh7) and GPC3-negative cells (SNU-182). The lower-affinity CAR (GPC3-1 BZ) showed equivalent cytotoxicity to the high-affinity (GPC3-2 BZ) CAR. See also Figure 5 .

[0194] GPC3-1 BZ demonstrates cytotoxicity against HCC cell lines expressing low levels of GPC3. All target cells tested were susceptible to GPC3-1 killing at E:T ratios of 3:1 and 0.3:1, with reduced killing observed only in one of the cell lines with lower GPC3 expression at an E:T ratio of 0.3:1. However, the completely comparable killing rates observed with our syngenes against GPC3-high and low Hep3B cell lines suggest that reduced antigen density itself is not a key factor limiting CAR-T-mediated cell lysis. See Figure 6.

[0195] Both GPC3-2 and GPC3-1 CAR T cells are pluripotent, regardless of the intracellular domains used. GPC3-1 BZ CAR T cells are pluripotent, with most cells exhibiting 2+ function. Furthermore, CAR T cells with CD28 exhibit lower pluripotency in vitro compared to CAR T cells with the 4-1BB intracellular domain. See also Figure 7 .

[0196] in conclusion

[0197] Treatment with GPC3-1 resulted in the lowest total cytokine production of the tested CAR. Both GPC3-1 and GPC3-2 are multifunctional and exhibit specific cytotoxicity against cells expressing GPC3.

[0198] Example 4: In vivo study of multiple CAR constructs in animal models of hepatocellular carcinoma

[0199] summary

[0200] In this example, the anti-GPC3 CAR construct was tested in vivo, and its effects on body weight, tumor size, and survival were compared.

[0201] method

[0202] 5x10 6 Hep3B cells were implanted into the flank of NSG mice (10 mice / group). When the tumor reached 150 mm... 3 At the mean volume, administer 4 million GPC3-2 BZ or GPC3-1 BZ to mice. Monitor body weight, tumor volume (2x / week), and survival. Supplement food for animals that lose 80% to 90% of their body weight; euthanize animals that lose less than 80% of their body weight. (The last sentence appears to be incomplete and possibly refers to a specific drug or treatment.) 3 Tumor size was used to determine survival events (death). Each experiment was performed twice.

[0203] result

[0204] Weight loss was observed using the high-affinity GPC3-2 construct (rather than the lower-affinity GPC3-1 construct), suggesting that the lower-affinity binder is less toxic in vivo. GPC3-2-based CAR T cells were intolerant at doses equivalent to those of GPC3-1-based CAR T cells in vivo. Greater toxicity of GPC3-2 BZ was associated with extensive infiltration of CAR T cells in the lungs of normal mice. Only moderate levels of infiltration were observed in the lungs of mice treated with GPC3-1 BZ. See also Figure 8A and 8B .

[0205] GPC3 CAR T induced Hep3B tumor regression in NSG mice. GPC3-1 BZ showed superior antitumor activity compared to GPC3-3 BZ and GPC3-4 BZ. See also Figure 9 .

[0206] GPC3-1 and GPC3-2 CAR T prolonged survival in tumor-bearing NSG mice to a greater extent than GPC3-3 or GPC3-4 CAR T; p < 0.01, relative to GPC3-3 BZ; Kaplan-Meier w / Mantel Cox log-rank. See also Figure 10 Similarly, in vitro and in vivo studies have determined that the absence of WPRE has no negative functional effect on GPC3-1 BZ cells.

[0207] in conclusion

[0208] Among the tested CARs, GPC3-1 BZ and GPC3-2 BZ showed the greatest antitumor activity and provided the greatest survival benefit. GPC3-1 BZ showed less toxicity and less infiltration into normal tissues than GPC3-2 BZ.

[0209] Example 5: In vivo comparison of GPC3-1 CAR constructs

[0210] summary

[0211] In this example, multiple GPC3-1 CAR constructs containing different signal transduction domains were tested and compared in vivo.

[0212] method

[0213] Differentiation and exhaustion analysis. Differentiation and exhaustion of multiple GPC3-1 CAR constructs were investigated. Mice with Hep3B tumors were treated with CAR-T cells containing different signal transduction domains (TZ = GPC3-1 TZ; BZ = GPC3-1 BZ; 28Z = GPC3-1 28Z), and the spleen and tumors were analyzed by flow cytometry 7 days after cell injection. Differentiation and exhaustion were determined using FACs that detected multiple markers in spleen and tumor cells. T cell differentiation status was analyzed by the co-expression of CD62L and CD45RO (CD62L+ / CD45RO- = naive; CD62L+ / CD45RO+ = central memory; CD62L- / CD45RO+ = effector memory; CD62L- / CD45RO- = effector memory cells reexpressing CD45RA (EMRA)). CD3% was used as a measure of persistence and expansion.

[0214] Inject mice with 5x10 6 One Hep3B cell was used to establish a cell line with an average size of 150 mm. 3 Tumors were treated. 4 million GPC3-1 BZ or GPC3-1 TZ T cells were administered to non-tumor-bearing mice or mice with Hep3B tumors. The effect on body weight in both tumor-bearing and non-tumor-bearing mice was measured until 35 days post-treatment. Tumor volume was also measured twice weekly. Animals were bled periodically post-treatment to analyze IFN-γ and TNF-α levels in the blood. Cytokines were analyzed in serum 8 days post-CAR-T administration. Each experiment was performed twice.

[0215] To investigate the potential for peripheral neurotoxicity in GPC3+ tumor-bearing (Hep3B HCC line) and non-tumor-bearing NSG mice, human anti-GPC3 CAR-T cells were administered to the animals. Histological examination was performed on tumor and enteric nerve tissue from animals with Hep3B tumors treated with GPC3-1 BZ.

[0216] Table 3. Research Design

[0217]

[0218] a Following the CAR-T response peak, but with harvestable tumors remaining; *Selected dose to completely eradicate Hep3B tumors; **GPC3-1 TZ contains a functional binding domain but lacks a signal transduction domain; **GPC3-1 BZ contains both functional binding and signal transduction domains.

[0219] result

[0220] In vivo, GPC3 CAR T cells with the 4-1BB / CD3ζ(BZ) signaling domain exhibited more central memory and less exhaustion than those with CD28 / CD3ζ(28Z). Results showed that GPC3-1BZ CAR T cells in the spleen were less differentiated than GPC3-128Z CAR T cells, while retaining the ability to be fully activated and differentiated in tumors in the presence of this antigen. See also Figure 11A-11D .

[0221] GPC3-1 BZ cells exhibit persistence. Expression of activation / exhaustion markers LAG3 and PD1 confirms that GPC3-1 BZCAR T cells maintain a lower percentage of activated / exhausted cells in the periphery. See also Figure 12 .

[0222] At tumor-regression doses, GPC3-1 BZ did not induce weight loss in either tumor-bearing or non-tumor-bearing mice. See also Figure 13 .

[0223] Complete tumor regression was observed only in mice treated with GPC3-1 BZ CAR T cells. See also Figure 14 .

[0224] Minimal systemic cytokines were detected (transiently elevated IFN-γ and TNF-α measured on day 8, 7 days post-infusion). Minimal and transient systemic cytokines were detected at effective CAR T doses, and no weight loss was observed. Following reduction-dose CAR treatment, human IFN-γ and TNF-α were the only cytokines detected transiently at elevated levels in serum. Other human or mouse cytokines (including hIL-2, mIL-10, mIL-6, mTNFα, and mIFNγ) were present at levels below the limit of detection (BDL). See also Figure 15 .

[0225] Tumor regression was accompanied by widespread T-cell infiltration and proliferation within the tumor. The tumor shrank (due to a reduction in neoplastic cells) and became necrotic and infiltrated by monocytes. The mice used lacked lymphocytes; therefore, any monocyte infiltration was assumed to be human CAR-T cells. See also Figure 16 The enteric nervous system, which expresses only low levels of GPC3, was unaffected. See also Figure 17 Minimal mononuclear cell infiltration was observed in the lungs and liver (data not shown).

[0226] in conclusion

[0227] Compared to constructs with other signal transduction domains, the GPC3-1 BZ construct exhibits persistence, promotes central memory responses, and demonstrates increased antitumor activity. Furthermore, treatment only induces a transient increase in some cytokines without causing weight loss. Following treatment, tumors become infiltrated by T cells and undergo necrosis, while normal tissue remains unaffected.

[0228] Example 6: Further characterization of GPC3-1 BZ CAR T cells

[0229] summary

[0230] In this example, GPC3-1 BZ CAR T cells were further characterized in response to treatment of various tumor types and cells with different GPC3 expression levels. Cytokine responses were analyzed.

[0231] method

[0232] Cytokine levels in response to GPC3-1 BZ CAR T cell treatment were investigated in tumor types with various GPC3 expression levels. Immunohistochemistry was also performed on representative Hep3B and Huh7 tumor xenografts.

[0233] GPC3 expression was also analyzed in cells within the tumor type. Staining intensity was graded from 1 to 4, where 1 is the lowest intensity and 4 is the highest intensity. Staining intensity 2 indicates low / intermediate intensity. Relative GPC3 expression was determined by FACs. GPC3 expression in Hep3B cells was determined by surface staining with a fluorophore-labeled anti-GPC3 antibody followed by flow cytometry analysis. Based on GPC3 expression, Hep3B cells were gated to low, intermediate, or high expression, and the frequency of GPC3 in each gate was plotted.

[0234] Cytokine levels were determined 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, the supernatant was 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 prior to cytokine analysis. Cytokine levels in different cell types were tested after GPC3-1 BZ treatment.

[0235] result

[0236] In GPC3-expressing cell lines, cytokine export induced by GPC3-1 BZ CAR T cells is proportional to surface GPC3 expression. See also Figure 18-22 .

[0237] GPC3-1 BZ CAR T cells did not elicit a cytokine response in GPC3-negative or normal tissues. See also Figure 23 and 24 .

[0238] in conclusion

[0239] GPC3-1 BZ CAR T cells induce cytokine output at levels proportional to GPC3 expression in the treated cells.

[0240] Example 7: Treatment with GPC3-1 CAR T cell construct and anti-cytokine antibody

[0241] summary

[0242] In this example, we attempted to combine GPC3-1 CAR T-cell therapy with an anti-cytokine antibody.

[0243] method

[0244] Tumors were treated with different combinations of GPC3-1 CAR T-cell therapy and anti-cytokine antibodies. Mice with Hep3B tumors (n=10 per group) were treated with 5 million GPC3-1 BZ or GPC3-1 TZ transduced cells (TZ = truncated CD3ζ, non-signal transduction negative control) in or without 100 μg of anti-human TNFα (golimumab, Janssen) or anti-mouse IL-6 (BioX Cell).

[0245] The drug resistance model of HCC, Huh7, was used to test high-dose (1e7-3e7) GPC3-1 CAR T combined with anti-TNF-α administration at two different times. Mice with Huh7 tumors (n=10 / group) were treated with a specified dose of GPC3-1BZ T cells (10 million or 30 million cells) and given 100 μg of anti-TNF-α on the same day as CAR T treatment (day 0) or two days after the start of treatment (day 2).

[0246] result

[0247] Blocking TNF-α but not IL-6 eliminated the efficacy of GPC3-1 BZ. See also Figure 25 .

[0248] Higher doses of CAR T cells were required to induce tumor growth inhibition in the drug-resistant HCC model Huh7, but higher doses were also associated with CRS-like symptoms and weight loss. Using delayed dosing reversed weight loss, allowing for tumor growth inhibition with anti-TNFα therapy. See also Figures 26A-26C .

[0249] in conclusion

[0250] Combining anti-TNFα therapy with GPC3-1 BZ therapy can mitigate the weight loss effects of high-dose CAR T-cell therapy.

[0251] The embodiments described herein can be practiced without the presence of any one or more elements or limitations not specifically disclosed herein. The terms and expressions used are descriptive and not restrictive, and are not intended to exclude any equivalents of the features shown and described or portions thereof when using such terms and expressions; however, it should be recognized that various modifications can be made within the scope of the claimed embodiments. Therefore, it should be understood that although the invention has been specifically disclosed by way of embodiments and optional features, modifications and variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to fall within the scope of the embodiments defined by the specification and appended claims. While some aspects of this disclosure may be considered particularly advantageous, it is contemplated that this disclosure is not limited to these specific aspects.

[0252] If one, more than one, or all members of the group are present in, used in, or otherwise related to the given product or method, then any claim or specification including "or" among one or more members of that group is considered satisfactory, unless otherwise specified or otherwise clearly apparent from the context. This disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to the given product or method. This disclosure includes embodiments in which more than one or all members of the group are present in, used in, or otherwise related to the given product or method.

[0253] Furthermore, this disclosure covers all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are incorporated into another claim. For example, any claim attached to another claim may be modified to include one or more limitations seen in any other claim attached to the same basic claim. Where elements are presented in a list (e.g., in Markush group form), each subgroup of elements is also disclosed, and any element may be removed from that group.

[0254] It should be understood that, generally, where a particular element and / or feature is referred to as being included in this disclosure or an aspect thereof, certain embodiments of this disclosure or an aspect thereof consist of or are substantially composed of such elements and / or features. For the purpose of brevity, these embodiments are not specifically described in the text herein.

[0255] All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as each individual patent and publication specifically and individually indicates that they are incorporated by reference. Any reference or designation of any reference in any part of this application should not be construed as an admission that such reference is prior art to this invention.

[0256] Table 3. Sequences used in the examples.

[0257]

[0258]

[0259] Table 4. Sequences

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

Claims

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

45.

2. The isolated nucleic acid as claimed in claim 1, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

3. The isolated nucleic acid as described in claim 2, wherein the antigen-binding domain is Fab or a single-stranded variable fragment (scFv).

4. The isolated nucleic acid as described in claim 3, wherein the antigen-binding domain is a scFv composed of the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO:

34.

5. The isolated nucleic acid as described in any of the preceding claims, wherein the isolated nucleic acid further encodes a transmembrane domain, a co-stimulatory domain, and a signaling domain.

6. The isolated nucleic acid of claim 5, wherein the encoded transmembrane domain comprises the CD28 transmembrane domain.

7. The isolated nucleic acid of claim 5, wherein the encoded co-stimulatory domain comprises one or more of the following co-stimulatory domains: CD28, 4-1BB, CD3ζ, OX-40, ICOS, CD27, GITR, and MyD88 / CD40.

8. The isolated nucleic acid of claim 5, wherein the encoded co-stimulatory domain comprises one or more of CD28, 4-1BB, and CD3ζ co-stimulatory domains.

9. The isolated nucleic acid of claim 5, wherein the encoded signal domain comprises a sequence encoding the CSFR2 signal peptide.

10. The isolated nucleic acid as described in any of the preceding claims, wherein the isolated nucleic acid further encodes a hinge / spacer subdomain.

11. The isolated nucleic acid of claim 10, wherein the encoded hinge / spacer domain is an IgG4P hinge / spacer.

12. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises 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.

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

45.

14. The anti-GPC3 CAR of claim 13, wherein the VH consists of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

15. The anti-GPC3 CAR of claim 13, wherein the VL consists of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

16. The anti-GPC3 CAR as described in any one of claims 13-15, wherein the CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain.

17. The anti-GPC3 CAR of claim 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.

18. A vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the nucleic acid comprises a 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.

19. A cell comprising the carrier as described in claim 18.

20. A cell comprising the isolated nucleic acid as claimed in any one of claims 1-12.

21. The cell of claim 20, wherein the nucleic acid comprises 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, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO:

34.

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

45.

23. The cell of claim 22, wherein the VH consists of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

24. The cell of claim 22, wherein the VL consists of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

25. The cell of any one of claims 22-24, wherein the CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain.

26. The cell according to any one of claims 22-24, 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.

27. The cell of any one of claims 19-24, wherein the cell is selected from the group consisting of T cells and natural killer (NK) cells.

28. The cell according to any one of claims 19-24, wherein the cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.

29. The cell of claim 27, wherein the cell exhibits antitumor immunity upon contact with tumor cells expressing GPC3.

30. The cell of claim 28, wherein the cell exhibits anti-tumor immunity upon contact with tumor cells expressing GPC3.

31. Use of a cell in the preparation of a drug for the treatment of hepatocellular carcinoma. The cell contains an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain, wherein the antigen-binding domain contains an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises CDR1, consisting of the amino acid sequence of SEQ ID NO: 37; CDR2, consisting of the amino acid sequence of SEQ ID NO: 38; and CDR3, consisting of the amino acid sequence of SEQ ID NO: 39; and The VL may contain CDR1 composed of the amino acid sequence of SEQ ID NO: 40, CDR2 composed of the amino acid sequence of SEQ ID NO: 41, and CDR3 composed of the amino acid sequence of SEQ ID NO: 42, or the VL may contain CDR1 composed of the amino acid sequence of SEQ ID NO: 43, CDR2 composed of the amino acid sequence of SEQ ID NO: 44, and CDR3 composed of the amino acid sequence of SEQ ID NO:

45.

32. The use as described in claim 31, further comprising inhibiting tumor growth, inducing tumor regression, and / or prolonging the survival of the subject.

33. The use as described in claim 31, wherein the cell is an autologous cell.

34. The use as described in claim 33, wherein the autologous cell is selected from the group consisting of T cells and natural killer (NK) cells.

35. The use as described in claim 33, wherein the autologous cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.

36. The use as described in any one of claims 31-35, wherein the drug is combined with an anti-TNFα antibody.

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