Compositions and methods for treating cancer with chimeric antigen receptors

Abstract

The present disclosure relates to compositions and methods for treating cancer using armored chimeric antigen receptor cells.

Description

[Technical Field]

[0001] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The above ASCII copy, created on April 12, 2021, is entitled CARTGPC(TGF)-WO-PCT_SL.txt and is 100,812 bytes in size.

[0002] The present disclosure relates to the treatment of cancer with chimeric antigen receptor T cells. [Background technology]

[0003] 1. Chimeric antigen receptor T cell therapy 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 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 function as living drugs, binding to and destroying cancer cells. If successful, the effects of CAR T-cell treatment tend to be long-lasting, as evidenced by the persistence and proliferation of CAR T cells detected in patients long after clinical remission.

[0004] 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. An ideal target antigen is widely expressed on tumor cells, allowing for targeting of a high percentage of cancer cells. Ideal candidate target antigens are also minimally expressed in normal tissues, limiting off-tumor 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.

[0005] The T cell activation domain of the CAR is intracellular and activates T cells in response to the antigen binding domain interacting with a target antigen. The T cell activation domain may contain one or more costimulatory domains, which are 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.

[0006] 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 the target antigen on tumor cells. The hinge domain can be used alone or in conjunction 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.

[0007] CAR T therapy directed against the B-lymphocyte antigen CD19 (Kymriah®, Novartis) has shown promise in 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 in 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. 124(18):2824-33, 2014), and mesothelin CAR T with pembrolizumab has demonstrated antitumor activity in mesothelioma. However, additional targets for treating solid tumors are needed.

[0008] 3. Challenges of CAR T cell therapy Unfortunately, the complexity of CAR T cell-based therapy can lead to unwanted and dangerous effects. Toxic effects, such as neurotoxicity and acute respiratory distress syndrome, are potential adverse and potentially fatal effects of CAR T cell therapy. 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. In patients with CRS, elevated serum levels of interleukin-2, interleukin-6, interleukin-1 beta, GM-CSF, and / or C-reactive protein are occasionally observed when assaying for these factors. CRS is classified by severity and diagnosed as grade 1 to 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 disease symptoms (Fitzgerald et al., Crit Care Med. 45(2):e124-e131(2017)).

[0009] Another challenge to the success of CAR T cell immunotherapy is immunosuppression caused by characteristics of the tumor microenvironment (TME) of solid tumors. For example, transforming growth factor-β (TGF-β) is a pleiotropic cytokine produced in large quantities by many cell types in the liver (e.g., liver sinusoidal endothelial cells, Kupffer cells, intrahepatic natural killer (NK) cells, and others) and within the tumor microenvironment (Dahmani et al., TGF-β in T Cell Biology: Implications for Cancer Immunotherapy. Cancers 2018, 10, 194, 1-21). TGF-β binds to TGFβR2, which recruits and phosphorylates TGFβR1. Upon phosphorylation, it phosphorylates receptor-regulated SMADs (R-SMADs). The phosphorylated SMAD complex with coSMADs translocates to the nucleus and helps regulate gene expression. In the context of T cells, TGF-β signaling suppresses the efficacy of CAR T cell therapy by inhibiting T cell proliferation, activation, and effector function and by favoring the differentiation of regulatory T cells. Thus, TGF-β-associated immunosuppression is a significant hurdle that must be overcome to achieve effective and durable CAR T cell therapy for solid tumors.

[0010] 4. Armoring A recent approach to producing CAR T cells that are more resistant to tumor-associated immunosuppression is called armoring. Armoring is the molecular engineering of CAR T cells to express one or more "armoring molecules" that can counteract immunosuppression. For example, investigators recently reported modifying CAR T cells to secrete a PD-1-blocking single-chain variable fragment (scFv), which binds PD-L1. +In mouse models of hematological and solid tumors, CAR T cell antitumor activity was improved (Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances antitumor efficacy in vivo. Nat Biotechnol 36, 847-856 (2018)). Other studies demonstrated the effectiveness of armoring T cells with a dominant-negative TGF-β receptor type 2 (TGFβRIIDN) armoring molecule to neutralize the inhibitory effects of TGF-β on T cells (Bollard et al., Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma, J Clin Oncol 36(11):1128-1139 (2018)). Currently, at least one clinical study is investigating the efficacy of arming anti-PSMA-CAR T cells with the TGFβRIIDN arming molecule (NCT03089203) to treat castration-resistant prostate cancer.

[0011] Therefore, additional CAR T cell therapies are needed to enhance the armourial basis of effective cancer treatment. Such therapies should include CAR T cells that effectively treat cancer while minimizing the risk of developing dangerous inflammatory responses, such as CRS. In addition, such therapies should include CAR T cells that can persist within the immunosuppressive TME of solid tumors. Summary of the Invention [Means for solving the problem]

[0012] The present disclosure describes compositions and methods for treating cancer using CAR T cells. As described below, in a first aspect, an isolated nucleic acid sequence is disclosed that encodes (a) a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for a cell surface antigen; and (b) an armoring molecule that counteracts cellular immune suppression when expressed on the surface of cells in the tumor microenvironment.

[0013] In another aspect, the present disclosure describes a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a TGFβRIIDN armoring molecule expressed on the surface of the cell.

[0014] In a further 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. Cells comprising the anti-GPC3 chimeric antigen receptor and TGFβRIIDN armoring molecule are described.

[0015] In yet another aspect, the present disclosure describes a method of treating cancer, comprising: administering to a subject in need of cancer treatment cells, the cells comprising (a) a chimeric antigen receptor (CAR) specific for a cell surface antigen, and (b) an armoring molecule that counteracts immune suppression of the cells in the tumor microenvironment of the cancer.

[0016] 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 description therein, and not by the specific discussion of the features and advantages described herein.

[0017] 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. [Brief explanation of the drawings]

[0018] [Figure 1A] Gene signatures of TGFβ1 and TGFβ, HCC progression, and survival. 1A. Fold change (log2) of TGFβ1 gene expression in normal solid tissue (left) and primary solid tumor of hepatocellular carcinoma (HCC, right). 1B. Gene signatures of TGFβ1 and TGFβ, HCC progression, and survival. Survival curves (OS = overall survival) of high vs. low TGFβ1 expression in HCC. 1C. Gene signatures of TGFβ1 and TGFβ, HCC progression, and survival. Fold change (log2) of TGFβ signaling in normal solid tissue (left) and solid tumor tissue (right). 1D. Gene signatures of TGFβ1 and TGFβ, HCC progression, and survival. Survival curves of high vs. low TGFβ signaling. [Figure 1B] This is a continuation of Figure 1A. [Figure 1C] This is a continuation of Figure 1B. [Figure 1D] This is a continuation of Figure 1C. [Figure 2] Human HCC samples are positive for TGF-β and TGF-β signaling. Semiquantitative pathological assessment of (2A) TGF-β1 and (2B) phosphorylated SMAD2 (pSMAD2) in FFPE normal liver and HCC specimens. Each circle represents data from one surgical resection. IHC intensity was defined as follows: score 0, negative staining; score 1, minimal staining; score 2, moderate staining; score 3, strong staining. [Figure 3]Armoring of GPC3 CAR T with TGFβRIIDN. As contemplated herein, armoring of GPC3 CAR T cells with TGFβRIIDN is believed to confer resistance to TGFβ, resulting in improved CAR T effector function and tumor control. 3A. Schematic of TGF-β-mediated immune suppression of non-armored CAR T cells (Figure adapted from Arrese et al, Current Protein & Peptide Science (2018) 19:1172). 3B. Diagram of an armored CAR T cell expressing a GPC3 CAR and a TGFβRIIDN armoring molecule. In some embodiments, the CAR and armoring molecule may be fused at the C-terminus or separated from the CAR by a spacer peptide. [Figure 4] Expression of TGFβRIIDN. Flow cytometry analysis of TGFβRII and CAR on the surface of unarmored and armored CAR T cells. [Figure 5] TGFβRIIDN suppresses TGF-β signaling in CAR T cells. 4B. Western blot analysis of phosphorylated SMAD2 / 3 and total SMAD2 / 3 in untransduced (UT); non-armored, and armored (TGFβRIIDN) CAR T cells 0-45 minutes after rhTGFβ (1 ng / ml) exposure. β-Actin served as a positive loading control for all samples. [Figure 6] Expression of TGFβRIIDN prevents TGF-β-induced suppression of effector cytokine transcription. (6A) IL2 and (6B) IFNG mRNA levels in purified TGFβRIIDN and non-armored GPC3 CAR T cells stimulated with the indicated concentrations of plate-bound recombinant GPC3 and TGF-β1 for 6 hours. Data are pooled from two independent experiments. [Figure 7]TGFβRIIDN reduces TGF-β-mediated differentiation of GPC3 CAR T cells toward a TRM phenotype. 7A. Flow cytometry analysis of TGF-β-mediated differentiation of GPC3 CAR T cells toward tissue-resident memory (TRM) cells in the absence (left scatter plot) and presence (right scatter plot) of TGF-β in non-armored (top scatter plot) and armored (bottom scatter plot) GPC3 CAR T cells. Y-axis = CD103 expression; X-axis = CAR expression. 7B. Percentage of TRM cell differentiation, evidenced by CD69+ / CD103+ co-expression, in non-armored (GPC3) CAR T cells and armored (GPC3 TGFβRIIDN) CAR T cells treated with TGF-β. [Figure 8] Exploratory in vitro readouts in non-armored and armored CAR T cells. 8A. Brightfield images of real-time monitoring of CAR T-mediated cytotoxicity during co-culture with GPC3+ tumor cells. CAR T cell proliferation was observed visually during co-culture with GPC3+ tumor cells. 8B. CAR T-mediated cytotoxicity during co-culture with GPC3+ tumor cells was assessed by xCelligence® real-time impedance-based killing assay (RTCA) and expressed as cell index. 8C. Quantification of CAR T cell proliferation induced by tumor cells (HEP3B or HUH-7 cells) in non-armored and armored GPC3 CAR T cells (AZ: in-house vector backbone; LN: Lentigen vector backbone) in the presence or absence of TGF-β. TGF-β suppresses tumor GPC3-induced proliferation of CAR T cells after 4 days in co-culture. Armored GPC3 CAR T cells were less susceptible to suppression. [Figure 9]In vivo reduction of tumor volume in a xenograft model using TGFβRIIDN-armored CAR T cells. Huh7-TGF-β overexpressing xenograft model. Untransduced T cells, unarmored GPC3 CAR T cells, or TGFβRIIDN-armored GPC3 CAR T cells were injected into Huh7 tumor-bearing mice engineered to overexpress TGF-β. Tumor volume was measured every two weeks (10 mice per group). [Figure 10] Increased number of tumor-infiltrating lymphocytes in mice treated with TGFβRIIDN-armored CAR T cells. Mice bearing Huh7-TGF-β-overexpressing tumors were administered 7×10 untransduced T cells, unarmored GPC3 CAR T, or TGFβRIIDN-armored GPC3 CAR T. 10A. Number of CAR+ cells in tumors of mice harvested at the indicated time points. 10B. Representative example of FACS data summarized in 10A. [Figure 11] Increased numbers of TGFβRIIDN-armed CAR T cells in the spleen. Huh7-TGF-β-overexpressing tumor-bearing mice were administered 7×106 untransduced T cells, unarmed GPC3 CAR T cells, or TGFβRIIDN-armed GPC3 CAR T cells. 11A. Number of CAR+ cells in the spleens of mice collected at the indicated time points. 11B. Representative example of FACS data summarized in 11A. [Figure 12] 12A. Ex vivo expression of TGFβRII on the surface of CAR+ cells. 12A. Expression of CAR and TGFβRII on the surface of spleen or tumor-infiltrating lymphocytes in Huh7-TGF-β overexpressing tumor-bearing mice 14 days after injection (FMO: fluorescence minus 1). 12B. Mean fluorescence intensity (MFI) of TGFβRII on the surface of CAR+ or CAR-CD8+ T cells in the tumor. [Figure 13]Reduced PD1 and LAG3 expression on the surface of TGFβRIIDN-armored CAR T cells within tumors. Expression of PD1 (A) and LAG3 (B) on the surface of tumor-infiltrating CD8+ and CD4+ CAR+ T cells in Huh7-TGF-β-overexpressing tumor-bearing mice 14 days after injection. [Figure 14] Decreased expression of CD70 and increased expression of CD27 on the surface of TGFβRIIDN-armored CAR T cells within tumors. Frequency of CD70+CD27- (A) and CD70+CD27- (B) on the surface of tumor-infiltrating CD8+ and CD4+CAR+ T cells 14 days after injection in Huh7-TGF-β-overexpressing tumor-bearing mice. [Figure 15] Immunophenotype of CAR T cells in the spleen. Expression of PD1 (15A), LAG3 (15B), and CD27 / CD70 (15C) on the surface of CD8+ and CD4+ CAR+ T cells in the spleen 14 days after injection in Huh7-TGF-β-overexpressing tumor-bearing mice. [Figure 16] Analysis of serum IFN-γ. Concentrations of IFN-γ detected in the serum of Huh7-TGF-β-overexpressing tumor-bearing mice injected with untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells (Baseline A: before tumor implantation; Baseline B: before CAR T injection). [Figure 17] Analysis of serum AFP. Concentrations of AFP detected in the serum of Huh7-TGF-β-overexpressing tumor-bearing mice injected with untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells (Baseline A: before tumor implantation; Baseline B: before CAR T injection). [Figure 18]In vivo reduction of tumor volume in TGFβRIIDN-armed CAR T cells and TGFβ(-) PDX models. Figures 18A-18C show tumor volumes in three different GPC3+TGF-β- hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5x106 untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells were injected into tumor-bearing mice, and tumor volumes were measured every two weeks (5 mice / group). [Figure 19-1] In vivo reduction of tumor volume in TGFβRIIDN-armored CAR T cells and TGFβ(+) PDX models. A-E represent tumor volumes of five different GPC3+TGF-β+ hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5×106 untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells were injected into tumor-bearing mice, and tumor volumes were measured every two weeks (5 mice / group). [Figure 19-2] This is a continuation of Figure 19-1. DETAILED DESCRIPTION OF THE INVENTION

[0019] 1.Definition 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 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 unless otherwise specified.

[0020] As used herein, the terms "comprise" and "include," and variations thereof (e.g., "comprises," "comprising," "includes," 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.

[0021] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] The percentages disclosed herein can vary in amounts of ±10, 20, or 30% from the disclosed values ​​and remain within the intended range of the disclosure.

[0023] 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, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0024] Ranges and amounts used herein 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 also "5%." The term "about" 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."

[0025] As used herein, the terms "or" and "and / or" can describe multiple elements in combination with each other or exclusively with 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."

[0026] 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 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 of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of or interchangeably with any of these terms.

[0027] 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.

[0028] An "isolated" material, e.g., an isolated nucleic acid, is a material that is not in its natural environment, but is 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 may be separated, fractionated, or at least partially purified by any suitable technique.

[0029] 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 a portion of the complementarity-determining regions (CDRs) of the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL), in the context of a typical antibody produced by a B cell. An antibody or antigen-binding fragment thereof may be, or may be derived from, a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, an epitope-binding fragment such as Fab, Fab', and F(ab'), Fd, Fv, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a fragment comprising the 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 a fragment 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. The numbering of amino acids within the variable domains, complementarity-determining regions (CDRs), and framework regions (FRs) of an antibody follows the Kabat definition as set forth in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise specified.

[0030] As used herein, the term "polynucleotide" includes single nucleic acids 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.

[0031] As used herein, the term "vector" may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain a nucleic acid sequence that enables it to replicate in a 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.

[0032] A "transformed" cell, or "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. All techniques by which a nucleic acid molecule can be introduced into such a cell are contemplated herein, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0033] 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 epitopes and paratopes (i.e., antigens and antigenic domains).

[0034] As used herein, the terms "treat," "treatment," or "treatment of," when used in the context of cancer treatment, refer to alleviating disease symptoms, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treatment can refer to the ability of a therapy, when administered to a subject, to alleviate symptoms, signs, or causes of disease. Treatment also refers to the alleviation or reduction of at least one clinical symptom, and / or the inhibition or delay of progression of symptoms, and / or the prevention or delay of the onset of a disease or disorder.

[0035] As used herein, the terms "subject," "individual," or "patient" refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Examples of mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0036] As used herein, an "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.

[0037] 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, for example, glypican 3 (GPC3). The CAR constructs, transformed cells expressing the constructs, and therapies utilizing the transformed cells disclosed herein can provide robust cancer treatments that minimize the risk of cytokine release syndrome (CRS) or indiscriminate cytokine release in non-GPC3-expressing cells.

[0038] 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 Wnt-Fzd interactions to stimulate Wnt signaling. GPC3 competes with patched for Hh binding, relieves smoothened inhibition, and induces GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) growth. GPC3 expression levels have also been shown to correlate with the stage and grade of HCC.

[0039] Furthermore, GPC3 is considered a promising target for CAR cell therapy, and therefore antibodies and CAR constructs derived from these antibodies are being developed as described herein.

[0040] Further embodiments of the present disclosure include CAR T cells, such as those targeting GPC3 or others, that are armored with TGFβRIIDN to protect the CAR T cells against TGF-β-associated immune suppression, e.g., of solid tumors.

[0041] 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. Selecting one component over another (i.e., selecting a particular costimulatory domain of one receptor over a costimulatory domain of a different receptor) can affect clinical efficacy and safety profile.

[0042] 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) containing the light and heavy chain variable regions from one or more antibodies specific for GPC3, linked either directly or via a flexible linker (e.g., one, two, three, or more repeats of GGGGS (SEQ ID NO: 48)).

[0043] The antigen-binding domain of the CAR disclosed herein may vary in binding affinity to the target 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)-CAR derived from a high-affinity scFv (dissociation constant 0.56 nM) showed an increased therapeutic index compared to low-affinity variants. Conversely, other examples have reported that engineering scFvs for lower affinity improves discrimination between cells with different antigen densities. This may be useful for improving therapeutic specificity for antigens differentially expressed in tumor versus normal tissues.

[0044] Various methods can be used to confirm the binding affinity of an antigen-binding domain. In some embodiments, a methodology can be used to exclude avidity effects. Avidity effects often require multiple antigen-binding sites, often in a multimerized structure, to simultaneously interact with multiple target epitopes. Therefore, avidity functionally represents the cumulative strength of multiple interactions. Examples of methodologies to exclude avidity effects include any approach 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.

[0045] 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, a proximal epitope may require a longer spacer, while a distal epitope may require a shorter epitope. 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 may have long, intermediate, or shorter spacers. A long spacer may include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (either native or with a modification common to therapeutic antibodies, such as the S228P mutation), although the CH3 region itself can be used to construct an intermediate spacer (approximately 120 amino acids). Shorter spacers can be derived from segments (<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.

[0046] 6. Hinge domain CARs may also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to a target antigen on a tumor cell. A flexible hinge domain can be used alone or in conjunction 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).

[0047] The CAR 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.

[0048] 7. Transmembrane domain The CAR may further comprise a sequence comprising a transmembrane domain. The transmembrane domain may comprise a hydrophobic α-helix spanning the cell membrane. The characteristics of the transmembrane domain have not been studied as extensively as other aspects of the CAR construct, but may potentially affect CAR expression and binding to endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0049] 8. Costimulatory Domain CARs may further comprise one or more sequences forming a costimulatory domain. A costimulatory domain is a domain that can enhance or modulate the response of immune effector cells. Costimulatory domains may include, for example, sequences derived 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 modified with CARs having a 4-1BB costimulatory domain tend to proliferate and persist longer in vivo, exhibit increased oxidative metabolism, are less susceptible to exhaustion, and have an increased ability to generate central memory T cells.

[0050] 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).

[0051] 10.CAR cell generation The CAR construct of the present disclosure can comprise any combination of the modular components described herein. For example, in some embodiments of the present disclosure, the CAR construct comprises a GPC3 scFv antigen-binding domain. In some embodiments, the CAR comprises a GPC3-2 scFv antigen-binding domain. In some embodiments of the present disclosure, the CAR construct comprises a CSFR2 signal peptide. In some embodiments, the CAR construct comprises an IgG4P hinge / spacer domain with an S228P mutation. In some embodiments, the CAR construct comprises a CD28 transmembrane domain.

[0052] 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.

[0053] 11. Evaluation of CAR Constructs Constructs of the present disclosure were compared and evaluated based on safety and persistence and establishment of central memory. GPC3, a lower affinity (higher off-rate) scFv, was favored due to its improved safety. The 4-1BB domain and CD3z costimulatory domain (both in the same construct) were favored based on their improved persistence and contribution to a favorable in vivo phenotype (greater central memory).

[0054] 12.CAR embodiment In some embodiments, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for a surface antigen on a tumor cell. In some embodiments, the cell surface antigen is a protein, a phosphorylated protein, a peptide-MHC, a carbohydrate, or a glycolipid molecule.

[0055] Examples of contemplated cell surface antigens include CD10, CD16, CD19, CD20, CD22, CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133, CD138, CD171, CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3, CDH3, CDH6, CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1, GPA33, GPC3, IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1 / EGFR, ERBB2 / Her-2, ERBB3, ERBB4, cMet, and EGFR. vIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV, HPV16-E6, HPV16-E7, IL13Ra2, Igκ chain, LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA. Other cell surface antigens are also contemplated herein.

[0056] In some embodiments, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for glypican 3 (GPC3). The antigen-binding domain has an equilibrium dissociation constant (K D ) is about 100 nanomolar (nM) or less, 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.

[0057] 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 CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40 costimulatory domains. In a 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.

[0058] In some embodiments, the isolated nucleic acid sequence may comprise a hinge / spacer domain. The hinge / spacer domain may be an IgG4P hinge / spacer.

[0059] 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.

[0060] In another embodiment, 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.

[0061] 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 comprise a transmembrane domain, a costimulatory domain, and a signaling domain.

[0062] In certain 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.

[0063] 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.

[0064] In other embodiments, the present 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.

[0065] In other embodiments, the present 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 D ) is about 100 nanomolar (nM) or less, and the CAR construct 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.

[0066] 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.

[0067] In some embodiments, the VH may have the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:29. In some embodiments, the VL may have the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30. The CAR may 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.

[0068] 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 may exhibit anti-tumor immunity when contacted with tumor cells that express GPC3.

[0069] 13. Treating 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 use described herein are particularly useful for inhibiting the proliferation or spread of neoplastic cells.In some aspects, they are particularly useful for inhibiting neoplastic cell proliferation in which GPC3 plays a role.

[0070] Neoplasms 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, examples of cancer types contemplated for treatment herein include NSCLC, advanced solid malignancies, bile duct neoplasms, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal neoplasms, 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 neoplasms, non-Hodgkin's lymphoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasms, pleural mesothelioma, prostate neoplasms, 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.

[0071] In one embodiment, the cancers intended to be treated herein include any cancer that expresses GPC3 on the cell surface of cancer cells. In one specific example, the cancers intended to be treated herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0072] 14. Armoring In some embodiments, the present disclosure provides "armored" cells, e.g., CAR T cells, with one or more genetic modifications that enhance or optimize cell function by protecting the cells from environmental insults, such as immunosuppressive cytokines or an immunosuppressive TME. Genetic modifications include, but are not limited to, cytokine secretion, enhanced expression of ligands that interact with immune cells, e.g., T cells, macrophages, and regulatory T cells, or altered functional characteristics. Those skilled in the art will appreciate that armoring cells, e.g., T cells, can provide many additional benefits not described herein that enable T cell survival in an immunosuppressive TME.

[0073] In some embodiments, the cells may comprise a chimeric antigen receptor (CAR) comprising a tumor-specific antigen-binding domain, the antigen-binding domain comprising an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); and a transforming growth factor beta (TGF-β) receptor type 2 dominant negative (TGFβRIIDN) armoring molecule.

[0074] In some embodiments, the armored cells may comprise 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 D ) is approximately 100 nanomolar (nM) or less, the CAR construct does not induce cytokine production in GPC3 cells, and the cells express the TGFβRIIDN armoring molecule.

[0075] In some embodiments, the armored cells may comprise an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain is a heavy chain variable region (VH) and a light chain variable region (VL) (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 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 antibody, Fab, or scFv; and a TGFβRIIDN armoring molecule.

[0076] 15. Treatment The CAR-modified cells of the present invention, such as CAR T cells, can be administered alone or as a pharmaceutical composition with a diluent and / or cytokines or other components associated with the cell population. Briefly, the pharmaceutical compositions of the present invention can include, for example, the CAR T cells 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 compositions of the present invention can be adapted for treatment (or prevention).

[0077] CAR-modified cells can also be administered in combination with one or more additional therapies. In one embodiment, the additional therapy can include an anti-cytokine antibody. For example, one or more anti-TNFα antibodies can be used to attenuate toxicity and promote anti-tumor activity at higher CAR T doses, which can be associated with CRS-like symptoms and weight loss.

[0078] The number of CAR cells administered per dose, the number of doses, and the frequency of administration will depend on various parameters, such as the patient's age, weight, clinical evaluation, tumor type, tumor burden, and / or other factors (including the judgment of the attending physician). Any acceptable route of administration is contemplated, including, but not limited to, intravenous (e.g., intravenous), parenteral, or subcutaneous routes of administration.

[0079] In certain embodiments, the contemplated treatment regimen may include one or more biological components, such as CAR T cells and anti-cancer antibodies, and / or chemotherapeutic components. For example, the treatment regimen could additionally include immune checkpoint inhibitors (ICIs), such as those targeting the PD-1 / PD-L1 axis (PDX), and other immuno-oncology (IO) treatments, such as immune system agonists.

[0080] Contemplated antibodies include anti-PD-L1 antibodies such as durvalumab (MEDI4736), avelumab, atezolizumab, KNO35, anti-PD-1 antibodies such as nivolumab, pembrolizumab, cemiplimab, 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. Any antibody subpart effective for treatment is also contemplated herein.

[0081] 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.

[0082] 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 (in which tremelimumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0083] Additional treatments (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 treatments that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora B protein kinase activity, and DNA-PK activity.

[0084] The therapeutic compositions or methods contemplated herein may be combined with any one or more of the other therapeutic compositions and methods provided herein.

[0085] 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 and an armoring molecule that, when expressed on the surface of the cell, counteracts cellular immunosuppression within the tumor microenvironment. In another aspect, the present disclosure provides that the antigen-binding domain can be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). The VH can 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 can 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 subject's survival.

[0086] In some embodiments, the armoring molecule is TGFβRIIDN.

[0087] 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.

[0088] In some embodiments, the cancer treated by this method is a solid tumor.For example, the cancer can be hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.In certain embodiments, the cancer is hepatocellular carcinoma.

[0089] It should be understood that the specific aspects described herein are not limited to the specific embodiments presented and may vary. It should also be understood that the terms used herein are for the purpose of describing specific aspects only and are 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 those skilled in the art. [Example]

[0090] The following examples illustrate specific embodiments of the present disclosure and various applications 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.

[0091] [Table 1]

[0092] Example 1: TGFβ gene expression and signal transduction in hepatocellular carcinoma overview In this example, TGFβ1 gene expression and TGFβ signaling were compared in normal liver versus hepatocellular carcinoma (LIHC).

[0093] method Data from the TCGA cohort were used in this analysis. TGFβ1 gene expression and TGFβ signaling signatures in normal liver and tumor tissues from TCGA were compared using t-tests. The TGFβ signaling signature was formed from the average expression levels of the following genes: TGFBR1, SMAD7, TGFB1, SMURF2, SMURF1, BMPR2, SKIL, SKI, ACVR1, PMEPA1, NCOR2, SERPINE1, JUNB, SMAD1, SMAD6, PPP1R15A, TGIF1, FURIN, SMAD3, FKBP1A, MAP3K7, BMPR1A, CTN. NB1, HIPK2, KLF10, BMP2, ENG, APC, PPM1A, XIAP, CDH1, ID1, LEFTY2, CDKN1C, TRIM33, RAB31, TJP1, SLC20A1, CD K9, ID3, NOG, ARID4B, IFNGR2, ID2, PPP1CA, SPTBN1, WWTR1, BCAR3, THBS1, FNTA, HDAC1, UBE2D3, LTBP2, and RHOA.

[0094] Kaplan-Meier analysis of overall survival (OS) was performed using LIHC data from TCGA. Data were grouped according to high (≥66th) and low (<66th) TGFβ1 gene expression and TGFβ signaling signature. P values ​​were calculated using the log-rank test.

[0095] result TGFβ1 gene expression was upregulated 1.74-fold in primary solid tumor cells (LIHC) compared with normal tissue. High expression of TGFβ1 in LIHC was associated with decreased OS (median, 47 months and 70 months, respectively) compared with LIHC expressing low levels of TGFβ1. See Figure 1A and Figure 1B.

[0096] TGFβ gene signaling was increased 1.1317-fold in primary solid tumors compared to normal tissues. High TGFβ signaling in LIHCs was associated with shorter cell survival compared to low TGFβ signaling (see Figure 1C and Figure 1D).

[0097] conclusion These results demonstrate a statistically significant correlation between increased TGFβ1 gene expression and TGFβ signaling with shorter overall survival in LIHC patients. Thus, greater TGFβ1 gene expression and TGFβ signaling may play a causative role in cancer-related death.

[0098] Example 2: TGF-β and signaling in hepatocellular carcinoma overview In this example, the expression of TGFβ and the intensity of TGFβ signaling (p-SMAD2) were compared in normal liver versus hepatocellular carcinoma (HCC) by immunohistochemical analysis.

[0099] method Three normal liver samples and 32 hepatocellular carcinoma samples were stained by immunohistochemistry and scored for intensity. TGF-β1 and pSMAD2 immunohistochemistry was performed on the Ventana Discovery platform using anti-TGF-β1 (Abcam) and anti-pSAMD2 (Cell Signaling Technology) antibodies. TGF-β1 and pSMAD2 expression in FFPE normal liver and HCC specimens was semiquantitatively scored by a pathologist. IHC staining intensity was defined as follows: score 0, negative staining; score 1, minimal staining; score 2, moderate staining; and score 3, strong staining.

[0100] result TGF-β was detected in 44% of tumors and 91% of stroma in HCC samples. TGF-β signaling (as determined by intensity phospo-SMAD2 (p-SMAD2)) was detected in 91% of HCC samples. Normal liver was negative for both TGF-β and p-SMAD2 (Figures 2A and 2B).

[0101] conclusion TGF-β was expressed and had active signaling in the majority of HCC samples, demonstrating that TGF-β is a common immunosuppressive factor in HCC tumors and that armoring may benefit a large population of HCC patients.

[0102] Example 3: Armoring of GPC3 CAR T cells by TGFβRIIDN overview In this example, we investigated the armoring of GPC3 BZ CAR T cells with TGFβRIIDN as a potential method to protect CAR T cells from TGFβ-mediated immune suppression and improve CAR T cell effector function and tumor control (see Figures 3A and 3B).

[0103] method TGFβRIIDN: A dominant-negative TGF-β receptor type 2 molecule was prepared by truncating the wild-type receptor at residue 194 so that the TGFβRIIDN receptor lacks the intracellular signaling domain.

[0104] Armored CAR T cells: GPC3 BZ CAR T cells were armored with TGFβRIIDN by expressing the TGFβRIIDN receptor as a C-terminal fusion to the GPC3 BZ CAR (a T2A peptide separates the GPC3 BZ CAR and the TGFβRIIDN receptor).

[0105] After expansion, CAR and TGFβRII expression was analyzed on the surface of unarmored and armored CAR T cells by flow cytometry. CAR expression was detected using the AF647 anti-idiotypic antibody against GPC3-CAR (Figure 4).

[0106] After staining with AF647 anti-idiotypic antibody against GPC3-CAR, CAR T cells were purified using anti-AF647 microbeads (Miltenyi). Purified cells were further expanded for 5 days, left overnight in the absence of IL-2 and serum, and stimulated with recombinant human TGF-β (1 ng / mL) for the indicated times. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and the expression of the indicated proteins was analyzed by Western blot (Figure 5).

[0107] Unarmored and armored CAR T cells were purified using anti-AF647 microbeads (Miltenyi) after staining with the AF647 anti-idiotypic antibody against GPC3-CAR. Purified cells were stimulated with plate-bound recombinant human GPC3 at the indicated concentrations (0.2 or 5 ng / mL) in the presence or absence of recombinant human TGF-β. After 6 hours, cells were harvested, and total RNA was obtained from the cells using the RNeasy Mini Kit (QIAGEN) and reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems) with the following TaqMan primers: GAPDH, Hs02758991_m1; IL2, Hs00174114_m1; and IFNG, Hs00989291_m1 (Figure 6).

[0108] CAR T cells were stimulated with anti-CD3 / CD28 beads (Dynabeads) in the presence of 20 ng / mL IL-15, without IL-2. After 3 days, TGFβ (50 ng / mL) was added, and cells were assessed for CD103 expression by flow cytometry after an additional 3 days of culture (Figure 7).

[0109] result Expression of TGFβRII on the surface of TGFβRIIDN CAR T. Surface staining of TGFβRII on unarmored and armored CAR T was assessed by flow cytometry. The antibody does not distinguish between endogenous TGFβRII and DN TGFβRII because they share the same extracellular moiety, but does distinguish between TGFβRIIDN CAR T co-expressed TGFβRII and CAR. This suggests that the antibody detects overexpressed DN receptors. See Figure 4.

[0110] TGFβRIIDN inhibits SMAD2 / 3 phosphorylation in CAR T cells after exposure to rhTGFβ: Unarmored CAR T cells demonstrated rhTGF-β-induced SMAD 2 / 3 phosphorylation similar to untransduced control cells at 0, 15, 30, and 45 minutes after rhTGF-β exposure. TGF-βRIIDN-armored T cells demonstrated attenuated SMAD 2 / 3 phosphorylation compared to unarmored CAR T cells and untransduced control cells at 15, 30, and 45 minutes after rhTGF-β exposure. Total SMAD 2 / 3 protein and β-actin expression were consistent between all groups. See Figure 5B.

[0111] Expression of TGFβRIIDN prevents TGF-β-mediated reduction in effector cytokine production. Stimulation with recombinant human GPC-3 induced transcription of the effector cytokines IFN-γ and IL-2 in CAR T cells. When present during stimulation, TGF-β reduced the levels of IFN-γ and IL-2 produced in non-armored CAR T cells, but not in armored CAR T cells. This result demonstrates that expression of TGFβRIIDN protects CAR T cells from the immunosuppressive effects of TGF-β. See Figures 6A and 6B.

[0112] Expression of TGFβRIIDN effectively inhibits TGFβ signaling. + T RM to CD103 -T cell differentiation requires IL-15 and TGF-β in vitro. Therefore, TGFβRIIDN CAR T cells are CAR - After incubation with TGF-β, T cells were significantly more potent than non-armored CAR T cells. RM These cells were unable to differentiate into TGFβRIIDN-induced cells. This result demonstrates that expression of TGFβRIIDN inhibits TGF-β-induced signaling during prolonged exposure (see Figures 7A and 7B).

[0113] conclusion TGFβRIIDN suppresses TGF-β signaling in armored CAR T cells, preventing the TGF-β-mediated decrease in effector cytokine production and enhancing the TGFβRIIDN-armored CAR T cells. RM Overall, these results demonstrate that expression of a dominant-negative TGFβRII is sufficient to inhibit TGFβ signaling and its biological effects.

[0114] Example 3: Cytotoxicity and proliferation of TGFβRIIDN-armed CAR T cells in co-culture with GPC3+ cells overview In this example, CAR T cell-mediated cytotoxicity and proliferation were compared between UT, non-armored CAR T cells, and TGFβRIIDN-armed CAR T cells during co-culture with GPC3+ hepatoma cells.

[0115] method T cells (20,000 CAR+ cells / well) were cocultured with a squamous cell carcinoma line engineered to express GPC3 (OE21 cells, 10,000 tumor cells per well) for 5 days on an xCELLigence eSight RTCA, and real-time tumor cell viability was simultaneously monitored by electrical impedance and CAR T cell density by microscopy (see Figure 8A). Subsequently, T cells (60,000 CAR+ cells / well) were cocultured with Hep3B (moderate / low GPC3 expression) or Huh7 (low GPC3 expression) cells (30,000 tumor cells / well) for 5 days on an xCELLigence RTCA-MP (no microscopy) (see Figure 8B). Non-adherent cells were then removed from the wells and viable CAR T were quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) (see Figure 8C).

[0116] result Dramatic proliferation of CAR T cells was observed during coculture with GPC3+ tumor cells (see Figure 8A). All CAR T cells efficiently killed GPC3+ tumor cells by coexpression of TGFβRIIDN or by the addition of exogenous TGF-β, and their cytolytic ability was not modulated (see Figure 8B). TGF-β suppressed tumor GPC3-induced proliferation of CAR T cells after 5 days of coculture. However, CAR T cells armed with TGFβRIIDN were less susceptible to this suppression (see Figure 8C).

[0117] conclusion TGFβRIIDN-armed CAR T cells exhibited significant cytotoxicity against GPC3+ tumor cells, indicating that expression of a dominant-negative receptor did not affect the ability of CAR T to kill target cells in vitro. Conversely, unlike non-armed CAR T cells, TGFβRIIDN-armed CAR T cells were significantly more cytotoxic to GPC3+ tumor cells. + The TGF-β-mediated inhibition of proliferation induced by α-glucan was not affected.

[0118] Example 4: In vivo armored CAR T cell xenograft model with TGFβRIIDN overview In this example, GPC3 + The efficacy of TGFβRIIDN-armed T cells against tumor cells was determined in vivo.

[0119] method The in vivo efficacy of TGFβRIIDN-armed T cells in reducing tumor volume was tested using the TGFβ-overexpressing hepatocellular carcinoma Huh7-TGFβ model. Tumor cells were implanted into the flank of NSG mice (10 mice / group). Tumors grew to 150 mm 3 When the average volume of 3, 7, or 21 x 10 6 Tumors were measured biweekly following administration of the indicated CAR T or 21 million untransduced T cells. Top graph: mean tumor volume per group. Bottom graph: tumor volume per individual mouse at the indicated doses (see Figure 9).

[0120] Ex vivo analysis was performed using 7 x 10 6 This was performed on Huh7-TGFβ tumor-bearing mice administered CAR T. Seven or 14 days after injection, tumors and spleens were collected from five mice per group. The number of CAR+ cells was calculated by flow cytometry after staining with AF647-labeled anti-idiotypic antibody against GPC3-CAR and using AccuCheck Counting Beads (see Figures 10 and 11).

[0121] Huh-7-TGFβ tumor-bearing mice received 7 × 10 6 CAR T cells were administered. 14 days after injection, tumors were harvested and TGFβRII expression was assessed by flow cytometry staining of CAR-positive and CAR-negative cells (see Figure 12).

[0122] Huh-7-TGFβ tumor-bearing mice received 7 × 10 6CAR T cells were administered. 14 days after injection, tumors were harvested and the expression of PD1, LAG3, CD27, and CD70 was assessed on the surface of the CAR T (see Figure 13 and Figure 14).

[0123] Huh-7-TGFβ tumor-bearing mice received 7 × 10 6 CAR T cells were administered. 14 days after infusion, spleens were collected and the expression of PD1, LAG3, CD27, and CD70 was assessed by flow cytometry staining (see Figure 15).

[0124] Huh-7-TGFβ tumor-bearing mice received 7 × 10 6 CAR T cells were administered. For serum cytokine and AFP analysis, blood was collected in small volumes at the indicated time points, and serum was separated using BD Microtainer Serum Separator Tubes. Cytokine levels were determined using an MSD assay, while AFP was assessed by sandwich ELISA. Five mice per group were bled before tumor implantation (baseline A), before CAR T infusion (baseline B), and 7 and 14 days after infusion (see Figures 16 and 17).

[0125] result Non-transduced T cells had no discernible effect on tumor growth. Treatment with unarmored CAR T cells resulted in a minor reduction in tumor volume at the lowest dose (3 million cells / mouse), while a more significant effect culminating in complete tumor regression was evident at higher doses. In contrast, administration of TGFβRIIDN-armored CAR T cells induced a significant reduction in tumor volume and complete regression at even lower doses. Furthermore, progression-free survival was significantly prolonged at all treatment doses of TGFβRIIDN-armored CAR T cells compared to unarmored CAR T (see Figure 9). Ex vivo analysis of CAR T cells was performed using 7x10 6This was performed in mice injected with TGFβRIIDN-armed CAR T cells. Consistent with the enhanced efficacy, an increased number of tumor-infiltrating lymphocytes (TILs) was detected in TGFβRIIDN-treated mice 7 days after injection, indicating an ongoing active immune response and associated proliferation that plateaued 14 days after treatment (see Figure 10). Conversely, an increased number of CAR T cells was observed in the spleens of mice treated with TGFβRIIDN-armed CAR T cells 14 days after injection, suggesting the increased number of CAR T cells and more CAR T cells. +This suggests that enhanced proliferation by TGFβRIIDN-mediated CAR T cells was therefore detectable in the circulation (see Figure 11). To investigate whether TGFβRIIDN is detectable on CAR T cells after in vivo expansion, we analyzed the expression of TGFβRII on TILs and lymphocytes in the spleen 14 days after infusion. While TGFβRII was barely detectable on non-armored cells, lymphocytes from mice treated with armored CAR T cells co-expressed CAR and TGFβRII. Therefore, it is reasonable to assume that TGFβRII expressed on TGFβRIIDN TILs is a dominant-negative receptor that is detectable ex vivo after an active immune response and associated antigen-dependent proliferation (see Figure 12). Notably, TGFβRIIDN TILs expressed low levels of the exhaustion markers LAG3 and PD1 (see Figure 13), the latter of which is directly regulated by TGF-β in a SMAD3-dependent manner (Park, BV, Freeman, ZT, Ghasemzadeh, A., Chattergoon, MA, Rutebemberwa, A., Steigner, J. et al. (2016). TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer. Cancer Discov, 6(12), 1366-1381). Furthermore, TGFβRIIDN TILs expressed less CD70 and more CD27 than non-armored TILs (see Figure 14).This result is consistent with previous evidence showing that TGF-β upregulates CD70 expression, inducing exhaustion of effector memory T cells, and reinforces the notion that expression of a dominant-negative receptor protects CAR T cells from TGF-β-mediated immunosuppression (Yang, ZZ., Grote, D., Xiu, B. et al. TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma. Leukemia 28, 1872-1884 (2014). https: / / doi.org / 10.1038 / leu.2014.84). In contrast to TILs, CAR T cells in the periphery did not express co-inhibitory markers or CD70 and were consistently mostly CD27 positive in the non-activated state (see Figure 15). Consistent with the enhanced efficacy and higher numbers of TILs, more IFN-γ was detected in the serum of mice injected with TGFβRIIDN CAR T 7 days after injection, and a dramatic decrease in serum concentrations of the tumor marker AFP 14 days after injection (see Figures 16 and 17).

[0126] These results demonstrate that expression of TGFβRIIDN increases the efficacy of CAR T therapy by counteracting the immunosuppressive effects of TGF-β in vivo.

[0127] conclusion TGFβRIIDN-armed CAR T cells show considerable promise as an effective in vivo treatment of GPC3+ tumors.

[0128] Example 5: In vivo armoring of CAR T cells with TGFβRIIDN - Hepatocellular carcinoma patient-derived xenograft model overview In this example, several GPC3 + The efficacy of TGFβRIIDN-armored CAR T cells in xenograft cells from hepatocellular carcinoma patients was determined in vivo.

[0129] method This study was conducted by Crown Bioscience Inc. Eight PDX models were selected based on the expression of GPC3 and TGF-β, assessed by IHC and RNA sequencing performed by Crown Bioscience. All selected models were high in GPC3 (IHC score >100), but three of them did not express TGF-β (IHC score <5), while the other five models were TGF-β positive (IHC score >20). Following the Crown Bioscience research protocol, tumor fragments from stock mice were collected and used for inoculation into NCG mice. Each mouse was subcutaneously inoculated with a specific PDX tumor fragment (3 x 3 x 3 mm) for tumor development in the right front flank. The average tumor size was approximately 150-250 mm. 3 Mice were randomized when tumor volume reached 5. Tumor-bearing mice were administered 5 million unarmored or armored CAR T cells and untransduced T cells provided by AstraZeneca (5 mice / group) and tumor volumes were measured every two weeks (see Figures 18 and 19).

[0130] result Tumors grew in mice that received non-transduced T cells. However, in the absence of TGF-β, non-armored and armored cells were equally effective, inducing rapid and complete regression in all mice included in the study (see Figure 18). When injected into a TGF-β-expressing model, non-armored CAR T cells were significantly less effective. In contrast, TGFβRIIDN-armed CAR T cells were consistently more potent and induced significant tumor regression.

[0131] conclusion PDX models simulate human tumor biology, allowing for natural cancer progression. Thus, these observations confirm and reinforce the evidence obtained with the Huh7-TGF-β xenograft model. Overall, these data demonstrate that TGFβRIIDN-armed GPC3 CAR T cells are able to effectively inhibit GPC3 + It may be an effective in vivo treatment for tumors and demonstrates that it can remain effective even in the presence of the immunosuppressive factor TGF-β.

[0132] 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 the embodiments as defined by the description and the appended claims. While some 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.

[0133] A claim or description including "or" between one or more members of a group is deemed to be satisfied if 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 the context makes clear otherwise. 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.

[0134] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms of one or more recited 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.

[0135] Generally, when the present disclosure or aspects of the present disclosure are referred to as comprising 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 have not been specifically described in these terms herein.

[0136] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or 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.

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Claims

1. a) a chimeric antigen receptor (CAR) comprising an scFv encoded by the nucleic acid sequence of SEQ ID NO: 33 specific for glypican 3 (GPC3); and b) An armoring molecule that counteracts immunosuppression of a cell when expressed on the surface of the cell in a tumor microenvironment, the armoring molecule being a TGF-β receptor type 2 dominant negative (TGFβRIIDN) consisting of the amino acid sequence of positions 478 to 671 in SEQ ID NO:

47. An isolated nucleic acid encoding

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

3. The isolated nucleic acid of claim 2 , wherein the transmembrane domain comprises a CD28 transmembrane domain.

4. 3. The isolated nucleic acid of claim 2, wherein the costimulatory domain comprises one or more of CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains.

5. 3. The isolated nucleic acid of claim 2, wherein the costimulatory domain comprises one or more of a CD28, 4-1BB, and CD3 zeta costimulatory domain.

6. 3. The isolated nucleic acid of claim 2, wherein the signal domain comprises a sequence encoding a CSFR2 signal peptide.

7. The isolated nucleic acid of any one of claims 1 to 6, further comprising a hinge / spacer domain.

8. 8. The isolated nucleic acid of claim 7, wherein the hinge / spacer domain is an IgG4P hinge / spacer.

9. The isolated nucleic acid of claim 1, encoded by the nucleic acid sequence of SEQ ID NO:

46.

10. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific to glypican 3 (GPC3) and an armoring molecule, wherein the nucleic acid sequence comprises SEQ ID NO:

46.

11. A cell comprising the vector of claim 10 or the isolated nucleic acid of any one of claims 1 to 9.

12. 10. The isolated nucleic acid of claim 1 encoding a chimeric antigen receptor (CAR), and TGFβRIIDN Armoring Molecules Expressed on the Surface of Cells Cells containing

13. The cell of claim 12 , wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain.

14. The cell of claim 13 , wherein the isolated nucleic acid comprises the sequence of SEQ ID NO:

46.

15. 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; 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; and A TGFβRIIDN armoring molecule comprising the amino acid sequence of positions 478 to 671 in SEQ ID NO:

47. Cells containing

16. The cell of claim 15, wherein the VH comprises the amino acid sequence of SEQ ID NO:

27.

17. The cell of claim 15, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 and the VH comprises the amino acid sequence of SEQ ID NO:

27.

18. The cell of any one of claims 15 to 17, wherein the anti-GPC3 chimeric antigen receptor (CAR) and TGFβRIIDN armoring molecule comprise the amino acid sequence of SEQ ID NO:

47.

19. The cell of any one of claims 15 to 18, 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.

20. 1. A pharmaceutical composition for treating cancer, comprising:

20. A pharmaceutical composition comprising the cells of claim 19, wherein the cells are administered to a subject in need of treatment for cancer.

21. 21. The pharmaceutical composition of claim 20, further for inhibiting tumor growth, inducing tumor regression, and / or prolonging survival in said subject.

22. 22. The pharmaceutical composition of claim 21, wherein the cells are autologous cells.

23. 23. The pharmaceutical composition of claim 22, 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.

24. The pharmaceutical composition according to any one of claims 20 to 23, wherein the cancer is a solid tumor.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

26. 24. The pharmaceutical composition of claim 23, wherein the cancer is hepatocellular carcinoma.

27. The pharmaceutical composition of any one of claims 20 to 26, wherein a therapeutically effective amount of an anti-cancer antibody and / or a chemotherapeutic component is further administered to the subject.

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