IgG4 hinge-containing chimeric antigen receptor targeting glypican-3 (GPC3) and uses thereof

Abstract

Described is an optimized chimeric antigen receptor (CAR) that targets glypican-3 (GPC3) and has a 12-amino acid hinge region derived from human IgG4.The optimized CAR also contains a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain derived from either CD8 or CD28.Immune cells or induced pluripotent stem cells expressing the optimized CAR can be used to treat GPC3-positive solid tumors.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 277,287, filed November 9, 2021, which is incorporated by reference in its entirety.

[0002] Field The present disclosure relates to an optimized chimeric antigen receptor (CAR) specific for the tumor antigen glypican-3 (GPC3) that contains an IgG4-derived hinge region. The present disclosure further relates to the use of GPC3-targeting IgG4 hinge-containing CARs, such as for the treatment of solid tumors.

[0003] Acknowledgement of Government Support This invention was made with Government support under Project No. Z01 BC010891 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] background Hepatocellular carcinoma (HCC) is a highly aggressive tumor with a poor prognosis. Immunotherapy using chimeric antigen receptor (CAR) engineered T lymphocytes has shown remarkable responses in hematopoietic malignancies. However, for solid tumors, there are significant barriers to clinical success. The main barrier in most solid tumors is high toxicity due to widespread biomarker expression both on cancer cells and on normal tissues. This is not the case for HCC, as one of the best-characterized tumor-associated antigens, glypican 3 (GPC3), is highly specific for HCC. GPC3 (cell surface oncofetal protein) is highly upregulated in HCC but is not expressed or expressed at low levels in normal tissues. Thus, targeting GPC3 is uniquely capable of delivering therapeutic agents exclusively to HCC sites and not to normal tissues.

[0005] GPC3-targeted CAR T cells have demonstrated limited efficacy in phase 1 clinical trials, likely due to both a lack of adequate tumor infiltration and an inability to induce a sustained and robust immune response within the tumor. Therefore, there is a need to develop CAR T cells with improved in vitro and in vivo potency. Summary of the Invention [Means for solving the problem]

[0006] Abstract Disclosed herein is an optimized GPC3-specific chimeric antigen receptor (CAR) that contains a hinge region derived from human IgG4 and a transmembrane domain derived from either human CD8 or human CD28. Demonstrated herein is that IgG4-hinge-containing CARs targeting GPC3 specifically lyse GPC3-positive cells in vitro and are highly effective in eradicating GPC3-positive tumors in animal models.

[0007] Provided herein is a CAR comprising an extracellular antigen-binding domain specific for GPC3; an IgG4 hinge region; a transmembrane domain; an intracellular costimulatory domain; and an intracellular signaling domain. In some embodiments, the CAR comprises a hinge region consisting of a modified IgG4 hinge sequence as set forth in SEQ ID NO: 43. In other embodiments, the CAR comprises a hinge region consisting of a wild-type IgG4 sequence as set forth in SEQ ID NO: 52. In some embodiments, the antigen-binding domain comprises the CDR sequence of the GPC3-specific single-domain antibody HN3 or the CDR sequence of the VH and VL of the GPC3-specific antibody hYP7, YP7, YP9, YP8, YP6, YP9.1, or HS20. In some examples, the transmembrane domain of the CAR is a CD28 transmembrane domain. In other examples, the transmembrane domain of the CAR is a CD8 transmembrane domain.

[0008] Further provided is a nucleic acid molecule encoding the disclosed CAR. In some embodiments, the nucleic acid molecule comprises, from 5' to 3', a nucleic acid encoding a first granulocyte macrophage colony stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding an antigen binding domain; a nucleic acid encoding an IgG4 hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding a costimulatory domain; a nucleic acid encoding a signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding a truncated human epidermal growth factor receptor (huEGFRt). In some examples, the nucleic acid molecule further comprises a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss. Further disclosed is a vector (such as a lentiviral vector) comprising the disclosed nucleic acid molecule.

[0009] Also provided are isolated immune cells (such as T cells, B cells, NK cells, or macrophages) and induced pluripotent stem cells (iPSCs) that express a CAR disclosed herein and / or contain an isolated nucleic acid molecule or vector disclosed herein.

[0010] Further provided is a composition comprising a pharma- ceutically acceptable carrier and a CAR, a nucleic acid molecule, a vector, or a cell disclosed herein.

[0011] Also provided is a method for treating GPC3 positive cancer in a subject or inhibiting tumor growth or metastasis of GPC3 positive cancer.In some embodiments, this method comprises administering to a subject a therapeutically effective amount of CAR, nucleic acid molecule, vector, cell or composition disclosed herein.In some examples, the GPC3 positive cancer is a solid tumor, such as hepatocellular carcinoma (HCC).

[0012] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0013] [Figure 1A]Figure 1A-1B: Jurkat binding assay. Jurkat T cells expressing hYP7-CD8H-CD8TM, HN3-CD8H-CD8TM, HN3-IgG4H-CD28TM, HN3-IgG4H-CD8TM, or HN3-CD8H-CD28TM CARs were exposed to GPC3-hFc (Figure 1A) or GPC1-hFc (Figure 1B). All hYP7 and HN3 CAR-expressing Jurkat cells bound specifically to GPC3-hFc. [Figure 1B] Same as above. [Figure 2A] Figures 2A-2D: (Figure 2A) Schematic representation of CAR T cell constructs. Constructs contained either HN3 or hYP7 with a hinge (H) of CD8 or IgG4, and a transmembrane (TM) domain of CD8 or CD28. (Figure 2B) Depiction of CAR T cell constructs in the cell membrane. (Figure 2C) Cell counts of CAR-expressing cells monitored over 11 days. (Figure 2D) Transduction efficiency of CAR constructs as measured by CAR-positive cells at day 8. These results demonstrate that engineered CARs can be expressed in donor T cells. [Figure 2B-C] Same as above. [Figure 2D] Same as above. [Figure 2E-F] Figures 2E-2H: Graphs showing cell killing induced by CAR T cells cultured with GPC3-expressing HCC cell lines Hep3B (Figure 2E), Huh7 (Figure 2G), and HepG2 (Figure 2H) or GPC3-negative Hep3B-GPC3-KO-C3 cells (Figure 2F). Specific lysis was measured at different effector-to-target ratios. Engineered CAR T cells potently killed Hep3B, Huh7, and HepG2 cells, but not Hep3B GPC3 knockout (KO) cells, demonstrating antigen-specific killing. [Figure 2G-H] Same as above. [Figure 3A-B]Figures 3A-3D: (Figure 3A) Schematic of the experimental study design using the NOD-SCID-Gamma (NSG) mouse model. Hep3B GFP / luciferase expressing cells (3 million) were injected IP into NSG mice and allowed to engraft for 12 days. Mice were treated with 5 million hYP7-CD8H-CD8TM, hYP7-IgG4H-CD28TM, HN3-IgG4H-CD8TM, or HN3-IgG4H-CE28TM CAR T cells on day 0 and imaged periodically. (Figure 3B) Bioluminescence images showing tumor size. (Figure 3C) Bioluminescence quantification of imaging results over 35 days. (Figure 3D) Survival curves over the course of the study. Results showed that within 10 days, HN3-IgG4H-CD28TM CAR T cells completely eradicated the tumor and mice remained tumor-free over the course of the study. [Figure 3C-D] Same as above. [Figure 4A-B] Figures 4A-4D: (Figure 4A) Schematic of the experimental study design using the NSG mouse model. Hep3B GFP / luciferase expressing cells (3 million) were injected IP into NSG mice and allowed to engraft for 12 days. Mice were treated with 5 million HN3-CD8H-CD8TM, HN3-IgG4H-CD28TM, HN3-IgG4H-CD28TM-M, or HN3-IgG4H-CD28TM-L CAR T cells on day 0 and imaged periodically. (Figure 4B) Bioluminescence imaging results showing tumor size. (Figure 4C) Bioluminescence quantification of imaging results over 15 days. (Figure 4D) Survival curve over the entire course of the study (67 days). Results showed that within 7 days, HN3-IgG4-CD28TM CAR T cells eliminated tumors and treated mice remained tumor-free for the duration of the study. Furthermore, mice treated with HN3-IgG4H-CD28TM CAR T cells exhibited the highest survival rate, with approximately 40% of mice in this group surviving and remaining tumor-free for at least the 67-day study period. [Figure 4C-D] Same as above. [Figure 5A-B]Figure 5A-5F: (Figure 5A-5B) Analysis of CD4 and CD8 T cells and T cell subsets. T cell subsets consist of stem cell memory cells (Tscm), central memory cells (Tcm), effector memory re-expressing CD45RA cells (Temra), and effector memory cells (Tem). (Figure 5A) At week 2, T cells had similar CD4 / CD8 ratios. Temra and Tem dominated the T cell landscape. (Figure 5B) At week 5, CD8 T cells were most abundant. Temra cells were enriched and involved in proliferation, memory, and effector functions. (Figure 5C) Time course of CAR T cell counts from mouse blood. (Figure 5D) Time course of PD1 expression on T cells from mouse blood. (Figure 5E) Exhaustion marker panel at week 5. (Figure 5F) Killing of Hep3B tumor cells induced by CAR-expressing T cells. The HN3-IgG4H-CD28TM construct showed the highest induction of tumor cell lysis. [Figure 5C-D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 6A-B] Figures 6A-6D: (Figure 6A) Schematic of experimental study design using NSG mouse model. Mice were IP injected with Huh7 GFP / luciferase expressing cells (day -12) and allowed to engraft for 12 days. Mice were IP injected with 15 million untransduced T cells or hYP7-CD8H-CD8TM or HN3-IgG4H-CD28TM CAR T cells on day 0 and imaged periodically over 4 weeks. (Figure 6B) Bioluminescence imaging results showing tumor size. (Figure 6C) Survival curve over the entire course of the study (28 days post-treatment). (Figure 6D) Tumor volume at the end of the 28-day study. [Figure 6C-D] Same as above. [Figure 7A] Figures 7A-7B: (Figure 7A) Western blot of active and total β-catenin, GPC3, and GAPDH in tumor cells exposed to CAR T cells for 30 minutes and 3 hours. (Figure 7B) NFAT signaling in CAR T cells when CAR T cells were cultured with Hep3B tumor cells. [Figure 7B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Sequence Listing The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. The sequence listing is submitted as an XML file (filename 4239-107164-02.xml (91,430 bytes), created October 25, 2022), and is incorporated herein by reference. In the attached sequence listing: SEQ ID NO:1 is the nucleic acid sequence encoding HN3-CD8H-CD8TM. SEQ ID NO:2 is the amino acid sequence of HN3-CD8H-CD8TM. SEQ ID NO:3 is the nucleic acid sequence encoding HN3-CD8H-CD28TM. SEQ ID NO:4 is the amino acid sequence of HN3-CD8H-CD28TM. SEQ ID NO:5 is the nucleic acid sequence encoding HN3-IgG4H-CD28TM. SEQ ID NO: 6 is the amino acid sequence of HN3-IgG4H-CD28TM. SEQ ID NO:7 is the nucleic acid sequence encoding HN3-IgG4H-CD8TM. SEQ ID NO: 8 is the amino acid sequence of HN3-IgG4H-CD8TM. SEQ ID NO: 9 is the nucleic acid sequence encoding HN3-IgG4H-CH3-CD28TM. SEQ ID NO: 10 is the amino acid sequence of HN3-IgG4H-CH3-CD28TM. SEQ ID NO:11 is the nucleic acid sequence encoding HN3-IgG4H-CH2CH3-CD28TM. SEQ ID NO: 12 is the amino acid sequence of HN3-IgG4H-CH2CH3-CD28TM. SEQ ID NO:13 is the nucleic acid sequence encoding hYP7-IgG4H-CD28TM. SEQ ID NO: 14 is the amino acid sequence of hYP7-IgG4H-CD28TM. SEQ ID NO:15 is the nucleic acid sequence encoding hYP7-CD8H-CD8TM. SEQ ID NO: 16 is the amino acid sequence of hYP7-CD8H-CD8TM. SEQ ID NO:17 is the nucleic acid sequence encoding the antibody HN3. SEQ ID NO: 18 is the amino acid sequence of antibody HN3. SEQ ID NO:19 is a nucleic acid sequence encoding the VH domain of antibody hYP7. SEQ ID NO:20 is the amino acid sequence of the VH domain of antibody hYP7. SEQ ID NO:21 is a nucleic acid sequence encoding the VL domain of antibody hYP7. SEQ ID NO: 22 is the amino acid sequence of the VL domain of antibody hYP7. SEQ ID NO:23 is a nucleic acid sequence encoding the VH domain of antibody YP9.1. SEQ ID NO: 24 is the amino acid sequence of the VH domain of antibody YP9.1. SEQ ID NO:25 is a nucleic acid sequence encoding the VL domain of antibody YP9.1. SEQ ID NO: 26 is the amino acid sequence of the VL domain of antibody YP9.1. SEQ ID NO:27 is a nucleic acid sequence encoding the VH domain of antibody YP8. SEQ ID NO: 28 is the amino acid sequence of the VH domain of antibody YP8. SEQ ID NO:29 is a nucleic acid sequence encoding the VL domain of antibody YP8. SEQ ID NO: 30 is the amino acid sequence of the VL domain of antibody YP8. SEQ ID NO:31 is a nucleic acid sequence encoding the VH domain of antibody YP9. SEQ ID NO: 32 is the amino acid sequence of the VH domain of antibody YP9. SEQ ID NO:33 is the nucleic acid sequence encoding the VL domain of YP9 clone 9. SEQ ID NO: 34 is the amino acid sequence of the VL domain of YP9 clone 9. SEQ ID NO:35 is the nucleic acid sequence encoding the VL domain of YP9 clone 10. SEQ ID NO:36 is the amino acid sequence of the VL domain of YP9 clone 10. SEQ ID NO:37 is the nucleic acid sequence encoding the VL domain of YP9 clone 1. SEQ ID NO:38 is the amino acid sequence of the VL domain of YP9 clone 1. SEQ ID NO:39 is a nucleic acid sequence encoding the VH domain of antibody HS20. SEQ ID NO: 40 is the amino acid sequence of the VH domain of antibody HS20. SEQ ID NO: 41 is a nucleic acid sequence encoding the VL domain of antibody HS20. SEQ ID NO: 42 is the amino acid sequence of the VL domain of antibody HS20. SEQ ID NO: 43 is the amino acid sequence of a modified IgG4 hinge region. SEQ ID NO: 44 is the amino acid sequence of GMSCFRss. SEQ ID NO: 45 is the amino acid sequence of the CD8 alpha hinge. SEQ ID NO: 46 is the amino acid sequence of the CD28 transmembrane domain. SEQ ID NO: 47 is the amino acid sequence of the CD8α transmembrane domain. SEQ ID NO:48 is the amino acid sequence of 4-1BB. SEQ ID NO: 49 is the amino acid sequence of CD3ζ. SEQ ID NO:50 is the amino acid sequence of the self-cleaving T2A peptide. SEQ ID NO:51 is the amino acid sequence of huEGFRt. SEQ ID NO: 52 is the amino acid sequence of the wild-type IgG4 hinge region. SEQ ID NO:53 is a nucleic acid sequence encoding the VH domain of antibody YP7. SEQ ID NO: 54 is the amino acid sequence of the VH domain of antibody YP7. SEQ ID NO:55 is a nucleic acid sequence encoding the VL domain of antibody YP7. SEQ ID NO: 56 is the amino acid sequence of the VL domain of antibody YP7. SEQ ID NO:57 is a nucleic acid sequence encoding the VH domain of antibody YP6. SEQ ID NO: 58 is the amino acid sequence of the VH domain of antibody YP6. SEQ ID NO:59 is the amino acid sequence of the modified CH2 domain. SEQ ID NO:60 is the amino acid sequence of the wild-type CH2 domain.

[0015] Detailed Description I. Abbreviations CAR Chimeric Antigen Receptor CDR Complementarity Determining Region EF1α Elongation factor 1 alpha EGF epidermal growth factor EGFR epidermal growth factor receptor GPC3 Glypican-3 GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence HCC hepatocellular carcinoma huEGFRt Human truncated epidermal growth factor receptor IP intraperitoneal iPSC induced pluripotent stem cells KO Knockout NK Natural Killer NSG NOD-SCID-Gamma scFv single chain variable fragment TM transmembrane T cm Central memory T cells T em Effector memory T cells T emra Effector memory T cells re-express CD45RA T scm Stem cell memory T cells VH Heavy chain variable VL variable light chain

[0016] II. Terminology Overview Unless otherwise stated, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones&Bartlett Learning,2017. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "an antigen" can be considered to include a single or multiple antigens and equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should be further understood that any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting of the present invention.

[0017] In order to facilitate review of the various embodiments, the following terms are explained:

[0018] 4-1BB: a costimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes and other cells, including natural killer cells. Ligation of 4-1BB induces a signal transduction cascade that results in the production of cytokines, the expression of anti-apoptotic molecules, and the enhancement of immune responses. An exemplary amino acid sequence of 4-1BB is set forth herein as SEQ ID NO: 48.

[0019] Administration: Providing or giving an agent (such as a CAR or CAR-expressing cell provided herein) to a subject by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraarterial (including intrahepatic artery), intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. In some examples, administration is local administration. In some examples, administration is systemic administration.

[0020] Antibody: A polypeptide ligand that includes at least one variable region that recognizes and binds (e.g., specifically recognizes and binds) an epitope of an antigen, such as GPC3. Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has a respective heavy chain variable (V H ) region and the light chain variable (V L ) region. H Area and V L The region is responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the primary antibody produced by birds and reptiles and is somewhat similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0021] Antibody variable regions contain "framework" regions and hypervariable regions known as "complementarity determining regions" or "CDRs". The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The boundaries of the amino acid sequence of a given CDR may be determined according to several numbering schemes (those described by Kabat et al. (Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1991; "Kabat" numbering scheme), those described by Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and those described by the ImMunoGeneTics (IMGT) database (Lefranc, Nucleic Acids Res. 29:207-9, 2001; the "IMGT" numbering scheme). The Kabat and IMGT databases are maintained online.

[0022] A "single domain antibody" refers to an antibody that contains a single domain (a variable domain) that is capable of specifically binding to an antigen or an epitope of an antigen, in the absence of further antibody domains. Single domain antibodies include, for example, H Domain antibodies, V NAR Antibody, Camelidae V H H antibodies, and V L Domain antibodies are examples. NAR Antibodies are produced by cartilaginous fishes, such as nurse sharks, scleractinian sharks, dogfish sharks, and bamboo sharks. HH antibodies are produced by several species that produce heavy chain antibodies that are naturally devoid of light chains, including camels, llamas, alpacas, dromedaries, and guanacos.

[0023] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or a cell into which a single antibody coding sequence has been transfected. Monoclonal antibodies are produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0024] A "chimeric antibody" has framework residues from one species (such as human) and CDRs (which generally confer antigen binding) from another species.

[0025] A "humanized" antibody is an immunoglobulin that includes a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, should be substantially identical to human immunoglobulin constant regions (i.e., at least about 85-90% identical, e.g., about 95% or more identical). Thus, all parts of a humanized immunoglobulin, with the possible exception of the CDRs, are substantially identical to the corresponding parts of a native human immunoglobulin sequence. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions.

[0026] Binding affinity: the affinity of an antibody or other antigen-binding molecule to an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In some embodiments, binding affinity is measured using the Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In other embodiments, Kd is measured using surface plasmon resonance assays using BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ). In other embodiments, antibody affinity is measured by flow cytometry. An antibody or CAR that "specifically binds" to an antigen (such as GPC3) is an antibody or CAR that binds to said antigen with high affinity and does not significantly bind to other unrelated antigens.

[0027] Chemotherapeutic agent: Any chemical agent that has therapeutic utility in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancers. In one embodiment, the chemotherapeutic agent is an agent useful in the treatment of GPC3-positive tumors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Exemplary chemotherapeutic agents that can be used with the methods provided herein include those described in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17 in Abeloff, Clinical Oncology 2 nded., (C)2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one drug to treat cancer. One example is the administration of GPC3-targeted CAR T cells in combination with radioactive or chemical compounds. In one example, the chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody), such as an anti-GPC3 antibody, and other anti-cancer antibodies, such as anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4). Combination chemotherapy is the administration of more than one agent to treat cancer.

[0028] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a single domain antibody or scFv) and a signaling domain (such as a signaling domain derived from a T cell receptor (e.g., CD3ζ)). In many examples, a CAR is composed of an antigen-binding portion, a hinge region, a transmembrane domain, and an endodomain. The endodomain typically includes a signaling chain (such as CD3ζ or FcεRIγ) with an immunoreceptor tyrosine-based activation motif (ITAM). In some cases, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain (such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, MYD88-CD40, KIR2DS2, and / or DAP10). In some examples, a CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule composed of at least two antigen binding domains (such as scFvs and / or single domain antibodies), each binding to a different antigen or different epitopes on the same antigen (see, e.g., U.S. Patent Application Publication No. 2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule with two antigen binding domains, each binding to a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen binding moiety that binds to a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules linked by a cleavable linker. Immune cells (such as T cells, B cells, NK cells, or macrophages) or iPSCs expressing bispecific or bicistronic CARs can bind to cells expressing both of the antigens to which the binding moieties are directed (see, e.g., Qin et al., Blood 130:810, 2017; and WO 2018 / 213337). In some embodiments, the CAR is a double-chain antibody-T cell receptor (AbTCR) as described by Xu et al. (Cell Discovery 4:62, 2018) or a synthetic T cell receptor and antigen receptor (STAR) as described by Liu et al. (Sci Transl Med 13(586):eabb5191, 2021).

[0029] Complementarity determining region (CDR): A hypervariable amino acid sequence region that defines the binding affinity and specificity of an antibody. Each of the light and heavy chains of mammalian immunoglobulins has three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Single domain antibodies contain three CDRs, referred to herein as CDR1, CDR2, and CDR3.

[0030] Conservative variant: In the context of this disclosure, a "conservative" amino acid substitution is one that does not substantially affect or reduce the affinity of a protein, such as an antibody, to GPC3. As an example, a monoclonal antibody that specifically binds to GPC3 can contain at most about 1, at most about 2, at most about 5, and at most about 10, or at most about 15 conservative substitutions and specifically bind to a GPC3 polypeptide. The term "conservative variant" also includes the use of a substituted amino acid in place of a non-substituted parent amino acid, provided that the variant maintains activity. A non-conservative substitution is one that reduces the activity (such as affinity) of a protein.

[0031] Conservative amino acid substitution tables providing functionally similar amino acids are known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0032] In some embodiments herein, amino acid sequences are provided that contain no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution compared to any amino acid sequence disclosed herein.

[0033] Contact: To be placed in direct physical association; includes both solids and liquids.

[0034] Degenerate variant: A polynucleotide encoding a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0035] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (provoke a specific immune response). Antibodies specifically bind to particular antigenic epitopes on a polypeptide.

[0036] Framework region: Amino acid sequences interposed between the CDRs. Framework regions include the light chain variable framework region and the heavy chain variable framework region. The framework regions serve to hold the CDRs in the proper orientation for antigen binding.

[0037] Fusion Protein: A protein that contains at least parts of two different (heterologous) proteins.

[0038] Glypican-3 (GPC3): A member of the glypican family of heparan sulfate (HS) proteoglycans attached to the cell surface by a glycosylphosphatidylinositol anchor (Filmus and Selleck, J Clin Invest 108:497-501, 2001). The GPC3 gene encodes an approximately 70 kD core protein that can be cleaved by furin to produce an N-terminal 40 kD fragment and a C-terminal 30 kD fragment. Two HS chains are attached to the C-terminal portion of GPC3. GPC3 and other glypican family proteins play a role in the control of cell division and cell growth. GPC3 is highly expressed in HCC and several other human cancers, including melanoma, squamous cell carcinoma of the lung, and clear cell carcinoma of the ovary (Ho and Kim, Eur J Cancer 47(3):333-338, 2011), but is not expressed in normal tissues. GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2.

[0039] Four isoforms of human GPC3 are known (isoforms 1 to 4) (Ho and Kim, Eur J Cancer 47(3):333-338, 2011). The nucleic acid and amino acid sequences of the four isoforms of GPC3 are known (GenBank accession numbers: NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (isoform 2); NM_001164618 and NP_001158090 (isoform 3); and NM_001164619 and NP_001158091 (isoform 4)).

[0040] GPC3 positive cancer: cancer that expresses or overexpresses GPC3. Examples of GPC3 positive cancer include, but are not limited to, HCC, melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms' tumor, lung squamous cell carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, Schwannoma, and embryonal tumor (Ho and Kim, Eur J Cancer 47(3):333-338,2011; Baumhoer et al., Am J Clin Pathol 129(6):899-906,2008; Saikali and Sinnett, Int J Cancer 89(5):418-422,2000).

[0041] Hepatocellular carcinoma (HCC): A primary malignant disease of the liver that typically occurs in patients with hepatitis due to viral hepatitis, hepatotoxins, or cirrhosis (often due to alcoholism). HCC is also called malignant hepatoma.

[0042] Heterologous: Derived from separate genetic sources or species.

[0043] Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be a prokaryotic or eukaryotic cell. In some examples, the prokaryotic cell is an E. coli cell. In some examples, the eukaryotic cell is a human cell (such as a human embryonic kidney (HEK) cell). The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny are included.

[0044] Immune response: A response of a cell of the immune system (such as a B cell, T cell, or monocyte) to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response (CD4 + Response or CD8 + In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0045] Isolated: An "isolated" biological component (such as a nucleic acid, protein (including an antibody), or organelle) has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles) in the environment (such as a cell) in which said component is present. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0046] Label: A detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or a protein, to facilitate detection of the molecule. Non-limiting examples of labels include fluorescent tags, enzyme conjugation, and radioisotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into the antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that can be detected by a marker-tagged avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or colorimetric methods). A variety of methods for labeling polypeptides and glycoproteins are known and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides ( 35 S, 11 C. 13 N, 15 O. 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C. 15 N, 90 Y, 99 Tc, 111 In, and 125I, fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors, etc.), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, etc.), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.), or magnetic agents (such as gadolinium chelates, etc.). In some embodiments, the labels are attached with spacer arms of various lengths to reduce potential steric hindrance.

[0047] Linker: In some cases, a linker is a peptide in an antibody-binding fragment (such as an Fv fragment) that serves to indirectly link the variable heavy chain to the variable light chain. A "linker" can also refer to a peptide that serves to link a targeting moiety (such as an antibody) to an effector molecule (such as a cytotoxin or detectable label). The terms "conjugation," "joining," "linking," or "linking" refer to the creation of one contiguous polypeptide molecule from two polypeptides, or the covalent attachment of a radionuclide or other molecule to a polypeptide (such as an scFv). In a specific context, the term includes reference to the conjugation of a ligand (such as an antibody moiety) to an effector molecule. Linking can be by either chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0048] Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects (such as mice, rats, cows, cats, dogs, pigs, and non-human primates).

[0049] Melanoma: A form of cancer derived from melanocytes (cells that produce the pigment melanin). Melanocytes are found primarily in the skin, but also in the intestine and eye. Cutaneous melanomas include superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, and lentigo maligna (melanoma). Any of the above types may produce melanin or be amelanotic. Similarly, any subtype may show desmoplasia (a neurotropic dense fibrous reaction), a marker of aggressive behavior and a tendency to local recurrence. Other melanomas include clear cell sarcoma, mucosal melanoma, and uveal melanoma.

[0050] Neoplasia, malignancy, cancer, or tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. As a neoplasm grows, it can produce a tumor. The amount of tumor in an individual is the "tumor burden" and can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are termed "benign." Tumors that can invade surrounding tissues and / or metastasize are termed "malignant."

[0051] Neuroblastoma: A solid tumor that arises from embryonic neural crest cells. Neuroblastomas commonly arise in or around the adrenal glands, but can arise anywhere sympathetic nervous tissue is found, such as in the abdomen, chest, neck, or in nervous tissue near the spine. Neuroblastomas typically occur in children under the age of 5.

[0052] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, are in the same reading frame.

[0053] Ovarian cancer: Cancer that forms in the tissues of the ovary, one of the paired female reproductive glands in which ova (i.e., eggs) are formed. Most ovarian cancers are either ovarian epithelial cancers (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in the egg cells).

[0054] Ovarian clear cell carcinoma: A distinctive histopathological subtype of epithelial ovarian cancer that accounts for less than 5% of all ovarian malignancies. When viewed under a microscope, the cells of this tumor type have clear interiors.

[0055] Pharmaceutically acceptable carriers: Useful pharma- ceutically acceptable carriers are conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013) describes compositions and formulations suitable for pharmaceutical delivery of CAR-expressing immune cells and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually include injectable fluids that contain pharma- ceutically and physiologically acceptable fluids (such as water, physiological saline, balanced salt solutions, aqueous dextrose, or glycerol) as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically natural carriers, the pharmaceutical composition to be administered can contain small amounts of non-toxic auxiliary substances (such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as, for example, sodium acetate or sorbitan monolaurate).

[0056] Prevention, treatment, or amelioration of a disease: "Prevention" of a disease refers to the complete inhibition of disease onset. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after onset has begun (such as reducing tumor burden or the number or size of metastases). "Amelioration" refers to a reduction in the number or severity of a sign or symptom of a disease (such as cancer).

[0057] Purified: The term purified does not necessarily mean absolute purity; rather, it is intended to be a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is enriched more than the peptide or protein is in its natural environment within a cell. In one embodiment, the preparation is purified such that the protein or peptide is present in at least 50% of the total peptide or protein content of the preparation. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Thus, in one non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

[0058] Recombinant: A recombinant nucleic acid has a sequence that is not found in nature or that is made by the artificial combination of two naturally separated segments of sequence, often by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids (e.g., genetic engineering techniques).

[0059] Sample (or biological sample): A biological specimen comprising genomic DNA, RNA (including mRNA), protein, or a combination thereof obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, the sample includes a tumor biopsy (such as a tumor tissue biopsy).

[0060] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between sequences, or is otherwise referred to as sequence identity. Sequence identity is often measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of polypeptides or nucleic acid molecules will have a relatively high degree of sequence identity when aligned using standard methods.

[0061] Methods for aligning sequences for comparison are known. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, discusses sequence alignment methods and homology calculations in detail.

[0062] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet. A description of how to determine sequence identity using this program is available on the Internet at the NCBI website.

[0063] Homologs and variants of antibodies or CARs that specifically bind to GPC3 are typically characterized by having at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity when counted by full-length alignment with the amino acid sequence of the antibody or CAR using gapped insertion blastp set to default parameters in NCBI Blast2.0. When comparing amino acid sequences of more than about 30 amino acids, use the Blast2 sequence function with the default BLOSUM62 matrix set to default parameters (gap extension cost of 11 and gap cost per residue of 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap penalty of 1). Proteins with even greater similarity to the reference sequence will have increased percent identity when assessed by this method (such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). When less than the entire sequence is to be compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over a short window of 10-20 amino acids, and may possess at least 85% or at least 90% or 95% sequence identity depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website. These sequence identity ranges provide guidance only; it is entirely possible that very significant homologs may be obtained outside the ranges provided.

[0064] Squamous cell carcinoma: A type of cancer derived from squamous cells (thin, flat cells that form the surfaces of the skin, eyes, various internal organs, and the lining of hollow organs and some glandular ducts). Squamous cell carcinoma is also called epidermoid carcinoma. One type of squamous cell carcinoma is squamous cell carcinoma of the lung. Squamous cell carcinoma is the most common type of skin cancer.

[0065] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals, such as pigs, mice, rats, rabbits, sheep, horses, cows, dogs, cats, and non-human primates.

[0066] Synthetic: Produced by artificial means in a laboratory. For example, a synthetic nucleic acid or protein (e.g., an antibody) can be chemically synthesized in a laboratory.

[0067] Therapeutically effective amount: A quantity of a particular substance sufficient to achieve a desired effect in a treated subject. For example, this may be the amount required to inhibit or suppress the growth of a tumor. In one embodiment, a therapeutically effective amount is the amount required to eliminate or reduce the size of a tumor or prevent metastasis (e.g., reduce the size and / or volume of a tumor by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, compared to the size / volume / number before treatment). A dosage will generally be used that, when administered to a subject, results in a target tissue concentration (e.g., in a tumor) that has been shown to produce the desired in vitro effect.

[0068] Vector: A nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that allows 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 known genetic elements. In some examples, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[0069] III. Overview of Certain Aspects CAR T cells have transformed the treatment of CD19+ B-cell malignancies; however, they have met with limited success in solid tumors due to several barriers, including a lack of tumor-specific antigens, inability of CAR T cells to efficiently expand at tumor sites, and heterogeneous antigen expression (Kochenderfer et al., Blood. 2012;119(12):2709-2720; Porter et al., N Eng J Med 2011;365(8):725-733; Jiang et al., Front Immunol 2017;7:690; Gao et al., Clin Cancer Res 2014;20(24):6418-6428; Ishiguro et al., Sci Transl Med 2017;9(410)aal4291; Ishiguro et al., Cancer Res 2008;68(23):9832-9838; Losic et al., Nat Commun 2020;11(1):291). The present disclosure addresses these challenges using rational engineering strategies to precisely alter the structural components of the CAR. Specifically, it is disclosed herein that immune cells expressing GPC3-targeted CARs were most effective when a short hinge sequence from IgG4 was used in the construct. Furthermore, combining an IgG4 hinge (IgG4H) with the CD28 transmembrane (CD28TM) domain yielded the most potent antitumor effects (Figures 3A-3D and 4A-4D). Additionally, the data disclosed herein demonstrate that GPC3-targeted IgG4H-CD28TM CAR immune cells induce potent CD8 T cells and T emra We demonstrate that the IL-16 / IL-16 induced a 3-fold increased IL-16 response (Figure 5B), leading to remarkably rapid and durable tumor eradication.

[0070] Provided herein is a CAR comprising an extracellular antigen binding domain that specifically binds to GPC3; a wild-type or modified IgG4 hinge region; a transmembrane domain; an intracellular costimulatory domain; and an intracellular signaling domain. In some embodiments, the hinge region comprises or consists of a modified IgG4 hinge sequence ESKYGPPCPPCP (SEQ ID NO: 43). In other embodiments, the hinge region comprises or consists of a wild-type IgG4 sequence ESKYGPPCPSCP (SEQ ID NO: 52). In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain.

[0071] In some embodiments, the antigen binding domain of the CAR specifically binds to GPC3 with high affinity. In some examples, the antigen binding domain comprises a GPC3-specific single domain antibody or a GPC3-specific scFv. In some examples, the antigen binding domain comprises one or more CDR sequences (such as one, two, or all three CDR sequences) from the GPC3-specific single domain antibody HN3. In other examples, the antigen binding domain comprises one or more CDR sequences (such as one, two, three, four, five, or all six CDR sequences) from the GPC3-specific monoclonal antibody hYP7, YP9.1, YP8, YP9, YP6, YP7, or HS20.

[0072] In some embodiments, the antigen binding domain of the CAR is a single domain antibody comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 (HN3). In some examples, the CDR1, CDR2, and CDR3 sequences comprise residues 31-35, 50-65, and 96-105 of SEQ ID NO: 18, respectively; or residues 26-33, 51-57, and 96-105 of SEQ ID NO: 18. In specific examples, the amino acid sequence of the single domain antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 18 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18). In certain non-limiting examples, the amino acid sequence of the single domain antibody comprises or consists of SEQ ID NO: 18.

[0073] In other embodiments, the antigen binding domain of the CAR comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:20 (the VH domain of hYP7), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:22 (the VL domain of hYP7). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO:20; or residues 26-33, 51-60, and 99-106 of SEQ ID NO:20. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO: 22; or residues 27-38, 56-58, and 95-103 of SEQ ID NO: 22. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 20); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 22). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 20; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 22. In other specific examples, the antigen-binding domain is an scFv comprising the amino acid sequence of residues 1 to 245 of SEQ ID NO: 14.

[0074] In other embodiments, the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:24 (the VH domain of YP9.1), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:26 (the VL domain of YP9.1). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO:24; or residues 26-33, 51-60, and 99-106, respectively, of SEQ ID NO:24. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO:26; or residues 27-38, 56-58, and 95-103, respectively, of SEQ ID NO:26. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:24); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:26 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:26). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:24; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:26.

[0075] In other embodiments, the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:28 (the VH domain of YP8), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:30 (the VL domain of YP8). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO:28; or residues 26-33, 51-60, and 99-106 of SEQ ID NO:28. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO:30; or residues 27-38, 56-58, and 95-103 of SEQ ID NO:30. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:28 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:28); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:30). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:28; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:30.

[0076] In other embodiments, the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:58 (the VH domain of YP6), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:30 (the VL domain of YP6). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO:58; or residues 26-33, 51-60, and 99-106 of SEQ ID NO:28. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO:30; or residues 27-38, 56-58, and 95-103 of SEQ ID NO:30. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:58 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:58); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:30 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:30). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:58; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:30.

[0077] In other embodiments, the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 32 (the VH domain of YP9), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, 36, or 38 (the VL domain of YP9 clone 9, YP9 clone 10, and YP9 clone 1). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO: 32; or residues 26-33, 51-60, and 99-106 of SEQ ID NO: 32. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103 of SEQ ID NO: 34, 36, or 38, respectively; or residues 27-38, 56-58, and 95-103 of SEQ ID NO: 34, 36, or 38. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 32 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 32); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 34, 36, or 38 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34, 36, or 38, respectively). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 32; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 34, 36, or 38, respectively.

[0078] In other embodiments, the antigen binding domain of the CAR comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 54 (the VH domain of YP7), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 56 (the VL domain of YP7). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO: 54; or residues 26-33, 51-60, and 99-106 of SEQ ID NO: 54. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO: 56; or residues 27-38, 56-58, and 95-103 of SEQ ID NO: 56. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 54 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 54); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 56 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 56). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:54; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:56.

[0079] In other embodiments, the antigen binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 40 (the VH domain of HS20), and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 42 (the VL domain of HS20). In some examples, the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-66, and 97-105, respectively, of SEQ ID NO: 40; or residues 26-33, 51-58, and 97-105, respectively, of SEQ ID NO: 40. In some examples, the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-34, 50-56, and 89-97, respectively, of SEQ ID NO: 42; or residues 27-32, 50-52, and 89-97, respectively, of SEQ ID NO: 42. In specific examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 40 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 40); and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 42 (and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 42). In specific, non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 40; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 42.

[0080] In some embodiments, the transmembrane domain of the CAR comprises a CD28 transmembrane domain. In some examples, the amino acid sequence of the CD28 transmembrane domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 46. In certain non-limiting examples, the amino acid sequence of the CD28 transmembrane domain comprises or consists of SEQ ID NO: 46. In other embodiments, the transmembrane domain of the CAR comprises a CD8 transmembrane domain. In some examples, the amino acid sequence of the CD8 transmembrane domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 47. In certain non-limiting examples, the amino acid sequence of the CD8 transmembrane domain comprises or consists of SEQ ID NO: 47.

[0081] In some embodiments, the costimulatory domain of the CAR comprises a 4-1BB signaling moiety. In some examples, the amino acid sequence of the 4-1BB signaling moiety is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 48. In certain non-limiting examples, the amino acid sequence of the 4-1BB signaling moiety comprises or consists of SEQ ID NO: 48.

[0082] In some embodiments, the signaling domain of the CAR comprises a CD3 zeta signaling domain. In some examples, the amino acid sequence of the signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49. In certain non-limiting examples, the amino acid sequence of the CD3 zeta signaling domain comprises or consists of SEQ ID NO: 49.

[0083] In some embodiments, the amino acid sequence of the CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In some examples, the amino acid sequence of the CAR comprises any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14. In specific examples, the amino acid sequence comprises SEQ ID NO: 6 (HN3-IgG4H-CD28TM) or SEQ ID NO: 14 (hYP7-IgG4H-CD28TM).

[0084] Further provided is a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter (such as an inducible or constitutive promoter). In some examples, the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13. In specific, non-limiting examples, the nucleic acid molecule comprises any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13. In specific, non-limiting examples, the nucleic acid molecule comprises SEQ ID NO: 5 (HN3-IgG4H-CD28TM) or SEQ ID NO: 13 (hYP7-IgG4H-CD28TM).

[0085] In some embodiments, the nucleic acid molecule includes, in a 5' to 3' direction, a nucleic acid encoding a first granulocyte macrophage colony stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding an antigen binding domain; a nucleic acid encoding an IgG4 hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding a costimulatory domain; a nucleic acid encoding a signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding a truncated human epidermal growth factor receptor (huEGFRt). In some examples, the nucleic acid molecule further includes a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss (see WO 2019 / 094482).

[0086] Further provided is a vector comprising the nucleic acid molecule disclosed herein. In some examples, the vector is a viral vector (such as a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector).

[0087] Also provided are isolated cells that comprise a nucleic acid molecule encoding a CAR disclosed herein and / or express a CAR disclosed herein. In some embodiments, the cell is an immune cell (such as a T cell, a B cell, a NK cell, or a macrophage). In other embodiments, the cell is an induced pluripotent stem cell (iPSC).

[0088] Further provided is a composition comprising a pharma- ceutically acceptable carrier (such as water or saline) and the CAR, nucleic acid molecule, vector, or cell disclosed herein.In some examples, the composition is frozen.In some examples, the composition is frozen and comprises cells and DMSO or other cryopreservative.In some examples, the composition is lyophilized.

[0089] Also provided is a method for treating GPC3-positive cancer in a subject or inhibiting tumor growth or metastasis of GPC3-positive cancer.In some embodiments, the method comprises administering to a subject a therapeutically effective amount of CAR, nucleic acid molecule, vector, cell or composition disclosed herein.In some examples, the GPC3-positive cancer is a solid tumor.In certain non-limiting examples, the GPC3-positive cancer is hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms' tumor, squamous cell carcinoma of the lung, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, Schwannoma, or embryonal tumor.In a specific example, the GPC3-positive cancer is HCC.

[0090] IV. GPC3-specific antibody sequences The CAR disclosed herein comprises an antibody (or an antigen-binding fragment thereof) that specifically binds to GPC3. In some embodiments, the antibody is HN3 (a human single domain (VH) monoclonal antibody) or hYP7 (a humanized mouse antibody) (scFv format). In other embodiments, the antibody is YP9, YP8, YP6, YP7, or YP9.1 (such as in scFv format), or a humanized version thereof. In yet other embodiments, the antibody is HS20 (such as in scFv format), which is a human antibody. HN3, hYP7, YP9, YP8, YP6, YP7, YP9.1, and HS20 antibodies are described in PCT Publication Nos. WO 2012 / 145469, WO 2012 / 145469, and WO 2019 / 094482. The nucleotide and amino acid sequences of HN3, hYP7, YP9, YP8, YP6, YP7, YP9.1, and HS20 are provided below. Tables 1-13 list the amino acid positions of CDR1, CDR2, and CDR3 of HN3 and the CDR1, CDR2, and CDR3 of the VH and VL domains of hYP7, YP9, YP8, YP6, YP7, YP9.1, and HS20 as determined using Kabat and IMGT. Alternative numbering schemes (such as the Paratome or Chothia numbering schemes) can be used to easily determine the boundaries of the CDRs.

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[0091] V. GPC3-targeted CAR sequence Several different CAR constructs were generated using the HN3 single domain antibody and hYP7 scFv, utilizing either a CD8 hinge (SEQ ID NO: 45), an IgG4 hinge (SEQ ID NO: 43), an IgG4-CH3 hinge, or an IgG4-CH2-CH3 hinge, and either a CD8 transmembrane (TM) domain (SEQ ID NO: 47) or a CD28 transmembrane domain (SEQ ID NO: 46). In the CAR amino acid sequences provided below, the antigen binding sequence (HN3 single domain or hYP7 VH-linker-VL) is underlined, the hinge region (CD8α, IgG4, IgG4-CH3, or IgG4-CH2-CH3) is in bold, and the TM domain (CD8α or CD28) is in italics. [ka] [ka] [Table 14] [ka] [Table 15] [ka] [ka] [Table 16] [ka] [Table 17] [ka] [ka] [Table 18] [ka] [ka] [Table 19]

[0092] The CH2 domain contained in the HN3-IgG4H-CH2CH3-CD28TM CAR is a modified CH2 domain having the following sequence (SEQ ID NO:59; substituted amino acid residues are underlined): [ka]

[0093] In some embodiments, a wild-type CH2 domain is used in place of the modified CH2 domain (e.g., a wild-type CH2 domain having the following sequence (SEQ ID NO: 60): [ka] [ka] [Table 20] [ka] [ka] [Table 21]

[0094] VI. Chimeric Antigen Receptors (CARs) Disclosed herein are GPC3-specific CARs and cells engineered to express the CARs (e.g., T cells, B cells, NK cells, macrophages, and iPSCs). In general, CARs include a binding moiety, an extracellular hinge / spacer element, a transmembrane region, and an intracellular domain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many examples, the binding moiety is an antigen-binding fragment of a monoclonal antibody (such as an scFv or single domain antibody). The spacer / hinge region typically includes sequences from IgG subclasses (such as IgG1, IgG4), IgD, and CD8 domains. In some embodiments herein, the hinge region includes (or consists of) a modified IgG4 hinge sequence as set forth in SEQ ID NO:43. In other embodiments herein, the hinge region comprises (or consists of) the wild-type IgG4 hinge sequence set forth in SEQ ID NO:52. The transmembrane domain can be derived from a variety of different T cell proteins (such as CD3ζ, CD4, CD8, CD28, or inducible T cell costimulatory molecule (ICOS)). Several different endodomains have been used to generate CARs. For example, the endodomain can consist of a signaling chain with ITAM (such as CD3ζ or FcεRIγ). In some examples, the endodomain further comprises the intracellular portion of at least one additional costimulatory domain (such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, MYD88-CD40, killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2), and / or DAP10).

[0095] The CAR can also include a signal peptide sequence, for example, at the N-terminus of the antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2. Although the signal peptide sequence can facilitate the expression of the CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not necessary for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence can be excised from the CAR. Thus, in some embodiments, the CAR lacks a signal peptide sequence.

[0096] In some embodiments, the CAR disclosed herein is expressed from a construct (such as a lentiviral vector) that also expresses a truncated version of human EGFR (huEGFRt; discussed in more detail in Section VII below). The CAR and huEGFRt are separated by a self-cleaving peptide sequence (such as T2A) such that upon expression in transduced cells, the CAR is cleaved from the huEGFRt (see WO 2019 / 094482).

[0097] In some embodiments discussed herein, the CAR construct encodes the following amino acid sequence from N-terminus to C-terminus: GMCSFRss:MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 44) NdeI:HM Antigen binding: scFv or single domain antibody sequences SpeI:TS Hinge: modified IgG4 (ESKYGPPCPPCP; SEQ ID NO: 43), wild type IgG4 (ESKYGPPCPSCP; SEQ ID NO: 52), or CD8α (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD; SEQ ID NO: 45) TM: CD28 (FWVLVVVGGVLACYSLLVTVAFIIFWV; SEQ ID NO: 46) or CD8α (IYIWAPLAGTCGVLLLSLVIT; SEQ ID NO: 47) [ka]

[0098] Immune cells (such as T cells, B cells, NK cells, or macrophages) or iPSCs expressing the CARs disclosed herein can be used to target specific cell types, such as tumor cells (e.g., GPC3-positive tumor cells). The use of immune cells (such as T cells) expressing CARs is more universal than standard CTL-based immunotherapy, as immune cells expressing CARs are not HLA-restricted and therefore can be used in any patient with a tumor expressing the target antigen.

[0099] Thus, CARs comprising GPC3-specific antibodies (or binding fragments thereof) are provided herein.Also provided are isolated nucleic acid molecules and vectors encoding CARs, and host cells expressing CARs (such as T cells, B cells, NK cells, macrophages, or iPSCs).Cells expressing CARs composed of GPC3-specific monoclonal antibodies can be used to treat cancers expressing GPC3 (such as HCC, melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms' tumor, lung squamous cell carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, Schwannoma, or embryonal tumors).

[0100] VII. Truncated human EGFR (huEGFRt) Human epidermal growth factor receptor is composed of four extracellular domains, a transmembrane domain, and three intracellular domains. The EGFR domains are found in the following order from N-terminus to C-terminus: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-juxtamembrane domain-tyrosine kinase domain-C-terminal tail. Domain I and domain III are leucine-rich domains involved in ligand binding. Domain II and domain IV are cysteine-rich domains and do not contact EGFR ligands. Domain II mediates the formation of homodimers or heterodimers with similar domains from other EGFR family members, and domain IV can form disulfide bonds with domain II. The EGFR TM domain spans the cell membrane once and may play a role in protein dimerization. The intracellular domain, which contains the juxtamembrane domain, the tyrosine kinase domain, and the C-terminal tail, mediates EGFR signaling (Wee and Wang, Cancers 9(52), doi:10.3390 / cancers9050052; Ferguson, Annu Rev Biophys 37:353-373, 2008; Wang et al., Blood 118(5):1255-1263, 2011).

[0101] The shortened version of human EGFR, referred to herein as "huEGFRt", contains only domain III, domain IV and TM domain.HuEGFRt therefore lacks domain I, domain II and all three intracellular domains.HuEGFRt cannot bind to EGF and lacks signal transduction activity.However, this molecule retains the ability to bind to certain EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894).

[0102] Transduction of immune cells (such as T cells, B cells, NK cells, or macrophages) or iPSCs with constructs (such as lentiviral vectors) encoding both the huEGFRt and GPC3-specific CARs disclosed herein allows the selection of transduced cells using labeled EGFR monoclonal antibody cetuximab (Erbitux™). For example, cetuximab can be labeled with biotin, and transduced cells can be selected using commercially available anti-biotin magnetic beads (such as from Miltenyi Biotec). Co-expression of huEGFRt also allows in vivo tracking of adoptively transferred CAR-expressing cells. Furthermore, binding of cetuximab to cells expressing huEGFRt induces cytotoxicity of ADCC effector cells, thereby providing a mechanism for in vivo elimination of transduced immune cells or iPSCs, such as at the end of treatment (Wang et al., Blood 118(5):1255-1263, 2011).

[0103] In some embodiments herein, the amino acid sequence of huEGFRt is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51. In some examples, the amino acid sequence of huEGFRt comprises or consists of SEQ ID NO: 51. In other embodiments, the amino acid sequence of huEGFRt contains no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitutions compared to SEQ ID NO: 51. In some examples, the amino acid substitutions are conservative substitutions.

[0104] VIII. CAR-Expressing Cell Compositions Compositions are provided that include CAR-expressing cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. CAR-expressing cells include iPSCs, T cells (e.g., CD3 + T cells, e.g., CD4 + and / or CD8 +The composition may be a T cell, a B cell, a NK cell, a macrophage, or any other suitable immune cell. Such compositions may include a buffer (such as neutral buffered saline, phosphate buffered saline, etc.); a carbohydrate (such as glucose, mannose, sucrose dextran, or mannitol); a protein; a polypeptide or amino acid (such as glycine); an antioxidant; a chelating agent (such as EDTA or glutathione); an adjuvant (e.g., aluminum hydroxide); and a preservative. In some examples, the cell-containing composition includes a cryopreservative (such as DMSO or glycerol). In some examples, the cell-containing composition includes a culture medium (such as DMEM or RPMI) and may further include serum (such as FBS). In some examples, the cell-containing composition is in frozen or liquid form. The cells may be autologous to the recipient. However, the cells may be xenogeneic (allogeneic).

[0105] For cells, various aqueous carriers (e.g., buffered saline, etc.) can be used to introduce the cells. These solutions are sterile and generally free of undesirable substances. These compositions can be sterilized by conventional sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary agents (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.) when necessary to approximate physiological conditions, such as pH adjusting and buffering agents, and toxicity adjusting agents. The concentrations in these formulations can vary widely and will be selected primarily based on the volume, viscosity, and weight of the fluid, etc., according to the particular mode of administration selected and the needs of the subject.

[0106] The exact dosage of the composition can be determined by a physician, taking into account individual differences in age, body weight, tumor size / tumor burden, extent of metastasis, and condition of the patient (subject). The pharmaceutical composition comprising the CAR-expressing immune cells (T cells, B cells, macrophages, and / or NK cells) or iPSCs described herein can be administered for 10 minutes or more. 4 ~10 9 Cells / kg body weight, e.g., 10 5 ~10 6It can be generally stated that the cells may be administered in a dosage of 1000 cells / kg body weight (including all integer values ​​within the aforementioned ranges). Exemplary doses are 10 6 cells / kg~about 10 8 Cells / kg, e.g., about 5 x 10 6 Cells / kg to approximately 7.5 x 10 7 Up to cells / kg, e.g., about 2.5 x 10 7 cells / kg, or approximately 5.0 x 10 7 cells / kg.

[0107] The composition can be administered at these dosages once or multiple times (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). The composition can be administered using known immunotherapeutic injection techniques (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The composition can be administered once a day, once a week, twice a month, or once a month. In some non-limiting examples, the composition is formulated for intravenous administration and administered multiple times. The appropriate dosage can be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the subject's symptoms and the type and severity of the subject's disease.

[0108] In some embodiments, a nucleic acid molecule encoding a CAR is introduced into a cell (such as a T cell, B cell, NK cell, macrophage, or iPSC) and the cell is administered to the subject initially, followed by one or more subsequent administrations of the cell, where the one or more subsequent administrations are administered less than 15 days (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days) after the previous administration. In one embodiment, the subject is administered a CAR-expressing cell more than once per week (e.g., the disclosed CAR-expressing cell is administered 2, 3, or 4 times per week). In one embodiment, the subject is administered CAR-expressing cells more than once per week (e.g., 2, 3, or 4 administrations per week) (also referred to as a cycle), followed by a week without administration of CAR-expressing cells, and then the subject is administered one or more additional administrations of CAR-expressing cells (e.g., more than one administration of CAR-expressing cells per week). In another embodiment, the subject (e.g., a human subject) is administered more than one cycle of CAR-expressing cells, with the period between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR-expressing cells are administered three times, every other day per week. In another embodiment, the CAR-expressing cells are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks, or more. The dosage of the above treatment to be administered to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosages for administration to humans can be routinely adjusted.

[0109] In some embodiments, the CAR-expressing cells can replicate in vivo, so that they can persist long term and maintain tumor control. In various embodiments, the iPSCs, T cells, B cells, macrophages, or NK cells administered to a subject, or the progeny of these cells, persist in the subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 1 year after administration of the cells to the subject. In other embodiments, the cells and their progeny exist for less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month (e.g., 3 weeks, 2 weeks, 1 week) after administration of the CAR-expressing cells to the subject.

[0110] The composition may be administered in any conventional manner, including injection, ingestion, infusion, implantation, and transplantation. The disclosed composition may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intralymph node, intramedullary, intramuscularly, intraarterially (including hepatic artery (such as HAI) or femoral artery), intravenous (iv) injection, intraprostatically (e.g., for prostate cancer), or intraperitoneally. In some embodiments, the composition is administered to a patient by intradermal or subcutaneous injection. In other embodiments, the disclosed composition is administered by iv injection. In other embodiments, the disclosed composition is administered by intraarterial injection. The composition may also be directly injected into a tumor or lymph node.

[0111] In some embodiments, a subject may undergo leukapheresis, in which white blood cells are harvested and enriched or depleted ex vivo to select and / or isolate cells of interest (e.g., iPSCs, T cells, B cells, macrophages, and / or NK cells). These cell isolates may be expanded by known methods and treated to allow for the introduction of one or more CAR constructs, thereby creating autologous cells expressing the CAR. In some embodiments herein, CAR-expressing cells are generated using a lentiviral vector expressing a CAR and a truncated form of human EGFR (huEGFRt). Co-expression of huEGFRt allows for the selection and purification of CAR-expressing immune cells using an antibody that recognizes huEGFRt as described in Section VIII above (e.g., cetuximab, see PCT Publication No. WO 2011 / 056894).

[0112] In some embodiments, immune cells (such as T cells, B cells, NK cells, and / or macrophages) are isolated from peripheral blood by lysing red blood cells and, in some cases, depleting monocytes, for example, by centrifugation through a Percoll™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells (such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells) can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28) conjugated beads (such as DYNABEADS® M-450 CD3 / CD28 T) for a time sufficient for positive selection of the desired T cells (see US Patent Publication No. 20140271635). In a non-limiting example, the time is about 30 minutes. In other non-limiting examples, the time ranges from 30 minutes to 36 hours or more and all integer values ​​therebetween. In further non-limiting examples, the time is at least 1, 2, 3, 4, 5, or 6 hours, 10 to 24 hours, 24 hours or more. Longer incubation times can be used to isolate T cells in any situation where there are few T cells compared to other cell types (such as isolation from immunocompromised individuals). Additionally, longer incubation times can be used to increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time, T cells can be bound to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected at will at the beginning of culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected at will at the beginning of culture or at other desired times. Multiple rounds of selection can also be used.

[0113] Enrichment of cell populations by negative selection can be performed in combination with antibodies directed to surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ T cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. T cell populations expressing one or more cytokines can be selected. Methods for screening cell expression are disclosed in PCT Publication WO 2013 / 126712.

[0114] For the isolation of desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied to ensure maximum cell and bead contact. In some embodiments, a concentration of 1 billion cells / ml is used. In further embodiments, more than 100 million cells / ml is used. In other embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 million cells / ml are used. Without being bound by theory, using high concentrations can increase cell yield, cell activation, and cell expression. Lower cell concentrations can also be used. Without being bound by theory, significantly diluting the mixture of T cells and a surface (e.g., a particle such as a bead) minimizes interactions between the particles and the cells. This selects for cells that express large amounts of the desired antigen to be bound to the particle. For example, CD4+ T cells express higher levels of CD28 than CD8+ T cells at diluted concentrations and are captured with greater efficiency. In some embodiments, the cell concentration used is 5×10 6 In other embodiments, the concentration used is about 1×10 5 / ml to 1×10 6 / ml and any integer value therebetween.

[0115] IX. Treatment Method Provided herein is a method for treating cancer in a subject by administering to the subject a therapeutically effective amount of the GPC3-targeting CAR-expressing immune cells (such as T cells, B cells, NK cells, or macrophages) or CAR-expressing iPSCs disclosed herein.Also provided herein is a method for inhibiting tumor growth or metastasis in a subject by administering to the subject a therapeutically effective amount of the GPC3-targeting CAR-expressing cells disclosed herein.Thus, in some examples, the method reduces the size, volume, and / or weight of the tumor by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, for example, compared to the size, volume, and / or weight of the tumor before treatment. In some examples, the method reduces the size, volume, and / or weight of metastases by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, for example, compared to the size, volume, and / or weight of metastases before treatment. In some examples, the method extends the survival time of a subject with GPC3-positive cancer by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least (at last) 36 months, at least 48 months, or at least 60 months, for example, compared to the survival time without the treatment provided herein. In some examples, a combination of these effects is achieved.

[0116] Specifically provided is a method for treating GPC3-positive cancer in a subject.In some embodiments, the method comprises administering to the subject a therapeutically effective amount of isolated immune cells or iPSCs comprising a nucleic acid molecule encoding GPC3-targeting CAR and huEGFRt, or administering to the subject a therapeutically effective amount of isolated immune cells or iPSCs co-expressing GPC3-targeting CAR and huEGFRt.In some embodiments, the GPC3-positive cancer is hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms' tumor, squamous cell carcinoma of the lung, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, Schwannoma, or embryonal tumor.In a specific embodiment, the cancer to be treated is HCC.

[0117] In some embodiments of the methods disclosed herein, the isolated immune cells are T lymphocytes.In some examples, the T lymphocytes are autologous T lymphocytes.In other embodiments, the isolated host cells are B cells, NK cells, or macrophages.

[0118] The therapeutically effective amount of CAR-expressing immune cells or iPSCs will depend on the severity of the disease, the type of disease, and the general health of the patient. The therapeutically effective amount of CAR-expressing cells and compositions thereof will provide either a subjective reduction in symptom(s) or an objectively identifiable improvement (such as a reduction in tumor volume or metastases) noted by a clinician or other qualified observer.

[0119] The CAR-expressing cells and compositions disclosed herein can also be administered by administration of other anti-cancer agents or therapeutic treatments (such as surgical removal of tumors). Any suitable anti-cancer agent can be administered in combination with the compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents (e.g., mitotic blockers, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g., anti-androgens), and anti-angiogenic agents, etc.). Other anti-cancer treatments include radiation therapy and antibodies (e.g., mAbs) that specifically target cancer cells or other cells (e.g., anti-PD-1, anti-CLTA4, anti-EGFR, or anti-VEGF). In one example, the cancer is treated by administration of the GPC3-targeted CAR immune cells disclosed herein (such as iPSCs, T cells, B cells, NK cells, or macrophages) and one or more therapeutic mAbs (such as one or more of a PD-L1 antibody (e.g., durvalumab, KN035, cosibelimab, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737), or a CLTA-4 antibody (e.g., ipilimumab or tremelimumab)).In one example, the cancer is treated by administration of a GPC3-targeted CAR immune cell (such as an iPSC, T cell, B cell, NK cell, or macrophage) disclosed herein and, for example, one or more of the following mAbs: 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafentox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glembatumumab vedotin, ibritumomab tiuxetan , igovomab, imciromab, intetumumab, inotuzumab ozogamicin, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, matuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentane, ofatumumab, olaratumab, oportuzumab monatox, oregon Bomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, zalutumumab, or combinations thereof.

[0120] In one example, the cancer is treated by administering the GPC3-targeted CAR immune cells disclosed herein (such as iPSCs, T cells, B cells, NK cells, or macrophages) and / or one or more alkylating agents, such as a nitrogen mustard (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), an alkyl sulfonate (such as busulfan), a nitrosourea (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0121] In one example, cancer is treated by administering a GPC3-targeted CAR immune cell (such as an iPSC, a T cell, a B cell, an NK cell, or a macrophage) disclosed herein and one or more antimetabolites, such as a folate analog (such as methotrexate), a pyrimidine analog (such as 5-FU or cytarabine), and a purine analog, such as mercaptopurine or thioguanine.

[0122] In one example, cancer is treated by administering the GPC3-targeted CAR immune cells disclosed herein (such as iPSCs, T cells, B cells, NK cells, or macrophages) and one or more natural products, including vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0123] In one example, cancer is treated by administering the GPC3-targeted CAR immune cells disclosed herein (such as iPSCs, T cells, B cells, NK cells, or macrophages) and one or more platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical suppressants (such as mitotane and aminoglutethimide).

[0124] In one example, cancer is treated by administering a GPC3-targeted CAR immune cell (such as an iPSC, a T cell, a B cell, a NK cell, or a macrophage) disclosed herein and one or more hormones or antagonists, such as a corticosteroid (such as prednisone), a progestin (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), an estrogen (such as diethylstilbestrol and ethinyl estradiol), an anti-estrogen (such as tamoxifen), and an androgen (such as testosterone propionate and fluoxymesterone).

[0125] In one example, cancer is treated with a GPC3-targeted CAR immune cell (such as an iPSC, T cell, B cell, NK cell, or macrophage) disclosed herein, as well as one or more chemotherapeutic agents, such as adriamycin, alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatinum, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, hydrea, idarubicin, ifosfamide, methotrexate, mithramycin, , mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, e.g., docetaxel), velban, vincristine, VP-16, gemcitabine (Gemzar), herceptin, irinotecan (Camptozar, CPT-11), leustatin, navelbine, rituximab STI-571, taxotere, topotecan (Hycamtin), Xeloda (Capecitabine), Zeveline, and calcitriol.

[0126] In one example, cancer is treated by administering the GPC3-targeted CAR immune cells disclosed herein (such as iPSCs, T cells, B cells, NK cells, or macrophages) and one or more immunomodulatory agents, such as AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), Imreg (Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0127] Another treatment that can be used in combination with the treatments provided herein is a surgical treatment, such as surgical removal of the cancer or a portion thereof. Another example of a treatment is radiation therapy, such as the administration of radioactive material or energy (such as external beam radiation therapy) to the tumor site to help cure the tumor or shrink the tumor before surgical removal. In a specific example, the method comprises administering to the subject a therapeutically effective amount of (1) an isolated immune cell or iPSC comprising a nucleic acid molecule encoding a GPC3-targeting CAR and huEGFRt, or administering a therapeutically effective amount of an isolated immune cell or iPSC co-expressing a GPC3-targeting CAR and huEGFRt, thereby treating HCC. In some examples, the method further comprises administering to the subject a therapeutically effective amount of one or more other chemotherapeutic agents or biological agents. In some embodiments, the one or more other chemotherapeutic agents or biological agents are one or more of 5-FU, cisplatin, gemcitabine, oxaliplatin, doxorubicin, capecitabine, floxuridine, or mitoxantrone, such as gemcitabine + oxaliplatin (GEMOS), floxuridine, cisplatin, and oxaliplatin, or 5-FU, oxaliplatin, and leucovorin (FOLFOX). In some embodiments, the one or more other chemotherapeutic or biological agents are one or more of sorafenib, lenvatinib, regorafenib, cabozantinib, and ramucirumab. In some embodiments, the one or more other chemotherapeutic or biological agents are immunotherapeutic agents, such as pembrolizumab and / or nivolumab.

[0128] The following examples are provided to illustrate certain particular features and / or aspects and are not to be construed as limiting the disclosure to the particular described features or aspects. EXAMPLES

[0129] Working Example The fully human HN3 nanobody has several attractive features, including the ability to alleviate immunogenicity concerns in humans; cross-species binding to mouse and human GPC3; and Wnt blocking ability (Li et al., Hepatology 70(4):1231-1245, 2019; Fleming et al., Hepatology 71(5):1696-1711, 2020; Feng et al., Proc Natl Acad Sci USA 110(12):E1083-E1091, 2013; Chen et al., Cancer Immunol Immunother 66(4):475-489, 2017; Phung et al., mAbs 4(5):592-599, 2012; Gao et al., Hepatology 60(1527-3350):576-587, 2014; Zhang and Ho, Sci Rep 6:633878,2016). The fully human HN3 features are less constrained by immunogenicity compared to mouse-derived scFv. The cross-species features allow rapid preclinical testing in mouse models and subsequent studies in humans without modifying the nanobody content. Finally, the HN3 nanobody specifically targets and inactivates the native Wnt-binding domain of GPC3. Utilizing natural binding regions is advantageous since upregulation of Wnt signaling is a key factor in HCC pathogenesis (de La Coste et al., Proc Natl Acad Sci USA 95(15):8847-8851, 1998; Cancer Genome Atlas Research Network, Cell 169(7):1327-1341, 2017; Laurent-Puig et al., Gastroenterology 120(7):1763-1773, 2001; Zucman-Rossi et al., Hepatology 43(3):515-524, 2006; Capurro et al., Cancer Res 65(14):6245-6254, 2005).

[0130] The following examples describe the rational design of GPC3-targeting CAR, using HN3 nanobody or hYP7 scFv as targeting element for delivery to GPC3-positive tumor cells.It is demonstrated herein that precise engineering of hinge and transmembrane domain maximizes antitumor efficacy.By analyzing the contribution of each hinge and transmembrane component, it is determined that hinge and transmembrane act synergistically.

[0131] The data below demonstrate that HN3 engineered T cells containing an IgG4 hinge and CD28 transmembrane induced rapid, strong and durable antitumor responses. Moreover, these antitumor responses were driven by specific CD8+ T cells not seen with other CAR T cells. emra correlated with the signature.

[0132] Example 1: Materials and Methods This example describes the materials and experimental procedures used in the studies described in Examples 2-6.

[0133] Cellular models Hep3B (HCC), HepG2 (hepatoblastoma), HEK-293T, and Jurkat cell lines were purchased from American Type Culture Collection (Manassas, VA). Hep3B and HepG2 were transduced with luciferase-expressing lentivirus. Hep3BKO GFP-luciferase cell lines were established using CRISPR / CAS9 knockout as previously described. Cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin. Peripheral blood mononuclear cells (PBMCs) from peripheral blood of healthy donors were isolated using Ficoll (GE Healthcare, Chicago, IL) according to the manufacturer's instructions. Jurkat cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin at 37° C. in a humidified atmosphere containing 5% CO 2 .

[0134] Jurkat binding and Jurkat NFκB reporter Jurkat cells were transduced with CAR-containing lentivirus at a multiplicity of infection (MOI) of 5. Seven days later, cell surface EGFRt was assessed using an anti-human IgG-phycoerythrin (APC)-conjugated antibody. Cell surface binding of hFc-tagged GPC3 protein (ACRO) to CAR-expressing Jurkat cells was assessed using an anti-human IgG-phycoerythrin (APC)-conjugated antibody (Jackson ImmunoResearch, West Grove, PA).

[0135] Generation of CAR T cells CAR T cells were generated as previously described (Li et al., Gastroenterology 158(8):2250-2265, 2020). Briefly, HEK-293T cells were co-transfected with packaging plasmid psPAX2 (Addgene #12260) and enveloped plasmid pMD2.G (Addgene #12259) using calfectin (SignaGen, Rockville, MD). Lentiviral particles were harvested, concentrated, and functional titers were assessed using EGFRt marker. PBMCs from healthy donors were stimulated for 24 hours in the presence of interleukin 2 (Invitrogen, Carlsbad, CA) using anti-CD3 / anti-CD28 antibody-coated beads. To track cell counts and viability, viable cells were counted using trypan blue.

[0136] Cell Death Research Cytolysis assays were performed using luciferase-expressing cell lines and T cells transduced with GPC3 CAR as previously described (Wang et al., Blood 118(5):1255-1263, 2011). Briefly, tumor cells were incubated with T cells at the indicated effector-target ratios for 24 h. Luminescence of lysates was analyzed in a plate spectrophotometer (Victor, Perkin Elmer) using a luciferase assay system (Promega). Specific lysis of each sample was calculated using the luminescence of target cells alone (corresponding to 0% lysis and 100% lysis, respectively).

[0137] animal research Xenograft mouse models were established by injecting 3 × 10 6 1 x 10 Hep3B cells or 1 x 10 6 Huh7 GFP-luciferase cells were injected intraperitoneally. The mean tumor bioluminescence was 1 × 10 8 When it reaches the mouse, 5 × 10 6 Non-transduced normal human donor T cells or engineered HN3 CAR T cells were injected intraperitoneally or intravenously. Treated mice were imaged with a Xenogen IVIS-200Spectrum camera twice weekly for weeks 1-4 and once weekly from week 4 onwards. Mice were euthanized when IACUC approved morbidity endpoints were reached, which included any signs of distress.

[0138] Example 2: HN3 Nanobody CAR Jurkat Cells Retain Antigen Specificity Several CAR constructs were generated using (1) HN3 or hYP7 scFv as the antigen binding domain, (2) modified IgG4-derived hinge region (IgG4H) or CD8-derived hinge region (CD8H), and (3) CD8 (CD8TM) or CD28 (CD28TM)-derived transmembrane domain: HN3-CD8H-CD8TM, HN3-IgG4H-CD28TM, HN3-IgG4H-CD8TM, HN3-CD8H-CD28TM, hYP7-CD8H-CD8TM, and hYP7-IgG4H-CD88TM. These CAR constructs are shown in Figure 2A and Figure 2B. T cells were transduced with CAR-containing lentivirus and cell counts were measured up to day 11 post-transfection (Figure 2C). The cell counts of transduced T cells were not significantly different from non-transduced T cells. The transduction efficiency of the CAR construct was also measured by detection of CAR-positive cells on day 8 (Figure 2D).

[0139] To test the antigen specificity and cell binding ability of the engineered GPC3-targeting CARs, we generated a T cell line (Jurkat) transfected with the CAR constructs and assessed binding to GPC3 tagged with human Fc (GPC3-hFc). First, successful transduction (95-98%) was confirmed for all CAR constructs using a truncated EGFR marker expressed by the CAR lentivirus. Second, it was demonstrated that all Jurkat-CAR constructs bound efficiently (75.8-98.6%) to GPC3-hFc at 1, 2, and 5 μg / mL (Figure 1A). All CAR T cells bound specifically to GPC3-hFc (Figure 1A) and not to GPC1-hFc (Figure 1B) at the same concentrations. Overall, the data indicates that HN3 and engineered HN3 CARs bind specifically to GPC3 with minimal non-specific binding to other glypicans.

[0140] Example 3: HN3-IgG4H-CD28TM CAR T cells have enhanced activity in settings of high and low antigen density in vitro To test the efficacy of CAR T cells in high versus low antigen density settings, multiple HCC cell lines (Hep3B, HepG2, and Huh7) with various antigen densities were used (Fu et al, Hepatology 70(2):563-576, 2019). These cell lines were isolated from various patients with different levels of tumor progression. Hep3B cells highly express GPC3, while Huh7 cells express GPC3 to a lower extent. It was found that HN3-IgG4H-CD28TM CAR T cells showed the highest cell killing in all HCC lines, while HN3-IgG4H-CD28TM CAR T cells showed high activity in Huh7 cells with low antigen density (Figures 2E-2H). HN3-CD8H-CD28TM CAR T cells demonstrated a 40% improved killing compared to HN3-CD8H-CD8TM CAR T cells, indicating that CD28TM increases CAR T potency under both low and high antigen density conditions. However, CD28TM alone is insufficient to induce 100% killing at lower ratios. Comparing HN3-CD8H-CD28TM to HN3-IgG4H-CD28TM, IgG4H increased potency by 10-20%. All CAR T cells were minimally reactive with the GPC3 knockout cell line (Figure 2F) and had low levels of alloantigen reactivity and antigen-specific cell killing under these conditions. Taken together, these results indicate that HN3-IgG4H-CD28TM is the most potent CAR construct tested. Both IgG4 hinge and CD28 TM independently enhance the antitumor efficacy of GPC3 CAR T cell therapy.

[0141] Example 4: Fc-free IgG4H and CD28TM domains enhance HN3 potency in vitro To test whether hinge length affects antigen binding, HN3 CAR T cells were tested incorporating Fc components and with short hinge (IgG4H), medium hinge (IgG4-CH3), and long hinge (IgG4-CH2CH3) (Smith et al., Sci Transl Med. 11(485):eaau7746, 2019; Jonnalagadda et al., Mol Ther 23(4):757-768, 2015). Cell counting and transduction assays showed that the long hinge constructs expanded slower and expressed less effectively at the same MOI. All constructs had minimal reaction with GPC3 knockout Hep3B cells (ell), indicating antigen-specific interaction. HN3-IgG4H-CD28TM CAR T cells outperformed medium and long hinge CAR T cells. These data demonstrate that Fc-based hinge extension is not required and can disrupt HN3-GPC3 interactions in vivo. Altogether, the data confirm that HN3 nanobodies can be engineered to induce potent killing in vitro using both IgG4H and CD28TM.

[0142] Example 5: HN3-IgGH-CD28TM CAR T cells eradicate GPC3-high HCC xenografts in mice Having demonstrated both the specificity and cytotoxicity of engineered CAR T cells in vitro, further studies were performed to test their in vivo antitumor activity. In the first study (Figure 3A), Hep3B GFP / luciferase expressing cells (3 million) were injected IP into NSG mice and allowed to engraft for 12 days. Mice were treated with 5 million hYP7-CD8H-CD8TM, hYP7-IgG4H-CD28TM, HN3-IgG4H-CD8TM, or HN3-IgG4H-CD28TM CAR T cells on day 0 and imaged periodically. Results are shown in Figures 3B-3D. Consistent with the in vitro findings, HN3-IgG4H-CD28TM CAR T cells demonstrated high antitumor activity, with tumors eradicated within 10 days. In contrast, tumors in the HN3-IgG4H-CD8TM group responded in part and continued to grow during the study. Median survival times for mice treated with non-transduced T cells, HN3-CD8H-CD8TM, and hYP7-CD8H-CD8TM CAR T cells were 27, 29, and 30 days, respectively. In contrast, three mice treated with HN3-IgG4H-CD28TM, and HN3-IgG4H-CD8TM survived until the end of the study (day 35). The HN3-IgG4H-CD28TM group showed a rapid reduction in tumor size starting from day 3 and ultimately remained tumor-free for the entire study period.

[0143] To address the issue of scFv and nanobody performance, we included hYP7-IgG4H-CD28TM CAR T cells for comparison with HN3-IgG4H-CD28TM. The hYP7-IgGH-CD28TM construct was difficult to transduce in vitro, with transduction rates ranging from 25-30%. Despite these challenges, the construct worked well in the cells tested, especially in the Hep3B and HepG2 lines. Surprisingly, in vivo, the engineered HN3 construct induced tumor regression within 3 days, while the hYP7 construct was delayed in inducing tumor regression until day 30. These data suggest that HN3-IgG4-CD28TM is more potent than hYP7-IgG4-CD28TM at identical dose levels.

[0144] In a second study using identical study conditions as above (Figure 4A), the in vivo antitumor activity of Fc-containing CAR T cells was tested. The results are shown in Figures 4B-4D. Consistent with the in vitro findings, in the HN3-IgG4H-CD28TM group, tumors regressed within 7 days. All tumors in the HN3-IgG4H-CD28TM group were completely eradicated and did not regrow after 67 days. Mice treated with CAR T cells containing Fc components showed partial responses. Notably, 4 of 5 mice in the HN3-IgG4H-CH3-CD28TM group ("HN3-IgG4H-CD28TM-M") were tumor-free at day 24, and 2 of 5 mice in the HN3-IgG4H-CH2CH3-CD28TM ("HN3-IgG4H-CD28TM-L") group were tumor-free at day 15. The HN3-CD8H-CD8TM group did not respond to CAR T cell treatment.

[0145] In an in vivo low antigen density model (Huh7), significant tumor regression was observed in 5 of 6 mice (Figures 6A-6D). Collectively, these data demonstrated that the HN3-IgG4H-CD28TM construct was the most potent in achieving rapid and sustained antitumor responses.

[0146] Example 6: Chimeric antigen receptor T cells targeting GPC3 express CD8+ and T emra Enriching subsets Blood from study mice was analyzed to monitor the number of CAR T cells, exhaustion markers, and T cell subsets (Chen et al., Cancer Discov 11(9):2186-2199, 2021; Ma et al., Cancer Discov 3(4):418-429, 2013; Xu et al., Blood 123(24):3750-3759, 2014). The results are shown in Figure 5A-5F. At day 28, mice from the HN3-IgG4H-CD28TM group had several log-fold higher CAR T cell numbers than the HN3-CD8H-CD8TM treatment group, indicating a greater proliferative response. In addition, PD1 expression was lowest in the HN3-IgG4H-CH3-CD28TM group (Guo et al., Front Pharmacol 9:1118, 2018). To understand the T cell response, CD8 versus CD4 expression was examined, as well as T cell subsets including Tscm, Tcm, Tem, and Temra. Engineered CAR T cells harbored large numbers of CAR-positive CD8 T cells. T cells displayed similar phenotypes early on, but by weeks 4 and 5, they displayed both memory and effector functions in vivo. By weeks 4 and 5, most CAR T cells in the HN3-IgG4H-CD28TM group had a CD8+ phenotype and were engaged in effector function. These data support the conclusion that over the course of 2–5 weeks, CAR T cell expansion resulted in the proliferation of CD8+ T cells. emra We show that CD8+ T cells are clearly shifted to CD8+ T cells, which appear to be crucial for antitumor responses. Central memory T cells were also maintained within a subset of the population, indicating that both T cell types are required for avoiding exhaustion and continuing trafficking to the tumor site. emra This predominance of CD8+ T cells was not observed in non-responders in the Huh7 tumor group. emra This supports the hypothesis that early cell migration is important for inducing regression and sustained responses.

[0147] Example 7: HN3-IgG4H-CD28TM blocks Wnt signaling and activates NFAT in HCC Given the in vivo efficacy, we tested the ability of the HN3 construct to block tumor Wnt signaling when exposed to GPC3-expressing Hep3B cells (Figure 7A). We observed a decrease in both active and total β-catenin levels in HN3-IgG4H-CD28TM CAR T cells when co-cultured with Hep3B cells for 30 min and 3 h. Engineered HN3 CAR T cells rapidly inhibited total β-catenin and completely abolished active β-catenin at 3 h (Figure 7A). Even at 30 min, engineered HN3 constructs had lower total β-catenin levels. These data confirmed that Wnt signaling is a direct target of HN3-based CAR T cells and that within 3 h, HN3-directed CAR T cells directly inhibit β-catenin activation.

[0148] To understand how CAR T cells work in vivo, we performed studies to determine whether NFAT plays a role in T cell signaling (Wilson et al., Nat Commun 6:6818, 2015; Li et al., Cancer Cell 31:383-395, 2017; Capurro et al., J Cell Sci 127:1565-1575, 2014). Over each time point, NFAT signaling was stepwise higher in HN3-IgG4H-CD28TM CAR T cells compared to the original construct containing CD8H-CD8TM. Maximal saturation of NFAT signaling was reached at 4 hours when CAR cells outnumbered Hep3B cells in a representative imaging window (Figure 7B). When tdTomato signal was quantified by cell count and size, the difference between NFAT signaling in HN3-IgG4H-CD28TM versus HN3-CD8H-CD8TM was striking. NFAT signaling in HN3-IgG4H-CD28TM treated Hep3B cells is initiated in a greater number of cells across the 200 range, doubling within 4 hours in terms of cell number and signal intensity. These findings indicated that NFAT-mediated signaling is more prevalent in T cell activation in HN3-IgG4H-CD28TM than in HN3-CD8H-CD8TM. Overall, it was observed that NFAT was robustly upregulated in HN3-IgG4H-CD28TM CAR T cells. Based on the overall data, it is proposed that fine-tuning the antigen-CAR interaction alters these mechanisms in the aforementioned cells, thereby potently and persistently eradicating tumors.

[0149] It will be apparent that the precise details of the methods or compositions described may be changed or modified without departing from the spirit of the disclosed embodiments described, and the inventors assert that all such modifications and variations are within the scope and spirit of the following claims.

Claims

1. A chimeric antigen receptor (CAR), an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3); a hinge region consisting of the IgG4 hinge region set forth in SEQ ID NO: 43 or SEQ ID NO: 52; transmembrane domain; an intracellular costimulatory domain; and Intracellular signaling domains A chimeric antigen receptor (CAR) comprising:

2. The CAR of claim 1, wherein the extracellular antigen-binding domain comprises a GPC3-specific single domain antibody.

3. The CAR of claim 2, wherein the single domain antibody comprises the complementarity determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO:

18.

4. the CDR1, CDR2, and CDR3 sequences are, respectively: Residues 31-35, 50-65, and 96-105 of SEQ ID NO: 18; or Residues 26-33, 51-57, and 96-105 of SEQ ID NO:18 The CAR of claim 3, comprising:

5. The CAR of claim 3, wherein the amino acid sequence of the single domain antibody is at least 90% identical to SEQ ID NO: 18 and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

18.

6. The CAR of claim 2, wherein the amino acid sequence of the single domain antibody comprises or consists of SEQ ID NO:

18.

7. The CAR of claim 1, wherein the antigen-binding domain comprises a GPC3-specific scFv.

8. The CAR of claim 7, wherein the scFv comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises complementarity determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 20, and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

22.

9. the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 31-35, 50-68, and 101-106, respectively, of SEQ ID NO:20, and the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 24-40, 56-62, and 95-103, respectively, of SEQ ID NO:22; or The CAR of claim 8, wherein the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 26-33, 51-60, and 99-106, respectively, of SEQ ID NO: 20, and the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 27-38, 56-58, and 95-103, respectively, of SEQ ID NO:

22.

10. the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO:20 and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:20; and The CAR of claim 8, wherein the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 22 and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

22.

11. the amino acid sequence of the VH domain comprises or consists of SEQ ID NO:20; and The CAR of claim 8, wherein the amino acid sequence of the VL domain comprises or consists of SEQ ID NO:

22.

12. The CAR of claim 8, wherein the amino acid sequence of the scFv comprises residues 1 to 245 of SEQ ID NO:

14.

13. The CAR of claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain.

14. The CAR of claim 1, wherein the costimulatory domain comprises a 4-1BB signaling moiety.

15. The CAR of claim 1, wherein the signaling domain comprises a CD3ζ signaling domain.

16. An isolated cell expressing the CAR according to any one of claims 1 to 15.

17. 17. The isolated cell of claim 16, which is an immune cell or an induced pluripotent stem cell (iPSC).

18. 18. The isolated cell of claim 17, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, or a macrophage.

19. A nucleic acid molecule encoding the CAR according to any one of claims 1 to 15.

20. 20. The nucleic acid molecule of claim 19 operably linked to a promoter.

21. In the 5' to 3' direction, a nucleic acid encoding a first granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding the antigen-binding domain; a nucleic acid encoding the IgG4 hinge region; a nucleic acid encoding the transmembrane domain; a nucleic acid encoding the costimulatory domain; a nucleic acid encoding the signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and Nucleic Acids Encoding Truncated Human Epidermal Growth Factor Receptor (huEGFRt) 20. The nucleic acid molecule of claim 19, comprising:

22. 22. The nucleic acid molecule of claim 21, further comprising a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss.

23. A vector comprising the nucleic acid molecule of claim 19.

24. 24. The vector of claim 23, wherein the vector is a lentiviral vector.

25. 20. An isolated cell comprising the nucleic acid molecule of claim 19.

26. 26. The isolated cell of claim 25, which is an immune cell or an induced pluripotent stem cell (iPSC).

27. 27. The isolated cell of claim 26, wherein the immune cell is a T cell, a B cell, a NK cell, or a macrophage.

28. A composition comprising a pharmaceutically acceptable carrier and the CAR according to any one of claims 1 to 15.

29. A composition for treating GPC3-positive cancer in a subject, comprising the CAR according to any one of claims 1 to 15.

30. A composition for inhibiting tumor growth or metastasis of GPC3-positive cancer in a subject, the composition comprising the CAR according to any one of claims 1 to 15.

31. 30. The composition of claim 29, wherein the GPC3-positive cancer is a solid tumor.

32. 30. The composition of claim 29, wherein the GPC3-positive cancer is hepatocellular carcinoma (HCC), melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms' tumor, squamous cell carcinoma of the lung, testicular nonseminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, or embryonal tumor.

33. 33. The composition of claim 32, wherein the GPC3-positive cancer is HCC.

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