Compositions and methods for treating cancer using chimeric antigen receptors

Abstract

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

Description

Background of the Invention Technical Field

[0002] The present disclosure relates to the use of chimeric antigen receptor T cells to treat cancer. Background Art

[0004] 1. Chimeric antigen receptor T cell therapy

[0005] Chimeric antigen receptor (CAR) T cell therapy is a specific form of cell-based immunotherapy that uses engineered T cells to fight cancer. In CAR-T cell therapy, T cells are harvested from the patient's blood, engineered in vitro to express CAR containing an antigen binding domain and a T cell activation domain, expanded to a larger population and administered to the patient. CAR-T cells are used as living drugs that bind to cancer cells and cause the destruction of these cancer cells. When successful, the effects of CAR-T cell therapy tend to last for a long time, as demonstrated by detecting the persistence and amplification of CAR-T cells in patients for a long time after clinical remission.

[0006] 2. CAR structure and function

[0007] The antigen binding domain of CAR is the extracellular region of the surface antigen on the target tumor cell. Suitable target antigens can be proteins, phosphorylated proteins, peptide-MHC, carbohydrates or glycolipid molecules. Ideal target antigens are widely expressed on tumor cells to be able to target a high percentage of cancer cells. Ideal candidate target antigens are also minimally expressed on normal tissues, thereby limiting the on-target toxicity outside the tumor. The antigen binding domain of CAR includes a targeting portion for the target antigen, such as an antibody single-chain variable fragment (scFv).

[0008] The T cell activation domain of the CAR is intracellular and activates T cells in response to the antigen binding domain that interacts with the target antigen. The T cell activation domain can contain one or more costimulatory domains, which are intracellular domains known to activate T cell receptors. Because costimulatory domains have different effects on CAR-T cell dynamics, cytotoxic function, and safety, the choice and location of the costimulatory domain within the CAR construct can affect the function and fate of the CAR-T cell.

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

[0010] CAR-T therapy targeting B lymphocyte antigen CD19 ( Novartis has shown promise in pediatric acute lymphoblastic leukemia, and its CAR-T therapy ("bb2121," and Collaboration) shows promise for relapsed / refractory multiple myeloma. Recent data suggest that CAR approaches can be effective against solid tumors. GD2 CAR natural killer T cell (NKT) therapy has shown activity in neuroblastoma (Heczey A et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood; 124(18): 2824-33, 2014), and mesothelin CAR-T with pembrolizumab has demonstrated anti-tumor activity in mesothelioma. However, additional targets for the treatment of solid tumors are needed.

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

[0012] Unfortunately, the complexity of CAR-T cell-based therapies may lead to undesirable and unsafe effects. Toxic effects, such as neurotoxicity and acute respiratory distress syndrome, are potential adverse effects of CAR-T cell therapies and may be fatal. Cytokine release syndrome (CRS) is the most common acute toxicity associated with CAR-T cells. When lymphocytes are highly activated and release excessive inflammatory cytokines, CRS occurs. When these factors are measured, serum levels of interleukin-2, interleukin-6, interleukin-1β, GM-CSF and / or C-reactive protein are sometimes observed to increase in patients with CRS. CRS is graded and diagnosed as one of grades 1-4 (mild to severe) according to severity, with the clinical features of more severe cases being high fever, hypotension, hypoxia and / or multi-organ toxicity in patients. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR-T cell therapy experienced CRS, and 50% of these patients had grade 3-4 symptoms (Fitzgerald et al., Crit Care Med. 45(2):e124-e131 (2017)).

[0013] Another challenge for successful CAR-T cell immunotherapy is the immunosuppression caused by the characteristics of the tumor microenvironment (TME) of solid tumors. For example, transforming growth factor β (TGF-β) is a pleiotropic cytokine produced by many cell types in the liver (e.g., liver sinusoidal endothelial cells, Kupffer cells, intrahepatic natural killer (NK) cells, etc.) and is present in large quantities in the cancer microenvironment (Dahmani et al., TGF-βin T Cell Biology: Implications for Cancer Immunotherapy. [TGF-β in T cell biology: Impact on cancer immunotherapy] Cancers [Cancer] 2018, 10, 194, 1-21). TGF-β binds to TGFβR2, which recruits and phosphorylates TGFβR1. Once phosphorylated, TGFβR1 then phosphorylates receptor-regulated SMAD (R-SMAD). The complex of phosphorylated SMAD and coSMAD is transferred to the nucleus to help regulate gene expression. In the context of T cells, TGF-β signaling inhibits the effectiveness of CAR-T cell therapy by suppressing T cell proliferation, activation, and effector function and by promoting the differentiation of regulatory T cells. Therefore, TGF-β-related immunosuppression is a major obstacle that must be overcome to achieve effective and durable CAR-T cell therapy for solid tumors.

[0014] 4. Armoring

[0015] The latest approach to making CAR-T cells more resistant to tumor-associated immunosuppression is called "armoring." Armoring is the molecular manipulation of CAR-T cells to express one or more "armor molecules" that can resist immunosuppression. For example, researchers recently reported modifying CAR-T cells to secrete a single-chain variable fragment (scFv) that blocks PD-1, which improved PD-L1 expression. +CAR-T cell antitumor activity in mouse models of hematological and solid tumors (Rafiq, S., Yeku, O., Jackson, H. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 36, 847-856 (2018)). Other studies have shown the effectiveness of armoring T cells with dominant negative type 2 TGF-β receptor (TGFβRIIDN) armor molecules to neutralize the inhibitory effects of TGF-β on T cells (Bollard et al., Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma, J Clin Oncol 36(11): 1128-1139 (2018). Currently, at least one clinical study is investigating the effectiveness of armoring anti-PSMA-CAR-T cells with TGFβRIIDN armor molecules for the treatment of castration-resistant prostate cancer (NCT03089203).

[0016] Therefore, additional CAR-T cell therapies are needed to enhance the armamentarium of effective cancer treatment. Such therapies should include CAR-T cell therapies that effectively treat cancer while minimizing the risk of dangerous inflammatory reactions such as CRS. Furthermore, such therapies should include CAR-T cells that can persist in the immunosuppressive TME of solid tumors. Summary of the Invention

[0017] The present disclosure describes compositions and methods for treating cancer using CAR-T cells. As described below, in a first aspect, an isolated nucleic acid sequence encodes (a) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cell surface antigen; and (b) an armored molecule, wherein the armored molecule resists immunosuppression of the cell when expressed on the surface of a cell in a tumor microenvironment.

[0018] In another aspect, the disclosure describes a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a TGFβRIIDN armor molecule expressed on the cell surface.

[0019] In another aspect, the disclosure describes a cell comprising: an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; and a TGFβRIIDN armor molecule.

[0020] In yet another aspect, the present disclosure describes a method of treating cancer, comprising administering cells to a subject in need thereof, wherein the cells comprise (a) a chimeric antigen receptor (CAR) specific for a cell surface antigen, and (b) an armored molecule, wherein the armored molecule resists immunosuppression of cells in the tumor microenvironment of the cancer.

[0021] These and other features and advantages of the present invention will be more fully understood from the following detailed description and appended claims.It should be noted that the scope of the claims is defined by the recitation therein rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure and together with the description serve to explain the principles and operations of the disclosure.

[0023] Figures 1A-1DTGFβ1 and the TGFβ gene signature and HCC development and survival. 1A. Fold change (log2) of TGFβ1 gene expression in normal solid tissues (left) and primary solid tumors of hepatocellular carcinoma (HCC, right). 1B. Survival curves (OS = overall survival) for high versus low TGFβ1 expression in HCC. 1C. Fold change (log2) of TGFβ signaling in normal solid tissues (left) and solid tumor tissues (right). 1D. Survival curves for high versus low TGFβ signaling.

[0024] Figure 2 A and 2B. Human HCC samples are positive for TGFβ and TGFβ signaling. Semi-quantitative pathological assessment of (2A) TGF-β1 and (2B) phosphorylated SMAD2 (pSMAD2) in FFPE normal liver and HCC samples. Each circle represents data from a single surgical resection. IHC intensity was defined as: score 0, negative staining; score 1, minimal staining; score 2, moderate staining; score 3, strong staining.

[0025] Figure 3 A and 3B. Armoring GPC3 CAR-T with TGFβRIIDN. As contemplated herein, armoring GPC3 CAR-T cells with TGFβRIIDN is believed to confer resistance to TGFβ, resulting in improved CAR-T effector function and tumor control rates. 3A. Schematic diagram of TGF-β-mediated immunosuppression of unarmored CAR-T cells (Figure modified from: Arrese et al., Current Protein & Peptide Science [Contemporary Protein and Peptide Science] (2018) 19: 1172). 3B. Diagram of armored CAR-T cells expressing GPC3 CAR and TGFβRIIDN armor molecules. In some embodiments, the CAR and armor molecules can be fused or separated from the CAR by a spacer peptide at the C-terminus.

[0026] Figure 4 . Expression of TGFβRIIDN. Flow cytometric analysis of TGFβRII and CAR on the surface of unarmored and armored CAR-T cells.

[0027] Figure 5 TGFβRIIDN inhibits TGF-β signaling in CAR-T cells. 4B. Western blot analysis of phosphorylated SMAD2 / 3 and total SMAD 2 / 3 in untransduced (UT), unarmored, and armored (TGFβRIIDN) CAR-T cells after 0-45 minutes of rhTGFβ (1 ng / ml) exposure. β-Actin served as a positive loading control for all samples.

[0028] Figure 6 A and 6B. Expression of TGFβRIIDN prevents TGF-β-induced repression of effector cytokine transcription. (6A) IL2 and (6B) IFNG mRNA levels in purified TGFβRIIDN and unarmored GPC3 CAR-T cells stimulated with the indicated concentrations of plate-bound recombinant GPC3 and TGF-β1 for 6 hours. Data are combined from two independent experiments.

[0029] Figure 7 A and 7B. TGFβRIIDN reduces TGF-β-mediated differentiation of GPC3 CAR-T cells into T cells. RM Phenotype. 7A. TGF-β-mediated differentiation of GPC3 CAR-T cells into tissue-resident memory (T) cells in the absence (left plot) and presence (right plot) of TGF-β in unarmored (upper plot) and armored (lower plot) GPC3 CAR-T cells. RM ) cells. Y axis = CD103 expression; X axis = CAR expression. 7B. T cell expression in unarmored (GPC3) and armored (GPC3 TGFβRIIDN) CAR-T cells treated with TGF-β, as demonstrated by CD69+ / CD103+ co-expression. RM Percentage of cell differentiation.

[0030] Figure 8 A, 8B, and 8C. Exploratory in vitro readouts in unarmored and armored CAR-T cells. 8A. Bright field images of real-time monitoring of CAR-T-mediated cytotoxicity during co-culture with GPC3+ tumor cells. 8B. Visual observation of CAR-T cell expansion during co-culture with GPC3+ tumor cells. A real-time impedance-based killing assay (RTCA) assessed CAR-T-mediated cytotoxicity during co-culture with GPC3+ tumor cells and expressed as a cell index. 8C. Quantification of tumor cell (HEP3B or HUH-7 cell)-induced CAR-T cell proliferation in unarmored and armored GPC3 CAR-T cells (AZ: internal vector backbone; LN: Lentigen vector backbone) in the presence or absence of TGF-β. After 4 days of co-culture, TGF-β inhibited tumor GPC3-induced CAR-T cell proliferation. Armored GPC3 CAR-T cells were less susceptible to inhibition.

[0031] Figure 9TGFβRIIDN-armored CAR-T cells and tumor volume reduction in an in vivo xenograft model. Huh7-TGF-β-overexpressing xenograft model. Untransduced T cells, unarmored GPC3 CAR-T cells, or TGFβRIIDN-armored GPC3 CAR-T cells were infused into mice bearing Huh7 tumors engineered to overexpress TGF-β. Tumor volume was measured weekly (10 mice / group).

[0032] Figure 10 The number of tumor-infiltrating lymphocytes increased in mice treated with armored TGFβRIIDN CAR-T cells. Mice bearing Huh7-TGF-β-overexpressing tumors were given 7x10 6 Untransduced T cells, unarmored or TGFβRIIDN-armored GPC3 CAR-T. 10A. CAR in mouse tumors harvested at the indicated time points. + 10B. Representative examples of FACS data are summarized in 10A.

[0033] Figure 11 The number of TGFβRIIDN-armored CAR-T cells in the spleen increased. 7x10 6 Untransduced T cells, unarmored or TGFβRIIDN-armored GPC3 CAR-T cells. 11A. CAR in the spleen of mice harvested at the indicated time points. + 11B. Representative examples of FACS data are summarized in 11A.

[0034] Figure 12 . Expression of TGFβRII on the surface of CAR+ cells in vitro. 12A. Expression of CAR and TGFβRII on the surface of lymphocytes infiltrating the spleen or tumor 14 days after infusion in mice bearing tumors overexpressing Huh7-TGF-β. (FMO: fluorescence minus one). 12B. Expression of TGFβRII on CAR in tumors + or CAR - CD8 + Mean fluorescence intensity (MFI) on the surface of T cells.

[0035] Figure 13 Reduced PD1 and LAG3 expression on the surface of TGFβRIIDN-armored CAR-T cells in tumors. PD1 (A) and LAG3 (B) expression on tumor-infiltrating CD8 + and CD4 + CAR + Expression on the surface of T cells.

[0036] Figure 14 . Reduced CD70 expression and increased CD27 expression on the surface of TGFβRIIDN-armored CAR-T cells in tumors. 14 days after infusion in mice bearing tumors overexpressing Huh7-TGF-β, CD70 + CD27 - (A) and CD70 + CD27 - (B) In tumor-infiltrating CD8 + and CD4 + CAR + frequency on the surface of T cells.

[0037] Figure 15 Immunophenotyping of CAR-T cells in the spleen. 14 days after infusion in mice bearing tumors overexpressing Huh7-TGF-β, PD1 (15A), LAG3 (15B), and CD27 / CD70 (15C) were expressed in the spleen of CD8 + and CD4 + CAR + Expression on the surface of T cells.

[0038] Figure 16 Analysis of serum IFN-γ. IFN-γ concentrations were detected in the serum of mice bearing tumors overexpressing Huh7-TGF-β and infused with untransduced T cells, unarmored, or armored GPC3 CAR-T cells (Baseline A: before tumor transplantation; Baseline B: before CAR-T infusion). Figure 17 Analysis of serum AFP. AFP concentrations were detected in the serum of mice bearing Huh7-TGF-β-overexpressing tumors and infused with untransduced T cells, unarmored, or armored GPC3 CAR-T cells (Baseline A: before tumor transplantation; Baseline B: before CAR-T infusion).

[0039] Figure 18 . CAR-T cells armored with TGFβRIIDN and reduction of tumor volume in the in vivo TGFβ(-) PDX model. Figure 18 AC represents three different GPC3 + TGF-β - Tumor volume of hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5x10 6 Untransduced T cells, unarmored, or armored GPC3 CAR-T cells were infused into tumor-bearing mice, and tumor volume was measured every two weeks (5 mice / group).

[0040] Figure 19 . CAR-T cells armored with TGFβRIIDN and reduction of tumor volume in TGFβ(+)PDX models in vivo. AE represents five different, GPC3 + TGF-β + Tumor volume of hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5x10 6 Untransduced T cells, unarmored, or armored GPC3 CAR-T cells were infused into tumor-bearing mice, and tumor volume was measured every two weeks (5 mice / group). DETAILED DESCRIPTION

[0041] 1. Definition

[0042] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide general definitions of multiple terms used in the present invention for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology [microbiology and molecular biology dictionary] (2nd edition 1994); The Cambridge Dictionary of Science and Technology [Cambridge Dictionary of Science and Technology] (Walker, 1988); The Glossary of Genetics [genetics vocabulary], 5th edition, R. Rieger et al. (eds.), Springer Verlag (Springer Verlag) (1991); and Hale and Marham, The Harper Collins Dictionary of Biology [Harper Collins biological dictionary] (1991). Unless otherwise indicated, the following terms as used herein have the following meanings assigned to them.

[0043] As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises / comprising," "includes / including") should be understood to imply the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning.

[0044] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0045] The percentages disclosed herein may vary from the disclosed values ​​by an amount of ±10%, 20%, or 30% and still be within the intended disclosed range.

[0046] Unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, the values ​​herein expressed as ranges in the various embodiments of the present disclosure may take any specific value or sub-range within the stated range, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0047] As used herein, ranges and amounts may be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%" and also refers to "5%." The term "about" can also refer to ±10% of a given value or range of values. Thus, for example, about 5% also refers to 4.5%-5.5%. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about."

[0048] As used herein, the terms "or" and "and / or" can describe multiple components that are combined or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z".

[0049] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may replace or be used interchangeably with any of these terms.

[0050] As used herein, "protein" may refer to a single polypeptide, ie, a single amino acid chain as defined above, but may also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to create a multimeric protein.

[0051] "Isolated" material, such as an isolated nucleic acid, is material that is not in its natural environment, although the isolated material is not necessarily purified. For example, an isolated nucleic acid is a nucleic acid that is not produced or located in its natural or native environment (e.g., a cell). The isolated material can be separated, fractionated, or at least partially purified by any suitable technique.

[0052] As used herein, the terms "antibody" and "antigen-binding fragment thereof" refer to at least the minimal portion of an antibody that is capable of binding to a designated antigen that the antibody targets, for example, in the case of a typical antibody produced by a B cell, the variable domain of the heavy chain (VH) and at least some of the complementarity determining regions (CDRs) of the variable domain of the light chain (VL). [RP1] ). The antibody or antigen-binding fragment thereof can be or be derived from a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody, a single chain antibody, an epitope binding fragment, e.g., Fab, Fab′ and F(ab′)2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), a fragment comprising a VL or VH domain (e.g., an entire VL domain and a partial VH domain having one, two or three CDRs) alone or in combination with a portion of the opposite domain, and fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The antibody molecules encompassed by the present disclosure can be or be derived from any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. Unless otherwise indicated, the amino acid numbering in the variable domains, complementarity determining regions (CDRs), and framework regions (FRs) of antibodies follows the Kabat definition as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0053] As used herein, the term "polynucleotide" includes single nucleic acids as well as multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any nucleic acid type, such as DNA or RNA.

[0054] As used herein, the term "vector" may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. Specific types of vectors contemplated herein may be associated with or incorporated into viruses to promote cellular transformation.

[0055] A "transformed" cell or "host" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. All techniques for introducing a nucleic acid molecule into such a cell are contemplated herein, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0056] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (e.g., an epitope) to its cognate binding domain (e.g., a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a population of epitopes and paratopes (i.e., antigens and antigen-binding domains).

[0057] As used herein, the terms "treat," "treatment," and "treatment of" when used in the context of treating cancer refer to alleviating disease pathology, alleviating or eliminating disease symptoms, promoting improved survival, and / or alleviating discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or disorder.

[0058] As used herein, the term "subject," "individual," or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

[0059] As used herein, the term "effective amount" or "therapeutically effective amount" of a therapeutic substance (such as CAR-T cells) administered is an amount sufficient to carry out a particular stated or intended purpose, such as treating cancer. An "effective amount" can be determined empirically in a routine manner according to the stated purpose.

[0060] 2. Overview

[0061] The present disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More specifically, the present disclosure relates to CAR cell therapy, in which transformed cells (such as T cells) express, for example, CAR targeting glypican-3 (GPC3). CAR constructs disclosed herein, cells expressing the transformation of these constructs, and the therapy utilizing these transformed cells can provide robust cancer treatment with cytokine release syndrome (CRS) or the risk of minimizing the release of indiscriminate cytokines in non-GPC3 expressing cells.

[0062] Without wishing to be bound by theory, GPC3 is believed to be a viable cancer target in a variety of forms, including bispecific T cell engagers, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). GPC3 is an oncofetal antigen and a GPI-linked heparin sulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction, thereby stimulating Wnt signaling. GPC3 competes with Patched for Hh binding, which relieves Smoothened inhibition and induces GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) growth. Furthermore, GPC3 expression levels have been shown to correlate with the stage and grade of HCC.

[0063] Furthermore, GPC3 is believed to be a promising target for CAR cell therapy. Accordingly, antibodies and CAR constructs derived from these antibodies have been developed as described herein.

[0064] Additional aspects of the disclosure include CAR-T cells, such as those targeting GPC3 and those armored with TGFβRIIDN to protect CAR-T cells from TGF-β-related immunosuppression, for example, against solid tumors.

[0065] 3. CAR Construct Design

[0066] The CAR construct of the present disclosure may have several components, many of which may be selected based on the desired or precise function of the resulting CAR construct. In addition to the antigen binding domain, the CAR construct may also have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. Selecting a component rather than another (i.e., selecting a specific costimulatory domain from a receptor, relative to the costimulatory domains from different receptors) may affect clinical efficacy and safety.

[0067] 4. Antigen Binding Domain

[0068] Antigen binding domains contemplated herein may include antibodies or one or more antigen-binding fragments thereof. A contemplated CAR construct targeting GPC3 comprises a single-chain variable fragment (scFv) containing light and heavy chain variable regions from one or more antibodies specific for GPC3, these variable regions being linked together directly or via a flexible linker (e.g., a repeating sequence of GGGGS with 1, 2, 3 or more repeats).

[0069] As disclosed herein, the binding affinity of the antigen binding domain of CAR to the target protein can vary. As compared with antibodies (generally these antibodies are expected to have higher affinity), in the case of CAR, the relationship between binding affinity and therapeutic effect may be more subtle. For example, when compared with low-affinity variants, preclinical studies of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CAR derived from high-affinity scFv (with a dissociation constant of 0.56nM) lead to an increase in therapeutic index. On the contrary, other examples have been reported, in which engineered scFv improves the distinction between cells with different antigen densities for lower affinity. This can be used to improve the therapeutic specificity of antigens differentially expressed in tumor tissue and normal tissue.

[0070] A variety of methods can be used to determine the binding affinity of an antigen binding domain. In some embodiments, methods that exclude avidity effects can be used. Avidity effects involve multiple antigen-binding sites that interact with multiple target epitopes simultaneously, typically involving multimeric structures. Therefore, avidity functionally represents the cumulative strength of multiple interactions. An example of a method that excludes avidity effects is any method in which one or both of the interacting proteins are monomeric / monovalent, because if one or both partners only contain a single interaction site, multiple simultaneous interactions are impossible.

[0071] 5. Spacer domain

[0072] The CAR construct of the present disclosure may have a spacer domain to provide conformational freedom, thereby promoting binding to the target antigen on the target cell. The optimal length of the spacer domain may depend on the proximity of the binding epitope to the target cell surface. For example, a proximal epitope may require a longer spacer, while a distal epitope may require a shorter spacer. In addition to promoting the binding of CAR to the target antigen, achieving the optimal distance between the CAR cell and the cancer cell can also help to spatially block the entry of large inhibitory molecules into the immune synapse formed between the CAR cell and the target cancer cell. CAR may have a long spacer, a medium spacer, or a short spacer. The long spacer may include a CH2CH3 domain (about 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural, or with common modifications in therapeutic antibodies, such as S228P mutations), and the CH3 region can be used alone to construct a medium spacer (about 120 amino acids). The short spacer may be derived from a segment (<60 amino acids) of CD28, CD8α, CD3, or CD4. The short spacer may also be derived from the hinge region of the IgG molecule. These hinge regions may be derived from any IgG isotype and may or may not contain mutations commonly found in therapeutic antibodies, such as the S228P mutation mentioned above.

[0073] 6. Hinge domain

[0074] CAR may also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to a target antigen on a tumor cell. It can be used alone or in combination with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably - for example, an IgG4 sequence can be considered a "hinge" sequence and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0075] CAR may further include a sequence comprising a signal peptide. The function of the signal peptide is to promote cells to transfer CAR to the cell membrane. Examples include IgG1 heavy chain signal peptide, Ig kappa or lambda light chain signal peptide, granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal peptide or CD33 signal peptide.

[0076] 7. Transmembrane domain

[0077] The CAR may further include a sequence comprising a transmembrane domain. The transmembrane domain may include a hydrophobic alpha helix that spans the cell membrane. The properties of the transmembrane domain have not been studied as carefully as other aspects of the CAR construct, but it may potentially affect CAR expression and association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0078] 8.Co-stimulatory domain

[0079] CAR may further include one or more sequences forming a costimulatory domain. A costimulatory domain is a domain that can enhance or regulate the response of immune effector cells. A costimulatory domain may include, for example, one or more sequences from CD3ζ (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The selection of costimulatory domains affects the phenotype and metabolic characteristics of CAR cells. For example, CD28 costimulatory domains produce an effective but transient effector-like phenotype with high levels of cytolytic ability, interleukin 2 (IL-2) secretion, and glycolysis. In contrast, T cells modified with CARs carrying 4-1BB costimulatory domains tend to expand and persist longer in vivo, have increased oxidative metabolism, are not easily exhausted, and have increased production of central memory T cells.

[0080] 9. Cells

[0081] CAR cell-based therapy can be used with a variety of cell types (such as lymphocytes). Available specific cell types include T cells, natural killer (NK) cells, natural killer T (NKT) cells, constant natural killer T (iNKT) cells, αβT cells, γδT cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg). In one embodiment, the CAR cells used to treat the subject are autologous. In other embodiments, the CAR cells can be from genetically similar but not identical donors (allogeneics).

[0082] 10. CAR cell production

[0083] The CAR constructs of the present disclosure may include some combinations of the modular components described herein. For example, in some embodiments of the present disclosure, the CAR construct comprises a GPC3 scFv antigen binding domain. In some embodiments, the CAR comprises a GPC3-2 scFv antigen binding domain. In some embodiments of the present disclosure, the CAR construct comprises a CSFR2 signal peptide. In some embodiments, the CAR construct comprises an IgG4P hinge / spacer domain carrying an S228P mutation. In some embodiments, the CAR construct comprises a CD28 transmembrane domain.

[0084] The different costimulatory domains that can be used are the CAR constructs of the present disclosure. In certain embodiments, the CAR construct comprises a costimulatory domain from the intracellular domain of CD3z. In certain embodiments, the CAR construct comprises a CD28 costimulatory domain. In certain embodiments, the CAR construct comprises a 4-1BB costimulatory domain. In certain embodiments, the CAR construct comprises a costimulatory domain from CD3z and CD28. In certain embodiments, the CAR construct comprises a costimulatory domain from CD3z and 4-1BB. In certain embodiments, the CAR construct comprises a costimulatory domain from all CD3z, CD28, and 4-1BB. In certain embodiments, the CAR construct comprises a costimulatory domain from ICOS, OX-40, and / or GITR.

[0085] 11.CAR Construct Evaluation

[0086] Based on the establishment of safety and persistence and central memory, the construct of the present disclosure is compared and evaluated. Due to its improved safety, low affinity (high dissociation rate) is advantageously assessed scFv, GPC3. Based on the contribution of its improved persistence and favorable in vivo phenotype (more central memory), 4-1BB and CD3z costimulatory domains (both in same construct) are advantageously assessed.

[0087] 12. CAR Examples

[0088] In some embodiments, the present disclosure provides a kind of isolated nucleic acid sequence, the isolated nucleic acid sequence encoding chimeric antigen receptor (CAR), the chimeric antigen receptor comprising a surface antigen on a tumor cell with specific antigen binding domains.In some embodiments, the cell surface antigen is a protein, a phosphorylated protein, a peptide-MHC, a carbohydrate or a glycolipid molecule.

[0089] Examples of contemplated cell surface antigens include CD10, CD16, CD19, CD20, CD22, CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133, CD138, CD171, CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3, CDH3, CDH6, CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1, GPA33, GPC3, IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1 / EGFR, ERBB2 / Her-2, ERBB3, ERBB4, cMet, EGFR vIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV, HPV16-E6, HPV16-E7, IL13Ra2, Ig ​​kappa chain, LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA. Other cell surface antigens are also contemplated herein.

[0090] In some embodiments, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for glypican 3 (GPC3). The antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D), and the CAR construct does not induce the production of cytokines in GPC3- cells. In some embodiments, the antigen binding domain comprises an antibody or an antigen binding fragment thereof. The antigen binding domain can be a Fab or a single-chain variable fragment (scFv). In some embodiments, the antigen binding domain is an scFv comprising the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0091] In certain embodiments, CAR further includes a transmembrane domain, a costimulatory domain and a signaling domain. The transmembrane domain can be a CD28 transmembrane domain. The costimulatory domain can be one or more of CD3 ζ (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β and MyD88 / CD40 costimulatory domains. In a specific embodiment, the costimulatory domain is one or more of CD28, 4-1BB and CD3 ζ costimulatory domains. The signaling domain can be a sequence encoding CSFR2 signal peptide.

[0092] In some embodiments, the isolated nucleic acid sequence may include a hinge / spacer domain. The hinge / spacer domain may be an IgG4P hinge / spacer.

[0093] In some specific embodiments, the isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) may have a sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 26.

[0094] In other embodiments, the present disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain. The antigen binding domain can be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH may have a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VL may have a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0095] In some embodiments, VH may be the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and VL may be the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the CAR may further have a transmembrane domain, a costimulatory domain, and a signaling domain.

[0096] In some specific embodiments, the anti-GPC3 CAR may have the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

[0097] In other embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence can be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0098] In other embodiments, the disclosure provides a cell comprising a vector having a nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0099] In other embodiments, the disclosure provides a cell having a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for glypican 3 (GPC3), wherein the antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D), and wherein the CAR construct does not induce cytokine production in GPC3 cells. For example, the nucleic acid sequence can be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0100] In other embodiments, the present disclosure provides a cell that expresses an anti-GPC3 chimeric antigen receptor (CAR) on its extracellular surface. The CAR may have an antigen binding domain, which may be an antibody, Fab, or scFv each having a heavy chain variable region (VH) and a light chain variable region (VL). The VH may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0101] In some embodiments, the VH may have an amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. In some embodiments, the VL may have an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The CAR may further include a transmembrane domain, a costimulatory domain, and a signaling domain. The cell expresses a CAR having an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

[0102] In some embodiments, the present disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells that express a CAR on their extracellular surface, and the CAR may have an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. Such cells can exhibit anti-tumor immunity after contact with tumor cells expressing GPC3.

[0103] 13. Using CARs to Treat Cancer

[0104] In some embodiments, the present disclosure provides CAR cells for treating cancer. Compositions (e.g., antibodies, CAR constructs, and CAR cells) with uses described herein and methods are particularly useful for suppressing the growth or spread of neoplastic cells. In some aspects, they are particularly useful for suppressing the growth of neoplastic cells in which GPC3 plays a role.

[0105] Neoplasms treatable by the compositions of the present disclosure include solid tumors, for example, solid tumors of the liver, lung, or ovary. However, the cancers listed herein are not intended to be limiting. For example, cancer types contemplated for treatment herein include, for example, NSCLC, advanced solid malignancies, biliary tract tumors, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal tumors, esophageal squamous cell carcinoma, extensive-stage small cell lung cancer, gastric adenocarcinoma, gastric cancer, gastroesophageal junction cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin's lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal carcinoma, metastatic melanoma, metastatic non-cutaneous melanoma, multiple myeloma, nasopharyngeal tumors, non-Hodgkin's lymphoma, ovarian cancer, fallopian tube cancer, peritoneal tumors, pleural mesothelioma, prostate tumors, recurrent or metastatic PD-L1 positive or negative SCCHN, recurrent squamous cell lung cancer, renal cell cancer (renal cell cancer / renal cell carcinoma), SCCHN, hypopharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, small cell lung cancer, head and neck squamous cell carcinoma o f the head and neck), squamous cell lung cancer, TNBC, transitional cell carcinoma, unresectable or metastatic melanoma, urothelial cancer / urothelialcarcinoma.

[0106] In one embodiment, cancers contemplated for treatment herein include any cancer that expresses GPC3 on the cell surface of cancer cells. In a specific example, cancers contemplated for treatment herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0107] 14. Armor

[0108] In some embodiments, the present disclosure provides "armored" cells, such as CAR-T cells, having one or more genetic modifications that enhance or optimize cell function by protecting cells from environmental damage (such as immunosuppressive cytokines or immunosuppressive TME). Genetic modifications include, but are not limited to, increased secretion of cytokines, expression of ligands that interact with immune cells (such as T cells, macrophages, and regulatory T cells), or changes in functional properties. It will be understood by those skilled in the art that armoring cells (such as T cells) can provide many additional benefits not described herein that allow T cells to survive in immunosuppressive TME.

[0109] In some embodiments, the cell may include a chimeric antigen receptor (CAR) comprising a tumor-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); and a dominant negative transforming growth factor beta (TGF-β) receptor type 2 (TGFβRIIDN) armored molecule.

[0110] In some embodiments, the armored cell may include a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for glypican 3 (GPC3), wherein the antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), and wherein the CAR construct does not induce cytokine production in GPC3 cells, and wherein the cells express the TGFβRIIDN armor molecule.

[0111] In some embodiments, the armored cell may include an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; and a TGFβRIIDN armored molecule.

[0112] 15. Treatment Methods

[0113] The CAR-modified cells of the present invention (such as CAR-T cells) can be administered alone or as a pharmaceutical composition with a diluent and / or other components associated with cytokines or cell colonies. In short, the pharmaceutical composition of the present invention may include, for example, CAR-T cells as described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, buffered saline, etc.; sulfates; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins, polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The pharmaceutical composition of the present invention may be suitable for treatment (or prevention).

[0114] CAR modified cells can also be administered together with one or more additional therapies. In one embodiment, additional therapies may include anti-cytokine antibodies. For example, one or more anti-TNF α can be used to reduce toxicity and promote anti-tumor activity at higher CAR-T doses (which may be associated with CRS-like symptoms and weight loss).

[0115] The number of CAR cells administered per dose, the number of dosages, and the frequency of administration will depend on different parameters, such as the patient's age, weight, clinical evaluation, tumor type, tumor load, and / or other factors (including the judgment of the attending physician). Any acceptable route of administration is contemplated, such as, but not limited to, intravenous administration (e.g., intravenous infusion), parenteral or subcutaneous administration.

[0116] In a particular embodiment, the intended treatment regimen may include one or more biological components, such as CAR-T cells and anti-cancer antibodies and / or chemotherapeutic components. For example, the intended treatment regimen may additionally include immune checkpoint inhibitors (ICIs), such as those targeting the PD-1 / PD-L1 axis (PDX), and other immuno-oncology (IO) therapies, such as immune system agonists.

[0117] Contemplated antibodies include anti-PD-L1 antibodies (e.g., durvalumab (MEDI4736), avelumab, atezolizumab, KNO35), anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, cemiplimab, SHR1210, IBI308, PDR001, anti-PD-1, BGB-A317, BCD-100, and JS001), and anti-CTLA4 antibodies (e.g., tremelimumab or ipilimumab). Additional antibodies are also contemplated herein. Any therapeutically effective antibody sub-portion is also contemplated herein.

[0118] Information about durvalumab (or a fragment thereof) for use in the methods provided herein can be found in U.S. Patent Nos. 8,779,108; 9,493,565; and 10,400,039, the disclosures of which are incorporated herein by reference in their entireties. In a specific aspect, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90PT antibody as disclosed in the aforementioned U.S. Patents.

[0119] Information regarding tremelimumab (or an antigen-binding fragment thereof) for use in the methods provided herein can be found in US Pat. No. 6,682,736 (where tremelimumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0120] Additional therapeutic agents (chemotherapeutic agents or biologics) contemplated herein include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, ADRIAN4YCINT M(doxorubicin), cisplatin (MVAC), a carboplatin-based regimen, or a single-agent taxane or gemcitabine, temozolomide, or dacarbazine, vinflunine, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Additional examples include drugs that target the DNA damage repair system, such as poly (ADP-ribose) polymerase 1 (PARP1) inhibitors and therapeutic agents that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora protein kinase B activity, and DNA-PK activity.

[0121] Any therapeutic composition or method contemplated herein can be combined with one or more of any other therapeutic compositions and methods provided herein.

[0122] In some embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject in need thereof an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain and an armored molecule that resists immunosuppression against the cell when expressed on the surface of a cell in the tumor microenvironment. On the other hand, the present disclosure describes an antigen binding domain, which can be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). VH may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. VL may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the method further inhibits tumor growth, induces tumor regression, and / or prolongs the subject's survival.

[0123] In some embodiments, the armor molecule is TGFβRIIDN.

[0124] In some embodiments, the cell is an autologous cell. For example, the autologous cell can be selected from the group consisting of: a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0125] In some embodiments, the cancer treated by the method is a solid tumor. For example, the cancer can be hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. In a specific embodiment, the cancer is hepatocellular carcinoma.

[0126] It should be understood that the specific aspects of the description described herein are not limited to the specific embodiments presented and may vary. It should also be understood that the terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting unless specifically defined herein. In addition, as will be appreciated by those skilled in the art, specific embodiments disclosed herein may be combined with other embodiments disclosed herein without limitation.

[0127] Examples

[0128] The following examples illustrate specific embodiments of the present disclosure and various uses thereof. They are set forth for illustrative purposes only and should not be construed in any way as limiting the scope of the present disclosure. Table 1 provides a description of the terms.

[0129] Table 1. Description of terms

[0130]

[0131] Example 1: TGFβ gene expression and signaling in hepatocellular carcinoma of the liver

[0132] Overview

[0133] In this example, TGFβ1 gene expression and TGFβ signaling were compared in normal liver and liver hepatocellular carcinoma (LIHC).

[0134] method

[0135] Data from the TCGA cohort were used for this analysis.TGFβ1 gene expression and TGFβ signaling signatures in normal liver and tumor tissues from TCGA were compared using t-test. The TGFβ signaling signature was formed by the average expression levels of the following genes: TGFBR1, SMAD7, TGFB1, SMURF2, SMURF1, BMPR2, SKIL, SKI, ACVR1, PMEPA1, NCOR2, SERPINE1, JUNB, SMAD1, SMAD6, PPP1R15A, TGIF1, FURIN, SMAD3, FKBP1A, MAP3K7, BMPR1A, CTNNB1, HIPK2, KLF10, BMP2, ENG, APC, PPM1A, XIAP, CDH1, ID1, LEFTY2, CDKN1C, TRIM33, RAB31, TJP1, SLC20A1, CDK9, ID3, NOG, ARID4B, IFNGR2, ID2, PPP1CA, SPTBN1, WWTR1, BCAR3, THBS1, FNTA, HDAC1, UBE2D3, LTBP2, and RHOA.

[0136] Kaplan-Meier analysis of overall survival (OS) was performed using LIHC data from TCGA. Data were grouped according to high (≥66th) and low (<66th) TGFβ1 gene expression and TGFβ signaling signatures. P values ​​were determined using the log-rank test.

[0137] result

[0138] TGFβ1 gene expression was upregulated 1.74-fold in primary solid tumor cells (LIHC) compared to normal tissues. High TGFβ1 expression in LIHC was associated with shorter OS compared to LIHC expressing low levels of TGFβ1 (median OS: 47 months vs. 70 months, respectively). Figure 1A and 1B .

[0139] TGFβ gene signaling was increased 1.1317-fold in primary solid tumors compared to normal tissues. High TGFβ signaling in LIHC was associated with shortened cell survival compared to low TGFβ signaling. Figure 1C and 1D .

[0140] in conclusion

[0141] These results suggest that increased TGFβ1 gene expression and TGFβ signaling are statistically significantly associated with shortened overall survival in patients with LIHC. Therefore, higher TGFβ1 gene expression and TGFβ signaling may contribute to cancer-related mortality.

[0142] Example 2: TGF-β and signaling in hepatocellular carcinoma of the liver

[0143] Overview

[0144] In this example, the expression of TGFβ and the intensity of TGFβ signaling (p-SMAD2) were compared in normal liver and hepatocellular carcinoma (HCC) of the liver by immunohistochemical analysis.

[0145] method

[0146] Three normal liver samples and 32 hepatocellular carcinoma samples were stained by immunohistochemistry and scored for intensity. TGF-β1 and pSMAD2 immunohistochemistry was performed on the Ventana discovery platform using anti-TGF-β1 (Abcam) and anti-pSAMD2 (Cell Signaling Technology) antibodies. Pathologists semi-quantitatively scored TGF-β1 and pSMAD2 expression in FFPE normal liver and HCC samples. IHC staining intensity was defined as: score 0, negative staining; score 1, minimal staining; score 2, moderate staining; score 3, strong staining.

[0147] result

[0148] TGF-β was detected in 44% of the tumors and 91% of the stroma of HCC samples. TGF-β signaling, as judged by phospo-SMAD2 (p-SMAD2) intensity, was detected in 91% of HCC samples. Normal liver was negative for TGF-β and pSMAD-2 ( Figure 2 A and 2B).

[0149] in conclusion

[0150] TGF-β is expressed and actively signals in the vast majority of HCC samples, suggesting that it is a common immunosuppressive factor in HCC tumors and targeting it could benefit a large number of HCC patients.

[0151] Example 3: Armoring GPC3 CAR-T cells with TGFβRIIDN

[0152] Overview

[0153] In this example, armoring GPC3 BZ CAR-T cells with TGFβRIIDN was investigated as a potential approach to protect CAR-T cells from TGFβ-mediated immunosuppression to improve CAR-T cell effector function and tumor control rates. Figure 3 A and 3B.

[0154] method

[0155] TGFβRIIDN: A dominant negative type 2 TGF-β receptor molecule was prepared by truncation of the wild-type receptor at residue 194, such that the TGFβRIIDN receptor lacks the intracellular signaling domain.

[0156] Armored CAR-T cells: GPC3 BZ CAR-T cells were armored with TGFβRIIDN by expressing the TGFβRIIDN receptor as a C-terminal fusion to the GPC3 BZ CAR, with a T2A peptide separating the GPC3 BZ CAR and the TGFβRIIDN receptor.

[0157] After expansion, the expression of CAR and TGFβRII on the surface of unarmored and armored CAR-T cells was analyzed by flow cytometry. CAR expression was detected by using AF647 anti-idiotypic antibody against GPC3-CAR ( Figure 4 ).

[0158] After staining with AF647 anti-idiotypic antibody against GPC3-CAR, CAR-T cells were purified with anti-AF647 microbeads (Miltenyi). The purified cells were expanded for more than 5 days, left to stand overnight in the absence of IL-2 and serum, and stimulated with recombinant human TGF-β (1 ng / mL) for the indicated time. The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and the expression of the indicated proteins was analyzed by Western blotting. Figure 5 ).

[0159] After staining with AF647 anti-idiotypic antibody against GPC3-CAR, unarmored or armored CAR-T cells were purified with anti-AF647 microbeads (Miltenyi). Purified cells were stimulated with plate-bound recombinant human GPC3 at a specified concentration in the presence or absence of recombinant human TGF-β (0.2 or 5 ng / mL). After 6 hours, cells were harvested and total RNA was obtained from the cells using the RNeasy mini kit (QIAGEN) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR was performed using the following TaqMan primers: GAPDH, Hs02758991_m1; IL2, Hs00174114_ml and IFNG, Hs00989291_m1 according to the protocol of TaqMan Gene Expression Master Mix (Applied Biosystems). Figure 6 )

[0160] In the presence of 20 ng / mL IL-15 (without IL-2), CAR-T cells were stimulated with anti-CD3 / CD28 beads (Dynabeads). After 3 days, TGFβ (50 ng / mL) was added, and the CD103 expression of cells cultured for more than 3 days was evaluated by flow cytometry (Figure 7).

[0161] result

[0162] Expression of TGFβRII on the surface of TGFβRIIDN CAR-T. Surface staining of TGFβRII on unarmored and armored CAR-T cells was assessed by flow cytometry. Since the extracellular portion is identical, the antibody cannot distinguish between endogenous and DNTGFβRII, but can distinguish between TGFβRII and CAR co-expressed by TGFβRIIDN CAR-T cells, indicating that the antibody is detecting overexpressed DN receptors. Figure 4 .

[0163] TGFβRIIDN inhibits SMAD 2 / 3 phosphorylation in CAR-T cells upon exposure to rhTGFβ: Unarmored CAR-T cells demonstrated rhTGF-β-induced SMAD 2 / 3 phosphorylation similar to that of untransduced control cells at 0, 15, 30, and 45 minutes after rhTGF-β exposure. CAR-T cells armored with TGF-βRIIDN showed attenuated SMAD 2 / 3 phosphorylation at 15, 30, and 45 minutes after rhTGF-β exposure compared to unarmored CAR-T cells and untransduced control cells. Expression of total SMAD 2 / 3 protein and β-actin was consistent across all groups. Figure 5 .

[0164] Expression of TGFβRIIDN prevents TGF-β-mediated reduction in effector cytokine production. Stimulation with recombinant human GPC-3 induced transcription of the effector cytokines IFN-γ and IL-2 in CAR-T cells. When present during stimulation, TGF-β reduced the levels of IFN-γ and IL-2 produced by unarmored CAR-T cells, but not in armored CAR-T cells. This result demonstrates that expression of TGFβRIIDN protects CAR-T cells from the immunosuppressive effects of TGF-β. Figure 6 A and 6B.

[0165] Expression of TGFβRIIDN effectively inhibits TGFβ signaling. CD103- T cells differentiate into CD103 in vitro + T RM IL-15 and TGF-β are required. Therefore, compared with CAR-cells or unarmored CAR-T cells, TGFβRIIDNCAR-T cells cannot differentiate into T cells after incubation with TGF-β. RM cells. This result demonstrates that expression of TGFβRIIDN inhibits TGF-β-induced signaling during prolonged exposure. Figure 7 A and 7B.

[0166] in conclusion

[0167] TGFβRIIDN inhibits TGF-β signaling in armored CAR-T cells, prevents TGF-β-mediated reduction in effector cytokine production, and attenuates TGF-β-mediated differentiation of armored CAR-T cells into T cells. RM Phenotype. Overall, these results demonstrate that expression of dominant negative TGFβRII is sufficient to inhibit TGF-β signaling and its biological effects. Example 3: CAR-T armored with TGFβRIIDN during co-culture with GPC3+ cells

[0168] Cell cytotoxicity and proliferation

[0169] Overview

[0170] In this example, CAR-T cell-mediated cytotoxicity and expansion were compared in UT, unarmored, and TGFβRIIDN-armored CAR-T cells during co-culture with GPC3+ hepatoma cells.

[0171] method

[0172] T cells (20,000 CAR+ cells / well) were co-cultured with a squamous cell carcinoma line engineered to express GPC3 (OE21 cells, 10,000 tumor cells / well) on the xCELLigence eSight RTCA for 5 days to simultaneously monitor real-time tumor cell viability (by electrical impedance) and CAR-T cell density (by microscopy) (see Figure 8 A). T cells (60,000 CAR+ cells / well) were then co-cultured with Hep3B cells (medium / low GPC3 expression) or Huh7 cells (low GPC3 expression) (30,000 tumor cells / well) on xCELLigence RTCA-MP (without microscope) for 5 days (see Figure 8 B) Non-adherent cells were then removed from the wells and assayed using CellTiter- Luminescent cell viability assay (Promega, Madison, WI) was used to quantify viable CAR-T cells (see Figure 8 C).

[0173] result

[0174] Dramatic expansion of CAR-T cells was observed when co-cultured with GPC3+ tumor cells. Figure 8 A. All CAR-T cells effectively killed GPC3+ tumor cells, and co-expression of TGFβRIIDN or addition of exogenous TGF-β did not modulate cytolytic capacity. See Figure 8B. After 5 days of co-culture, TGF-β inhibited the proliferation of tumor GPC3-induced CAR-T cells. However, CAR-T cells armored with TGFβRIIDN were not susceptible to this inhibition. See Figure 8B. Figure 8 C.

[0175] in conclusion

[0176] Armored TGFβRIIDN CAR-T cells showed significant cytotoxicity against GPC3+ tumor cells, indicating that the expression of the dominant negative receptor did not affect the ability of CAR-T to kill target cells in vitro. Conversely, unlike unarmored CAR-T cells, armored TGFβRIIDN CAR-T cells were not susceptible to TGF-β-mediated inhibition of GPC3. + Induced inhibition of proliferation.

[0177] Example 4: In vivo armored TGFβRIIDN CAR-T cell xenograft model

[0178] Overview

[0179] In this example, we determined in vivo that T cells armored with TGFβRIIDN were responsive to GPC3 + The effectiveness of tumor cells.

[0180] method

[0181] The Huh7-TGF-β model of hepatocellular carcinoma overexpressing TGF-β was used to test the in vivo effectiveness of armored TGFβRIIDN T cells in reducing tumor volume. Tumor cells were implanted into the flanks of NSG mice (10 mice / group). When the average tumor volume reached 150 mm 3 When 3, 7 or 21x10 6 The indicated CAR-T cells or 21 million untransduced T cells were injected and tumors were measured every two weeks. Upper panel: Mean tumor volume per group. Lower panel: Tumor volume of each individual mouse at the indicated dose (see Figure 9 ).

[0182] Give 7x10 6 Huh7-TGFβ tumor-bearing mice with CAR-T were analyzed ex vivo. Seven or fourteen days after infusion, tumors and spleens were collected from 5 mice / group. After staining with AF647-labeled anti-idiotypic antibody against GPC3-CAR, the number of CAR+ cells was counted by flow cytometry using AccuCheck counting beads. (See Figure 10 and Figure 11).

[0183] Huh-7-TGFβ tumor-bearing mice were given 7x10 6 Fourteen days after infusion, tumors were harvested and TGFβRII expression on CAR-positive and CAR-negative cells was assessed by flow cytometry. (See Figure 12 ).

[0184] Huh-7-TGFβ tumor-bearing mice were given 7x10 6Fourteen days after infusion, tumors were harvested and the expression of PD1, LAG3, CD27, and CD70 on the surface of CAR-T cells was assessed (see Figure 13 and Figure 14 ).

[0185] Huh-7-TGFβ tumor-bearing mice were given 7x10 6 Fourteen days after infusion, spleens were collected and the expression of PD1, LAG3, CD27, and CD70 was assessed by flow cytometry. (See Figure 15 ).

[0186] Huh-7-TGFβ tumor-bearing mice were given 7x10 6 CAR-T cells. For serum cytokine and AFP analysis, small volumes of blood were harvested at the indicated time points and serum was separated using BD Microtainer serum separator tubes. Cytokine levels were determined using MSD assays, while AFP was assessed by sandwich ELISA. Five mice / group were bled before tumor implantation (baseline A), before CAR-T infusion (baseline B), and on days 7 and 14 after infusion. (See Figure 16 and Figure 17 ).

[0187] result

[0188] Untransduced T cells had no significant effect on tumor growth. At the lowest dose (3 million cells / mouse), treatment with unarmored CAR-T cells resulted in a small reduction in tumor volume, while at higher doses there was a more pronounced effect, leading to complete tumor regression. In contrast, administration of armored TGFβRIIDN CAR-T cells induced a significant reduction in tumor volume and complete regression even at the lowest dose. In addition, progression-free survival was significantly prolonged at all treatment doses of armored TGFβRIIDN CAR-T cells compared to unarmored CAR-T (see Figure 9 ). For infusion, there are 7x10 6 Consistent with the enhanced efficacy, increased numbers of tumor-infiltrating lymphocytes (TILs) were detected in TGFβRIIDN-treated mice 7 days after infusion, indicating a sustained active immune response and associated proliferation (reaching a plateau at day 14 after treatment) (see Figure 10 In contrast, an increase in the number of CAR-T cells was observed in the spleens of mice treated with armored TGFβRIIDN CAR-T cells 14 days after infusion, suggesting that the proliferation of CAR-T cells was enhanced and, therefore, more CARs were detectable in the circulation. + Cells (see Figure 11 ). To investigate whether TGFβRIIDN is detectable on CAR-T cells after in vivo expansion, we analyzed the expression of TGFβRII on TILs and lymphocytes in the spleen 14 days after infusion. Although TGFβRII was barely detectable on unarmored cells, lymphocytes from mice treated with armored CAR-T cells co-expressed CAR and TGFβRII. Therefore, it is reasonable to assume that TGFβRII expressed on TGFβRIIDN TILs is a dominant negative receptor that can be detected in vitro after an active immune response and associated antigen-dependent proliferation (see Figure 12 ). Notably, TGFβRIIDN TILs expressed low levels of the exhaustion markers LAG3 and PD1 (see Figure 13 ), which is directly regulated by TGF-β in a SMAD3-dependent manner (Park, BV, Freeman, ZT, Ghasemzadeh, A., Chattergoon, MA, Rutebemberwa, A., Steigner, J. et al. (2016). TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer. Cancer Discov, 6(12), 1366-1381). In addition, TGFβRIIDN TILs express less CD70 and conversely more CD27 than unarmored TILs (see Figure 14). This result is consistent with previous evidence showing that TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells and reinforces the concept that expression of a dominant negative receptor protects CAR-T cells from TGF-β-mediated immunosuppression (Yang, ZZ., Grote, D., Xiu, B. et al. TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma. [In B-cell non-Hodgkin's lymphoma, TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells] Leukemia [Leukemia] 28, 1872-1884 (2014). https: / / doi.org / 10.1038 / leu.2014.84). Compared with TILs, CAR-T cells in the periphery do not express co-inhibitory markers or CD70, and most are CD27 positive, which is always in an inactive state (see Figure 15 Consistent with the enhanced efficacy and higher TIL numbers, more IFN-γ was detected in the serum of mice infused with TGFβRIIDN CAR-T 7 days after infusion, and the serum concentration of the tumor marker AFP was significantly reduced 14 days after infusion (see Figure 16 and Figure 17 ). This result suggests that expression of TGFβRIIDN increases the efficacy of CAR-T therapy by eliminating the immunosuppressive effects of TGF-β in vivo.

[0189] in conclusion

[0190] Armored TGFβRIIDN-expressing CAR-T cells showed promising results in effectively treating GPC3+ tumors in vivo.

[0191] Example 5: In vivo TGFβRIIDN-armored CAR-T cell-derived hepatocellular carcinoma xenograft model

[0192] Overview

[0193] In this example, we determined that armored TGFβRIIDN CAR-T cells were expressed in many GPC3 + Effectiveness in hepatocellular carcinoma patient-derived xenograft cells.

[0194] method

[0195] This study was conducted by Crown Bioscience Inc. Eight PDX models were selected based on the expression of GPC3 and TGF-β (assessed by IHC and RNA sequencing performed by Crown Bioscience). All the selected models were GPC3 high (IHC score >100), but three of the models did not express TGF-β (IHC score <5), while the other five models were TGF-β positive (IHC score >20). According to Crown Bioscience's research protocol, tumor fragments were harvested from stock mice and used to inoculate NCG mice. Each mouse was inoculated with a specific PDX tumor fragment (3x3x3mm) subcutaneously in the right anterior flank to develop tumors. When the average tumor size reached approximately 150-250mm 3 At 4 hr, mice were randomized into groups. Tumor-bearing mice were given 5 million unarmored or armored CAR-T cells and non-transduced T cells provided by AstraZeneca (5 mice / group), and tumor volume was measured every two weeks. (See Figure 18 and Figure 19 ).

[0196] result

[0197] Tumors grew in mice that received non-transduced T cells. However, in the absence of TGF-β, unarmored and armored cells were equally effective and induced rapid and complete regression in all mice included in the study (see Figure 18 When infused in a TGF-β expression model, unarmored CAR-T cells had a less pronounced effect; in contrast, CAR-T cells armored with TGFβRIIDN were consistently more effective and induced significant tumor regression.

[0198] in conclusion

[0199] PDX models mimic human tumor biology that allows for natural cancer progression. Therefore, these observations confirm and strengthen the evidence obtained with the Huh7-TGF-β xenograft model. Overall, these data suggest that GPC3 CAR-T cells armored with TGFβRIIDN may be a promising candidate for GPC3 + It is an effective in vivo therapy for tumors and can maintain its efficacy even in the presence of the immunosuppressive factor TGF-β.

[0200] The embodiments described herein can be practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. The terms and expressions that have been adopted are used as descriptive terms, rather than restrictive, and are not intended to exclude any equivalents of the features shown and described or parts thereof when using such terms and expressions, but it should be recognized that various modifications can be made within the scope of the claimed embodiments. Therefore, it should be understood that although the present invention has been specifically disclosed through the embodiments, optional features, those skilled in the art may modify and change the concepts disclosed herein, and it is believed that such modifications and changes may be within the scope of these embodiments defined by the specification and the appended claims. Although some aspects of the present disclosure may be considered particularly advantageous, it is contemplated that the present disclosure is not limited to these specific aspects of the disclosure.

[0201] If one, more than one, or all members of a group are present in, used in, or otherwise relevant to a given product or method, a claim or specification that includes "or" between one or more members of the group is considered satisfied, unless otherwise indicated or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or method. The present disclosure includes embodiments in which more than one or all members of the group are present in, used in, or otherwise relevant to a given product or method.

[0202] In addition, the present disclosure covers all variations, combinations and permutations in which one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim attached to another claim can be amended to include one or more limitations found in any other claim attached to the same base claim. Where elements are presented as a list (e.g., in Markush group form), each subgroup of elements is also disclosed, and any element can be removed from the group.

[0203] It will be understood that, in general, where the present disclosure or aspects of the present disclosure are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features. For the sake of brevity, these embodiments are not specifically set forth herein in words.

[0204] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any part of this application shall not be construed as an admission that such reference is available as prior art with respect to the present invention.

[0205] Table 5. Sequences used in the Examples.

[0206]

[0207]

[0208] Table 6. Sequence

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224] Sequence Listing <110> ImmunoMedical LLC <120> Compositions and methods for treating cancer using chimeric antigen receptors <130> CARTGPC(TGF)-CN-NP <140> <141> <150> 63 / 014,831 <151> 2020-04-24 <160> 48 <170> PatentIn Version 3.5 <210> 1 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polypeptide <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4​​​​​​​​​​​​​​​​​​​​​Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile 145 150 155 160 Gly Ser Asn Thr Val Asn Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Val Tyr Phe Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly 195 200 205 Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp 210 215 220 Asp Ser Leu Asn Ala Pro Val Phe Gly Gly Gly Thr Lys Val Thr Val 225 230 235 240 Leu <210> 2 <211> 241 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asp Ile 145 150 155 160 Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 210 215 220 Asp Arg Met Tyr Ser Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 3 <211> 460 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 305 310 315 320 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 325 330 335 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 340 345 350 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 355 360 365 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 370 375 380 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 385 390 395 400 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 405 410 415 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 420 425 430 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 435 440 445 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 4 <211> 318 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic multi-peptide <400> 4 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 305 310 315 <210> 5 <211> 459 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 305 310 315 320 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 325 330 335 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser 340 345 350 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 355 360 365 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 370 375 380 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 385 390 395 400 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 405 410 415 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 420 425 430 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 435 440 445 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 6 <211> 501 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic multi-peptide <400> 6 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 305 310 315 320 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 325 330 335 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys 340 345 350 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 355 360 365 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 370 375 380 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 385 390 395 400 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 405 410 415 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 420 425 430 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 435 440 445 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 450 455 460 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 465 470 475 480 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 485 490 495 Ala Leu Pro Pro Arg 500 <210> 7 <211> 456 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 290 295 300 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 305 310 315 320 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 325 330 335 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 340 345 350 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 355 360 365 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 370 375 380 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 385 390 395 400 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 405 410 415 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 420 425 430 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 435 440 445 His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 8 <211> 314 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Val 290 295 300 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 305 310 <210> 9 <211> 455 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 290 295 300 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 305 310 315 320 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 325 330 335 Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360 365 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 370 375 380 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 385 390 395 400 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 405 410 415 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 420 425 430 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 435 440 445 Met Gln Ala Leu Pro Pro Arg 450 455 <210> 10 <211> 497 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic multi-peptide <400> 10 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 290 295 300 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 305 310 315 320 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 325 330 335 Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr 340 345 350 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 355 360 365 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 370 375 380 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 385 390 395 400 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 405 410 415 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 420 425 430 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg <210> 11 <211> 1383 <212> DNA <213> Synthetic sequence <220> <223> Description of synthetic sequence: Synthetic polynucleotide <​​​attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaaggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttatt ctctgctggt cacagtggct 900 ttcatcatct tctgggtcaa gcgaggccgg aagaaactgc tgtacatctt caaacagcct 960 tttatgcgcc cagtgcagac aactcaggag gaagacggct gctcttgtcg gttccccgag 1020 gaagaggaag ggggatgtga gctgcgcgtg aagttttctc gaagtgccga tgctcctgca 1080 tatcagcagg gacagaacca gctgtacaac gagctgaatc tgggccggag agaggaatac 1140 gacgtgctgg ataagaggcg cggcagagac ccagaaatgg gcgggaagcc acgacggaaa 1200 aacccccagg aggggctgta taatgaactg cagaaggaca aaatggccga ggcttacagc 1260 gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggact gtatcagggc 1320 ctgagcactg ccaccaagga cacctacgat gctctgcaca tgcaggcact gccacccagg 1380 tga 1383 <210> 12 <211> 957 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 12 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaaggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttatt ctctgctggt cacagtggct 900 ttcatcatct tctgggtccg cgtgaagttt tctcgaagtg ccgatgctcc tgcatga 957 <210> 13 <211> 1380 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 13 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaagggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttat ccctgctggt cactgtggcc 900 ttcatcatct tctgggtgcg gagcaagcgg agccggctgc tgcactctga ctacatgaac 960 atgactccac ggagacccgg ccctacccgg aaacattatc agccctacgc cccacccaga 1020 gattttgccg cttataggtc cagggtgaag ttttctcgca gtgcagatgc ccctgcttat 1080 cagcagggac agaatcagct gtacaacgag ctgaatctgg gcaggcgcga ggaatacgac 1140 gtgctggata agcgacgggg cagagacccc gaaatgggag ggaagcccag aaggaaaaac 1200 cctcaggagg ggctgtataa tgaactgcag aaggacaaaa tggcagaggc ctacagtgaa 1260 atcgggatga agggagagcg ccgacgggga aaaggccacg atggactgta tcagggcctg 1320 tctactgcca ccaaggacac ctacgatgcc ctgcacatgc aggctctgcc tccacgctga 1380 <210> 14 <211> 1506 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 14 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaagggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttat ccctgctggt cactgtggcc 900 ttcatcatct tctgggtgcg gagcaagcgg agccggctgc tgcactctga ctacatgaac 960 atgactccac ggagacccgg ccctacccgg aaacattatc agccctacgc cccacccaga 1020 gattttgccg cttataggtc caagcgcggc cgaaagaaac tgctgtacat cttcaaacag 1080 cccttcatga gacccgtcca gacaactcag gaggaagacg gctgcagctg taggttcccc 1140 gaggaagagg aagggggatg tgagctgagg gtgaagtttt ctcgcagtgc agatgcccct 1200 gcttatcagc agggacagaa tcagctgtac aacgagctga atctgggcag gcgcgaggaa 1260 tacgacgtgc tggataagcg acggggcaga gaccccgaaa tgggagggaa gcccagaagg 1320 aaaaaccctc aggaggggct gtataatgaa ctgcagaagg acaaaatggc agaggcctac 1380 agtgaaatcg ggatgaaggg agagcgccga cggggaaaag gccacgatgg actgtatcag 1440 ggcctgtcta ctgccaccaa ggacacctac gatgccctgc acatgcaggc tctgcctcca 1500 cgctga 1506 <210> 15 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 15 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gacacagcca 480 cctagcgcct ccggaacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gacattggga gcaacaccgt gaattggtat cagcagctgc ctggaacagc tccaaagctg 600 ctgatctact ataacaatca gaggccctcc ggcgtccctg atcgcttctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctggtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaaactgctg tacatcttca aacagccttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tcttgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggagag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaaatgggc gggaagccac gacggaaaaa cccccaggag 1200 gggctgtata atgaactgca gaaggacaaa atggccgagg cttacagcga aatcgggatg 1260 aagggagaga gaaggcgcgg aaaaggccac gatggactgt atcagggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacccaggtg a 1371 <210> 16 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 16 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gacacagcca 480 cctagcgcct ccggaacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gacattggga gcaacaccgt gaattggtat cagcagctgc ctggaacagc tccaaagctg 600 ctgatctact ataacaatca gaggccctcc ggcgtccctg atcgcttctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttactcc ctgctggtca ctgtggcctt catcatcttc 900 tgggtgcggg tgaagttttc tcgcagtgcc gacgctcccg catga 945 <210> 17 <211> 1368 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 17 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gandacagcca 480 cctagcgcct ccggacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gatattggga gcaaccgt gattggtat cagcagctgc ctggaacagc tccaagctg 600 ctgatctact atacaatca gaggccctcc ggcgtccctg atcgctctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtccttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattcc ctgctgtca cagtggcctt catcatcttc 900 tgggtgcgga gcaagcggag ccggctgctg cactctgact acatgaacat gaccccgg 960 agacccggcc ctacaagaaa gcattatcag ccttaccgccc cacccaggga cttcgcagct 1020 tatcgctccc gagtgaaattt ttcgcagt gcagatgccc ccgcttatca gcagggccag 1080 aatcagctgt acacgagct gatctgggg aggcgcgagg atacgacgt gctggataag 1140 cgacggggcc gggaccccga aatgggagga aagcctagaa ggaaaaaccc acaggagggc 1200 ctgtataatg aactgcagaa ggacaaaatg gcagaggcct acagcgaaat cggaatgaag 1260 ggagagcgcc gacggggcaa aggacacgat ggcctgtatc aggggctgag caccgccaca 1320 aaggacacct acgatgccct gcacatgcag gctctgcctc cacgctga 1368 <210> 18 <211> 1494 ]<212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 18 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaagt acttgatta tggggacag ggcactatgg tgaccgtc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gandacagcca 480 cctagcgcct ccggacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gatattggga gcaaccgt gattggtat cagcagctgc ctggaacagc tccaagctg 600 ctgatctact atacaatca gaggccctcc ggcgtccctg atcgctctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtccttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattcc ctgctgtca ctgtggcctt catcatcttc 900 tgggtgcgga gcaagcggag ccggctgctg cactctgact acatgaacat gactccacgg 960 agacccggcc ctacccggaa acatcag ccctaccgccc cacccagaga tttgccgct 1020 tataggtcca agcgcggccg aaagaaactg ctgtacatct tcaacagcc cttcatgaga 1080 cccgtccaga caactcagga ggaagacggc tgcagctgta ggttccccga ggaagaggaa 1140 gggggatgtg agctgagggt gaagttttct cgcagtgcag atgcccctgc ttatcagcag 1200 ggacagaatc agctgtacaa cgagctgaat ctgggcaggc gcgaggaata cgacgtgctg 1260 gataagcgac ggggcagaga ccccgaaatg ggagggaagc ccagaaggaa aaaccctcag 1320 gaggggctgt ataatgaact gcagaaggac aaaatggcag aggcctacag tgaaatcggg 1380 atgaagggag agcgccgacg gggaaaaggc cacgatggac tgtatcaggg cctgtctact 1440 gccaccaagg acacctacga tgccctgcac atgcaggctc tgcctccacg ctga 1494 <210> 19 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 19 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110 Tyr Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro 260 265 270 Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 275 280 285 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 290 295 300 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 305 310 315 320 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 325 330 335 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360 365 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 370 375 380 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 385 390 395 400 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 405 410 415 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 420 425 430 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 435 440 445 His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 20 <211> 497 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic multi-peptide <400> 20 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110 Tyr Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro 260 265 270 Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 275 280 285 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 290 295 300 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 305 310 315 320 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 325 330 335 Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 340 345 350 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 355 360 365 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg <210> 21 <211> 457 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty one Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Asp Asp 100 105 110 Thr Ala Leu Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu Pro Leu Ser 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln 145 150 155 160 Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys 165 170 175 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln 180 185 190 Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln 195 200 205 Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr 210 215 220 Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp 225 230 235 240 Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly 245 250 255 Thr Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys 260 265 270 Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 275 280 285 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys 290 295 300 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 305 310 315 320 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 325 330 335 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 340 345 350 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 355 360 365 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 370 375 380 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 385 390 395 400 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 405 410 415 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 420 425 430 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 435 440 445 Leu His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 22 <211> 1371 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 22 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgatg tcgtgatgac gcagagccct ctctctcttc ccgttacccc tggtgaaccc 120 gcatcaataa gttgccgctc cagtcaatca cttgtacatt caaatcgcaa tacctacctg 180 cactggtatt tgcagaagcc gggacaatcc cctcaattgt tgatatataa ggtatccaat 240 cgcttttctg gagttcctga tagattcagc ggatccgggt ctggtactga tttcactctg 300 aaaatatcca gggtcgaagc tgaggacgta ggcgtatatt attgctctca gaacacgcat 360 gtcccgccga ctttcggcca gggcactaaa cttgagatca agggtggggg gggcagcggt 420 ggtggaggct ctggtggagg agggagccag gtccaactcg ttcaaagtgg cgcagaggtc 480 aaaaagccag gcgcgagcgt taaagtatca tgtaaggcca gcggttatac tttcactgat 540 tatgaaatgc actgggtgcg acaagccccc gggcaaggtc ttgagtggat gggtgcactt 600 gatccaaaaa ctggggatac tgcctatagc cagaattca aagggcgcgt cacactcact 660 gccgacaaaa gcgagcac agcttatatg gaattgagtt cactgacgag cgaggatacg gcagtttatt actgtacgcg cttctactct tacacttatt gggggcaagg cactttggtt 780 actgtgtcct ctgacaagac ccatacgtgt ccaccgtgtc ccttctgggt attggttgtg 840 gtcggcggtg tccttgcttg ttacagcctt ctcgtgacag tcgcattcat aattttttgg 900 gtgaaagag gtcggaaaaa gttgctgtat attttcaaac aaccctttat gagacctgta caaacgactc aggaagga tggttgtagt tgcaggtttc cggaggagga ggaaggtggg tgcgaactgc gggtgaatt taggedc gctgacgcac cagcttacca acaaggacag aaccaattgt acaacgagct taacttgggt aggagggagg aatatgatgt actggacaaa aggcgaggtc gcgatccgga aatgggaggc aagccacagc gccggaaaaa cccgcagga ggcttgtaca acgaacttca gaaagataaa atggcagaag catactccga aatagggatg aaaggtgaac ggcggcgagg caagggccac gacggtctgt accaagggtt gtcaacggca actaaagaca cgtatgatgc acttcatatg caagctctgc cacccaggtg a 1371 <210> 23 <211> 1494 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 23 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgatg tcgtgatgac gcagagccct ctctctcttc ccgttacccc tggtgaaccc 120 gcatcaataa gttgccgctc cagtcaatca cttgtacatt caaatcgcaa tacctacctg 180 cactggtatt tgcagaagcc gggacaatcc cctcaattgt tgatatataa ggtatccaat 240 cgcttttctg gagttcctga tagattcagc ggatccgggt ctggtactga tttcactctg 300 aaaatatcca gggtcgaagc tgaggacgta ggcgtatatt attgctctca gaacacgcat 360 gtcccgccga ctttcggcca gggcactaaa cttgagatca agggtggggg gggcagcggt 420 ggtggaggct ctggtggagg agggagccag gtccaactcg ttcaaagtgg cgcagaggtc 480 aaaaagccag gcgcgagcgt taaagtatca tgtaaggcca gcggttatac tttcactgat 540 tatgaaatgc actgggtgcg acaagccccc gggcaaggtc ttgagtggat gggtgcactt 600 gatccaaaaa ctggggatac tgcctatagc cagaaattca aagggcgcgt cacactcact 660 gccgacaaaa gtacgagcac agcttatatg gaattgagtt cactgacgag cgaggatacg 720 gcagtttatt actgtacgcg cttctactct tacacttatt gggggcaagg cactttggtt 780 actgtgtcct ctgacaagac ccatacgtgt ccaccgtgtc ccttctgggt attggttgtg 840 gtcggcggtg tccttgcttg ttacagcctt ctcgtgacag tcgcattcat aatttttgg 900 gtgcggagca agcggagccg gctgctgcac tctgactaca tgaacatgac tccacggaga 960 cccggcccta cccggaaaca ttatcagccc tacgccccac ccagagattt tgccgcttat 1020 aggtccaaaa gaggtcggaa aaagttgctg tatattttca aaaccctt tatgagacct 1080 gtacaaacga ctcaggaaga ggatggttgt agttgcaggt ttccggagga ggaggaaggt 1140 gggtgcgaac tgcgggtgaa atttagtaga agcgctgacg caccagctta ccaacaagga 1200 cagaaccaat tgtacaacga gcttaacttg ggtaggaggg aggaatatga tgtactggac 1260 aaaaggcgag gtcgcgatcc ggaaatggga ggcaagccac agcgccggaa aaacccgcag 1320 gaaggcttgt acaacgaact tcagaaagat aaaatggcag aagcatactc cgaaataggg 1380 atgaaaggtg aacggcggcg aggcaagggc cacgacggtc tgtaccaagg gttgtcaacg 1440 gcaactaaag acacgtatga tgcacttcat atgcaagctc tgccacccag gtga 1494 <210> 24 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 24 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctcagg tccagcttgt gcaaagcgga ggaggagtgg tacagcctgg ccgctctttg 120 agactgtctt gtgcggccag tggatttaca ttctcttctt atgggttgca ttgggtcaga 180 caagcaccgg gcaaaggatt ggaatgggtc gcggccatta gctatgatgg ctcaaagaaa 240 tattatgccg attccgtaaa agggaggttg acaataagcc gggataacag caagaacact 300 ttgtatcttc agatgaatag cctccgaccg gacgacacgg cactgtattt ttgcgcacgc 360 gggtggtttg tagaacccct gagttgggga caaggtactc ttgtcacggt atcttctggc 420 ggaggtggga gtggtggggg tggcagtggc gggggtgggt cacaaagcgt gcttacacaa 480 cctccttctg cgagcggac tccggggaca cgggttacga tttcatgctc cggctcaagt 540 agcaatatag gatcaatac agtcaatac tatcaacaac tccctggcac agcgcccaag ctgctgatct actctaataa ccagaggccg agtggtgtgc cagataggtt cagtggctct aaatcaggta ctagcgcgag cctcgccatt tcaggacttc aatcagagga tgaagcggac 720 tactactgtg ccgcgtggga tgattcactt aatggatatg ttttcgggac cggaacaaaa 780 ttgacggtat tggagagcaa atatggacca ccatgccctc catgtccttt ttgggtcctg 840 gtggtcgtgg gaggcgtgct ggcatgttat tctctgctgg tcacagtggc tttcatcatc 900 ttctgggtca agcgaggccg gaagaaactg ctgtacatct tcaaacagcc ttttatgcgc ccagtgcaga caactcagga ggagacggc tgctcttgtc ggttccccga ggagagga gggggatgtg agctgcgcgt gaagttttct cgaagtgccg atgctcctgc atatcagcag 1080. ggacagaacc agctgtacaa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 1140 gataagaggc gcggcagaga cccagaaatg ggcgggaagc cacgacggaa aaacccccag 1200 gaggggctgt ataatgaact gcagaaggac aaaatggccg aggcttacag cgaaatcggg 1260 atgaagggag agagaaggcg cggaaaaggc cacgatggac tgtatcaggg cctgagcact 1320 gccaccaagg acacctacga tgctctgcac atgcaggcac tgccacccag gtga 1374 <210> 25 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 25 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Phe Arg Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 290 295 300 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 305 310 315 320 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 325 330 335 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 340 345 350 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 355 360 365 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 370 375 380 Arg Asp Pro Glu Meth Gly Gly Light Pro Arg Arg Light Asn Pro Gln Glue 385 390 395 400 Gly Leo Tyr Asn Gly Leo Gln Lys Asp Lys Met White White White Ser 405 410 415 Glu Ile Gly Met Light Gly Glu Arg Arg Arg Gly Light Gly His Asp Gly 420 425 430 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu Free Mp3 Download 435 440 445 His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 26 <211> 1368 <212> DNA <213> The snowstorm <220> <223> Make a snowflake: a snowflake <400> 26 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg attcctgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 120 agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 180 caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 240 tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 300 ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 360 ggaaagcgat actttgacta ctggggccag gggacaatgg tcaccgtctc gagtggtggg 420 gggggcagcg gtggtggagg ctctggtgga ggagggagct cctatgagct gactcagcca 480 ccctcagcgt ctgggacccc cgggcagagg gtcaccatct cttgttctgg aggcagctcc 540 aacatcggaa gtaatactgt aaactggttc cggcagctcc caggaacggc ccccaaactc 600 ctcgtttatt ttaataatca gcgaccctca ggggtccctg accgattctc tggctccaag 660 tctggcacct cggcctccct ggccatcggt gggctccagt ctgacgatga ggctgactat 720 tactgtgtag catgggatga ctctctgaat gctccggtgt tcggcggagg gaccaaggtc 780 accgtcctag agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctggtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaaactgctg tacatcttca aacagccttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tcttgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggagag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaaatgggc gggaagccac gacggaaaaa cccccaggag 1200 gggctgtata atgaactgca gaaggacaaa atggccgagg cttacagcga aatcgggatg 1260 aagggagaga gaaggcgcgg aaaaggccac gatggactgt atcagggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacccagg 1368 <210> 27 <211> 116 <212> PRT <213> Synthetic Sequence[[ID=二十一]] [[ID=二十二]]<220>[[ID=二十三]] [[ID=二十四]]<223> Description of Synthetic Sequence: Synthetic Polypeptide[[ID=二十五]] [[ID=二十六]]<400> 27[[ID=二十七]] [[ID=二十八]]Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly[[ID=二十九]] [[ID=三十]]1 5 10 15[[ID=三十一]] [[ID=三十二]]Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr[[ID=三十三]] [[ID=三十四]]20 25 30[[ID=三十五]] [[ID=三十六]]Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val[[ID=三十七]] 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 28 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Val Tyr Phe Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln 65 70 75 80 Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu 85 90 95 Asn Ala Pro Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 29 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 29 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 30 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asp Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg Met 85 90 95 Tyr Ser Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 31 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 31 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 [[ID=D26]]Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser 130 135 140 Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu 145 150 155 160 Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 165 170 175 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 180 185 190 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 195 200 205 Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220 Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser <210> 32 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 32 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Gly Trp Phe Val Glu Pro Leu Ser Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly 130 135 140 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn 145 150 155 160 Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala 165 170 175 Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro 180 185 190 Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile 195 200 205 Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp 210 215 220[[ID=l2]] Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr 225 230 235 240 Val Leu <210> 33 <211> 723 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 33 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaggaaag 300 cgatactttg actactgggg ccaggggaca atggtcaccg tctcgagtgg tggggggggc 360 cgatactttg actactgggg ccaggggaca atggtcaccg tctcgagtgg tggggggggc 360 agcggtggtg gaggctctgg tggaggaggg agctcctatg agctgactca gccaccctca 420 agcggtggtg gaggctctgg tggaggaggg agctcctatg agctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgtt ctggaggcag ctccaacatc 480 gcgtctggga cccccgggca gagggtcacc atctcttgtt ctggaggcag ctccaacatc 480 ggaagtaata ctgtaaactg gttccggcag ctcccaggaa cggcccccaa actcctcgtt 540 ggaagtaata ctgtaaactg gttccggcag ctcccaggaa cggcccccaa actcctcgtt 540 tattttaata atcagcgacc ctcaggggtc cctgaccgat tctctggctc caagtctggc 600 tattttaata atcagcgacc ctcaggggtc cctgaccgat tctctggctc caagtctggc 600 acctcggcct ccctggccat cggtgggctc cagtctgacg atgaggctga ctattactgt 660 acctcggcct ccctggccat cggtgggctc cagtctgacg atgaggctga ctattactgt 660 gtagcatggg atgactctct gaatgctccg gtgttcggcg gagggaccaa ggtcaccgtc 720 gtagcatggg atgactctct gaatgctccg gtgttcggcg gagggaccaa ggtcaccgtc 720 cta 723 cta 723 <210> 34<210> 34 <211> 723<211> 723 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220><220> <223> Description of artificial sequence: Synthetic polynucleotide<223> Description of artificial sequence: Synthetic polynucleotide <400> 34 <400> 34 , gaggtccagc tgctggagag cggaggagga ctggtgcagc ctggaggaag tctgcgactg 60 gaggtccagc tgctggagag cggaggagga ctggtgcagc ctggaggaag tctgcgactg 60 tcatgcgccg ctagcggctt caccttcagc tcctatgcaa tgagctgggt gcgacaggca 120 tcatgcgccg ctagcggctt caccttcagc tcctatgcaa tgagctgggt gcgacaggca 120 ccaggcaagg ggctggagtg ggtctccgct atctccggct ctggaggctc tacttactat gcagacagtg tgaaggggcg gttcacaatc tccagagata actctaagaa cactctgtac ctgcagatga actctctgag agctgaggac accgcagtgt actattgcgc caagggcaaa aggtactttg attattgggg acagggcact atggtgaccg tctctagtgg aggaggagga 360 agcgaggag gaggatccgg cggaggaggc agtcagtcag tgctgacaca gccacctagc 420. gcctccgga ccccaggaca gcgggtcaca atctcttgta gtgggggatc aagcgacatt 480 gggagcaaca ccgtgaattg gtatcagcag ctgcctggaa cagctccaaa gctgctgatc tactataaca atcagaggcc ctccggcgtc cctgatcgct tctcaggcag caaatccgggg acttctgcaa gtctggccat tagtggcctg cagtcagagg acgaagccga ttactattgt gctacctggg acgataggat gtactctccc gtgttcggcg ggggaacaaa gctgactgtc 720 ctg 723 <210> 35 <211> 726 <212> DNA <213> The snowstorm <220> <223> Make a snowflake: a snowflake <400> 35 60. gatgtcgtga tgacgcagag cccctctctct cttcccgtta cccctggtga acccgcatca ataagttgcc gctccagtca atcacttgta cattcaaatc gcaataccta cctgcactgg 120 tatttgcaga agccgggaca atcccctcaa ttgttgatat ataaggtatc caatcgcttt 180 tctggagttc ctgatagatt cagcggatcc gggtctggta ctgatttcac tctgaaaata 240 tccagggtcg aagctgagga cgtaggcgta tattattgct ctcagaacac gcatgtcccg 300 ccgactttcg gccagggcac taaacttgag atcaagggtg gggggggcag cggtggtgga 360 ggctctggtg gaggagggag ccaggtccaa ctcgttcaaa gtggcgcaga ggtcaaaaag 420 ccaggcgcga gcgttaaagt atcatgtaag gccagcggtt atactttcac tgattatgaa 480 atgcactggg tgcgacaagc ccccgggcaa ggtcttgagt ggatgggtgc acttgatcca 540 aaaactgggg atactgccta tagccagaaa ttcaaagggc gcgtcacact cactgccgac 600 aaaagtacga gcacagctta tatggaattg agttcactga cgagcgagga tacggcagtt 660 tattactgta cgcgcttcta ctcttacact tattgggggc aaggcacttt ggttactgtg 720 tcctct 726 <210> 36 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 36 caggtccagc ttgtgcaaag cggaggagga gtggtacagc ctggccgctc tttgagactg 60 tcttgtgcgg ccagtggatt tacattctct tcttatgggt tgcattgggt cagacaagca 120 ccgggcaaag gattggaatg ggtcgcggcc attagctatg atggctcaaa gaaatattat 180 gccgattccg taaaagggag gttgacaata agccgggata acagcaagaa cactttgtat 240 cttcagatga atagcctccg accggacgac acggcactgt atttttgcgc acgcgggtgg 300 tttgtagaac ccctgagttg gggacaaggt actcttgtca cggtatcttc tggcggaggt 360 gggagtggtg ggggtggcag tggcgggggt gggtcacaaa gcgtgcttac acaacctcct 420 tctgcgagcg gaactccggg acaacgggtt acgatttcat gctccggctc aagtagcaat 480 ataggatcaa atacagtgaa ttggtatcaa caactccctg gcacagcgcc caagctgctg 540 atctactcta ataaccagag gccgagtggt gtgccagata ggttcagtgg ctctaaatca 600 ggtactagcg cgagcctcgc catttcagga cttcaatcag aggatgaagc ggactactac 660 tgtgccgcgt gggatgattc acttaatgga tatgttttcg ggaccggaac aaaattgacg 720 gtattg 726 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 37 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 38 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 38 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 39 Gly Lys Arg Tyr Phe Asp Tyr 1 5 <210> 40 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 40 Ser Gly Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 41 Phe Asn Asn Gln Arg Pro Ser 1 5 <210> 42 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 42 Val Ala Trp Asp Asp Ser Leu Asn Ala Pro Val 1 5 10 <210> 43 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 43 Ser Gly Gly Ser Ser Ser Asp Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 44 Tyr Asn Asn Gln Arg Pro Ser 1 5 <210> 45 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 45 Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val 1 5 10 <210> 46 <211> 2013 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 46 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 120 agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 180 caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 240 tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 300 ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 360 ggaaagcgat actttgacta ctggggccag gggacaatgg tcaccgtctc gagtggtggg 420 gggggcagcg gtggtggagg ctctggtgga ggagggagct cctatgagct gactcagcca 480 ccctcagcgt ctgggacccc cgggcagagg gtcaccatct cttgttctgg aggcagctcc 540 aacatcggaa gtaatactgt aaactggttc cggcagctcc caggaacggc ccccaaactc 600 ctcgtttatt ttaataatca gcgaccctca ggggtccctg accgattctc tggctccaag 660 tctggcacct cggcctccct ggccatcggt gggctccagt ctgacgatga ggctgactat 720 tactgtgtag catgggatga ctctctgaat gctccggtgt tcggcggagg gaccaaggtc 780 accgtcctag agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctggtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaaactgctg tacatcttca aacagccttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tcttgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggagag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaaatgggc gggaagccac gacggaaaaa ccccaggag 1200 gggctgtata atgaactgca gaaggaaa atggccgagg cttacagcga aatcgggatg 1260 aagggagaga gaaggcgcgg aaaaggccac gatggactgt atcagggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacccagggg tagcggcgag 1380 ggcagagaga gtcttctaac atgcggtgac gtgggagaga atcccggcccc tatgggtcgg 1440 gggctgctca ggggcctgtg gccgctgcac atcgtcctgt ggacgcgtat cgccagcacg 1500 atcccaccgc acgttcagaa gtcggttaat aacgacatga tagtcactga caacaacggt 1560 1620 cagaaatcct gcatgagcaa ctgcagcatc acctccatct gtgagaagcc acaggaagtc 1680 1740 cccaagctcc cctaccatga ctttattctg gaagatgctg cttctccaaa gtgcattatg 1800 agaaaaaaaaa aaaagcctgg tgagactttc ttcatgtgtt cctgtagctc tgatgagtgc 1860 aatgacaaca tcatcttctc agaagaatat aacaccagca atcctgactt gttgctagtc 1920 atatttcaag tgacaggcat cagcctcctg ccaccactgg gagttgccat atctgtcatc 1980 atcatcttct actgctaccg cgttaaccgg cag 2013 <210> 47 <211> 671 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 47 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Phe Arg Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 290 295 300 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 305 310 315 320 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 325 330 335 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 340 345 350 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 355 360 365 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 370 375 380 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 385 390 395 400 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 405 410 415 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 420 425 430 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 435 440 445 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser 450 455 460 Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Arg 465 470 475 480 Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu Trp Thr Arg 485 490 495 Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp 500 505 510 Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys 515 520 525 Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys 530 535 540 Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val 545 550 555 560 Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr 565 570 575 Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp 580 585 590 Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu 595 600 605 Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile 610 615 620 Ile Phe Ser Glu Glu Tyr Asn Thr Ser Ser Asn Pro Asp Leu Leu Leu Val 625 630 635 640 Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala 645 650 655 Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln 660 665 670 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> Site <222> (1)..(15) <223> This sequence can contain 1-3 repeating units of "Gly Gly Gly Gly Ser" <220> <223> Detailed description of alternative and preferred embodiments is given in the filed specification. <400> 48 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15

Claims

1. An isolated nucleic acid molecule encoding a) a chimeric antigen receptor (CAR), wherein the CAR comprises a scFv specific for GPC3 encoded by the nucleic acid sequence of SEQ ID NO: 33; and b) an armored molecule, wherein the armored molecule resists immunosuppression against cells in the tumor microenvironment when expressed on the surface of the cells, wherein the armored molecule is a dominant negative type 2 TGF-β receptor (TGFβRIIDN) consisting of a nucleic acid encoding amino acids 478-671 of SEQ ID NO:

47.

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

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

4. The isolated nucleic acid molecule of claim 2, wherein the costimulatory domain comprises one or more of CD28, 4-1BB, CD3ζ, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains.

5. The isolated nucleic acid molecule of claim 2, wherein the costimulatory domain comprises one or more of a CD28, 4-1BB, and CD3ζ costimulatory domain.

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

7. The isolated nucleic acid molecule of any preceding claim, further comprising a hinge or spacer domain.

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

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

46.

10. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and an armor molecule, wherein the CAR comprises an antigen binding domain specific for a cell surface antigen, wherein the nucleic acid sequence comprises SEQ ID NO:

46.

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

12. A cell comprising: an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises a scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; and wherein the VL comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 42; and TGFβRIIDN armored molecule consisting of amino acids 478-671 of SEQ ID NO:

47.

13. The cell of claim 12, wherein the VH consists of the amino acid sequence of SEQ ID NO:

27.

14. The cell of claim 12, wherein the VL consists of the amino acid sequence of SEQ ID NO: 28, and the VH consists of the amino acid sequence of SEQ ID NO:

27.

15. The cell of any one of claims 12-14, wherein the anti-GPC3 chimeric antigen receptor (CAR) and TGFβRIIDN armored molecule comprises the amino acid sequence of SEQ ID NO:

47.

16. The cell of any one of claims 12-14, wherein the cell is selected from the group consisting of a T cell and a natural killer (NK) cell.

17. The cell of any one of claims 12-14, wherein the cell is selected from the group consisting of: cytotoxic T lymphocytes (CTLs) and regulatory T cells.

18. Use of the cell according to any one of claims 12 to 17 in the preparation of a medicament for treating hepatocellular carcinoma.

19. The use of claim 18, further comprising inhibiting tumor growth, inducing tumor regression, and / or prolonging the survival of a subject in need thereof.

20. The use of claim 19, wherein the cell is an autologous cell.

21. The use of any one of claims 18-20, further comprising administering a therapeutically effective amount of an anti-cancer antibody and / or a chemotherapeutic component to a subject in need thereof.

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