Compositions and methods for treating cancer with chimeric antigen receptors

Abstract

This invention provides compositions and methods for treating cancer using CAR T cells. [Solution] Provided are a) a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific to a cell surface antigen; and b) an isolated nucleic acid sequence encoding an armoring molecule that, when expressed on the surface of a cell in the tumor microenvironment, counteracts the immunosuppression of the cell. In another embodiment, a cell is provided comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a TGFβRIIDN armoring molecule expressed on the surface of the cell.

Description

[Technical Field]

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

[0002] This disclosure relates to the treatment of cancer using chimeric antigen receptor T cells. [Background technology]

[0003] 1. Chimeric antigen receptor T cell therapy Chimeric antigen receptor (CAR) T-cell therapy is a specific form of cell-based immunotherapy that uses engineered T cells to fight cancer. In CAR T-cell therapy, T cells are collected from the patient's blood, engineered ex vivo to express CARs containing both antigen-binding and T-cell activation domains, grown into larger populations, and then administered to the patient. CAR T cells function as living drugs, binding to cancer cells and causing their destruction. When successful, the effects of CAR T-cell treatment tend to be long-lasting, as evidenced by the detection of CAR T-cell persistence and proliferation in the patient long after clinical remission.

[0004] 2. Structure and Function of CAR The antigen-binding domain of a CAR is an extracellular region that targets surface antigens on tumor cells. Suitable target antigens can be proteins, phosphorylated proteins, peptide-MHCs, carbohydrates, or glycolipid molecules. An ideal target antigen is widely expressed on tumor cells, enabling targeting of a high percentage of cancer cells. Ideal candidate target antigens are also minimally expressed in normal tissues, limiting off-tumor-on-target toxicity. The antigen-binding domain of a CAR contains a targeting moiety, such as an antibody single-chain variable fragment (scFv), directed towards the target antigen.

[0005] The T cell activation domain of a CAR is located intracellularly and activates T cells in response to an antigen-binding domain that interacts with the target antigen. The T cell activation domain may contain one or more costimulatory domains, which are intracellular domains of known activated T cell receptors. Since costimulatory domains have different effects on the dynamics, cytotoxic function, and safety profile of CAR T cells, the selection and positioning of costimulatory domains within a CAR construct influences the function and fate of CAR T cells.

[0006] The extracellular antigen-binding domain and intracellular T-cell activation domain of a CAR are linked by a transmembrane domain, a hinge, and possibly a spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on tumor cells. The hinge domain can be used alone or in conjunction with a spacer domain that causes the scFv to protrude from the T-cell surface. The optimal length of the spacer is determined by the proximity of the binding epitope to the cell surface.

[0007] CAR T therapy targeting B lymphocyte antigen CD19 (Kymriah®, Novartis) is considered promising for pediatric acute lymphoblastic leukemia, while CAR T therapy targeting B cell maturation antigen ("bb2121," a collaboration between Celgene® and bluebirdbio®) is considered promising for relapsed / refractory multiple myeloma. More recent data suggest that CAR approaches may be effective against solid tumors. GD2 CAR natural killer T cell (NKT) therapy has shown activity in neuroblastoma (Heczey A, et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. 124(18):2824-33, 2014), and mesoserine CAR T with pembrolizumab has demonstrated antitumor activity in mesothelioma. However, additional targets are needed to treat solid tumors.

[0008] 3. Challenges of CAR T-cell therapy Unfortunately, the complexity of CAR T cell-based therapies can lead to undesirable and dangerous side effects. Neurotoxicity and acute respiratory distress syndrome (CRS) are potential adverse effects of CAR T cell therapy and are potentially lethal. Cytokine release syndrome (CRS) is the most common acute toxicity associated with CAR T cells. CRS occurs when lymphocytes become highly activated and release excessive amounts of inflammatory cytokines. In patients with CRS, elevated serum levels of interleukin-2, interleukin-6, interleukin-1 beta, GM-CSF, and / or C-reactive protein are occasionally observed when these factors are assayed. CRS is classified by severity and diagnosed as one of four grades (mild to severe), with more severe cases clinically characterized by high fever, hypotension, hypoxia, and / or multi-organ toxicity in the patient. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR T-cell therapy experienced CRS, and 50% of these patients presented with grade 3-4 symptoms (Fitzgerald et al., Crit Care Med. 45(2):e124-e131 (2017)).

[0009] Another challenge to the success of CAR T-cell immunotherapy is immunosuppression caused by the characteristics of the tumor microenvironment (TME) of solid tumors. For example, transforming growth factor β (TGF-β) is a pleomorphic cytokine that is produced in large quantities by many cell types in the liver (e.g., hepatic sinusoidal endothelial cells, Kupfer cells, intrahepatic natural killer (NK) cells, etc.) and within the cancer microenvironment (Dahmani et al., TGF-β in T Cell Biology: Implications for Cancer Immunotherapy. Cancers 2018, 10, 194, 1-21). TGF-β binds to TGFβR2, which recruits TGFβR1 to phosphorylate it. When phosphorylated, it phosphorylates receptor-modulating SMAD (R-SMAD). The phosphorylated SMAD complex with coSMAD transposes to the nucleus to assist in regulating gene expression. In the context of T cells, TGF-β signaling suppresses the effectiveness of CAR T cell therapy by inhibiting T cell proliferation, activation, and effector function, and by prioritizing the differentiation of regulatory T cells. Therefore, TGF-β-related immunosuppression is a significant hurdle that must be overcome to obtain effective and lasting CAR T cell therapy for solid tumors.

[0010] 4. Armoring A recent approach to producing CAR T cells that are more resistant to tumor-associated immunosuppression is called armoring. Armoring is the molecular manipulation of CAR T cells to express one or more "armoring molecules" that can counteract immunosuppression. For example, researchers recently reported modifications of CAR T cells to secrete PD-1 blocking single-chain variable fragments (scFv). This is PD-L1 +In mouse models of hematological tumors and solid tumors, CAR T cells enhanced antitumor activity (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 demonstrated the effectiveness of neutralizing the inhibitory effects of TGF-β on T cells by armoring T cells with the dominant-negative TGF-β receptor type 2 (TGFβRIIDN) armoring molecule (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 using the TGFβRIIDN armoring molecule (NCT03089203) to armor anti-PSMA-CAR T cells for the treatment of castration-resistant prostate cancer.

[0011] Therefore, additional CAR T-cell therapy is needed to enhance the armamentealium of effective cancer treatment. Such therapies should include CAR T cells that effectively treat cancer while minimizing the risk of developing dangerous inflammatory responses, such as CRS. In addition, such therapies should include CAR T cells that can survive within the immunosuppressive TME of solid tumors. [Overview of the Initiative] [Means for solving the problem]

[0012] This disclosure describes compositions and methods for treating cancer using CAR T cells. In a first embodiment, as described below, (a) a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific to a cell surface antigen; and (b) an isolated nucleic acid sequence encoding an armoring molecule that, when expressed on the surface of a cell in the tumor microenvironment, counteracts cellular immunosuppression.

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

[0014] In a further embodiment, the Disclosure describes an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45, and a cell comprising a TGFβRIIDN armoring molecule.

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

[0016] These and other features and advantages of this disclosure will be better understood from the following detailed description together with the attached claims. Note that the claims are defined by their content and not by the specific consideration of the features and advantages described herein.

[0017] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles and operations of the present disclosure.

Brief Description of the Drawings

[0018] [Figure 1A] TGFβ1 and TGFβ gene signatures, HCC progression, and survival. 1A. Fold change (log2) in TGFβ1 gene expression in normal solid tissue (left) and primary solid tumors of hepatocellular carcinoma (HCC, right). 1B. TGFβ1 and TGFβ gene signatures, HCC progression, and survival. Survival curve for high TGFβ1 expression versus low TGFβ1 expression in HCC (OS = overall survival period). 1C. TGFβ1 and TGFβ gene signatures, HCC progression, and survival. Fold change (log2) in TGFβ signaling in normal solid tissue (left) and solid tumor tissue (right). 1D. TGFβ1 and TGFβ gene signatures, HCC progression, and survival. Survival curve for high TGFβ signaling versus low TGFβ signaling. [Figure 1B] Continuation of Figure 1A. [Figure 1C] Continuation of Figure 1B. [Figure 1D] Continuation of Figure 1C. [Figure 2] Human HCC samples are positive for TGFβ and TGFβ signaling. Semi - quantitative pathological evaluation of (2A) TGF - b1 and (2B) phosphorylated SMAD2 (pSMAD2) in FFPE normal liver and HCC specimens. Each circle represents data from one surgical resection. IHC intensity was defined as follows: score 0, negative staining; score 1, minimal staining; score 2, moderate staining; score 3, strong staining. [Figure 3]Armoring of GPC3 CAR T cells by TGFβRIIDN. As intended herein, armoring of GPC3 CAR T cells by TGFβRIIDN is thought to confer resistance to TGFβ, leading to improved CAR T effector function and tumor control. 3A. Schematic diagram of TGF-β-mediated immunosuppression of non-armored CAR T cells (modified from Arrese et al, Current Protein & Peptide Science (2018) 19:1172). 3B. Diagram of armored CAR T cells expressing GPC3 CAR and TGFβRIIDN armoring molecules. In some embodiments, the CAR and armoring molecules may be fused at the C-terminus or separated from the CAR by a spacer peptide. [Figure 4] Expression of TGFβRIIDN. Flow cytometry analysis of TGFβRII and CAR on the surface of non-armoring and armored CAR T cells. [Figure 5] TGFβRIIDN suppresses TGF-β signaling in CAR T cells. Western blot analysis of phosphorylated SMAD2 / 3 and total SMAD2 / 3 in untransduced (UT), unarmored, and armored (TGFβRIIDN) CAR T cells 0–45 minutes after exposure to 4B.rhTGFβ (1 ng / ml). β-actin served as a positive loading control for all samples. [Figure 6] Expression of TGFβRIIDN inhibits the TGF-β-induced repression of effector cytokine transcription. mRNA levels of (6A)IL2 and (6B)IFNG in purified TGFβRIIDN and non-armoring GPC3 CAR T cells stimulated for 6 hours with plate-bound recombinant GPC3 and TGF-β1 concentrations are shown. Data are pooled from two independent experiments. [Figure 7]TGFβRIIDN reduces TGF-β-mediated differentiation of GPC3 CAR T cells to the TRM phenotype. 7A. Flow cytometry analysis of TGF-β-mediated differentiation of GPC3 CAR T cells to tissue resident memory (TRM) cells in non-armoring GPC3 CAR T cells (top scatter plot) and armored (bottom scatter plot) GPC3 CAR T cells in the absence of TGF-β (left scatter plot) and in the presence of TGF-β (right scatter plot). Y axis = CD103 expression; X axis = CAR expression. 7B. Percentage of TRM cell differentiation, as demonstrated by CD69+ / CD103+ coexpression, in non-armoring (GPC3) CAR T cells and armored (GPC3 TGFβRIIDN) CAR T cells treated with TGF-β. [Figure 8] Exploratory in vitro readouts in non-armored and armored CAR T cells. 8A. Bright-field images of real-time monitoring of CAR T-mediated cytotoxicity during co-culture with GPC3+ tumor cells. CAR T cell proliferation was visually observed during co-culture with GPC3+ tumor cells. 8B. CAR T-mediated cytotoxicity during co-culture with GPC3+ tumor cells, expressed as a cellular index, as evaluated by xCelligence® Real-Time Impedance-Based Killing Assay (RTCA). 8C. Quantification of tumor cell (HEP3B cells or HUH-7 cells)-induced CAR T cell proliferation in non-armored GPC3 CAR T cells and armored GPC3 CAR T cells (AZ: proprietary vector scaffold; LN: Lentigen vector scaffold) in the presence or absence of TGF-β. TGF-β suppressed tumor GPC3-induced proliferation of CAR T cells after 4 days in co-culture. Armored GPC3 CAR T cells were less susceptible to suppression. [Figure 9]In vivo reduction of tumor volume in a xenograft model using CAR T cells armored with TGFβRIIDN. Huh7-TGF-β was overexpressed in the xenograft model. Untransduced T cells, unarmored GPC3 CAR T cells, or GPC3 CAR T cells armored with TGFβRIIDN were injected into Huh7 tumor-carrying mice engineered to overexpress TGF-β. Tumor volume was measured every two weeks (10 mice / group). [Figure 10] Increased number of tumor-infiltrating lymphocytes in mice treated with TGFβRIIDN-armored CAR T cells. Huh7-TGF-β overexpressing tumor-carrying mice were administered 7 × 10⁶ untransduced T cells, unarmored GPC3 CAR T cells, or TGFβRIIDN-armored GPC3 CAR T cells. 10A. Number of CAR+ cells in the tumor of mice collected at the indicated time point. 10B. Representative example of FACS data summarized in 10A. [Figure 11] Increased number of TGFβRIIDN-armored CAR T cells in the spleen. Huh7-TGF-β overexpressing tumor-carrying mice were administered 7 × 10⁶ untransduced T cells, unarmored GPC3 CAR T cells, or TGFβRIIDN-armored GPC3 CAR T cells. 11A. Number of CAR+ cells in the spleen of mice collected at the indicated time point. 11B. Representative example of FACS data summarized in 11A. [Figure 12] Ex vivo expression of TGFβRII on the surface of CAR+ cells. 12A. Expression of CAR and TGFβRII on the surface of lymphocytes infiltrating the spleen or tumor 14 days after injection in Huh7-TGF-β overexpressing tumor-carrying mice (FMO: fluorescence minus 1). 12B. Mean fluorescence intensity (MFI) of TGFβRII on the surface of CAR+ or CAR-CD8+ T cells in tumors. [Figure 13]Decreased expression of PD1 and LAG3 on the surface of CAR T cells armored with TGFβRIIDN within tumors. Expression of PD1(A) and LAG3(B) on the surface of tumor-infiltrating CD8+ and CD4+ CAR+ T cells 14 days after injection in Huh7-TGF-β overexpressing tumor-carrying mice. [Figure 14] Decreased CD70 expression and increased CD27 cell expression on the surface of CAR T cells armored with TGFβRIIDN within tumors. Frequency of CD70+CD27-(A) and CD70+CD27-(B) on the surface of tumor-infiltrating CD8+ and CD4+CAR+ T cells 14 days after injection in Huh7-TGF-β overexpressing tumor-carrying mice. [Figure 15] Immunophenotyping of CAR T cells in the spleen. Expression of PD1 (15A), LAG3 (15B), and CD27 / CD70 (15C) on the surface of CD8+ and CD4+ CAR+ T cells in the spleen 14 days after injection in Huh7-TGF-β overexpressing tumor-carrying mice. [Figure 16] Analysis of serum IFN-γ. Concentrations of IFN-γ detected in the serum of Huh7-TGF-β overexpressing tumor-carrying mice injected with untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells (baseline A: before tumor transplantation; baseline B: before CAR T injection). [Figure 17] Analysis of serum AFP. Concentrations of AFP detected in the serum of Huh7-TGF-β overexpressing tumor-carrying mice injected with untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells (baseline A: before tumor transplantation; baseline B: before CAR T injection). [Figure 18]In vivo reduction of tumor volume in TGFβRIIDN-armored CAR T cells and TGFβ(-)PDX models. Figures 18A–18C show tumor volumes from three different GPC3+TGF-β-hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5 × 10⁶ untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells were injected into tumor-bearing mice, and tumor volume was measured every two weeks (5 mice / group). [Figure 19-1] In vivo reduction of tumor volume in TGFβRIIDN-armored CAR T cells and TGFβ(+) PDX models. A-E represent tumor volumes from five different GPC3+TGF-β+ hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models. For each model, 5 × 10⁶ untransduced T cells, unarmored GPC3 CAR T cells, or armored GPC3 CAR T cells were injected into tumor-bearing mice, and tumor volume was measured every two weeks (5 mice / group). [Figure 19-2] This is a continuation of Figure 19-1. [Modes for carrying out the invention]

[0019] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton, et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms have the meanings described below.

[0020] The terms “comprise” and “include” as used herein, and their variations (e.g., “comprises,” “comprising,” “includes,” and “including”), shall be understood to include the described component, feature, element, or process, or group of components, features, elements, or processes, but not to exclude any other component, feature, element, or process, or group of components, features, elements, or processes. The terms “to include,” “essentially consisting of,” and “consisting of” may be substituted for any of the other two terms while retaining their usual meanings.

[0021] In this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0022] The percentages disclosed herein may vary by ±10, 20, or 30% from the disclosed values, and may remain within the intended scope of disclosure.

[0023] Unless otherwise indicated, or unless otherwise evident from the context and the understanding of those skilled in the art, values ​​expressed herein as ranges may be considered any specific value or subrange within the ranges described in the various embodiments of this disclosure, up to one-tenth of the lower limit of the range, unless the context clearly indicates otherwise.

[0024] The ranges and quantities used herein may be expressed as "about" a particular value or range. The term "about" also includes the exact quantity. For example, "about 5%" means "about 5%" and also "5%." Furthermore, the term "about" can refer to ±10% of a given value or range of values. Therefore, about 5% could also mean, for example, 4.5% to 5.5%. Unless otherwise clearly stated in the context, all numerical values ​​provided herein are modified by the term "about."

[0025] As used herein, the terms "or" and "and / or" can describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" may refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z".

[0026] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids. Therefore, the terms peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used instead of or interchangeably for any of these terms.

[0027] As used herein, "protein" may refer to a single polypeptide, that is, a single amino acid chain as defined above, but may also refer to two or more polypeptides that are linked together, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to form a polymer protein.

[0028] An "isolated" substance, such as an isolated nucleic acid, is a substance that does not exist in its natural environment, but is not necessarily purified. For example, an isolated nucleic acid is a nucleic acid that is not produced or located in its natural or natural environment, such as a cell. An isolated substance may be separated, fractionated, or at least partially purified by any appropriate technique.

[0029] As used herein, the terms “antibody” and “its antigen-binding fragment” refer to at least the smallest portion of an antibody capable of binding to a specific antigen targeted by the antibody, for example, at least a portion of the complementarity-determining regions (CDRs) of the variable domains of the heavy chain (VH) and the light chain (VL), in relation to a typical antibody produced by B cells. An antibody or its antigen-binding fragment may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, an epitope-binding fragment, for example, Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv(scFv), a single-chain antibody, a disulfide-bound Fv(sdFv), a fragment containing a VL or VH domain alone or in combination with a portion of the corresponding domain (e.g., the entire VL domain and a partial VH domain having one, two, or three CDRs), and fragments generated by a Fab expression library, or may be derived from them. The scFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019. The antibody molecules encompassed by this disclosure may be of 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, or derived therefrom. The numbering of amino acids within the variable domain, complementarity-determining region (CDR), and framework region (FR) of the antibody follows the Kabat definitions given in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise specified.

[0030] As used herein, the term "polynucleotide" includes single nucleic acids and multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes all types of nucleic acids, such as DNA or RNA.

[0031] As used herein, the term "vector" may refer to a nucleic acid molecule that, upon introduction into a host cell, generates a transformed host cell. A vector may include a nucleic acid sequence, such as an origin of replication, that enables replication within the host cell. A vector may also include one or more select marker genes and other genetic elements known in this art. Certain types of vectors envisioned herein may be bound to or incorporated into a virus in order to facilitate cell transformation.

[0032] A “transformed” cell, or “host” cell, is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. All methods that may introduce nucleic acid molecules into such cells are intended herein, including viral vector transduction, plasmid vector transformation, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0033] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (such as an epitope) to its homologous binding domain (such as a paratope). As used herein, the term "avidence" refers to the overall stability of the complex between the epitope and paratope populations (i.e., the antigen and the antigenic domain).

[0034] As used herein, the terms “to treat,” “treatment,” or “treatment of” mean, when used in connection with the treatment of cancer, to alleviate symptoms, reduce or eliminate symptoms, promote increased survival rates, and / or reduce discomfort. For example, treatment may mean the ability of a therapy, when applied to a subject, to alleviate symptoms, signs, or causes. Treatment may also mean the alleviation or reduction of at least one clinical symptom, and / or the inhibition or delay of symptom progression, and / or the prevention or delay of the onset of disease or illness.

[0035] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject, particularly mammalian subjects, for which diagnosis, prognosis, or treatment is desired. Examples of mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and others.

[0036] The terms used herein, the administered treatment substance, and, for example, the “effective dose” or “therapeutic effective dose” of CAR T cells, refer to amounts sufficient to accomplish a particular stated or intended purpose, such as the treatment of cancer. The “effective dose” can be determined based on routine experiments with respect to the stated purpose.

[0037] 2. Overview This disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More specifically, this disclosure relates to CAR cell therapy in which transformed cells, such as T cells, express CARs that target, for example, glypican 3 (GPC3). The CAR constructs, transformed cells expressing the constructs, and therapies utilizing the transformed cells disclosed herein may provide robust cancer treatment that minimizes the risk of cytokine release syndrome (CRS) or indiscriminate cytokine release in non-GPC3 expressing cells.

[0038] While we do not wish to be constrained by theory, GPC3 is considered a viable cancer target across multiple modalities, including bispecific T cell engagers, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). The carcinoembryonic antigen GPC3 is a GPI-conjugated heparin sulfate proteoglycan. GPC3 stimulates Wnt signaling by stabilizing the Wnt-Fzd interaction. GPC3 competes with patched for Hh binding, mitigating smoothed inhibition and inducing GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) proliferation. Furthermore, GPC3 expression levels have been shown to correlate with the stage and grade of HCC.

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

[0040] Further aspects of this disclosure include targeting GPC3 and others, such as CAR T cells, for example, solid tumor cells, which are armored with TGFβRIIDN to protect CAR T cells against TGF-β-related immunosuppression.

[0041] 3. Design of CAR structures The CAR constructs of this disclosure may have several components, many of which can be selected based on the desired or refined function of the resulting CAR construct. In addition to the antigen-binding domain, the CAR construct may have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. The selection of one component in preference to another (i.e., selecting a particular costimulatory domain of one receptor for a costimulatory domain of a different receptor) may affect the clinical efficacy and safety profile.

[0042] 4. Antigen-binding domain The antigen-binding domains intended herein may include an antibody or one or more antigen-binding fragments thereof. A one-CAR construct intended to target GPC3 includes a single-chain variable fragment (scFv) containing light-chain variable regions and heavy-chain variable regions derived from one or more antibodies specific to GPC3, either directly linked or linked via a flexible linker (e.g., repeats of GGGGS (SEQ ID NO: 48) having 1, 2, 3, or more repeats).

[0043] The antigen-binding domains of the CARs disclosed herein may exhibit variability in their binding affinity to target proteins. The relationship between binding affinity and potency may be more nuanced in relation to CARs compared to antibodies, where higher affinity is generally desirable. For example, preclinical studies on receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CARs derived from high-affinity scFv (dissociation constant 0.56 nM) showed increased treatment index compared to low-affinity variants. Conversely, other examples have reported that manipulating scFv for lower affinity improves the differentiation between cells with different antigen densities. This may be useful in improving therapeutic specificity for antigens differentially expressed in tumor versus normal tissue.

[0044] Various methods can be used to confirm the binding affinity of the antigen-binding domain. In some embodiments, methodologies that exclude the avidity effect can be used. The avidity effect often requires multiple antigen-binding sites that interact simultaneously with multiple target epitopes in a multimerized structure. Therefore, avidity functionally represents the cumulative strength of multiple interactions. An example of a methodology that excludes the avidity effect is any approach in which one or both of the interacting proteins are monomeric / monovalent. This is because if one or both partners contain only a single interaction site, multiple simultaneous interactions are not possible.

[0045] 5. Spacer Domain The CAR constructs of this disclosure may have spacer domains that provide conformational freedom to facilitate binding to target antigens on target cells. The optimal length of the spacer domain may be determined by the proximity of the binding epitope to the target cell surface. For example, proximal epitopes may require longer spacers, and distal epitopes may require shorter epitopes. In addition to facilitating binding of the CAR to the target antigen, achieving the optimal distance between the CAR cell and the cancer cell may also help sterically block large inhibitory molecules from the immunological synapse formed between the CAR cell and the target cancer cell. The CAR may have long spacers, intermediate spacers, or shorter spacers. Long spacers may include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (either natural or having modifications common to therapeutic antibodies such as the S228P mutation), although the CH3 region can itself be used to construct an intermediate spacer (approximately 120 amino acids). Shorter spacers may originate from the CD28, CD8α, CD3, or CD4 segments (<60 amino acids). Short spacers may also originate from the hinge region of the IgG molecule. These hinge regions may originate from any IgG isotype and may or may not contain mutations common to therapeutic antibodies, such as the S228P mutation mentioned above.

[0046] 6. Hinged Domain CARs may also possess hinge domains. Flexible hinge domains are short peptide fragments that provide conformational freedom to facilitate binding to target antigens on tumor cells. Flexible hinge domains can be used alone or in combination with spacer sequences. The terms “hinge” and “spacer” are often used interchangeably; for example, an IgG4 sequence can be considered both a “hinge” and a “spacer” sequence (i.e., a hinge / spacer sequence).

[0047] CARs may further contain sequences containing signal peptides. These signal peptides prompt cells to translocate the CAR to the cell membrane. Examples include IgG1 heavy chain signal polypeptide, Ig kappa or lambda light chain signal peptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal polypeptide, or CD33 signal peptide.

[0048] 7. Transmembrane domain CARs may further contain sequences including a transmembrane domain. This transmembrane domain may include a hydrophobic α-helix spanning the cell membrane. While the properties of the transmembrane domain have not been studied in as much detail as other aspects of CAR constructs, they may potentially influence CAR expression and binding to endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0049] 8. Co-stimulatory domain CARs may further contain one or more sequences that form a co-stimulatory domain. A co-stimulatory domain is a domain that can enhance or modulate the response of immune effector cells. Co-stimulatory domains may include, for example, sequences derived from one or more of the following: CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The selection of the co-stimulatory domain affects the phenotype and metabolic signature of CAR cells. For example, CD28 co-stimulation results in a potent but short-lived effector-like phenotype with high levels of cytolytic ability, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells modified with CARs containing the 4-1BB co-stimulatory domain tend to proliferate and survive longer in vivo, exhibit increased oxidative metabolism, reduced depletion, and increased ability to generate central memory T cells.

[0050] 9.Cells CAR-based cell therapy can be used with various cell types, such as lymphocytes. Specific cell types that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, alpha-beta T cells, gamma-delta T cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells (Tregs). In one embodiment, the CAR cells used to treat the subject are autologous. In other embodiments, the CAR cells may be derived from a genetically similar but not identical donor (allogeneic).

[0051] 10.CAR cell generation The CAR constructs of this disclosure may include several combinations of the modular components described herein. For example, in some embodiments of this disclosure, the CAR construct includes a GPC3 scFv antigen-binding domain. In some embodiments, the CAR includes a GPC3-2 scFv antigen-binding domain. In some embodiments of this disclosure, the CAR construct includes a CSFR2 signal peptide. In some embodiments, the CAR construct includes an IgG4P hinge / spacer domain having an S228P mutation. In some embodiments, the CAR construct includes a CD28 transmembrane.

[0052] In the CAR constructs of this disclosure, different co-stimulatory domains can be utilized. In some embodiments, the CAR construct includes a co-stimulatory domain derived from the intracellular domain of CD3z. In some embodiments, the CAR construct includes a CD28 co-stimulatory domain. In some embodiments, the CAR construct includes a 4-1BB co-stimulatory domain. In some embodiments, the CAR construct includes co-stimulatory domains derived from CD3z and CD28. In some embodiments, the CAR construct includes co-stimulatory domains derived from CD3z and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains derived from all of CD3z, CD28, and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains derived from ICOS, OX-40, and / or GITR.

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

[0054] 12. Embodiment of CAR In some embodiments, the disclosure provides isolated nucleic acid sequences encoding chimeric antigen receptors (CARs) that include antigen-binding domains specific to surface antigens on tumor cells. In some embodiments, the cell surface antigen is a protein, a phosphorylated protein, a peptide-MHC, a carbohydrate, or a glycolipid molecule.

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

[0056] In some embodiments, the disclosure provides isolated nucleic acid sequences encoding a chimeric antigen receptor (CAR) containing an antigen-binding domain specific to glypican 3 (GPC3). The antigen-binding domain has an equilibrium dissociation constant (K). D The CAR construct has a concentration of approximately 100 nanomoles (nM) or less and does not induce cytokine production in GPC3 cells. In some embodiments, the antigen-binding domain includes an antibody or its antigen-binding fragment. The antigen-binding domain may be a Fab or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain is an scFv containing the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0057] In some embodiments, the CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain. The transmembrane domain may be a CD28 transmembrane domain. The co-stimulatory domain may be one or more of the CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40 co-stimulatory domains. In a particular embodiment, the co-stimulatory domain is one or more of the CD28, 4-1BB, and CD3 zeta co-stimulatory domains. The signaling domain may be a sequence encoding a CSFR2 signal peptide.

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

[0059] In some specific embodiments, the isolated nucleic acid sequence encoding the chimeric antigen receptor (CAR) may have the sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 26.

[0060] In other embodiments, the disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain. The antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, VH may have CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, VL may have CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0061] 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, CAR may further have a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0062] 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.

[0063] In other embodiments, the disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence may be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0064] In another embodiment, the disclosure provides cells comprising a vector having the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0065] In other embodiments, the disclosure relates to a cell having a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K D The CAR construct provides cells in which the CAR construct does not induce cytokine production in GPC3 cells, and the CAR construct has a concentration of approximately 100 nanomoles (nM) or less. For example, the nucleic acid sequence may be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0066] In other embodiments, the disclosure provides cells expressing an anti-GPC3 chimeric antigen receptor (CAR) on the extracellular surface. The CAR may have an antigen-binding domain which may be an antibody, Fab, or scFv having a heavy chain variable region (VH) and a light chain variable region (VL). The VH may include CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39. The VL may include CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0067] In some embodiments, VH may have the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. In some embodiments, VL may have the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. CAR may further include a transmembrane domain, a costimulatory domain, and a signaling domain. Cells express CAR having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

[0068] In some embodiments, the Disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells expressing CAR on their extracellular surface, the CAR may have the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. Such cells may exhibit anti-tumor immunity upon contact with tumor cells expressing GPC3.

[0069] 13. Cancer treatment with CAR In some embodiments, this disclosure provides CAR cells for the treatment of cancer. The compositions described herein (e.g., antibodies, CAR constructs, and CAR cells) and methods of use thereof are useful, among other things, for inhibiting the proliferation or spread of neonatal cells. In some embodiments, they are particularly useful for inhibiting neonatal cell proliferation in which GPC3 plays a role.

[0070] Examples of neoplasms treatable with the compositions of this disclosure include solid tumors, such as tumors of the liver, lungs, or ovaries. However, the cancers listed herein are not intended to be limiting. For example, examples of cancer types intended to be treated herein include NSCLC, advanced solid malignant tumors, cholangiocarcinoma, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal neoplasm, esophageal squamous cell carcinoma, advanced small cell lung cancer, gastric adenocarcinoma, gastric cancer, gastroesophageal junction cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal cancer, metastatic melanoma, metastatic noncutaneous melanoma, multiple myeloma, nasopharyngeal neoplasm, non-Hodgkin lymphoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasm, pleural mesothelioma, prostate neoplasm, recurrent or metastatic PD-L1 positive or negative SCCHN, recurrent squamous cell lung cancer, renal cell carcinoma, renal cell carcinoma Examples include carcinoma, SCCHN, pharyngeal-pituitary squamous cell carcinoma, laryngeal squamous cell carcinoma, small cell lung cancer, squamous cell carcinoma of the head and neck, squamous cell lung cancer, TNBC, transitional cell carcinoma, unresectable or metastatic melanoma, urothelial cancer, and urothelial carcinoma.

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

[0072] 14. Armoring In some embodiments, the Disclosure provides “armored” cells, such as CAR T cells having one or more genetic modifications that enhance or optimize cellular function by protecting the cells from environmental disturbances, such as immunosuppressive cytokines or immunosuppressive TMEs. Genetic modifications include, but are not limited to, cytokine secretion, enhanced expression of ligands that interact with immune cells, such as T cells, macrophages, and regulatory T cells, or alteration of functional characteristics. Those skilled in the art will understand that arming cells, such as T cells, can provide many additional benefits not described herein, such as enabling T cell survival in immunosuppressive TMEs.

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

[0074] In some embodiments, armored cells may contain a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain having an equilibrium dissociation constant (K D The concentration is approximately 100 nanomoles (nM) or less, the CAR construct does not induce cytokine production in GPC3 cells, and the cells express the TGFβRIIDN armoring molecule.

[0075] In some embodiments, the armored cells may 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) (VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39; VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45); and a TGFβRIIDN armoring molecule.

[0076] 15. Treatment Method CAR-modified cells of the present invention, such as CAR T cells, may be administered alone or as part of a pharmaceutical composition having diluents and / or other components associated with cytokines or cell populations. Briefly, a pharmaceutical composition of the present invention may, for example, contain CAR T cells as described herein together with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such a composition may include buffers such as neutral buffered saline or buffered saline; sulfates; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; amino acids such as proteins, polypeptides, or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. A pharmaceutical composition of the present invention can be adapted for treatment (or prevention).

[0077] CAR-modified cells may also be administered in combination with one or more additional therapies. In one embodiment, an additional therapy may be an anti-cytokine antibody. For example, one or more anti-TNFα antibodies may be used to reduce toxicity and enhance antitumor activity at higher CAR T doses that may be associated with CRS-like symptoms and weight loss.

[0078] The number of CAR cells administered per dose, the number of administrations, and the frequency of administration will be determined by various parameters, such as the patient's age, weight, clinical assessment, tumor type, tumor load, and / or other factors (including the physician's judgment). Any acceptable route of administration, such as intravenous (e.g., IV), parenteral, or subcutaneous, is intended but not limited to these.

[0079] In certain embodiments, 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, additional treatment regimens may include immune checkpoint inhibitors (ICIs), such as those targeting the PD-1 / PD-L1 axis (PDX), and other immuno-oncology (IO) treatments, such as immune system agonists.

[0080] Antibodies intended include anti-PD-L1 antibodies such as durvalumab (MEDI4736), avelumab, atezolizumab, and KNO35; anti-PD-1 antibodies such as nivolumab, pembrolizumab, cemiprimab, SHR1210, IBI308, PDR001, anti-PD-1, BGB-A317, BCD-100, and JS001; and anti-CTLA4 antibodies such as tremelimumab or ipilimumab. Additional antibodies are also intended herein. Furthermore, any antibody subpart effective for treatment is intended herein.

[0081] Information relating to durvalumab (or its fragments) used in the methods provided herein can be found in U.S. Patent No. 8,779,108, U.S. Patent No. 9,493,565, and U.S. Patent No. 10,400,039, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, durvalumab or its antigen-binding fragment used in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H9OPT antibody disclosed in the aforementioned U.S. patents.

[0082] Information relating to tremelimumab (or its antigen-binding fragment) used in the methods provided herein can be found in U.S. Patent No. 6,682,736 (in which tremelimumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0083] Additional treatment agents (chemotherapeutic agents or biologics) intended herein include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, ADRIAMYCIN® (doxorubicin), cisplatin (MVAC), carboplatin-based regimens, or monotherapy with taxanes or gemcitabine, temozolomide, or dacarbazine, vinflunin, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Further examples include drugs that target the DNA damage repair system, such as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors, and treatment agents that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora B protein kinase activity, and DNA-PK activity.

[0084] The therapeutic compositions or therapeutic methods intended herein may be combined with one or more other therapeutic compositions and therapeutic methods provided herein.

[0085] In some embodiments, the Disclosure provides a method for treating cancer, comprising administering to a subject requiring cancer treatment an effective amount of cells containing an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, and an armoring molecule that, when expressed on the cell surface, addresses cellular immunosuppression within the tumor microenvironment. In another embodiment, the Disclosure states that the antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). The VH may comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the Method further inhibits tumor growth, induces tumor regression, and / or extends the survival of the subject.

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

[0087] In some embodiments, the cells are autologous cells. For example, autologous cells may be selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

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

[0089] It should be understood that the specific embodiments described herein are not limited to the specific embodiments presented and are subject to change. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting unless specifically defined herein. Furthermore, the specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation, as will be recognized by those skilled in the art. [Examples]

[0090] The following examples illustrate specific embodiments of the present disclosure and their various uses. They are provided for illustrative purposes only and should not be construed as limiting the scope of the present disclosure. A glossary of terms is provided in Table 1.

[0091] [Table 1]

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

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

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

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

[0096] TGFβ gene signaling was increased 1.1317-fold in primary solid tumors compared to normal tissue. High TGFβ signaling within LIHCs was associated with shortened cell survival compared to low TGFβ signaling. See Figures 1C and 1D.

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

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

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

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

[0101] conclusion TGF-β was expressed and actively signaled in the majority of HCC samples. This demonstrates that TGF-β is a common immunosuppressive factor in HCC tumors and that armoring could benefit a large population of HCC patients.

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

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

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

[0105] After proliferation, CAR and TGFβRII expression were analyzed on the surface of non-armoring and armored CAR T cells by flow cytometry. CAR expression was detected by using the AF647 anti-idiotype antibody on GPC3-CAR (Figure 4).

[0106] After staining GPC3-CAR with an AF647 anti-idiotype antibody, CAR T cells were purified using anti-AF647 microbeads (Miltenyi). The purified cells were grown for a further 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).

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

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

[0109] result Expression of TGFβRII on the surface of TGFβRIIDN CAR T cells. Surface staining of TGFβRII on non-armored and armored CAR T cells was evaluated by flow cytometry. The antibody did not distinguish between endogenous TGFβRII and DN TGFβRII, as their extracellular portions are identical, but it did distinguish between TGFβRII and CAR cells co-expressed on TGFββRIIDN CAR T cells. This suggests that the antibody is detecting the overexpressed DN receptor. See Figure 4.

[0110] TGFβRIIDN inhibits SMAD2 / 3 phosphorylation in CAR T cells after exposure to rhTGFβ: Non-armored CAR T cells exhibited rhTGF-β-induced SMAD2 / 3 phosphorylation similar to that of non-transduced control cells at 0, 15, 30, and 45 minutes after rhTGF-β exposure. T cells armored with TGF-βRIIDN exhibited attenuated SMAD2 / 3 phosphorylation at 15, 30, and 45 minutes after rhTGF-β exposure compared to non-armored CAR T cells and non-transduced control cells. Total SMAD2 / 3 protein and β-actin expression were consistent across all groups. See Figure 5B.

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

[0112] TGFβRIIDN expression efficiently inhibits TGFβ signaling. CD103 + T RM CD103 -Differentiation of T cells requires IL-15 and TGF-β in vitro. Therefore, TGFβRIIDN CAR T cells, after incubation with TGF-β, were unable to differentiate into T cells compared to CAR - cells or non-arming CAR T cells. This result demonstrates that the expression of TGFβRIIDN inhibits the signal transduction induced by TGF-β during long-term exposure. See Figures 7A and 7B. RM

[0113] Conclusion TGFβRIIDN suppresses TGF-β signal transduction in armed CAR T cells, prevents the TGF-β-mediated decrease in effector cytokine production, and attenuates the TGF-β-mediated differentiation of CAR T cells armed with TGFβRIIDN into the T RM phenotype. Overall, these results demonstrate that the expression of dominant-negative TGFβRII is sufficient to inhibit TGF-β signal transduction and its biological effects.

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

[0115] Method ​T cells (20,000 CAR+ cells / well) were co-cultured for 5 days on xCELLigence eSight RTCA with squamous cell carcinoma cells engineered to express GPC3 (OE21 cells, 10,000 tumor cells per well), and real-time tumor cell viability was simultaneously monitored by electrical impedance and CAR T cell density observed by microscopy (see Figure 8A). Subsequently, T cells (60,000 CAR+ cells / well) were co-cultured for 5 days on xCELLigence RTCA-MP with Hep3B cells (moderate / low GPC3 expression) or Huh7 cells (low GPC3 expression) (30,000 tumor cells / well) (without microscopy) (see Figure 8B). Next, non-adherent cells were removed from the wells, and viable CAR T was quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) (see Figure 8C).

[0116] result Dramatic proliferation of CAR T cells was observed during co-culture with GPC3+ tumor cells. See Figure 8A. All CAR T cells efficiently killed GPC3+ tumor cells by TGFβRIIDN co-expression or by the addition of exogenous TGF-β, and their cytolytic capacity was not regulated. See Figure 8B. TGF-β suppressed tumor GPC3-induced proliferation of CAR T cells after 5 days of co-culture. However, CAR T cells armored with TGFβRIIDN were less affected by this suppression. See Figure 8C.

[0117] conclusion CAR T cells armored with TGFβRIIDN showed considerable GPC3+ tumor cytotoxicity. This indicates that dominant-negative receptor expression did not affect the ability of CAR T cells to kill target cells in vitro. Conversely, unlike non-armored CAR T cells, CAR T cells armored with TGFβRIIDN showed significant GPC3+ tumor cytotoxicity. + It was less susceptible to the TGF-β-mediated suppression of proliferation induced by [the relevant factor].

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

[0119] method Using a Huh7-TGF-β model of hepatocellular carcinoma that overexpresses TGF-β, the in vivo efficacy of T cells armored with TGFβRIIDN in reducing tumor volume was tested. Tumor cells were transplanted into the flanks of NSG mice (10 mice / group). The tumor size was 150 mm. 3 When the average volume reaches 3, 7, or 21 × 10 6 Tumors were measured every two weeks after administration of the indicated CAR T cells or 21 million untransduced T cells. Top graph: Mean tumor volume per group. Bottom graph: Tumor volume for each individual mouse at the indicated dose (see Figure 9).

[0120] Ex vivo analysis, 7 x 10 6 The procedure was performed on Huh7-TGFβ tumor-carrying mice administered with CAR T cells. Tumors and spleens were collected from 5 mice per group 7 or 14 days after injection. The number of CAR+ cells was calculated by flow cytometry after staining with AF647-labeled anti-idiotype antibody against GPC3-CAR and using AccuCheck Counting Beads (see Figures 10 and 11).

[0121] In mice carrying Huh-7-TGFβ tumors, 7 × 10 6 Individual CAR T cells were administered. Fourteen days after infusion, tumors were collected, and TGFβRII expression was evaluated by flow cytometry staining of CAR-positive and CAR-negative cells (see Figure 12).

[0122] In mice carrying Huh-7-TGFβ tumors, 7 × 10 6Individual CAR T cells were administered. Fourteen days after infusion, tumors were collected and the expression of PD1, LAG3, CD27, and CD70 was evaluated on the surface of the CAR T cells (see Figures 13 and 14).

[0123] In mice carrying Huh-7-TGFβ tumors, 7 × 10 6 Individual CAR T cells were administered. Fourteen days after infusion, the spleen was collected and the expression of PD1, LAG3, CD27, and CD70 was evaluated by flow cytometry staining (see Figure 15).

[0124] In mice carrying Huh-7-TGFβ tumors, 7 × 10 6 Each mouse was administered CAR T cells. For serum cytokine and AFP analysis, blood was collected in small volumes at the indicated time points, and serum was separated using a BD Microtainer Serum Separator Tube. Cytokine levels were determined using an MSD assay, while AFP was evaluated by sandwich ELISA. Blood was collected from 5 mice per group before tumor transplantation (baseline A), before CAR T infusion (baseline B), and 7 and 14 days after infusion (see Figures 16 and 17).

[0125] result Untransduced T cells had no discernible effect on tumor growth. Treatment with non-armored CAR T cells resulted in a minor reduction in tumor volume at the lowest dose (3 million cells / mouse), while higher doses demonstrated a significant effect leading to complete tumor regression. In contrast, administration of TGFβRIIDN-armored CAR T cells induced a significant reduction in tumor volume and complete regression at even lower doses. Furthermore, progression-free survival was significantly longer with TGFβRIIDN-armored CAR T cells at all treatment doses compared to non-armored CAR T cells (see Figure 9). Ex vivo analysis of CAR T cells was performed at 7 × 10⁶ 6The procedure was performed on mice injected with individual cells. In conjunction with enhanced efficacy, an increase in the number of tumor-infiltrating lymphocytes (TILs) was detected in TGFβRIIDN-treated mice 7 days after injection. This indicates a sustained active immune response and associated proliferation, which plateaued at 14 days post-treatment (see Figure 10). Conversely, an increase in the number of CAR T cells was observed 14 days after injection in the spleen of mice treated with CAR T cells armored with TGFβRIIDN. This suggests an increase in CAR T cells and further CAR T cells. +The enhanced proliferation by these cells therefore suggests that it was detectable in the circulatory system (see Figure 11). To investigate whether TGFβRIIDN is detectable by CAR T cells after in vivo proliferation, the inventors analyzed the expression of TGFβRII on TILs in the spleen and on lymphocytes 14 days after infusion. TGFβRII was barely detectable on non-armoring cells, while lymphocytes from mice treated with armored CAR T cells co-expressed CAR and TGFβRII. Therefore, it is reasonable to assume that TGFβRII expressed on TILs is a dominant-negative receptor that is detectable ex vivo after an active immune response and associated antigen-dependent proliferation (see Figure 12). Notably, TGFβRIIDN TILs expressed low levels of fatigue markers LAG3 and PD1 (see Figure 13), the latter of which is directly regulated by TGF-β in a SMAD3-dependent manner (Park, BV, Freeman, ZT, Ghasemzadeh, A., Chattergoon, MA, Rutebemberwa, A., Steigner, J. et al. (2016). TGFβ1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer. Cancer Discov, 6(12), 1366-1381). Furthermore, TGFβRIIDN TILs expressed less CD70 and more CD27 compared to non-armoring TILs (see Figure 14).These results are consistent with previous evidence that TGF-β upregulates CD70 expression, induces exhaustion of effector memory T cells, and reinforces the concept that dominant-negative receptor expression protects CAR T cells from TGF-β-mediated immunosuppression (Yang, ZZ., Grote, D., Xiu, B. et al. TGF-β upregulates CD70 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin's lymphoma. Leukemia 28, 1872-1884 (2014). https: / / doi.org / 10.1038 / leu.2014.84). In contrast to TILs, peripheral CAR T cells did not express co-suppressive markers or CD70 and were consistently nearly CD27-positive in the inactivation state (see Figure 15). In conjunction with enhanced efficacy and a greater number of TILs, increased levels of IFN-γ were detected in the serum of mice injected with TGFβRIIDN CAR T 7 days after injection, and a dramatic decrease in serum concentrations of the tumor marker AFP was detected 14 days after injection (see Figures 16 and 17).

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

[0127] conclusion CAR T cells armored with TGFβRIIDN show considerable promise as an effective in vivo treatment for GPC3+ tumors.

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

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

[0130] result Tumors proliferated in mice that received untransduced T cells. However, in the absence of TGF-β, both unarmored and armored cells were equally effective, inducing rapid and complete regression in all mice included in this study (see Figure 18). When injected into a TGF-β expression model, unarmored CAR T cells showed significantly lower efficacy. In contrast, CAR T cells armored with TGFβRIIDN were consistently more potent, inducing significant tumor regression.

[0131] conclusion The PDX model simulates human tumor biology that enables natural cancer progression. Therefore, these observations confirm and reinforce the evidence obtained from the Huh7-TGF-β xenograft model. Overall, these data suggest that GPC3 CAR T cells armored with TGFβRIIDN are GPC3 + It may be an effective in vivo treatment for tumors, and it has been demonstrated that it can maintain its effectiveness even in the presence of the immunosuppressive factor TGF-β.

[0132] The embodiments described herein may be implemented in the absence of any elements, sets of elements, limitations, or sets of limitations not specifically disclosed herein. These terms and expressions used are for illustrative purposes only and are not intended to be limitations, nor are they intended to exclude any equivalents of the shown and described features or parts thereof, and it should be recognized that various modifications are possible within the scope of the claimed embodiments. Therefore, although this description is specifically disclosed by embodiments, those skilled in the art should understand that any features, modifications, and variations of the concepts disclosed herein may be adopted, and such modifications and variations should be considered within the scope of the embodiments as defined by the description and the accompanying claims. While certain aspects of this disclosure may be identified as particularly advantageous herein, this disclosure is not intended to be limited to these specific aspects.

[0133] A claim or statement containing "or" between one or more members of a group is deemed satisfied if one, more, or all members of that group are present in, used in, or otherwise related to a given product or process, unless the opposite meaning is stated or the context makes a different interpretation obvious. This disclosure includes embodiments in which exactly one of the members of that group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which more or all members of that group are present in, used in, or otherwise related to a given product or process.

[0134] Furthermore, this disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms of one or more enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented, for example, as a list in Markush group form, each subgroup of elements is also disclosed, and any element may be excluded from that group.

[0135] In general, where the Disclosure or aspects thereof are referred to as including certain elements and / or features, it should be understood that certain embodiments or aspects of the Disclosure consist of or are essentially such elements and / or features. For brevity, those embodiments are not specifically described herein using these terms.

[0136] All patents and publications described herein are incorporated herein by reference as if each separate patent and publication were explicitly and individually indicated to be incorporated by reference. Any citation or confirmation of any reference in any section of this application should not be construed as an admission that such reference is available as prior art to the present invention.

[0137] Table 2

[0138] Table 3

[0139] Table 4

[0140] Table 5

[0141] Table 6

[0142] Table 7

[0143] Table 8

[0144] Table 9

[0145] Table 10

[0146] Table 11

[0147] Table 12

[0148]

Table 13

[0149]

Table 14

[0150]

Table 15

[0151]

Table 16

[0152] <110> MedImmune, LLC <120> COMPOSITIONS AND METHODS OF TREATING CANCER WITH CHIMERIC ANTIGEN RECEPTORS <130> PA26-007 <140> JP 2022-563890 <141> 2021-04-23 <150> US 63 / 014,831 <151> 2020-04-24 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 241 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="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 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 Arg 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 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 11 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 gcatgttatt ctctgctggt cacagtggct 900 ttcatcatct tctggtca gcgaggccgg aagaactgc tgtacatct caacagcct 960 tttatgcgcc cagtgcagac aactcaggag gaagacggct gctctgtcg gttccccgag 1020 gaagaggaag ggggatgtga gctgcgcgtg aagttttctc gaagtgccga tgctcctgca 1080 tatcagcagg gagaacca gctgtacac gagctgaatc tggggccggg gaggaatac 1140 gacgtgctgg atagaggcg cggcagagac ccagaatgg gcgggaagcc acgacggaaa 1200 aacccccagg aggggctgta taatgactg cagaaggaca aaatggccga ggcttacagc 1260 gaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggact gtatcagggc 1320 ctgagcactg ccaccagga cacctacgat gctctgcaca tgcaggcact gccaccagg 1380 made 1383 <210> 12 <211> 957 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="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 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 ttttaataat 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> <221> source <223> / note="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 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 ttttaataat 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 tttctcgca gtgcagatgc ccctgcttat 1080 cagcagggac agaatcagct gtacaacgag ctgaatctgg gcaggcgcga ggaatacgac 1140 gtgctggata agcgacgggg cagagacccc gaaatgggag ggaagcccag aaagaaaac 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> <221> source <223> / note="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 gcatgttatt 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> <221> source <223> / note="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 aaaaagctg 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 16 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc ccatcccgc 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 aacaaagctg 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> <221> source <223> / note="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 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 aacaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattcc ctgctggtca cagtggcctt catcatcttc 900 tgggtgcgga gcaagcggag ccggctgctg cactctgact acatgaacat gaccccccgg 960 agacccggcc ctacaagaaa gcattatcag ccttacgccc cacccaggga cttcgcagct 1020 tatcgctccc gagtgaaatt ttctcgcagt gcagatgccc ccgcttatca gcagggccag 1080 aatcagctgt acaacgagct gaatctgggg aggcgcgagg aatacgacgt 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> <221> source <223> / note="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 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 atgttattcc ctgctggtca ctgtggcctt catcatcttc 900 tgggtgcgga gcaagcggag ccggctgctg cactctgact acatgaacat gactccacgg 960 agacccggcc ctacccggaa acattatcag ccctacgccc cacccagaga ttttgccgct 1020 tataggtcca agcgcggccg aaagaaactg ctgtacatct tcaaacagcc 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> <221> source <223> / note="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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 21 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> <221> source <223> / note="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 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 aattttttgg 900 gtgaaaagag gtcggaaaaa gttgctgtat attttcaaac aaccctttat gagacctgta 960 caaacgactc aggaagagga tggttgtagt tgcaggtttc cggaggagga ggaaggtggg tgcgaactgc gggtgaatt taggc 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 <210> 23 <211> 1494 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="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 agggtgggg gggcagcggt 420 ggtggaggct ctggtggagg agggagccag gtccaactcg ttcaaagtgg cgcagaggtc 480 aaaaagccag gcgcgagcgt taaagtatca tgtaaggcca gcggttatac tttcactgat 540 tatgaaatgc actgggtgcg acaagcccccc 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 aattttttgg 900 gtgcggagca agcggagccg gctgctgcac tctgactaca tgaacatgac tccacggaga 960 cccggcccta cccggaaaca ttatcagccc tacgccccac ccagagattt tgccgcttat 1020 aggtccaaaa gaggtcggaa aaagttgctg tatattttca aacaaccctt 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 24 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctcagg tccagcttgt gcaaagcgga ggaggagtgg tacagcctgg ccgctctttg 120 agactgtctt gtgcggccag tggattaca 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 gggtggttg tagaacccct gagttgggga caaggtactc ttgtcacggt atcttctggc 420 ggaggtggga gtggtggggg tggcagtggc gggggtgggt cacaaagcgt gcttacacaa 480 cctccttctg cgagcggaac tccgggacaa cgggttacga tttcatgctc cggctcaagt 540 agcaatatag gatcaaatac agtgaattgg tatcaacaac tccctggcac agcgcccaag 600 ctgctgatct actctaataa ccagaggccg agtggtgtgc cagataggtt cagtggctct 660 aaatcaggta ctagcgcgag cctcgccatt tcaggacttc aatcagagga tgaagcggac 720 tactactgtg ccgcgtggga tgattcactt aatggatatg tttcgggac cggaacaaaa 780 ttgacggtat tggagagcaa atatggacca ccatgccctc catgtcctttt ttgggtcctg 840 gtggtcgtgg gaggcgtgct ggcatgttat tctctgctgg tcacagtggc tttcatcatc 900 ttctgggtca agcgaggccg gaagaaactg ctgtacatct tcaaacagcc tttatgcgc 960 ccagtgcaga caactcagga ggaacggc tgctcttgtc ggttccccga ggaagaggaa 1020 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> <221> source <223> / note="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 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> 26 <211> 1368 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <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 actcgcag actccgtgaa gggccggttc accatctcca gagacaattc caagacacg ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 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> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 27 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 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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> <221> source <223> / note="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 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> <221> source <223> / note="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 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> <221> source <223> / note="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 agcggtggtg gaggctctgg tggaggaggg agctcctatg agctgactca gccaccctca 420 gcgtctggga cccccgggca gagggtcacc atctcttgtt ctggaggcag ctccaacatc 480 ggaagtaata ctgtaaactg gttccggcag ctcccaggaa cggcccccaa actcctcgtt 540 tattttaata atcagcgacc ctcaggggtc cctgaccgat tctctggctc caagtctggc 600 acctcggcct ccctggccat cggtgggctc cagtctgacg atgaggctga ctattactgt 660 gtagcatggg atgactctct gaatgctccg gtgttcggcg gagggaccaa ggtcaccgtc 720 cta 723 <210> 34 <211> 723 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 34 gaggtccagc tgctggagag cggaggagga ctggtgcagc ctggaggaag tctgcgactg 60 tcatgcgccg ctagcggctt caccttcagc tcctatgcaa tgagctgggt gcgacaggca 120 ccaggcaagg ggctggagtg ggtctccgct atctccggct ctggaggctc tacttactat 180 gcagacagtg tgaaggggcg gttcacaatc tccagagata actctaagaa cactctgtac 240 ctgcagatga actctctgag agctgaggac accgcagtgt actattgcgc caagggcaaa 300 aggtactttg attattgggg acagggcact atggtgaccg tctctagtgg aggaggagga 360 agcggaggag gaggatccgg cggaggaggc agtcagtcag tgctgacaca gccacctagc 420 gcctccggaa ccccaggaca gcgggtcaca atctcttgta gtgggggatc aagcgacatt 480 gggagcaaca ccgtgaattg gtatcagcag ctgcctggaa cagctccaaa gctgctgatc 540 tactataaca atcagaggcc ctccggcgtc cctgatcgct tctcaggcag caaatccggg 600 acttctgcaa gtctggccat tagtggcctg cagtcagagg acgaagccga ttactattgt 660 gctacctggg acgataggat gtactctccc gtgttcggcg ggggaacaaa gctgactgtc 720 ctg 723 <210> 35 <211> 726 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 35 gatgtcgtga tgacgcagag ccctctctct cttcccgtta cccctggtga acccgcatca 60 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> <221> source <223> / note="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 300 360. tttgtagaac ccctgagttg gggacaaggt actcttgtca cggtatcttc tggcggaggt gggagtggtg ggggtggcag tggcgggggt gggtcacaaa gcgtgcttac acaacctcct 420 tctgcgagcg gaactccggg acaacggggtt acgatttcat gctccggctc aagtagcaat attack attack attack ttggtat caactccctg gcacagcgcc caagctgctg atctactcta ataaccagag gccgagtggt gtgccagata ggttcagtgg ctctaatca ggtactagcg cgagcctcgc catttcagga cttcaatcag aggatgaagc ggactactac tgtgccgcgt gggatgattc acttaatgga tatgttttcg ggaccggac aaaattgacg 720 gtattg 726 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Gly Lys Arg Tyr Phe Asp Tyr 1 5 <210> 40 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Phe Asn Asn Gln Arg Pro Ser 1 5 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Ser Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Tyr Asn Asn Gln Arg Pro Ser 1 5 <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <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 aggcaaata tggaccacca tgccctccat gtccttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctgtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaactgctg tacatctca aacagcctttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tctgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaatgggc gggaagccac gacggaaaaa cccccaggag 1200 gggctgtata atgactgca gaaggacaa atggccgagg cttacagcga atcgggatg 1260 aagggagaga gaaggcgcgg aaaaggccac gatggactgt atcaggggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacchagggg 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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 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> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> SITE <222> (1)..(15) <223> / note="This sequence may encompass 1-3 'Gly Gly Gly Gly Ser' repeating units" <220> <221> source <223> / note="See specification as filed for detailed description of substitutions and preferred embodiments" <400> 48 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15

Claims

1. a) Chimeric antigen receptors (CARs) containing an antigen-binding domain specific to cell surface antigens; and b) Armoring molecules that, when expressed on the surface of cells in the tumor microenvironment, counteract the immunosuppression of said cells. An isolated nucleic acid sequence that codes for something.

2. The isolated nucleic acid sequence according to claim 1, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

3. The isolated nucleic acid sequence according to claim 2, wherein the antigen-binding domain is a Fab or a single-chain variable fragment (scFv).

4. The isolated nucleic acid sequence according to claim 3, wherein the antigen-binding domain is an scFv containing the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO:

34.

5. An isolated nucleic acid sequence according to any one of claims 1 to 4, further comprising a transmembrane domain, a costimulatory domain, and a signaling domain.

6. The isolated nucleic acid sequence according to claim 5, wherein the transmembrane domain includes a CD28 transmembrane domain.

7. The isolated nucleic acid sequence according to claim 5, wherein the co-stimulatory domain comprises one or more of the following: CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 co-stimulatory domains.

8. The isolated nucleic acid sequence according to claim 5, wherein the aforementioned co-stimulatory domain comprises one or more CD28, 4-1BB, and CD3 zeta co-stimulatory domains.

9. The isolated nucleic acid sequence according to claim 5, wherein the signal domain includes a sequence encoding a CSFR2 signal peptide.

10. An isolated nucleic acid sequence according to any one of claims 1 to 9, further comprising a hinge / spacer domain.

11. The isolated nucleic acid sequence according to claim 10, wherein the hinge / spacer domain is an IgG4P hinge / spacer.

12. The isolated nucleic acid sequence according to claim 1, wherein the armoring molecule is a TGF-β receptor type 2 dominant-negative (TGFβRIIDN).

13. The isolated nucleic acid sequence according to claim 1, encoded by the nucleic acid sequence of sequence number 46.

14. The cell surface antigens are 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, Igκ chain, LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STE The isolated nucleic acid according to claim 1, comprising one or more of AP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesoserine (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA.

15. A vector comprising a chimeric antigen receptor (CAR) containing an antigen-binding domain specific to a cell surface antigen, and a nucleic acid sequence encoding an armoring molecule, wherein the nucleic acid sequence includes SEQ ID NO:

46.

16. A cell comprising the vector according to claim 15 or the isolated nucleic acid according to any one of claims 1 to 14.

17. Nucleic acid sequences encoding chimeric antigen receptors (CARs), and TGFβRIDN armoring molecules expressed on the surface of cells Cells containing this substance.

18. The cell according to claim 17, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain.

19. The cell according to claim 18, wherein the antigen-binding domain is Fab or a single-chain variable fragment (scFv).

20. An anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned VH includes CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO:

39. The VL comprises an anti-GPC3 chimeric antigen receptor containing CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; and TGFβRIDN Armoring Molecules Cells containing this substance.

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

29.

22. The cell according to claim 20, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

23. The cell according to any one of claims 20 to 22, wherein the anti-GPC3 chimeric antigen receptor (CAR) and TGFβRIDN armoring molecule comprises the amino acid sequence of SEQ ID NO:

47.

24. The cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells, as described in any one of claims 16 to 23.

25. A method of treating cancer: This includes administering cells to a subject requiring treatment for cancer, wherein the cells are a) Chimeric antigen receptors (CARs) specific to cell surface antigens, and b) Armoring molecules that counteract the immunosuppression of cells in the tumor microenvironment of the cancer. Methods that include...

26. The cell surface antigens are 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, Igκ chain , LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, ST The method according to claim 25, wherein one or more of EAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesoserine (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA.

27. The method according to claim 26, wherein the armoring molecule is a TGFβRIDN armoring molecule.

28. The method according to claim 27, further comprising inhibiting tumor growth, inducing tumor regression, and / or extending survival time.

29. The method according to claim 31, wherein the cells are autologous cells.

30. The method according to claim 29, wherein the self-cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

31. The method according to any one of claims 25 to 30, wherein the cancer is a solid tumor.

32. The method according to claim 31, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

33. The method according to claim 32, wherein the cancer is hepatocellular carcinoma.

34. The method according to any one of claims 26 to 33, further comprising administering a therapeutically effective amount of an anti-cancer antibody and / or a chemotherapy component to the subject.

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