Compositions and methods for treating cancer using chimeric antigen receptors

Abstract

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

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on June 7, 2021, is named CARTGPC(HIF)-110-WO-PCT_SL.txt, and is 97,462 bytes in size. Background of the Invention Technical Field

[0004] The present disclosure relates to the use of chimeric antigen receptor T cells to treat cancer. Background Art

[0005] 1. Chimeric antigen receptor T cell therapy

[0006] Chimeric antigen receptor (CAR) T cell therapy is a specific form of cell-based immunotherapy that uses engineered T cells to fight cancer. In CAR T cell therapy, T cells are harvested from the patient's blood, engineered in vitro to express CAR containing an antigen binding domain and a T cell activation domain, expanded to a larger population and administered to the patient. CAR T cells are used as living drugs that bind to cancer cells and cause the destruction of these cancer cells. When successful, the effects of CAR T cell therapy tend to last for a long time, as demonstrated by detecting the persistence and amplification of CAR T cells in patients for a long time after clinical remission.

[0007] 2. CAR structure and function

[0008] The antigen binding domain of CAR is the extracellular region of the surface antigen on the target tumor cell.Suitable target antigen can be protein, phosphorylated protein, peptide-MHC, carbohydrate or glycolipid molecule.The ideal target antigen is widely expressed on tumor cells to enable targeting of a high percentage of cancer cells.The ideal candidate target antigen is also minimally expressed on normal tissue, thereby limiting the on-target toxicity outside the tumor.The antigen binding domain of CAR includes a targeting portion for the target antigen, such as an antibody single-chain variable fragment (scFv).

[0009] The T cell activation domain of the CAR is intracellular and activates T cells in response to the antigen binding domain that interacts with its target antigen. The T cell activation domain can contain one or more costimulatory domains, which are intracellular domains known to activate T cell receptors. Because costimulatory domains have different effects on CAR T cell dynamics, cytotoxic function, and safety characteristics, the choice and location of the costimulatory domain within the CAR construct can affect the function and fate of the CAR T cell.

[0010] The extracellular antigen binding domain and the intracellular T cell activation domain of CAR are connected by a transmembrane domain, a hinge, and optionally a spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to promote binding to the target antigen on the tumor cell. It can be used alone or in combination with a spacer domain that designs scFv away from the T cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface.

[0011] CAR T therapy targeting B lymphocyte antigen CD19 ( Novartis) has shown promise in pediatric acute lymphoblastic leukemia, and a CAR T therapy targeting a B-cell maturation antigen ("bb2121," and Collaboration) shows promise for relapsed / refractory multiple myeloma. Recent data suggest that CAR approaches may be effective for solid tumors. GD2 CAR natural killer T cell (NKT) therapy has shown activity in neuroblastoma (Heczey A et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood; 124(18): 2824-33, 2014), and mesothelin CAR T with pembrolizumab has been shown to have anti-tumor activity in mesothelioma. However, additional targets for the treatment of solid tumors are needed.

[0012] 3. Challenges of CAR T-cell therapy

[0013] Unfortunately, the complexity of CAR T cell-based therapies may lead to undesirable and unsafe effects. Toxic effects, such as neurotoxicity and acute respiratory distress syndrome, are potential adverse effects of CAR T cell therapy and may be fatal. Cytokine release syndrome (CRS) is the most common acute toxicity associated with CART cells. When lymphocytes are highly activated and release excessive inflammatory cytokines, CRS occurs. When these factors are measured, serum levels of interleukin 2, interleukin 6, interleukin 1β, GM-CSF and / or C-reactive protein are sometimes observed to increase in patients with CRS. CRS is graded and diagnosed as one of grades 1-4 (mild to severe) according to severity, with the clinical features of more severe cases being high fever, hypotension, hypoxia and / or multi-organ toxicity in patients. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR-T cell therapy experienced CRS, and 50% of these patients had grade 3-4 symptoms (Fitzgerald et al., Crit Care Med. 45(2):e124-e131 (2017)).

[0014] Another challenge for successful CAR T cell immunotherapy is the immunosuppression caused by the characteristics of the tumor microenvironment (TME) of solid tumors. Due to limited nutrient supply, the tumor microenvironment (TME) of solid tumors can be metabolically unfavorable to CAR T cells. This disadvantage leads to nutrient competition, loss of T cell metabolic adaptability and reduced oxygen content (i.e., "hypoxia"). Hypoxia reduces T cell activation and proliferation, and reduces cytokine and lytic enzyme production. The key role in these effects is hypoxia-inducible factor-1α (HIF-1α), which is the α subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1α is the main regulator of the cellular and systemic homeostatic response to hypoxia, which makes it a key factor in oxygen homeostasis. HIF-1α also controls genes involved in energy metabolism, angiogenesis, apoptosis, and promotes the transcription of other genes that adapt to the metabolism of hypoxia. HIF-1α also plays an important role in T cell activation. It causes T cells to exhibit a complete effector phenotype, including cytokine and lytic granule production, high glycolysis, high metabolic activity and high reactive oxygen species (ROS) production. Such a phenotype results in a reduction in the number of effector T cells at the tumor site, a reduction in the maintenance of effector characteristics, and low persistence. It has been shown that the low effector phenotype results in an increased number of effector cells with increased activity and greater persistence at the tumor site (Kishton, RJ, Sukumar, M., and Restifo, NP (2017). Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy [Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy]. Cell Metab [Cell Metabolism], 26 (1), 94-109). Therefore, HIF-1α-mediated immunosuppression is also an important obstacle that must be overcome in order to obtain effective and durable CAR T cell therapy for solid tumors.

[0015] 4. Armor

[0016] The latest approach to making CAR T cells more resistant to tumor-associated immunosuppression is called “armoring”. Armoring is the molecular manipulation of CAR T cells to express one or more “armor molecules” that can resist immunosuppression. For example, the expression of dominant negative HIF-1α (HIF1αDN) armor molecules in pancreatic cancer cells that are resistant to apoptosis induced by hypoxia and glucose deprivation caused by HIF-1α makes the cells sensitive to apoptosis and growth inhibition induced by hypoxia and glucose deprivation. (Che et al., Dominant-Negative Hypoxia-Inducible Factor-1αReduces Tumorigenicityof Pancreatic Cancer Cells through the Suppression of Glucose Metabolism [Dominant negative hypoxia-inducible factor-1α reduces the tumorigenicity of pancreatic cancer cells by inhibiting glucose metabolism], Am J Pathol [American Journal of Pathology], 162 (4), 1283-1291 (2003)).

[0017] Therefore, additional CAR T cell therapies are needed to enhance the armamentarium of effective cancer treatments. Such therapies should include CAR T cells that effectively treat cancer while minimizing the risk of developing dangerous inflammatory reactions (such as CRS). Furthermore, such therapies should include CAR T cells that can persist in the immunosuppressive TME of solid tumors. Summary of the Invention

[0018] The present disclosure describes compositions and methods for treating cancer using CAR T cells. As described below, in a first aspect, an isolated nucleic acid sequence encoding (a) a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for a cell surface antigen; and (b) an armor molecule, wherein the armor molecule resists immunosuppression of the cell when expressed on the surface of a cell in a tumor microenvironment.

[0019] In another aspect, the disclosure describes a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a HIF1α DN armor molecule expressed on the cell surface.

[0020] In another aspect, the disclosure describes a cell comprising: an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; and a HIF1α DN armor molecule.

[0021] In yet another aspect, the present disclosure describes a method of treating cancer, comprising administering to a subject in need thereof a cell, wherein the cell comprises (a) a chimeric antigen receptor (CAR) specific for a cell surface antigen, and (b) an armored molecule, wherein the armored molecule resists immunosuppression against the cell in the tumor microenvironment of the cancer.

[0022] These and other features and advantages of the present invention will be more fully understood from the following detailed description and appended claims.It should be noted that the scope of the claims is defined by the recitation therein rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure. The drawings illustrate one or more embodiments of the present disclosure and together with the description serve to explain the principles and operations of the disclosure.

[0024] Figure 1 Armoring of GPC3 CAR-T with HIF1α DN. Schematic diagram of the HIF1a DN construct.

[0025] Figure 2 A-2C. HIF1α DN is overexpressed relative to endogenous HIF1α.

[0026] Untransduced (UT) cells, unarmored GPC3 CART cells, and GPC3 HIF1α DN CAR T cells were amplified under normoxia (20% O2) or hypoxia (1% O2). 2A. The graph shows the expression of endogenous HIF1α mRNA for each sample, expressed as a fold induction relative to UT amplified under (20% O2). 2B. Endogenous HIF1α protein expression in untransduced, unarmored GPC3 CAR T cells and GPC3 HIF1α DN CAR T cells under normoxia (20% O2) and hypoxia (1% O2). β-actin was used as a loading control. 2C. Fold induction of HIF1α DN relative to endogenous HIF1α mRNA expression in untransduced (UT), GPC3 CAR T cells, and GPC3 HIF1α DN CAR T cells amplified under normoxia (20% O2) and hypoxia (1% O2).

[0027] Figure 3 CAR-T cells expressing HIF1αDN showed a low degree of differentiation. Differentiation of untransduced (UT), GPC3, and GPC3 HIF1αDN CAR T cells under normoxic or hypoxic conditions was analyzed by flow cytometry. The bar graph represents CD62L expression assessed by flow cytometry. 低 CD45RO 高 Effector memory T cells (T EM ) frequency.

[0028] Figure 4 Nanostring analysis of HIF1αDN CAR T cell stemness. Expression of genes associated with stemness (upper arrows) and effector function (lower arrows). Genes are expressed as GPC3 HIF1αDN relative to GPC3.

[0029] Figure 5GPC3-CAR-modified T cells expressing dominant-negative HIF1a exhibit enhanced oxidative phosphorylation after acute activation. (A) The upper panel shows the OCR of GPC3-CAR-modified and dominant-negative HIF1a GPC3-CAR-modified T cells under basal culture conditions and in response to activated CD3 / CD28-coated beads and oligomycin. The lower panel shows the basal OCR of resting T cells and the OCR gain after acute activation. Data are shown as mean ± SD. (B) The upper panel shows the ECAR of GPC3-CAR-modified and dominant-negative HIF1a GPC3-CAR-modified T cells under basal culture conditions and in response to activated CD3 / CD28-coated beads and exogenous glucose. The lower panel shows the basal ECAR of resting T cells and the ECAR gain after acute activation. Data are shown as mean ± SD. (**p < .01, ***p < .001; Wilcoxon matched pairs signed rank test).

[0030] Figure 6 .HIF1αDN CAR T cells degranulate after antigen exposure. After CAR-T cells were expanded under normoxic (left) and hypoxic (right) culture conditions, they were cultured in Hep3B (GPC3 + ) or SNU182(GPC3 - Flow cytometric analysis of CD107a expression (a marker of degranulation and toxicity) on UT, GPC3, and GPC3 HIF1α DN CAR T cells was performed in the presence of 1% (1%) cells. CAR-T cells degranulate in response to antigen exposure in vitro, even when expanded under hypoxia. HIF1α DN CAR T cells degranulate efficiently even when quiescent and poorly activated and differentiated.

[0031] Figure 7 HIF1α DN CAR T cells produced less IFNγ and IL-2 but similar TNFα after antigen exposure. Concentrations of IFNγ, IL-2, and TNFα in the culture supernatants of UT, GPC3, and GPC3HIF1α DN CAR T cells expanded under normoxic or hypoxic conditions 24 hours after antigen (GPC3) exposure under normoxic conditions.

[0032] Figure 8 Ectopic dominant negative HIF1a generates polyfunctional T cells after stimulation under hypoxic conditions. A: Representative flow cytometry staining of IFN-γ, IL-2, and TNF-α intracellular cytokines in GPC3-CAR-modified and dominant negative HIF1a GPC3-CAR-modified T cells after 6 hours of stimulation with PMA and ionomycin. B: Depicts CD4 +and CD8 + Bar graph showing the quality of T cell cytokine responses, as measured by the recognition of IFN-γ + IL-2 + and TNF-α + The gates of cells are determined by a Boolean combination. The numbers on the Y-axis represent the percentage of cells.

[0033] Figure 9 HIF1α DN CAR T cells rapidly and efficiently kill target cells. Targeting and killing of Hep3B target cells by untransduced (UT), GPC3, and GPC3 HIF1α DN CAR T cells by cytolysis were measured under normoxic and hypoxic conditions. Real-time impedance killing assay (RTCA) measures the % cell lysis of Hep3B cells over time. UT cells had minimal cytolysis of target cells, while both GPC3 and HIF1αDN CAR T cells had significant cytolysis. Unexpectedly, under normoxic and hypoxic conditions, HIF1αDN CAR T cells exhibited faster target cell killing compared to unarmored CAR T cells. These surprising results indicate that armoring CAR T cells with HIF1αDN provides CAR T cells with additional advantages beyond hypoxia resistance and suggest that HIF1αDN can be used to improve the efficiency of other CAR T cells regardless of the CAR target antigen.

[0034] Figure 10 The rate and efficiency of target cell cytolysis according to the E:T ratio under normoxic and hypoxic conditions. The cytolytic capacity of UT, GPC3, and GPC3 HIF1α DN CAR T cells against Hep3B cells expanded under normoxic (left) and hypoxic (right) conditions at different effector to target ratios (E:T). The real-time impedance killing assay (RTCA) measures the values ​​and expresses them as KT80 (time required to kill 80% of the target).

[0035] Figure 11 Visualization of Hep3B target cell cytolysis. Micrographs of Hep3B target cell cytolysis by UT, GPC3, and GPC3 HIF1α DN CAR T cells 3 hours after cell introduction under normoxic conditions (lower panels). The upper panels show T cells in the absence of target cells.

[0036] Figure 12Cytolysis of target cells. Cytolysis of HUH7 and PLC-PRF15 target cells by UT, GPC3, and GPC3 HIF1α DN CAR T cells under normoxic conditions with an E:T ratio of 1:1. GPC3 and GPC3 HIF1α DN CAR T cells exhibited similar rates of cytolysis against each cell type.

[0037] Figure 13 Cell proliferation in the presence of target antigen. Antigen-dependent cell proliferation measured by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

[0038] Figure 14 Expression of HIF1αDN enhances CAR-T efficacy in vivo (HUH7). NSG mice were subcutaneously inoculated with HUH7 cells and the tumor volume reached approximately 150 mm. 3 When 7×10 6 UT, GPC3, or GPC3 HIF1α DNCAR T cells were added. Tumor volume was measured twice a week.

[0039] Figure 15 .HIF1αDN expression enhances CAR-T efficacy in vivo (Hep3B). NSG mice were subcutaneously inoculated with Hep3B cells, and when the tumor volume reached approximately 150 mm 3 When 7×10 6 UT, GPC3, or GPC3 HIF1α DN CART cells were used. Tumor volume was measured twice a week.

[0040] Figure 16 The expression of HIF1αDN increases the ability of CAR-T to infiltrate solid tumors. NSG mice were subcutaneously inoculated with Hep3B cells, and when the tumor volume reached approximately 175 mm 3 When 7×10 6 The frequency and number of T cells in the tumor were assessed by flow cytometry 7 days after infusion.

[0041] Figure 17 High levels of IFNγ in the serum of mice treated with HIF1α DN CAR-T cells. IFN-γ concentrations detected in the serum of mice bearing Hep3B tumors and infused with untransduced T cells, unarmored, or armored GPC3 CAR-T cells.

[0042] Figure 18HIF1αDN CAR-T cells maintain a less differentiated phenotype in the periphery. CD70 in the spleens of mice bearing Hep3B tumors and infused with untransduced T cells, unarmored, or armored GPC3 CAR-T cells + CD27 - The frequency of CAR-T cells. DETAILED DESCRIPTION

[0043] 1. Definition

[0044] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide a general definition of a plurality of terms used in the present invention for technical personnel: Singleton et al., Dictionary of Microbiology and Molecular Biology [microbiology and molecular biology dictionary] (2nd edition 1994); The Cambridge Dictionary of Science and Technology [Cambridge dictionary of science and technology] (Walker writes, 1988); The Glossary of Genetics [genetics vocabulary], 5th edition, R. Rieger et al. (eds.), Springer Verlag (Springer Verlag) (1991); and Hale and Marham, The Harper Collins Dictionary of Biology [Harper Collins biological dictionary] (1991). Unless otherwise indicated, the following terms as used herein have the following meanings given to them.

[0045] As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises / comprising," "includes / including") should be understood to imply the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning.

[0046] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] The percentages disclosed herein may vary from the disclosed values ​​by an amount of ±10%, 20%, or 30% and still be within the intended disclosed range.

[0048] Unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, the values ​​herein expressed as ranges in the various embodiments of the present disclosure may take any specific value or sub-range within the stated range, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0049] As used herein, ranges and amounts may be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%" and also refers to "5%." The term "about" can also refer to ±10% of a given value or range of values. Thus, for example, about 5% also refers to 4.5%-5.5%. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about."

[0050] As used herein, the terms "or" and "and / or" can describe multiple components that are combined or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z".

[0051] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may replace or be used interchangeably with any of these terms.

[0052] As used herein, "protein" may refer to a single polypeptide, ie, a single amino acid chain as defined above, but may also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to create a multimeric protein.

[0053] "Isolated" material, such as an isolated nucleic acid, is material that is not in its natural environment, although the isolated material is not necessarily purified. For example, an isolated nucleic acid is a nucleic acid that is not produced or located in its natural or native environment (e.g., a cell). The isolated material can be separated, fractionated, or at least partially purified by any suitable technique.

[0054] As used herein, the terms "antibody" and "antigen-binding fragment thereof" refer to at least the smallest portion of an antibody that is capable of binding to a specified antigen targeted by the antibody, such as at least some of the complementarity determining regions (CDRs) of the variable domains of the heavy chain (VH) and the light chain (VL) in the context of a typical antibody produced by a B cell. The antibody or its antigen-binding fragment can be or be derived from a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody, a single-chain antibody, an epitope-binding fragment, such as Fab, Fab' and F(ab')2, Fd, Fv, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (sdFv), a fragment comprising a VL or VH domain (e.g., an entire VL domain and a partial VH domain with one, two or three CDRs) alone or in combination with a portion of an opposite domain, and a fragment produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Antibody molecules encompassed by the present disclosure can be of or derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0055] As used herein, the term "polynucleotide" includes single nucleic acids as well as multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any nucleic acid type, such as DNA or RNA.

[0056] As used herein, the term "vector" may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. Specific types of vectors contemplated herein may be associated with or incorporated into viruses to promote cellular transformation.

[0057] A "transformed" cell or "host" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. All techniques for introducing a nucleic acid molecule into such a cell are contemplated herein, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0058] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (e.g., an epitope) to its cognate binding domain (e.g., a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a population of epitopes and paratopes (i.e., antigens and antigen-binding domains).

[0059] As used herein, the terms "treat," "treatment," and "treatment of" when used in the context of treating cancer refer to alleviating disease pathology, alleviating or eliminating disease symptoms, promoting improved survival, and / or alleviating discomfort. For example, treatment can refer to the ability of a therapy to reduce disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or disorder.

[0060] As used herein, the term "subject," "individual," or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

[0061] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic substance (such as CAR T cells) is an amount sufficient to carry out a particular stated or intended purpose, such as treating cancer. An "effective amount" can be determined empirically in a routine manner according to the stated purpose.

[0062] 2. Overview

[0063] The present disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More particularly, the present disclosure relates to CAR cell therapy, in which transformed cells (such as T cells) express, for example, CAR targeting glypican-3 (GPC3). CAR constructs disclosed herein, cells expressing the transformation of these constructs, and the therapy utilizing these transformed cells can provide robust cancer treatment with cytokine release syndrome (CRS) or the risk of minimizing the release of indiscriminate cytokines in non-GPC3 expressing cells.

[0064] Without wishing to be bound by theory, GPC3 is considered to be a viable cancer target in a variety of forms, including bispecific T cell engagers, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). GPC3 is a carcinoembryonic antigen and a GPI-linked heparin sulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction, thereby stimulating Wnt signaling. GPC3 competes with Patched for Hh binding, which relieves Smoothened inhibition and induces GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) growth. Furthermore, GPC3 expression levels have been shown to correlate with the stage and grade of HCC.

[0065] Furthermore, GPC3 is believed to be a promising target for CAR cell therapy. Accordingly, antibodies and CAR constructs derived from these antibodies have been developed as described herein.

[0066] Additional aspects of the disclosure include CAR T cells, such as those targeting GPC3 and others armored with HIF1α DN to protect CAR T cells from immunosuppression associated with, for example, the hypoxic tumor microenvironment (TME) of solid tumors.

[0067] 3. CAR Construct Design

[0068] The CAR construct of the present disclosure may have several components, many of which may be selected based on the desired or precise function of the resulting CAR construct. In addition to the antigen binding domain, the CAR construct may also have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. Selecting a component rather than another (i.e., selecting a specific costimulatory domain from a receptor, relative to a costimulatory domain from different receptors) may affect clinical efficacy and safety characteristics.

[0069] 4. Antigen Binding Domain

[0070] Antigen binding domains contemplated herein may include antibodies or one or more antigen binding fragments thereof. A contemplated CAR construct targeting GPC3 comprises a single-chain variable fragment (scFv) containing light and heavy chain variable regions from one or more antibodies specific for GPC3, these variable regions being linked together directly or via a flexible linker (e.g., a repeat sequence of GGGGS with 1, 2, 3 or more repeats (SEQ ID NO: 48)).

[0071] As disclosed herein, the binding affinity of the antigen binding domain of CAR to the target protein can vary. Compared with antibodies (generally these antibodies are expected to have higher affinity), in the context of CAR, the relationship between binding affinity and therapeutic effect may be more subtle. For example, when compared with low-affinity variants, preclinical studies of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CAR derived from high-affinity scFv (with a dissociation constant of 0.56nM) lead to an increase in therapeutic index. On the contrary, other examples have been reported, wherein, engineered scFv improves the distinction between cells with different antigen densities for lower affinity. This can be used to improve the therapeutic specificity of differentially expressed antigens in tumor tissue and normal tissue.

[0072] A variety of methods can be used to determine the binding affinity of an antigen binding domain. In some embodiments, a method that excludes avidity effects can be used. Avidity effects involve multiple antigen binding sites that interact with multiple target epitopes simultaneously, typically involving multimeric structures. Therefore, avidity functionally represents the cumulative strength of multiple interactions. An example of a method that excludes avidity effects is any method in which one or both of the interacting proteins are monomeric / monovalent, because if one or both partners only contain a single interaction site, multiple simultaneous interactions are impossible.

[0073] 5. Spacer domain

[0074] The CAR construct of the present disclosure may have a spacer domain to provide conformational freedom, thereby promoting binding to the target antigen on the target cell. The optimal length of the spacer domain may depend on the proximity of the binding epitope to the target cell surface. For example, a proximal epitope may require a longer spacer, while a distal epitope may require a shorter spacer. In addition to promoting the binding of CAR to the target antigen, achieving the optimal distance between CAR cells and cancer cells can also help to spatially block the entry of large inhibitory molecules into the immune synapse formed between the CAR cells and the target cancer cells. CAR may have a long spacer, a medium spacer or a shorter spacer. The long spacer may include the CH2CH3 domain (about 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural, or with common modifications in therapeutic antibodies, such as S228P mutations), and the CH3 region can be used alone to construct a medium spacer (about 120 amino acids). The shorter spacer may be derived from a segment (<60 amino acids) of CD28, CD8α, CD3 or CD4. The short spacer may also be derived from the hinge region of the IgG molecule. These hinge regions may be derived from any IgG isotype and may or may not contain mutations commonly found in therapeutic antibodies, such as the S228P mutation mentioned above.

[0075] 6. Hinge domain

[0076] CAR may also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to a target antigen on a tumor cell. It can be used alone or in combination with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably - for example, an IgG4 sequence can be considered a "hinge" sequence and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0077] CAR may further include a sequence comprising a signal peptide. The function of the signal peptide is to promote cells to transfer CAR to the cell membrane. Examples include IgG1 heavy chain signal peptide, Igκ or λ light chain signal peptide, granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal peptide, or CD33 signal peptide.

[0078] 7. Transmembrane domain

[0079] The CAR may further include a sequence comprising a transmembrane domain. The transmembrane domain may include a hydrophobic alpha helix that spans the cell membrane. The properties of the transmembrane domain have not been studied as carefully as other aspects of the CAR construct, but it may potentially affect CAR expression and association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0080] 8.Co-stimulatory domain

[0081] CAR may further include one or more sequences forming a co-stimulatory domain.Co-stimulatory domain is a domain that can enhance or regulate the response of immune effector cells.Co-stimulatory domain may include, for example, one or more sequences from CD3ζ (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ and MyD88 / CD40. The selection of co-stimulatory domain affects the phenotype and metabolic characteristics of CAR cells. For example, CD28 co-stimulation produces an effective but transient effector-like phenotype with high levels of cytolytic ability, interleukin 2 (IL-2) secretion and glycolysis. In contrast, T cells modified with CAR carrying 4-1BB co-stimulatory domains tend to expand and last longer in vivo, have increased oxidative metabolism, are not easily exhausted, and have the ability to produce increased central memory T cells.

[0082] 9. Cells

[0083] CAR-based cell therapy can be used with a variety of cell types (such as lymphocytes). Available specific cell types include T cells, natural killer (NK) cells, natural killer T (NKT) cells, constant natural killer T (iNKT) cells, αβT cells, γδT cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg). In one embodiment, the CAR cells for treating the subject are autologous. In other embodiments, the CAR cells can be from genetically similar but not identical donors (allogeneics).

[0084] 10. CAR cell production

[0085] The CAR constructs of the present disclosure may include some combinations of the modular components described herein. For example, in some embodiments of the present disclosure, the CAR construct comprises a GPC3 scFv antigen binding domain. In some embodiments, the CAR comprises a GPC3-2 scFv antigen binding domain. In some embodiments of the present disclosure, the CAR construct comprises a CSFR2 signal peptide. In some embodiments, the CAR construct comprises an IgG4P hinge / spacer domain carrying an S228P mutation. In some embodiments, the CAR construct comprises a CD28 transmembrane domain.

[0086] The different costimulatory domains that can be used are the CAR constructs of the present disclosure. In certain embodiments, the CAR construct includes the costimulatory domain from the intracellular domain of CD3z. In certain embodiments, the CAR construct includes the CD28 costimulatory domain. In certain embodiments, the CAR construct includes the 4-1BB costimulatory domain. In certain embodiments, the CAR construct includes the costimulatory domain from CD3z and CD28. In certain embodiments, the CAR construct includes the costimulatory domain from CD3z and 4-1BB. In certain embodiments, the CAR construct includes the costimulatory domain from all CD3z, CD28 and 4-1BB. In certain embodiments, the CAR construct includes the costimulatory domain from ICOS, OX-40 and / or GITR.

[0087] 11.CAR Construct Evaluation

[0088] Based on the establishment of safety and persistence and central memory, the construct of this disclosure is compared and evaluated.Due to its improved safety, the scFv, GPC3 of lower affinity (high dissociation rate) are advantageously assessed.Based on the contribution of its improved persistence and favorable in vivo phenotype (more central memory), 4-1BB and CD3z costimulatory domains (both in same construct) are advantageously assessed.

[0089] 12. CAR Examples

[0090] In some embodiments, the present disclosure provides a kind of isolated nucleic acid sequence, the isolated nucleic acid sequence encoding chimeric antigen receptor (CAR), the chimeric antigen receptor comprising a surface antigen on a tumor cell with specific antigen binding domains.In some embodiments, the cell surface antigen is a protein, a phosphorylated protein, a peptide-MHC, a carbohydrate or a glycolipid molecule.

[0091] Examples of contemplated cell surface antigens include CD10, CD16, CD19, CD20, CD22, CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133, CD138, CD171, CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3, CDH3, CDH6, CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1, GPA33, GPC3, IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1 / EGFR, ERBB2 / Her-2, ERBB3, ERBB4, cMet, EGFRvIII, F AP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV, HPV16-E6, HPV16-E7, IL13Ra2, Ig ​​kappa chain, LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA. Other cell surface antigens are also contemplated herein.

[0092] In some embodiments, the present disclosure provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for glypican 3 (GPC3). The antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D), and the CAR construct does not induce the production of cytokines in GPC3- cells. In some embodiments, the antigen binding domain comprises an antibody or an antigen binding fragment thereof. The antigen binding domain can be a Fab or a single-chain variable fragment (scFv). In some embodiments, the antigen binding domain is an scFv comprising a nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0093] In certain embodiments, CAR further includes a transmembrane domain, a costimulatory domain and a signaling domain. The transmembrane domain can be a CD28 transmembrane domain. The costimulatory domain can be one or more of CD3 ζ (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2R β and MyD88 / CD40 costimulatory domains. In a specific embodiment, the costimulatory domain is one or more of CD28, 4-1BB and CD3 ζ costimulatory domains. The signaling domain can be a sequence encoding CSFR2 signal peptide.

[0094] In some embodiments, the isolated nucleic acid sequence may include a hinge / spacer domain. The hinge / spacer domain may be an IgG4P hinge / spacer.

[0095] In some specific embodiments, the isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR) may have a sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 26.

[0096] In other embodiments, the present disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain. The antigen binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, VH may have a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39. In some embodiments, VL may have a CDR1 comprising an amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0097] In some embodiments, VH may be the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and VL may be the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the CAR may further have a transmembrane domain, a costimulatory domain, and a signaling domain.

[0098] 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.

[0099] In other embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence can be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0100] In other embodiments, the present disclosure provides a cell comprising a vector having a nucleic acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:33, or SEQ ID NO:34.

[0101] In other embodiments, the disclosure provides a cell having a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for glypican 3 (GPC3), wherein the antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D), and wherein the CAR construct does not induce cytokine production in GPC3- cells. For example, the nucleic acid sequence can be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0102] In other embodiments, the present disclosure provides a cell that expresses an anti-GPC3 chimeric antigen receptor (CAR) on its extracellular surface. The CAR may have an antigen binding domain, which may be an antibody, Fab, or scFv each having a heavy chain variable region (VH) and a light chain variable region (VL). The VH may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may include a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0103] In some embodiments, the VH may have an amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. In some embodiments, the VL may have an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The CAR may further include a transmembrane domain, a costimulatory domain, and a signaling domain. The cell expresses a CAR having an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

[0104] In some embodiments, the present disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells that express a CAR on their extracellular surface, and the CAR may have an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. Such cells can exhibit anti-tumor immunity after contact with tumor cells expressing GPC3.

[0105] 13. Using CARs to Treat Cancer

[0106] In some embodiments, the present disclosure provides CAR cells for treating cancer. Compositions (e.g., antibodies, CAR constructs, and CAR cells) and methods with purposes described herein are particularly useful for suppressing the growth or spread of neoplastic cells. In some aspects, they are particularly useful for suppressing the growth of neoplastic cells in which GPC3 plays a role.

[0107] Neoplasms treatable by the compositions of the present disclosure include solid tumors, for example, solid tumors of the liver, lung, or ovary. However, the cancers listed herein are not intended to be limiting. For example, cancer types contemplated for treatment herein include, for example, NSCLC, advanced solid malignancies, biliary tract tumors, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal tumors, esophageal squamous cell carcinoma, extensive-stage small cell lung cancer, gastric adenocarcinoma, gastric cancer, gastroesophageal junction cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin's lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal carcinoma, metastatic melanoma, metastatic non-cutaneous melanoma, multiple myeloma, nasopharyngeal tumors, non-Hodgkin's lymphoma, ovarian cancer, fallopian tube cancer, peritoneal tumors, pleural mesothelioma, prostate tumors, recurrent or metastatic PD-L1 positive or negative SCCHN, recurrent squamous cell lung cancer, renal cell cancer (renal cell cancer / renal cell carcinoma), SCCHN, hypopharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, small cell lung cancer, squamous cell carcinoma of the head and neck, squamous cell lung cancer, TNBC, transitional cell carcinoma, unresectable or metastatic melanoma, urothelial cancer (urothelial carcinoma).

[0108] In one embodiment, cancers contemplated for treatment herein include any cancer that expresses GPC3 on the cell surface of cancer cells. In a specific example, cancers contemplated for treatment herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0109] 14. Armor

[0110] In some embodiments, the present disclosure provides "armored" cells, such as CAR T cells, having one or more genetic modifications that enhance or optimize cell function by protecting cells from environmental damage (such as immunosuppressive cytokines or immunosuppressive TME). Genetic modifications include, but are not limited to, increased secretion of cytokines, expression of ligands that interact with immune cells (such as T cells, macrophages, and regulatory T cells), or changes in functional properties. It will be understood by those skilled in the art that armoring cells (such as T cells) can provide many additional benefits not described herein that allow T cells to survive in immunosuppressive TME.

[0111] In some embodiments, the cell may include a chimeric antigen receptor (CAR) comprising a tumor-specific antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); and a hypoxia-inducible factor 1 alpha (HIF-1 alpha) dominant negative (HIF1 alpha DN) armored molecule.

[0112] In some embodiments, the armored cell may include a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain specific for glypican 3 (GPC3), wherein the antigen binding domain has an equilibrium dissociation constant (K) of about 100 nanomolar (nM) or less. D ), and wherein the CAR construct does not induce cytokine production in GPC3-cells, and wherein the cells express the HIF1α DN armor molecule.

[0113] In some embodiments, the armored cell may include an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain, wherein the antigen binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; a HIF1α DN armored molecule.

[0114] 15. Treatment Methods

[0115] The CAR-modified cells of the present invention (such as CAR T cells) can be administered alone or as a pharmaceutical composition with a diluent and / or other components associated with cytokines or cell colonies. In short, the pharmaceutical composition of the present invention may include, for example, CAR T cells as described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer, such as neutral buffered saline, buffered saline, etc.; sulfates; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins, polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The pharmaceutical composition of the present invention may be suitable for treatment (or prevention).

[0116] CAR modified cells can also be administered in combination with one or more other therapies. In one embodiment, additional therapies may include anti-cytokine antibodies. For example, one or more anti-TNF α can be used to reduce toxicity and promote anti-tumor activity at higher CAR T doses (which may be associated with CRS-like symptoms and weight loss).

[0117] The number of CAR cells administered per dose, the number of dosages, and the frequency of administration will depend on different parameters, such as the patient's age, weight, clinical evaluation, tumor type, tumor load, and / or other factors (including the judgment of the attending physician). Any acceptable route of administration is contemplated, such as, but not limited to, intravenous administration (e.g., intravenous infusion), parenteral or subcutaneous administration.

[0118] In a particular embodiment, the intended treatment regimen may include one or more biological components, such as CAR T cells and anti-cancer antibodies and / or chemotherapeutic components. For example, the intended treatment regimen may additionally include immune checkpoint inhibitors (ICIs), such as those targeting the PD-1 / PD-L1 axis (PDX), and other immuno-oncology (IO) therapies, such as immune system agonists.

[0119] Antibodies contemplated include anti-PD-L1 antibodies (e.g., durvalumab (MEDI4736), avelumab, atezolizumab, KNO35), anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, REGN2810, SHR1210, IBI308, PDR001, anti-PD-1, BGB-A317, BCD-100, and JS001), and anti-CTLA4 antibodies (e.g., tremelimumab or ipilimumab). Additional antibodies are also contemplated herein. Any therapeutically effective antibody sub-portion is also contemplated herein.

[0120] Information about durvalumab (or a fragment thereof) for use in the methods provided herein can be found in U.S. Patent Nos. 8,779,108; 9,493,565; and 10,400,039, the disclosures of which are incorporated herein by reference in their entireties. In a specific aspect, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90PT antibody as disclosed in the aforementioned U.S. Patents.

[0121] Information regarding tremelimumab (or an antigen-binding fragment thereof) for use in the methods provided herein can be found in U.S. Patent No. 6,682,736 (where tremelimumab is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety.

[0122] Additional therapeutic agents (chemotherapeutic agents or biologics) contemplated herein include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, ADRIAMYCIN TM(doxorubicin), cisplatin (MVAC), a carboplatin-based regimen, or a single-agent taxane or gemcitabine, temozolomide, or dacarbazine, vinflunine, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Additional examples include drugs that target the DNA damage repair system, such as poly (ADP-ribose) polymerase 1 (PARP1) inhibitors and therapeutic agents that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora protein kinase B activity, and DNA-PK activity.

[0123] Any therapeutic composition or method contemplated herein can be combined with one or more of any other therapeutic compositions and methods provided herein.

[0124] In some embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject in need thereof an effective amount of cells comprising an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen binding domain and an armored molecule that resists immunosuppression of the cell when expressed on the surface of a cell in the tumor microenvironment. On the other hand, the present disclosure describes an antigen binding domain, which may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). VH may include a CDR1 comprising an amino acid sequence of SEQ ID NO: 37, a CDR2 comprising an amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 39. VL may include a CDR1 comprising an amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the method further inhibits tumor growth, induces tumor regression, and / or prolongs the survival of the subject.

[0125] In some embodiments, the armor molecule is HIF1α DN.

[0126] In some embodiments, the cell is an autologous cell.For example, the autologous cell can be selected from the group consisting of: a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0127] In some embodiments, the cancer treated by the method is a solid tumor. For example, the cancer can be hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. In a specific embodiment, the cancer is hepatocellular carcinoma.

[0128] It should be understood that the specific aspects of the description described herein are not limited to the specific embodiments presented and may vary. It should also be understood that the terminology used herein is for the purpose of describing specific aspects only and is not intended to be limiting unless specifically defined herein. In addition, as will be appreciated by those skilled in the art, specific embodiments disclosed herein may be combined with other embodiments disclosed herein without limitation.

[0129] Examples

[0130] The following examples illustrate specific embodiments of the present disclosure and various uses thereof. They are set forth for illustrative purposes only and should not be construed in any way as limiting the scope of the present disclosure. Table 1 provides a description of the terms.

[0131] Table 1. Description of terms

[0132]

[0133]

[0134] Example 1: Armoring GPC3 CAR T cells with HIF1α DN

[0135] Produces less differentiated cells

[0136] summary

[0137] In this example, armoring GPC3BZ CAR T cells with a dominant-negative HIF-1α (HIF1αDN) molecule was investigated as a potential approach to protect CAR T cells from hypoxia-associated immunosuppression to improve CAR T effector function and tumor control.

[0138] method

[0139] Figure 1HIF1α DN: A dominant-negative HIF-1α molecule was prepared by truncating the wild-type HIF1α protein at the N- and C-termini so that the protein lacks the DNA binding domain, oxygen-dependent degradation domain, and transactivation domain. (Chen J, Zhao S, Nakada K, et al. Dominant-negative hypoxia-inducible factor-1alpha reduces tumorigenicity of pancreatic cancer cells through the suppression of glucose metabolism. Am J Pathol. 2003; 162(4): 1283-1291. doi: 10.1016 / s0002-9440(10)63924-7). The truncated HIF1α DN sequence (SEQ ID NO: 49) corresponds to residues 30-389 of the wild-type HIF1α sequence. Armored CART cells: GPC3 BZ CART cells were armored with HIF1α DN by expressing HIF1α DN as a C-terminal fusion with GPC3 BZ CAR (where the T2A peptide separates the GPC3 BZ CAR and HIF1α DN). In the HIF1α DN armored GPC3BZ CAR-T construct, the CD33 signal peptide is used to direct the secretion of the CAR, and the CAR is modified with an N-terminal HA epitope tag to facilitate detection of CAR expression on the cell surface.

[0140] Figure 2 T cells were either untransduced (UT) or transduced with GPC3-1 or GPC3-1 HIF1α DN constructs and expanded for 11 days under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. Figure 2 A and 2C. mRNA was extracted from frozen pellets using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions and reverse transcribed into cDNA. Endogenous or dominant-negative HIF1α was amplified by real-time PCR and expressed as fold induction calculated by the comparative C(T) method (Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3(6): 1101-1108). Figure 2 B. Detection of endogenous HIF1α expression by Western blotting. Figure 2 Cells were expanded as described in A and 2C and lysed in RIPA buffer containing protease and phosphatase inhibitors.

[0141] Figure 3 7 days after transduction, CD62L on the surface of UT, GPC3-1, or GPC3-1 HIF1α DN CAR-T cells expanded under normoxia (20% O2) or hypoxia (1% O2) was analyzed by flow cytometry 低 CD45RO 高 The frequency of T effector memory cells.

[0142] Figure 4 UT, GPC3-1, or GPC3-1 HIF1α DN CAR-T cells were expanded under normoxic conditions for 11 days. Cellular mRNA was extracted from frozen cell pellets using the RNeasy Micro Kit (Qiagen). Gene expression analysis was performed using the Nanostring CAR-T Characterization Panel according to the manufacturer's instructions. Gene expression is depicted as Log2 fold change relative to UT.

[0143] Figure 5 The GPC3-CAR modified T cell products were resuspended in serum-free unbuffered DMEM medium supplemented with L-glutamine (200 mM) and NaCl (143 mM) for glycolytic stress test or D-glucose (25 mM) and sodium pyruvate (1 mM) for mitochondrial stress test. The cells were then plated on Seahorse cell plates (1 × 10 6 Cells / well) were coated with Cell-Tak (Corning) to promote T cell attachment. The glycolytic stress test was performed by measuring ECAR (mpH / min) at steady state and after sequential injection of anti-CD3 / CD28 beads (cell to bead ratio of 1: 1) followed by injection of D-glucose (10mM). The mitochondrial stress test was performed by measuring OCR (pmol / min) at steady state and after sequential injection of anti-CD3 / CD28 beads (cell to bead ratio of 1: 1) followed by injection of oligomycin (0.5μM). The experiment using the Seahorse system adopted the following assay conditions: 2 minutes of mixing; 2 minutes of waiting; and 3 minutes of measurement.

[0144] result

[0145] The expression of dominant negative HIF1α was confirmed by real-time PCR, while the expression of endogenous HIF1α was investigated by Western blotting. Compared with endogenous HIF1α, dominant negative HIF1a was overexpressed. In addition, the expression of DN did not alter the stabilization of endogenous HIF1α induced by hypoxia as detected by Western blotting ( Figure 2 ).

[0146] T cell adaptability and persistence in the tumor microenvironment are related to their differentiation state (Kishton, RJ, Sukumar, M., and Restifo, NP (2017). Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metatab, 26(1), 94-109.). To investigate the effect of HIF1αDN expression on T cell differentiation, we analyzed CD62L in GPC3-1 or GPC3-1 HIF1αDN CAR-T cells after 6 days of expansion under normoxia (20% O2) or hypoxia (1% O2). 低 CD45RO 高 The frequency of T effector memory cells. GPC3-1 HIF1αDN maintained a less differentiated phenotype, as demonstrated by the decrease in T cells under hypoxic and normoxic conditions compared to unarmored CAR-T cells. EM Frequency reduction judgment (see Figure 3 Notably, cells expanded under hypoxia were more differentiated than those expanded under normoxia, yet even under these conditions, expression of HIF1α DN conferred a less differentiated phenotype similar to UT T cells.

[0147] HIF1α is not only a major regulator of the hypoxic response, but also a key intermediate in T cell activation under normoxia and is involved in the regulation of multiple signal transduction and metabolic pathways. To fully understand the genetic changes induced by the expression of HIF1αDN, we performed Nanostring analysis on purified GPC3-1 or GPC3-1 HIF1αDN CAR-T expanded under normoxic (20% O2) conditions. Compared with unarmored CAR T cells, HIF1αDN CAR T cells had increased expression of genes associated with stemness and decreased expression of genes associated with effector function. Therefore, this result confirms that the expression of HIF1αDN is associated with a less activated and differentiated phenotype (see Figure 4 ).

[0148] Resting T cells utilize an energy-efficient oxidative metabolism but switch to a highly glycolytic metabolism when stimulated for growth or after pathogen exposure (Michalek, RD, and Rathmell, JC (2010). The metabolic life and times of a T-cell. Immunol Rev, 236, 190-202.). To investigate the impact of HIF1αDN expression on the metabolic state of CAR-T cells, we assessed glycolysis by analyzing the extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation based on the oxygen consumption rate (OCR) under basal conditions and after antigen exposure using a real-time live cell assay (Seahorse XF). Under basal conditions, HIF1αDN CAR T cells exhibited a lower OCR and higher ECAR, indicating that they were more glycolytic. In contrast, after antigen exposure, HIF1αDN achieved a 60% OCR, while unarmored cells only achieved a 20% OCR. Conversely, unarmored cells achieved a 340% gain in ECAR after activation, while armored CAR-T cells achieved only 100%. Thus, expression of HIF1αDN restores cellular metabolism and confers the ability of CAR-T cells to respond to increased energy demands under stress (see Figure 5 ).

[0149] in conclusion

[0150] The characteristics of T cells used to generate anti-cancer immune responses are key factors in determining clinical outcomes. In particular, the use of T cells with cell lifespan extension characteristics to treat cancer is associated with improved anti-tumor responses (Kishton, RJ, Sukumar, M., and Restifo, NP (2017). Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy [Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy]. Cell Metatab [Cell Metabolism], 26 (1), 94-109). Hypoxia and the HIF signaling pathway affect the fate and function of immune cells, including the upregulation of glycolytic gene expression. Therefore, hypoxia drives cells towards a more differentiated state, which is associated with short-term persistence and poor outcomes in tumors. (Kishton, RJ, Sukumar, M., and Restifo, NP (2017). Metabolic Regulation of T Cell Longevity and Function in Tumor Immunotherapy. Cell Metab, 26(1), 94-109.; Krzywinska, E., and Stockmann, C. (2018). Hypoxia, Metabolism and Immune Cell Function. Biomedicines, 6(2)). Our data showed that CAR-T cells expressing HIF1α DN maintained naivety compared to unarmored CAR T cells. These findings suggest that HIF1α DN CAR T cells are well-adapted to tolerate the immunosuppressive effects associated with the severely hypoxic TME of solid tumors.

[0151] Example 2 Functional response of HIF1α DN CAR T cells after target exposure

[0152] summary

[0153] In this example, degranulation, cytotoxicity, effector cytokine production, ex vivo killing, and proliferative responses were measured in HIF1α DN CAR T cells in response to exposure to the target antigen GPC3.

[0154] method

[0155] Figure 6CAR T cell degranulation in response to GPC3. The indicated CAR-T cells were co-cultured with Hep3B or A375 for 6 hours in the presence of GolgiStop and an antibody against the degranulation marker CD107a directly labeled with a fluorescent dye. Target engagement induced CAR-T degranulation and subsequent binding to fluorescently labeled anti-CD107 present in the culture medium. CD107 accumulation, detected by flow cytometry, was proportional to the extent of degranulation and indicated target cell lysis. After 6 hours of incubation, cells were analyzed by flow cytometry.

[0156] Figure 7 . HIF1α DN CAR T cell effector cytokine production in response to GPC3: 5×10 4 CAR-T cells were co-cultured with target cells at a 1:1 ratio in RPMI 10% FCS. After 24 hours, the supernatant was collected and analyzed for cytokines using the Meso ScaleDiscovery 4-plex kit.

[0157] Figure 8 GPC3-1 or GPC3-1 HIF1α DN CAR-T cells were exposed to GPC3-expressing target cells under hypoxia or normoxia. After 6 hours of culture in the presence of PMA and ionomycin, intracellular cytokine staining was performed for GPC3-CAR-modified and dominant-negative HIF1a GPC3-CAR-modified T cells. The cells were then stained with Blue Live / Dead Fixable Dye and CD3 antibody, followed by fixation and permeabilization with BD Fixation and Permeabilization Buffer (eBioscience) according to the manufacturer's protocol. The cells were then incubated with IFN-γ, IL-2, and TNF-α (eBioscience) antibodies at 4°C for 30 minutes. Flow cytometry data were compensated and analyzed using FlowJo software (TreeStar). Antibodies used: anti-CD3 (SK7), anti-IL-2 (MQ1-17H12), anti-IFN-γ (4S.B3), and anti-TNFα (MAb11) were purchased from eBioscience.

[0158] Figure 9 CAR-T kills target-expressing cells. Cytotoxicity studies were conducted using cell impedance monitoring technology (xCELLigence). 3×10 4Target cells were plated and CAR-T cells cultured under hypoxic and normoxic conditions were added after 24 hours at the specified effector: target (E:T) ratio. The percentage of cell lysis and KT80 were calculated using RTCA software Pro. Images were acquired using RTCAeSight (xCELLigence instrument equipped with live cell imaging capabilities). Target cells used: Hep3B: GPC3 高 ;HUH7:GPC3 中等 ;PLC-PRF-15:GPC3 低 ;SNU-182GPC3 - .

[0159] Figure 10 . RDCA software Pro was used to analyze the E:T ratios of the samples under normoxia or hypoxia. Figure 9 Killing time 80 (KT) was calculated for the studies performed as indicated.

[0160] Figure 11 Bright-field images of real-time monitoring of CAR-T-mediated cytotoxicity during co-culture with GPC3+ tumor cells. Images were taken 3 hours after co-culture. Apoptotic features are already evident in the presence of armored CAR-T cells. The upper panel shows T cells in the absence of target cells.

[0161] Figure 12 .Use RDCA Pro software to Figure 9 Cytotoxicity studies were performed as indicated and percentage cell lysis was calculated using the indicated target cells (HUH7 and PLC / PRF / 5).

[0162] Figure 13 CAR-T cells were labeled with CFSE according to the manufacturer's instructions and incubated with the indicated cell lines. After 3 days, the dilution of CFSE was analyzed by flow cytometry.

[0163] Figure 14 HUH7 cells were implanted into the flanks of NSG mice (10 mice / group). 3 Figure 3. ...

[0164] Figure 15 Hep3B cells were implanted into the flanks of NSG mice (9 mice / group). 3 Figure 3. ...

[0165] result

[0166] HIF1αDN CAR T cells have a less differentiated and less activated phenotype, so we wanted to test whether they could generate effective antigen-specific immune responses. GPC3-1 CAR-T cells expanded under normoxia were exposed to GPC3 + Hep3B cells degranulated shortly after incubation, and the extent of degranulation was slightly reduced if the cells were expanded under hypoxic conditions. HIF1α DN CAR T cells expanded under normoxic conditions were able to degranulate similarly to unarmored CAR-T cells; however, degranulation was not reduced if expanded under hypoxic conditions. Degranulation was antigen-specific, as evidenced by the absence of CD107 after incubation with SNU-182. + This result indicates that HIF1α DN CAR T cells can degranulate efficiently and specifically in response to antigen contact even when expanded under hypoxia (see Figure 6 ).

[0167] Cytokine secretion is a hallmark of T cell activation; therefore, we wanted to investigate the impact of HIF1αDN expression on antigen-dependent effector cytokine production. Compared to unarmored CAR T cells, HIF1αDN CAR T cells produced less IFNγ and IL-2 but similar amounts of TNF-α after exposure to GPC3 under normoxic and hypoxic conditions. These data suggest that even though HIF1αDN CAR T cells have a less differentiated phenotype, they can secrete significant amounts of effector cytokines in an antigen-dependent manner. Figure 7 .

[0168] T cells that produce multiple cytokines, so-called "polyfunctional" T cells, provide a more effective immune response than cells that produce only a single cytokine. When cells are exposed to antigens under normoxic conditions, they produce three major effector cytokines, IFN-γ, after antigen stimulation. + / IL-2 + / TNFa + The frequencies of unarmored and armored CD4 and CD8 T cells were similar between unarmored and armored cells. Hypoxic stimulation significantly reduced the frequency of unarmored polyfunctional T cells. In contrast, the frequency of polyfunctional HIF1α DN CAR-T cells was only slightly affected by hypoxia (see Figure 8 ), indicating that HIF1αDN expression protects CAR-T from hypoxia-driven immunosuppression.

[0169] Killing target cells is the most important characteristic of CAR-T cells; therefore, we analyzed the ability of GPC3HIF1αDN CAR T to kill target cells expressing various levels of GPC3. Expression of HIF1αDN is associated with a less differentiated phenotype; therefore, we hypothesized that HIF1αDN would persist longer than their unarmored counterparts but would also kill target cells more slowly. Unexpectedly, HIF1αDN CAR T cells killed Hep3B cells faster than unarmored CAR T cells under normoxic or hypoxic conditions. Hep3B target cells displayed a clear and prominent pro-apoptotic appearance after only 3 hours of exposure to HIF1αDN CAR T (see Figure 9-11 ). GPC3 HIF1α DN CAR T kills other GPC3 cells with lower GPC3 expression + The cell lines HUH7 and PLC-PRF15 showed similar capacities to their unarmored counterparts. Figure 12 These results indicate that GPC3 HIF1α DN CAR T kills cells expressing higher levels of GPC3 at a faster rate than unarmored CAR T.

[0170] HIF1α is stabilized in T cells after antigen recognition and is an important mediator of T cell activation, so we asked whether the expression of DN leads to altered T cell proliferation. CFSE-labeled UT, GPC3, and GPC3 HIF1αDN CAR T cells were left unstimulated or co-cultured with Hep3B or HUH7. CFSE is gradually diluted within the daughter cells after each cell division, and the dilution is proportional to the extent of proliferation. Flow cytometric analysis after 3 days showed that HIF1αDN CAR T cells proliferated similarly to unmodified CAR T cells in response to target cells expressing high and low GPC3. Expression of HIF1αDN restored the nonspecific proliferation of unmodified CAR T cells in the absence of antigen, consistent with their less differentiated phenotype. This result indicates that blocking the HIF1α pathway with ectopic expression of a dominant negative molecule does not impair antigen-induced CAR-T cell proliferation (see Figure 13 ).

[0171] in conclusion

[0172] HIF1αDN CAR T cells were able to kill GPC3+ cancer cells while maintaining a less active and more naive phenotype. In addition, expression of HIF1αDN partially protected CAR-T cells from hypoxia-induced loss of polyfunctionality. Surprisingly, under normoxic and hypoxic conditions, HIF1αDN CAR T cells exhibited enhanced ability to kill certain target cells in vitro compared to unarmored CAR T cells. These surprising results suggest that armoring CAR T cells with HIF1αDN can provide CAR T cells with improved cytolytic efficacy in a manner independent of providing hypoxia resistance.

[0173] Example 3: Expression of HIF1αDN improves CAR-T efficacy in vivo

[0174] summary

[0175] In this example, the efficacy of HIF1α DN-armored CAR-T cells against GPC3 was determined in vivo in two different xenograft models. + The effectiveness of tumor cells.

[0176] method

[0177] The effectiveness of HIF1α DN-armored CAR-T cells in reducing tumor volume in vivo was tested using hepatocellular carcinoma Huh7. Tumor cells were implanted into the flanks of NSG mice (10 mice / group). When the tumor reached 150 mm 3 When the average volume of 7×10 6 CAR-T cells or 7 million untransduced T cells were used, and tumors were measured every two weeks (see Figure 14 ).

[0178] Hepatocellular carcinoma Hep3B was used to test the in vivo effectiveness of HIF1α DN armored CAR-T cells in reducing tumor volume. Tumor cells were implanted into the flanks of NSG mice (10 mice / group). When the tumor reached 150 mm 3 When the average volume of 7×10 6 CAR-T cells or 7 million untransduced T cells were used, and tumors were measured every two weeks (see Figure 15 ).

[0179] Give 7×10 6 Four days after infusion, tumors were harvested from five mice in each group. The number of CD45+ cells was counted by flow cytometry using AccuChec counting beads. (See Figure 16 ).

[0180] Mice bearing Hep3B tumors were given 7×10 6 CAR-T cells. For IFNγ analysis, blood was harvested in a small volume seven days after CAR-T infusion and serum was separated using BD Microtainer serum separator tubes. IFNγ levels were determined using the MSD assay (see Figure 17 ).

[0181] Mice bearing Hep3B tumors were given 7×10 6 Four days after infusion, spleens and tumors were harvested and evaluated for CD27 and CD70 expression by flow cytometry. (See Figure 18 ).

[0182] result

[0183] Compared with their unarmored counterparts, GPC3 HIF1α DN CAR-T cells were able to more effectively control HUH7 and induce faster Hep3B tumor regression (see Figure 14 and 15 We hypothesized that HIF1αDN expression confers CAR-T cells with an increased ability to infiltrate hypoxic solid tumors. Analysis of Hep3B tumors four days after T cell infusion showed that the number of T cells in HIF1αDN-treated mice was indeed increased 2-3 fold, indicating that HIF1αDN expression allows CAR-T cells to better infiltrate and proliferate in solid tumors (see Figure 16 Consistent with the increased number of cells in the tumor, we also detected more IFN-γ in the serum of mice infused with HIF1αDN-armored GPC3 CAR-T. Notably, although both armored and unarmored CAR-T cells displayed a CD70+ activation phenotype in the tumor, HIF1αDN was less activated in the spleen with na expression of CD70, similar to UT cells.

[0184] Conclusion GPC3 CAR-T cells expressing HIF1α DN induced faster tumor regression compared to unarmored GPC3 CAR-T cells. Tumor regression was associated with a significant increase in the cells' ability to infiltrate and proliferate in the tumor. Furthermore, although armored cells were fully activated in the tumor, they expressed less CD70 in the spleen than unarmored CAR-T cells, suggesting that they can maintain a less activated state even in vivo. Together, these observations suggest that blocking the HIF1α pathway through expression of dominant-negative HIF1α improves the in vivo efficacy of CAR-T cells while maintaining a less activated and differentiated phenotype.

[0185] The embodiments described herein can be practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. The terms and expressions that have been adopted are used as descriptive terms, rather than restrictive, and are not intended to exclude any equivalents of the features shown and described or parts thereof when using such terms and expressions, but it should be recognized that various modifications can be made within the scope of the claimed embodiments. Therefore, it should be understood that although the present invention has been specifically disclosed through the embodiments, optional features, those skilled in the art can modify and change the concepts disclosed herein, and it is believed that such modifications and changes can be within the scope of these embodiments defined by the specification and the appended claims. Although some aspects of the present disclosure may be considered particularly advantageous, it is contemplated that the present disclosure is not limited to these specific aspects of the disclosure.

[0186] If one, more than one, or all members of a group are present in, used in, or otherwise relevant to a given product or method, a claim or specification that includes "or" between one or more members of the group is considered satisfied, unless otherwise indicated or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or method. The present disclosure includes embodiments in which more than one or all members of the group are present in, used in, or otherwise relevant to a given product or method.

[0187] In addition, the present disclosure covers all variations, combinations and permutations in which one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim attached to another claim can be amended to include one or more limitations found in any other claim attached to the same base claim. Where elements are presented as a list (e.g., in Markush group form), each subgroup of elements is also disclosed, and any element can be removed from the group.

[0188] It will be understood that, in general, where the present disclosure or aspects of the present disclosure are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features. For the sake of brevity, these embodiments are not specifically set forth herein in words.

[0189] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any part of this application shall not be construed as an admission that such reference is available as prior art with respect to the present invention.

[0190] Table 2. Sequences used in the Examples.

[0191]

[0192]

[0193]

[0194] Table 3. Sequences

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] Sequence Listing <110> AstraZeneca (Sweden) Ltd. <120> Compositions and methods for treating cancer using chimeric antigen receptors <130> CARTGPC(HIF)-110-WO-PCT <140> PCT / EP2021 / 060620 <141> 2021-04-23 <150> 63 / 014,831 <151> 2020-04-24 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 241 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="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 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> / Comment="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 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> / Comment="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> / Comment="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> / Comment="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 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> / Comment="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 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> / Comment="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> / Comment="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> / Comment="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 [[ID= forty-five]]<220> <221> Source <223> / Comment="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 It should be noted that there seems to be a formatting issue in the original text where the tag <( has an extra parenthesis. This might be a typo. The translation is done as accurately as possible based on the provided text. tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttat ctctgctggt cacagtggct 900 ttcatcatct tctgggtcaa gcgaggccgg aagaaactgc tgtacatctt caaacagcct 960 tttatgcgcc cagtgcagac aactcaggag gaagacggct gctcttgtcg gttccccgag 1020 gaagaggaag ggggatgtga gctgcgcgtg aagttttctc gaagtgccga tgctcctgca 1080 tatcagcagg gacagaacca gctgtacaac gagctgaatc tgggccggag agaggaatac 1140 gacgtgctgg ataagaggcg cggcagagac ccagaaatgg gcgggaagcc acgacggaaa 1200 aacccccagg aggggctgta taatgaactg cagaaggaca aaatggccga ggcttacagc 1260 gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggact gtatcagggc 1320 ctgagcactg ccaccaagga cacctacgat gctctgcaca tgcaggcact gccacccagg 1380 tga 1383 <210> 12 <211> 957 <212> DNA <213> Artificial Sequence <220> <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> Synthetic Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: Synthetic polynucleotide" <400> 13 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaagggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta 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 areacaaaa 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 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 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 ggcaaaagt acttgatta tggggacag ggcactatgg tgaccgtc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gandacagcca 480 cctagcgcct ccggacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gatattggga gcaaccgt gattggtat cagcagctgc ctggaacagc tccaagctg 600 ctgatctact atacaatca gaggccctcc ggcgtccctg atcgctctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtccttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc 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 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> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 16 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gacacagcca 480 cctagcgcct ccggaacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gacattggga gcaacaccgt gaattggtat cagcagctgc ctggaacagc tccaaagctg 600 ctgatctact ataacaatca gaggccctcc ggcgtccctg atcgcttctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg 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 It should be noted that there seems to be a small error in the original text where "720" in line 12 should probably be "720". The above translation has been corrected accordingly. <223> / Comment="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> / Comment="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> / Comment="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> twenty one <211> 457 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polypeptide" <400> twenty one Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Asp Asp 100 105 110 Thr Ala Leu Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu Pro Leu Ser 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln 145 150 155 160 Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys 165 170 175 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln 180 185 190 Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln 195 200 205 Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr 210 215 220 Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp 225 230 235 240 Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly 245 250 255 Thr Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys 260 265 270 Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 275 280 285 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys 290 295 300 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 305 310 315 320 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 325 330 335 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 340 345 350 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 355 360 365 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 370 375 380 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 385 390 395 400 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 405 410 415 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 420 425 430 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 435 440 445 Leu His Met Gln Ala Leu Pro Pro Arg 450 455 <210> twenty two <211> 1371 <212> DNA <213> Artificial sequence <220> <221> source <223> / Annotation="Description of artificial sequence: synthetic polynucleotide" <400> twenty two 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 gcagtttat 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 1020 tgcgaactgc gggtgaaatt tagtagaagc gctgacgcac cagcttacca acaaggacag 1080 aaccaattgt acaacgagct taacttgggt aggagggagg aatatgatgt actggacaaa 1140 aggcgaggtc gcgatccgga aatgggaggc aagccacagc gccggaaaaa cccgcaggaa 1200 ggcttgtaca acgaacttca gaaagataaa atggcagaag catactccga aatagggatg 1260 aaaggtgaac ggcggcgagg caagggccac gacggtctgt accaagggtt gtcaacggca 1320 actaaagaca cgtatgatgc acttcatatg caagctctgc cacccaggtg a 1371 <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 aatttttgg 900 gtgcggagca agcggagccg gctgctgcac tctgactaca tgaacatgac tccacggaga 960 cccggcccta cccggaaaca ttatcagccc tacgccccac ccagagattt tgccgcttat 1020 aggtccaaaa gaggtcggaa aaagttgctg tatattttca aaaccctt tatgagacct 1080 gtacaaacga ctcaggaaga ggatggttgt agttgcaggt ttccggagga ggaggaaggt 1140 gggtgcgaac tgcgggtgaa atttagtaga agcgctgacg caccagctta ccaacaagga 1200 cagaaccaat tgtacaacga gcttaacttg ggtaggaggg aggaatatga tgtactggac 1260 aaaaggcgag gtcgcgatcc ggaaatggga ggcaagccac agcgccggaa aaacccgcag 1320 gaaggcttgt acaacgaact tcagaaagat aaaatggcag aagcatactc cgaataggg 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 tggatttaca ttctcttctt atgggttgca ttgggtcaga 180 caagcaccgg gcaaaggatt ggaatgggtc gcggccatta gctatgatgg ctcaaagaaa 240 tattatgccg attccgtaaa agggaggttg acaataagcc gggataacag caagaacact 300 ttgtatcttc agatgaatag cctccgaccg gacgacacgg cactgtattt ttgcgcacgc 360 gggtggtttg tagaacccct gagttgggga caaggtactc ttgtcacggt atcttctggc 420 ggaggtggga gtggtggggg tggcagtggc gggggtgggt cacaaagcgt gcttacacaa 480 cctccttctg 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> / Comment="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 60 attcctgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 120 agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 180 caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 240 tactacgcag actccgtgaa gggccggttc accatctcca gagacaattc caagaacacg 300 ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga 360 ggaaagcgat actttgacta ctggggccag gggacaatgg tcaccgtctc gagtggtggg 420 gggggcagcg gtggtggagg ctctggtgga ggagggagct cctatgagct gactcagcca 480 ccctcagcgt ctgggacccc cgggcagagg gtcaccatct cttgttctgg aggcagctcc 540 aacatcggaa gtaatactgt aaactggttc cggcagctcc caggaacggc ccccaaactc 600 ctcgtttatt ttaataatca gcgaccctca ggggtccctg accgattctc tggctccaag 660 tctggcacct cggcctccct ggccatcggt gggctccagt ctgacgatga ggctgactat 720 tactgtgtag catgggatga ctctctgaat gctccggtgt tcggcggagg gaccaaggtc 780 accgtcctag agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctggtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaaactgctg tacatcttca aacagccttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tcttgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggagag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaaatgggc gggaagccac gacggaaaaa 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[[ID=X]] 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> / Comment="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> / Comment="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 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> / Comment="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> / Comment="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> / Comment="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> / Annotation="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> [[ID=​​​​​​​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 < <220> <221> Source <223> / Comment="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 tttgtagaac ccctgagttg gggacaaggt actcttgtca cggtatcttc tggcggaggt 360 gggagtggtg ggggtggcag tggcgggggt gggtcacaaa gcgtgcttac acaacctcct 420 tctgcgagcg gaactccggg acaacgggtt acgatttcat gctccggctc aagtagcaat 480 ataggatcaa atacagtgaa ttggtatcaa caactccctg gcacagcgcc caagctgctg 540 atctactcta ataaccagag gccgagtggt gtgccagata ggttcagtgg ctctaaatca 600 ggtactagcg cgagcctcgc catttcagga cttcaatcag aggatgaagc ggactactac 660 tgtgccgcgt gggatgattc acttaatgga tatgttttcg ggaccggaac aaaattgacg 720 gtattg 726 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="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> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <400> 40 Ser Gly Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="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> / Comment="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> / Comment="Description of artificial sequence: synthetic peptide" <400> 43 Ser Gly Gly Ser Ser Ser Asp Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="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> / Comment="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> 1083 <212> DNA <213> Artificial sequence <220> <221> source <223> / Annotation="Description of artificial sequence: synthetic polynucleotide" <400> 46 atgcggagca aagaaagcga ggtgttctac gagctggccc accaactgcc tctgcctcac 60 aatgtgtcca gccacctgga taaggccagc gtgatgagac tgaccatcag ctacctgaga 120 gtgcggaagc tgctggatgc cggcgatctg gacatcgagg acgatatgaa ggcccagatg 180 aactgcttct acctgaaggc cctggacggc ttcgtgatgg tgctgaccga tgacggcgac 240 atgatctaca tcagcgacaa cgtgaacaag tacatggggc tgacccagtt cgagctgaca 300 ggccacagcg tgttcgactt cacacacccc tgcgaccacg aagagatgag agagatgctg 360 acccaccgga acggcctggt caagaagggc aaagagcaga atacccagcg gtcattcttc 420 ctgcggatga agtgcaccct gaccagcagg ggcagaacca tgaacatcaa gagcgccaca 480 tggaaggtgc tgcactgcac cggacacatc cacgtgtacg acaccaacag caaccagcct 540 cagtgcggct acaagaaacc tcctatgacc tgcctggtgc tgatctgcga gcccattcct 600 catcctagca acatcgagat ccctctggac agcaagacct tcctgagcag acacagcctg 660 gacatgaagt tcagctactg cgacgagcgg atcaccgagc tgatgggcta tgagcctgaa 720 gaactgctgg gccgcagcat ctacgagtac tatcacgcccc tggacagcga ccacctgacc 780 aagacacacc acgacatgtt caccaagggc caagtgacca ccggccagta cagaatgctg 840 gccaagcgcg gaggctacgt gtgggttgaa acacaggcca ccgtgatcta caacaccaag 900 aactcccagc cacagtgcat cgtgtgcgtg aactacgtgg tgtccggcat catccagcac 960 gacctgatct tcagcctgca gcagaccgag tgcgtgctga agcctgtgga aagcagcgac 1020 atgaagatga cccagctgtt taccaaggtg gaatccgagg acaccagcag cctgttcgac 1080 aag 1083 <210> 47 <211> 427 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic polypeptide" <400> 47 Met Arg Ser Lys Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu 1 5 10 15 Pro Leu Pro His Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met 20 25 30 Arg Leu Thr Ile Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly 35 40 45 Asp Leu Asp Ile Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr 50 55 60 Leu Lys Ala Leu Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp 65 70 75 80 Met Ile Tyr Ile Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln 85 90 95 Phe Glu Leu Thr Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp 100 105 110 His Glu Glu Met Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys 115 120 125 Lys Gly Lys Glu Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys 130 135 140 Cys Thr Leu Thr Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr 145 150 155 160 Trp Lys Val Leu His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn 165 170 175 Ser Asn Gln Pro Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu 180 185 190 Val Leu Ile Cys Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro 195 200 205 Leu Asp Ser Lys Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe 210 215 220 Ser Tyr Cys Asp Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu 225 230 235 240 Glu Leu Leu Gly Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser 245 250 255 Asp His Leu Thr Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val 260 265 270 Thr Thr Gly Gln Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp 275 280 285 Val Glu Thr Gln Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro 290 295 300 Gln Cys Ile Val Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His 305 310 315 320 Asp Leu Ile Phe Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val 325 330 335 Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser 340 345 350 Glu Asp Thr Ser Ser Leu Phe Asp Lys Ile Tyr Asn Thr Lys Asn Ser 355 360 365 Gln Pro Gln Cys Ile Val Cys Val Asn Tyr Val Val Ser Gly Ile Ile 370 375 380 Gln His Asp Leu Ile Phe Ser Leu Gln Gln Thr Glu Cys Val Leu Lys 385 390 395 400 Pro Val Glu Ser Ser Asp Met Lys Met Thr Gln Leu Phe Thr Lys Val 405 410 415 Glu Ser Glu Asp Thr Ser Ser Leu Phe Asp Lys 420 425 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <220> <221> Site <222> (1)..(15) <223> / comment="This sequence may include 1-3 repeating units of 'Gly Gly Gly Gly Ser'" <400> 48 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15

Claims

1. An isolated nucleic acid molecule encoding a) a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen binding domain specific for a cell surface antigen, wherein the antigen binding domain comprises an scFv comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; and wherein the light chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 42; and b) a dominant negative armored molecule of hypoxia-inducible factor-1α corresponding to amino acid residues 30-389 of wild-type hypoxia-inducible factor-1α, wherein the armored molecule resists immunosuppression of cells in the tumor microenvironment when expressed on the surface of the cells.

2. The isolated nucleic acid molecule of claim 1, wherein the antigen binding domain is a scFv encoded by the nucleic acid molecule of SEQ ID NO: 33 or SEQ ID NO:

34.

3. The isolated nucleic acid molecule of any one of the preceding claims, wherein the chimeric antigen receptor further comprises a transmembrane domain, a co-stimulatory domain, and a signal peptide domain.

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

5. The isolated nucleic acid molecule of claim 3, wherein the costimulatory domain comprises one or more of CD28, 4-1BB, CD3ζ, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 costimulatory domains.

6. The isolated nucleic acid molecule of claim 3, wherein the costimulatory domain comprises one or more of CD28, 4-1BB, and CD3ζ costimulatory domains.

7. The isolated nucleic acid molecule of claim 3, wherein the signal peptide domain comprises a CSFR2 signal peptide.

8. The isolated nucleic acid molecule of claim 1 or 2, wherein the chimeric antigen receptor further comprises a hinge or spacer domain.

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

10. The isolated nucleic acid molecule of claim 1, wherein the cell surface antigen comprises one or more of the following: 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, EGFRvIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV, HPV16-E6, HPV16-E7, IL13Ra2, Igκ chain, LGR5, LMP1, LeY, LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesothelin, MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA and PSCA.

11. A cell comprising the isolated nucleic acid molecule of any one of claims 1-10.

12. A cell comprising: A nucleic acid molecule encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen binding domain specific for a cell surface antigen, wherein the antigen binding domain comprises an scFv comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; and wherein the light chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 42; and The cell surface expresses a dominant negative armored molecule of hypoxia-inducible factor-1α corresponding to amino acid residues 30-389 of wild-type hypoxia-inducible factor-1α.

13. The cell of claim 12, wherein the chimeric antigen receptor further comprises a transmembrane domain, a costimulatory domain, and a signal peptide domain.

14. A cell comprising: an anti-GPC3 chimeric antigen receptor comprising an antigen binding domain, wherein the antigen binding domain comprises an scFv comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; and wherein the light chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 42; and A dominant negative armored molecule of hypoxia-inducible factor-1α corresponding to amino acid residues 30-389 of wild-type hypoxia-inducible factor-1α.

15. The cell of claim 14, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

16. The cell of claim 14 or 15, wherein the light chain variable region consists of the amino acid sequence of SEQ ID NO:

28.

17. The cell of any one of claims 11-15, wherein the cell is selected from the group consisting of a T cell and a natural killer cell.

18. The cell of any one of claims 11-15, wherein the cell is selected from the group consisting of: a cytotoxic T lymphocyte and a regulatory T cell.

19. Use of a cell in preparing a drug for treating cancer, wherein the cell comprises a) a chimeric antigen receptor specific for a cell surface antigen, wherein the chimeric antigen receptor comprises an anti-GPC3 chimeric antigen receptor comprising an antigen binding domain, wherein the antigen binding domain comprises an scFv comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 39; and wherein the light chain variable region comprises a CDR1 consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 41, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 42, and b) a dominant negative armored molecule of hypoxia-inducible factor-1α corresponding to amino acid residues 30-389 of wild-type hypoxia-inducible factor-1α, wherein the armored molecule resists immunosuppression against cells in the tumor microenvironment of the cancer, wherein the cancer is hepatocellular carcinoma.

20. The use of claim 19, wherein the cell surface antigen is one or more of the following: 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, STEAP1, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1, MAGE-A3, MAGE-A4, mesothelin, MUC1, MUC16, NY-ESO-1, WT1, PDL1, CAIX, CD70, PSMA, and PSCA.

21. The use of claim 20, wherein the medicament is combined with a drug that inhibits tumor growth, induces tumor regression and / or prolongs the survival of a subject.

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

23. The use of claim 22, wherein the autologous cells are selected from the group consisting of T cells and natural killer (NK) cells.

24. The use of claim 22, wherein the autologous cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs) and regulatory T cells.

25. The use of any one of claims 20-24, wherein the medicament is combined with an anti-cancer antibody and / or a chemotherapeutic component.

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