Composition and method for treating cancer using a chimeric antigen receptor targeting glypican 3

Abstract

This new chimeric antigen receptor (CAR) T-cell therapy effectively treats cancer while minimizing the risk of dangerous inflammatory responses such as cytokine release syndrome. [Solution] An isolated nucleic acid sequence encoding a CAR, wherein the CAR includes an antigen-binding domain specific to glypican 3 (GPC3), the antigen-binding domain has an equilibrium dissociation constant (KD) of approximately 100 nanomoles (nM) or less, and the CAR construct provides an isolated nucleic acid sequence that does not induce cytokine production in GPC3-expressing cells.

Description

[Technical Field]

[0001] This disclosure relates to the treatment of cancer using chimeric antigen receptor T cells. [Background technology]

[0002] 1. Chimeric antigen receptor T cell therapy Chimeric antigen receptor (CAR) T-cell therapy is a specific form of cell-based immunotherapy that uses genetically modified T cells to fight cancer. In CAR T-cell therapy, T cells are collected from the patient's blood, ex vivo engineered to express a CAR containing both an antigen-binding domain and a T-cell activation domain, expanded into a larger population, and administered to the patient. The CAR T cells act as living drugs, binding to cancer cells and causing their destruction. When successful, the effects of CAR T-cell therapy tend to be long-lasting, as evidenced by the detection of persistent and expanding CAR T cells in patients long after clinical remission.

[0003] 2. Structure and Function of CAR The antigen-binding domain of a CAR is an extracellular region that targets surface antigens on tumor cells. Suitable target antigens can be proteins, phosphorylated proteins, peptide-MHCs, carbohydrates, or glycolipid molecules. Ideal target antigens are widely expressed on tumor cells to enable targeting at a high rate of cancer cell activity. Ideal candidate target antigens are also typically expressed minimally in normal tissues, limiting off-tumor and on-target toxicity. The antigen-binding domain of a CAR contains a targeting moiety, such as an antibody single-chain variable region fragment (scFv), which is directed towards the target antigen.

[0004] The T cell activation domain of a CAR is located intracellularly and activates T cells in response to an antigen-binding domain that interacts with its target antigen. The T cell activation domain can contain one or more costimulatory domains, which are intracellular domains of known activated T cell receptors. Since costimulatory domains have different effects on the dynamics, cytotoxic function, and safety profile of CAR T cells, the selection and positioning of costimulatory domains within a CAR construct influences the function and fate of CAR T cells.

[0005] The extracellular antigen-binding domain and intracellular T-cell activation domain of a CAR are linked by a transmembrane domain, a hinge, and an optional spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on tumor cells. It can be used alone or in conjunction with a spacer domain that causes the scFv to protrude from the T-cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface.

[0006] CAR T therapy targeting B lymphocyte antigen CD19 (Kymriah®, Novartis) is considered promising for pediatric acute lymphoblastic leukemia, while CAR T therapy targeting B cell maturation antigen ("bb2121," a joint research project by Celgene® and Bluebirdbio®) is considered promising for relapsed / refractory multiple myeloma. More recent data suggest that CAR approaches may be effective against solid tumors. GD2 CAR natural killer T cell (NKT) therapy has shown activity in neuroblastoma (Heczey A, et al. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood;124(18):2824-33, 2014), and pembrolizumab-mediated mesothelin CAR T has shown antitumor effects against mesothelioma. However, further targets for treating solid tumors are needed.

[0007] 3. Challenges of CAR T-cell therapy Unfortunately, the complexity of CAR T cell-based therapies can lead to undesirable and dangerous consequences. Off-tumor effects such as neurotoxicity and acute respiratory distress syndrome are potential adverse effects of CAR T cell therapy and can be fatal. Cytokine release syndrome (CRS) is the most common acute toxicity associated with CAR T cells. CRS occurs when lymphocytes become highly activated and release excessive amounts of inflammatory cytokines. In patients with CRS, elevated levels of interleukin-2, interleukin-6, interleukin-1 beta, GM-CSF, and / or C-reactive protein in the serum are sometimes observed when these factors are measured. CRS is classified by severity and diagnosed as one of four grades (mild to severe), with more severe cases clinically characterized by high fever, hypotension, hypoxia, and / or multi-organ toxicity in the patient. One study reported that 92% of patients with acute lymphoblastic leukemia treated with anti-CD19 CAR T-cell therapy experienced CRS, and 50% of these patients developed grade 3–4 symptoms.

[0008] Therefore, further CAR T cell-based therapies are needed to enhance the capabilities of effective cancer treatment. However, new CAR T cell therapies must be devised that effectively treat cancer while minimizing the risk of dangerous inflammatory responses such as CRS. [Overview of the project] [Means for solving the problem]

[0009] This disclosure describes compositions and methods for treating cancer using CAR T cells.

[0010] As described below, in the first aspect, the disclosure is an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain specific to glypican 3 (GPC3), the antigen-binding domain having an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. Dhaving, the CAR construct provides an isolated nucleic acid sequence that does not induce cytokine production in GPC3-expressing cells.

[0011] In some embodiments of the first aspect, the antigen-binding domain of the CAR comprises an antibody or an antigen-binding fragment thereof.

[0012] In some embodiments of the first aspect, the antigen-binding domain is a Fab or a single-chain variable region fragment (scFv).

[0013] In some embodiments of the first aspect, the antigen-binding domain is an scFv comprising the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0014] In some embodiments of the first aspect, the isolated nucleic acid further encodes a transmembrane domain, a co-stimulatory domain, and a signal domain.

[0015] In some embodiments of the first aspect, the transmembrane domain comprises the CD28 transmembrane domain.

[0016] In some embodiments of the first aspect, the co-stimulatory domain comprises one or more of the CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 co-stimulatory domains.

[0017] In some embodiments of the first aspect, the co-stimulatory domain comprises one or more of the CD28, 4-1BB, and CD3 zeta co-stimulatory domains.

[0018] In some embodiments of the first aspect, the signal domain comprises a sequence encoding the CSFR2 signal peptide.

[0019] In some embodiments of the first aspect, the anti-GPC3 CAR further comprises a hinge / spacer domain.

[0020] In some embodiments of the first aspect, the hinge / spacer domain is an IgG4P hinge / spacer.

[0021] In some embodiments of the first aspect, the nucleic acid sequence comprises 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.

[0022] In a second aspect, the present disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and VL comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0023] In some embodiments of the second aspect, VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29.

[0024] In some embodiments of the second aspect, VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

[0025] In some embodiments of the second aspect, the anti-GPC3 CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0026] In some embodiments of the second aspect, the anti-GPC3 CAR comprises 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.

[0027] In a third aspect, the disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises 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.

[0028] In some embodiments, the present disclosure provides cells comprising a vector of a third embodiment.

[0029] In a fourth aspect, the disclosure relates to a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. D The CAR construct has ) and provides cells that do not induce cytokine production in GPC3 cells.

[0030] In some embodiments of the fourth aspect, the nucleic acid sequence includes 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.

[0031] In a fifth aspect, the disclosure provides cells 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), the VH comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0032] In some embodiments of the fifth aspect, VH comprises the amino acid of SEQ ID NO: 27 or SEQ ID NO: 29.

[0033] In some embodiments of the fifth aspect, VL includes the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

[0034] In some embodiments of the fifth aspect, the CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0035] In some embodiments of the fifth aspect, the CAR includes 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.

[0036] In some embodiments of the fifth aspect, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0037] In some embodiments of the fifth aspect, cells exhibit anti-tumor immunity upon contact with tumor cells expressing GPC3.

[0038] In a sixth aspect, the disclosure provides a method for treating cancer, comprising administering cells containing an effective amount of an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain to a subject in need of cancer treatment, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39, and the VL comprises CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0039] In some embodiments of the sixth aspect, the method further includes inhibiting tumor growth, inducing tumor regression, and / or extending survival.

[0040] In some embodiments of the sixth aspect, the cells are self-cells.

[0041] In some embodiments of the sixth aspect, the autologous cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0042] In some embodiments of the sixth aspect, the cancer is a solid tumor.

[0043] In some embodiments of the sixth aspect, the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

[0044] In some embodiments of the sixth aspect, the cancer is hepatocellular carcinoma.

[0045] In some embodiments of the sixth aspect, the method further comprises administering an effective amount of anti-TNFα antibody to the target.

[0046] These and other features and advantages of this disclosure will be better understood from the following detailed description together with the attached claims. Note that the claims are defined by reference thereto and not by the specific consideration of the features and advantages described herein.

[0047] The accompanying drawings are included to provide a further understanding of the methods and compositions of this disclosure. The drawings illustrate one or more embodiments of this disclosure and, together with the description, help to illustrate the principles and operation of this disclosure. This invention also relates to the following: [Item 1] An isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. D The CAR construct has an isolated nucleic acid sequence that does not induce cytokine production in GPC3 cells. [Item 2] The antigen-binding domain of the encoded CAR is an isolated nucleic acid sequence as described in item 1, comprising an antibody or its antigen-binding fragment. [Item 3] The antigen-binding domain of the encoded CAR is a Fab or a single-chain variable region fragment (scFv) of the isolated nucleic acid sequence as described in item 2. [Item 4] The antigen-binding domain is an scFv containing the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, as described in item 3. [Item 5] An isolated nucleic acid sequence described in any one of items 1-4, further encoding a transmembrane domain, a costimulatory domain, and a signaling domain. [Item 6] The encoded transmembrane domain is an isolated nucleic acid sequence as described in item 5, comprising the CD28 transmembrane domain. [Item 7] The encoded co-stimulatory domain is an isolated nucleic acid sequence as described in item 5, comprising one or more of the CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 co-stimulatory domains. [Item 8] The encoded co-stimulatory domain is an isolated nucleic acid sequence as described in item 5, comprising one or more CD28, 4-1BB, and CD3 zeta co-stimulatory domains. [Item 9] The encoded signal domain is an isolated nucleic acid sequence as described in item 5, comprising a sequence encoding the CSFR2 signal peptide. [Item 10] An isolated nucleic acid sequence described in any one of items 1-9 that further encodes the hinge / spacer domain. [Item 11] The encoded hinge / spacer domain is the IgG4P hinge / spacer, as described in item 10 of the isolated nucleic acid sequence. [Item 12] The nucleic acid sequence is the isolated nucleic acid sequence described in item 1, comprising 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. [Item 13] An anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL); The aforementioned VH includes CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The VL is an anti-GPC3 CAR comprising CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. [Item 14] The VH is the anti-GPC3 CAR according to item 13, comprising the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. [Item 15] The VL is the anti-GPC3 CAR described in item 13, comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. [Item 16] The CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain, as described in any one of items 13 to 15. [Item 17] The anti-GPC3 CAR according to item 16, wherein the CAR comprises 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. [Item 18] A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises 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. [Item 19] Cells containing the vector described in item 18. [Item 20] A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. D ) and the CAR construct does not induce cytokine production in GPC3 cells. [Item 21] The cell according to item 20, wherein the nucleic acid sequence includes 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. [Item 22] Cells comprising an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned VH includes CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The VL is a cell comprising CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. [Item 23] The VH is the cell described in item 22, comprising the amino acids of SEQ ID NO: 27 or SEQ ID NO: 29. [Item 24] The VL is the cell described in item 22, comprising the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. [Item 25] The CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain, as described in any one of items 22-24. [Item 26] The CAR is a cell according to any one of items 22 to 25, comprising 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. [Item 27] The cells are those selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells, as described in any one of items 19 to 26. [Item 28] The aforementioned cells are the cells described in item 27, which exhibit anti-tumor immunity upon contact with tumor cells expressing GPC3. [Item 29] A method of treating cancer, The treatment for cancer involves administering cells containing an anti-GPC3 chimeric antigen receptor (CAR) containing an effective amount of antigen-binding domain to a subject requiring cancer treatment, wherein the antigen-binding domain includes an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). The aforementioned VH includes CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The method wherein the VL comprises CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. [Item 30] The method according to item 29, further comprising inhibiting tumor growth, inducing tumor regression, and / or extending survival of the subject. [Item 31] The cell is the autologous cell, as described in item 29. [Item 32] The method according to item 31, wherein the self-cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. [Item 33] The cancer is a solid tumor, as described in any one of items 29-32. [Item 34] The method according to item 33, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. [Item 35] The cancer is hepatocellular carcinoma, as described in item 34. [Item 36] The method according to any one of items 29 to 35, further comprising administering an effective amount of anti-TNFα antibody to the subject. [Brief explanation of the drawing]

[0048] [Figure 1A] GPC3 expression in cancer and normal tissue. 1A. Anti-GPC3 antibody staining in hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and ovarian cancer. 1B. Results of immunohistochemical staining (IHC) of human colonic ganglion tissue. [Figure 1B] This is a continuation of Figure 1A. [Figure 2] Comparison of the heavy-chain and light-chain variable regions of a single-chain variable region fragment (scFv). GPC3-1 and GPC3-2 are shown. [Figure 3A] Binding of cell surface GPC3 CARs to soluble GPC3 protein. KD values ​​are shown (fitted lines are shown). [Figure 3B] Surface plasmon resonance binding of anti-GPC3 scFv-Fc to soluble GPC3 protein. For both interactions, the mean values ​​of ka, kd, and KD are reported (fits are shown as solid lines). [Figure 3C] This is a continuation of Figure 3B. [Figure 4A]Cytokine production when chimeric antigen receptor (CAR) constructs are administered in vitro to cells with or without the target antigen. GPC3 CAR T induces antigen-specific cytokine production. Cell lines are listed from top to bottom in the legend, and shown from left to right for each construct. 4A. Results for three cytokines are shown. UT: Untransduced T cells (donor T cells that are activated and proliferate, but do not have the CAR transgene introduced). 4B. Results for interferon-gamma (IFN-γ) of a subset of the constructs are shown. Cell types (all negative except HEPG2) are shown in the legend on the right. [Figure 4B] This is a continuation of Figure 4A. [Figure 5] Cytotoxicity of CARs in HCC cell lines. The constructs used are shown in the legend on the right. E:T ratio:Effector:Target ratio. UT:Untransduced T cells. [Figure 6-1] Cytotoxicity of GPC3-1 in HCC cell lines expressing low levels of GPC3. 6A GPC3 expression was evaluated by flow cytometry in the indicated cell lines. 6B. Receptor density on the indicated cell lines. 6C. Cytotoxicity of GPC3-1 against the indicated cell lines, effector:target ratio 3:1 (upper graph) and 0.3:1 (lower graph). 6D. KT50 (time to kill 50% of the target) of GPC3-1 against the indicated cell lines at two different effector:target ratios. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7] Multifunctional studies of GPC3-2 CAR T constructs and GPC3-1 CAR T constructs. The scFv is shown to the left of each row, and the co-stimulatory domain is shown at the top of each chart. [Figure 8A] 8A. Effect of chimeric antigen receptor T cell (CAR T) transplantation on body weight. The constructs used are shown in the legend on the right. BW: Body weight. ACT: Adoptive T cell therapy. UT: Untransduced T cells. PBS: Phosphate-buffered saline. 7B. IHC showing CAR-T accumulation in lung tissue of GPC3 CAR-T treated mice. [Figure 8B] This is a continuation of Figure 8A. [Figure 9] Effect of CAR T cell administration on tumor volume. The constructs used are shown in the legend on the right. ACT: Adoptive T cell therapy. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 10] The effect of CAR T cell therapy on survival. The constructs used are shown in the legend on the right. ACT: Adoptive T cell therapy. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 11A] Fluorescence-activated cell sorting (FACs) studies of GPC3-1 CAR T cell differentiation and depletion using different co-stimulatory domains. Results for spleen (11A and 11B) and tumor cells (11C and 11D) are shown. Dot plots show the frequency of CD3+ T cells infiltrating each organ for each construct (11A and 11C). GPC3 CAR-T cells with the 4-1BB / CD3 zeta (BZ) signaling domain exhibit greater central memory and less depletion than CD28 / CD3 zeta (28Z) cells in vivo. The co-stimulatory domains used are shown at the top of each panel. Assay markers are shown on the x and y axes. FSC: Forward scattered light. EM: Effector memory. CM: Central memory. TN: T-naive. [Figure 11B] This is a continuation of Figure 11A. [Figure 11C] This is a continuation of Figure 11B. [Figure 11D] This is a continuation of Figure 11C. [Figure 12] Persistence of GPC3-1 CAR T cells in Hep3B and HepG2 tumors. The constructs used are shown in the legend on the right. CD3 percentages are shown. ACT: Adoptive T cell therapy. UT: Untransduced T cells. [Figure 13] Effect of GPC3-1BZ treatment on body weight in non-tumor-bearing and tumor-bearing mice. The constructs used are shown in the legend on the right. BW: Body weight. ACT: Adoptive T cell therapy. TZ is GPC3-1 TZ. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 14]Tumor volume and blood sampling time in cytokine analysis of GPC3-1 BZ and GPC3-1 TZ. Arrows indicate blood sampling time for subsequent cytokine response testing. The constructs used are shown in the legend on the right. ACT: Adoptive T-cell therapy. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 15] Maximum systemic cytokine response (IFN-γ) GPC3-1 CAR T cell therapy. Blood sample data from day 8, when the maximum cytokine response was observed, is shown. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 16] Histological features of Hep3B tumor tissue in NOD scid gamma (NSG) immunodeficient mice. Top: Untreated control. Bottom: Animals treated with GPC3-1 BZ CAR T cells. Images are shown in 20× resolution. [Figure 17] Histological features of enteric nerve tissue in NOD scid gamma (NSG) immunodeficient mice. Left: Untreated control. Right: Animals treated with GPC3-1 BZ CAR T cells. [Figure 18] Quantification of GPC3 on the cell surface. From top to bottom: A375 cells (GPC3 negative), HepG2 cells (high GPC3), Hep3B cells (medium / low GPC3), and Huh7 cells (low GPC3). The area below the peak indicates the population of cells expressing the protein at the level shown on the x-axis. APC: Allophycocyanin. [Figure 19] Results of cytokine enzyme-linked immunosorbent assay (ELISA) after 24-hour exposure to GPC3-1 BZ T cells. Cell lines are listed from left to right in the order listed from top to bottom in the legend. [Figure 20] Immunohistochemical staining for GPC3 in representative tumor xenografts from two HCC cell lines. Both xenografts were scored with an intensity of 2. The data indicate that tumors with moderate intensity and at least 25% positive GPC3 expression respond to GPC3-1 BZ CAR-T. [Figure 21-1]Determination of relative surface GPC3 expression. FSC: Forward scattered light. APC: Allophycocyanin. MFI: Mean fluorescence intensity. The frequency of GPC3 in each gate is shown in the dot plot on the left (12.7%, 24%, and 9.34%, respectively). The histogram on the right shows the expression of GPC3 in the selected population, confirming purity and uniformity. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22] Cytokine ELISA after 24-hour exposure to GPC3-1 BZ. Results are shown for T cells only, A375 cells (GPC3-negative), and GPC3 low-expression, moderate-expression, and high-expression cells. Results are shown from left to right in each panel for each cell type, in the order listed from top to bottom in the legend. UT: Untransduced T cells. TZ and BZ: GPC3-1 TZ and GPC3-1 BZ. [Figure 23] Interferon-gamma (IFNγ) levels in different cell types after CAR T treatment. The construct used is shown on the x-axis. Results are shown from left to right for each construct in each cell type, in the order listed from top to bottom in the legend. TZ: GPC3-1 TZ. UT: Untransduced T cells. Moderate: Treatment with moderately affected cells only. [Figure 24] Cytokine levels in neuronal tissue cell types after treatment with GPC3-1 CAR T cells. The constructs used are shown on the x-axis. Results are shown from left to right for each construct in each cell type, in the order listed from top to bottom in the legend. UT: Untransduced T cells. Moderate: Treated with moderately elevated cells only. [Figure 25] Tumor volume after treatment with CAR T cells and anti-CRS-related cytokine antibodies. CRS = Cytokine Release Syndrome. Top: Tumor volume after treatment with different schemes of CAR and antibodies. Bottom: Studies of individual subjects treated with GPC3-1 BZ + PBS, GPC3-1 BZ + anti-IL-6, and GPC3-1 BZ + anti-TNF-α. MEDI7028 is GPC3-1 BZ. ACT: Adoptive T cell therapy. UT: Untransduced T cells. PBS: Phosphate-buffered saline. [Figure 26A]A study of higher-dose CAR-T and anti-TNFα therapy in a resistant HCC model (Huh7). Anti-TNFα was used to reduce toxicity and enhance antitumor activity with high-dose CAR-T. The constructs used are shown in the legend on the right. 26A. Study scheme. BW: Body weight. 26B. Tumor growth. iv: Intravenous administration. 26C. Change in body weight. [Figure 26B] This is a continuation of Figure 26A. [Figure 26C] This is a continuation of Figure 26B. [Modes for carrying out the invention]

[0049] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton, et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings attributed to them below.

[0050] As used herein, the terms “comprise” and “include,” and their variations (e.g., “comprises,” “comprising,” “include,” and “including”), shall be understood to include the described component, feature, element, or process, or group of components, features, elements, or processes, but not to exclude any other component, feature, element, or process, or group of components, features, elements, or processes. The terms “comprising,” “consisting essentially of,” and “consisting” may be substituted for any of the other two terms while retaining their usual meanings.

[0051] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0052] The percentages disclosed herein may vary by ±10, 20, or 30% from the disclosed values, but will remain within the intended scope of disclosure.

[0053] Unless otherwise indicated or unless otherwise evident from the context and the understanding of those skilled in the art, any value expressed as a range herein may be considered any specific value or subrange within the range described in the various embodiments of this disclosure, up to one-tenth of the lower limit of the range, unless otherwise clearly indicated by the content.

[0054] As used herein, ranges and quantities may be expressed as “approximately” a particular value or range. The term “approximately” also includes exact quantities. For example, “approximately 5%” means “approximately 5%,” and the term “approximately” also means “5%.” It can also refer to ±10% of a given value or range of values. Thus, approximately 5% also means, for example, 4.5% to 5.5%. Unless otherwise made clear from the context, all numerical values ​​provided herein are modified by the term “approximately.”

[0055] As used herein, the terms “or” and “and / or” can be used to describe multiple components in combination or mutually exclusive. For example, “x, y, and / or z” may refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0056] 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 one or more chains of two or more amino acids. Thus, the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to one or more chains of two or more amino acids, and the term “polypeptide” can be used instead of or interchangeably for any of these terms.

[0057] As used herein, "protein" may refer to a single polypeptide, i.e., a single amino acid chain as defined above, but may also refer to two or more polypeptides linked together by disulfide bonds, hydrogen bonds, or hydrophobic interactions to form a polymer protein.

[0058] An "isolated" substance, such as isolated nucleic acid, is a substance that is not found in its natural environment, although it is not necessarily purified. For example, isolated nucleic acid is a nucleic acid that is not produced or located in its natural or natural environment (e.g., a cell). An isolated substance can be separated, fractionated, or at least partially purified by any appropriate technique.

[0059] As used herein, the terms “antibody” and “its antigen-binding fragment” refer to at least the smallest portion of an antibody capable of binding to a specific antigen targeted by the antibody, for example, in relation to a typical antibody produced by B cells, at least some of the complementarity-determining regions (CDRs) of the heavy chain (VH) variable domain and the light chain (VL) variable domain. Antibodies or their antigen-binding fragments may be polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv(scFv), single-chain antibodies, disulfide-bonded Fv(sdFv), fragments containing a VL or VH domain alone or in combination with a portion of the opposite domain (e.g., the entire VL domain and a partial VH domain having one, two, or three CDRs), as well as fragments produced by or derived from Fab expression libraries. The scFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019. The antibody molecules included in this disclosure may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, or derived therefrom.

[0060] As used herein, the term “polynucleotide” includes single nucleic acids and multiple nucleic acids and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term “nucleic acid” includes any nucleic acid type, such as DNA or RNA.

[0061] 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, such as an origin of replication, that enables replication in the host cell. A vector may also include one or more select marker genes and other genetic elements known in the art. Certain types of vectors envisioned herein may be conjugated to or incorporated into a virus to facilitate cell transformation.

[0062] "Transformed" cells, or "host" cells, are cells into which nucleic acid molecules have been introduced using molecular biological techniques. Techniques for introducing nucleic acid molecules into such cells include, for example, transfection using viral vectors, transformation using plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0063] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (such as an epitope) to its homozygous domain (such as a paratope). As used herein, the term "avidence" refers to the overall stability of the complex between the population of epitopes and paratopes (i.e., the antigen and the antigenic domain).

[0064] As used herein, the terms “to treat,” “to treat,” or “treatment of” mean, when used in connection with the treatment of cancer, to alleviate disease symptoms, reduce or eliminate disease symptoms, promote increased survival rates, and / or reduce discomfort. For example, treatment may mean the ability of a therapy to alleviate the symptoms, signs, or causes of a disease when administered to a subject. Treatment may also mean the alleviation or reduction of at least one clinical symptom and / or the inhibition or delay of disease progression and / or the prevention or delay of the onset of disease or illness.

[0065] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject, particularly mammalian subjects, for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and others.

[0066] As used herein, the terms “effective dose” or “therapeutic effective dose” of a therapeutic substance administered, for example, CAR T cells, are sufficient to accomplish a particular stated or intended purpose, such as the treatment of cancer. The “effective dose” can be determined based on routine experiments with respect to the stated purpose.

[0067] 2. Overview This disclosure relates to compositions and methods for treating cancer using chimeric antigen receptor (CAR) cell therapy. More specifically, this disclosure relates to CAR cell therapy in which transformed cells, such as T cells, express CARs that target glypican 3 (GPC3). Furthermore, the CAR constructs, transformed cells expressing the constructs, and therapies utilizing the transformed cells disclosed herein may provide robust cancer treatment with minimal risk of cytokine release syndrome (CRS) or indiscriminate cytokine release in non-GPC3 expressing cells.

[0068] While we do not wish to be constrained by theory, GPC3 appears to be a viable cancer target across multiple modalities, including bispecific T cell engagers, CAR cells, and monoclonal antibodies and antibody-drug conjugates (ADCs). The carcinoembryonic antigen GPC3 is a GPI-conjugated heparin sulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction and stimulates Wnt signaling. GPC3 competes with patched for Hh binding, mitigates smoothed inhibition, and induces GPC3 degradation. Both pathways have been shown to stimulate hepatocellular carcinoma (HCC) proliferation. Furthermore, GPC3 expression levels have been shown to correlate with the stage and malignancy of HCC.

[0069] Furthermore, GPC3 is considered a promising target for CAR cell therapy. Therefore, antibodies and CAR constructs derived from these antibodies are being developed, as described herein.

[0070] 3. Design of CAR structures The CAR constructs of this disclosure may have several components, many of which can be selected based on the desired or refined function of the resulting CAR construct. In addition to the antigen-binding domain, the CAR construct may have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. Selecting one component from another (i.e., selecting a specific costimulatory domain of one receptor for a costimulatory domain of a different receptor) may affect the clinical efficacy and safety profile.

[0071] 4. Antigen-binding domain The antigen-binding domains intended herein may include an antibody or one or more antigen-binding fragments thereof. One intended CAR construct targeting GPC3 includes a single-chain variable region fragment (scFv) containing light and heavy chain variable regions derived from one or more antibodies specific to GPC3, which are either directly linked or linked via a flexible linker (e.g., repeats of GGGS having 1, 2, 3 or more repeats).

[0072] The antigen-binding domain of GPC3-targeting CARs, such as those disclosed herein, may exhibit altered binding affinity to the GPC3 protein. The relationship between binding affinity and potency can be more nuanced in relation to CARs compared to antibodies, where higher affinity is generally desirable. For example, preclinical studies on receptor tyrosine kinase-like orphan receptor 1 (ROR1)-CARs derived from high-affinity scFv (dissociation constant 0.56 nM) showed increased therapeutic index compared to low-affinity variants. Conversely, other examples have reported that manipulating scFv to lower affinity improves the identification of cells with altered antigen density. This may be useful for improving therapeutic specificity for antigens differentially expressed in tumor versus normal tissue.

[0073] Various methods can be used to confirm the binding affinity of the antigen-binding domain. In some embodiments, methodologies that exclude the avidity effect can be used. The avidity effect often involves multiple antigen-binding sites that interact simultaneously with multiple target epitopes in a multimerized structure. Thus, avidity functionally represents the cumulative strength of multiple interactions. An example of a methodology that excludes the avidity effect is that one or both of the interacting proteins are monomeric / monovalent, since multiple simultaneous interactions are not possible if one or both partners contain only a single interaction site.

[0074] 5. Spacer Domain The CAR constructs of this disclosure may have a spacer domain that provides conformational freedom to facilitate binding to the target antigen on the target cell. The optimal length of the spacer domain may depend on the proximity of the binding epitope to the target cell surface. For example, a proximal epitope may require a longer spacer, and a distal epitope may require a shorter epitope. In addition to promoting the binding of the CAR to the target antigen, achieving the optimal distance between the CAR cell and the cancer cell may also help sterically occlude large inhibitory molecules from the immunological synapse formed between the CAR cell and the target cancer cell. CARs targeting GPC3 may have a long spacer, an intermediate spacer, or a shorter spacer. The long spacer may contain the CH2CH3 domain (about 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (natural or with modifications common to therapeutic antibodies such as the S228P mutation), while the CH3 region can itself be used to construct an intermediate spacer (about 120 amino acids). Shorter spacers may originate from segments (less than 60 amino acids) of CD28, CD8α, CD3, or CD4. Short spacers may also originate from hinge regions of the IgG molecule. These hinge regions may originate from any IgG isotype and may or may not contain mutations common in therapeutic antibodies, such as the S228P mutation mentioned above.

[0075] 6. Hinged Domain CARs targeting GPC3 may also possess a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on tumor cells. It can be used alone or in conjunction with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably, and for example, an IgG4 sequence can be considered both a "hinge" and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0076] CARs targeting GPC3 may further contain sequences containing signal peptides. These signal peptides stimulate cells to translocate the CAR to the cell membrane. Examples include IgG1 heavy chain signal polypeptide, Ig kappa or lambda light chain signal peptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal polypeptide, or CD33 signal peptide.

[0077] 7. Transmembrane domain CARs targeting GPC3 may further contain sequences including a transmembrane domain. This transmembrane domain may include a hydrophobic α-helix spanning the cell membrane. While the properties of the transmembrane domain have not been studied in as much detail as other aspects of CAR constructs, they may potentially influence CAR expression and its association with endogenous membrane proteins. The transmembrane domain may originate from, for example, CD4, CD8α, or CD28.

[0078] 8. Co-stimulatory domain CARs targeting GPC3 may further contain one or more sequences that form a co-stimulatory domain. A co-stimulatory domain is a domain that can enhance or modulate the response of immune effector cells. Co-stimulatory domains may include sequences from one or more of the following, for example: CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The selection of the co-stimulatory domain affects the phenotype and metabolic signature of CAR cells. For example, CD28 co-stimulation results in a potent but short-lived effector-like phenotype with high levels of cytolytic ability, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells modified with CARs having a 4-1BB co-stimulatory domain tend to enlarge and persist longer in vivo, have increased oxidative metabolism, are less prone to depletion, and have an increased ability to generate central memory T cells.

[0079] 9.Cells CAR-based cell therapy can be used with various cell types, including lymphocytes. Specific cell types that can be used include T cells, natural killer (NK) cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, alpha-beta T cells, gamma-delta T cells, virus-specific T (VST) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells (Tregs). In one embodiment, the CAR cells used to treat the target are autologous. In other embodiments, the CAR cells may be derived from a genetically similar but not identical donor (allogeneic).

[0080] 10. CAR cell production The CAR constructs of this disclosure may include some combination of the modular components described herein. For example, in some embodiments of this disclosure, the CAR construct includes a GPC3-1 scFv antigen-binding domain. In some embodiments, the CAR includes a GPC3-2 scFv antigen-binding domain. In some embodiments of this disclosure, the CAR construct includes a CSFR2 signal peptide. In some embodiments, the CAR construct includes an IgG4P hinge / spacer domain having an S228P mutation. In some embodiments, the CAR construct includes a CD28 transmembrane.

[0081] In the CAR constructs of this disclosure, different co-stimulatory domains can be utilized. In some embodiments, the CAR construct includes a co-stimulatory domain derived from the intracellular domain of CD3z. In some embodiments, the CAR construct includes a CD28 co-stimulatory domain. In some embodiments, the CAR construct includes a 4-1BB co-stimulatory domain. In some embodiments, the CAR construct includes co-stimulatory domains derived from CD3z and CD28. In some embodiments, the CAR construct includes co-stimulatory domains derived from CD3z and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains derived from all of CD3z, CD28, and 4-1BB. In some embodiments, the CAR construct includes co-stimulatory domains derived from ICOS, OX-40, and / or GITR.

[0082] 11. Evaluation of CAR structures The constructs of this disclosure were compared and evaluated based on safety and the persistence and establishment of central memory. The low-affinity (high offrate) scFv GPC3-1 was well-rated for its good safety. The 4-1BB and CD3z co-stimulatory domains (both in the same construct) were well-rated based on their good persistence and contribution to a favorable in vivo phenotype (greater central memory). The GPC3-1 CAR and GPC3-2 CAR of this disclosure were comparable to published GPC3-targeted CAR-based constructs. Further evaluation details can be found in the examples.

[0083] 12. Embodiments In some embodiments, this disclosure provides isolated nucleic acid sequences encoding chimeric antigen receptors (CARs). The CARs include an antigen-binding domain specific to glypican 3 (GPC3). The antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. D The CAR construct has ) and does not induce cytokine production in GPC3 cells. In some embodiments, the antigen-binding domain includes an antibody or its antigen-binding fragment. The antigen-binding domain may be a Fab or a single-chain variable region fragment (scFv). In some embodiments, the antigen-binding domain is an scFv containing the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0084] In some embodiments, the CAR further comprises a transmembrane domain, a co-stimulatory domain, and a signaling domain. The transmembrane domain may be a CD28 transmembrane domain. The co-stimulatory domain may be one or more of the CD28, 4-1BB, CD3 zeta, OX-40, ICOS, CD27, GITR, and MyD88 / CD40 co-stimulatory domains. In one particular embodiment, the co-stimulatory domain is one or more of the CD28, 4-1BB, and CD3 zeta co-stimulatory domains. The signaling domain may be a sequence encoding a CSFR2 signal peptide.

[0085] In some embodiments, the isolated nucleic acid sequence may contain a hinge / spacer domain. The hinge / spacer domain may be an IgG4P hinge / spacer.

[0086] In some specific embodiments, the isolated nucleic acid sequence encoding the chimeric antigen receptor (CAR) may have the sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 26.

[0087] In other embodiments, the disclosure provides an anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain. The antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the VH may have CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the VL may have CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0088] In some embodiments, VH may be the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29, and VL may be the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, CAR may further have a transmembrane domain, a co-stimulatory domain, and a signaling domain.

[0089] 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.

[0090] In other embodiments, the disclosure provides a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). The nucleic acid sequence may be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0091] In other embodiments, the disclosure provides cells comprising a vector having the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0092] In other embodiments, the disclosure relates to cells having a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. D The CAR construct has ) and provides cells that do not induce cytokine production in GPC3 cells. For example, the nucleic acid sequence may be SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

[0093] In other embodiments, the disclosure provides cells expressing an anti-GPC3 chimeric antigen receptor (CAR) on their extracellular surface. The CAR may have an antigen-binding domain which may be an antibody, Fab, or scFv having a heavy chain variable region (VH) and a light chain variable region (VL). The VH may include CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39. The VL may include CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

[0094] In some embodiments, VH may have the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 29. In some embodiments, VL may have the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. CAR may further include a transmembrane domain, a costimulatory domain, and a signaling domain. The cell expresses a CAR having the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

[0095] In some embodiments, the disclosure provides T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and / or regulatory T cells expressing CAR on their extracellular surface, the CAR of which may have the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25. Such cells may exhibit anti-tumor immunity upon contact with tumor cells expressing GPC3.

[0096] 13. Cancer treatment with CAR In some embodiments, this disclosure provides CAR cells for the treatment of cancer. The compositions described herein (e.g., antibodies, CAR constructs, and CAR cells) and methods of use thereof are particularly useful for inhibiting the proliferation or spread of tumor cells, in particular for inhibiting the proliferation of tumor cells in which GPC3 plays a role.

[0097] Tumors treatable by the compositions of this disclosure include solid tumors, such as tumors of the liver, lungs, or ovaries. However, the cancers listed herein are not intended to be limiting. For example, the types of cancer intended for treatment in this specification include, for example, NSCLC, advanced solid malignant tumors, cholangiocarcinoma, bladder cancer, colorectal cancer, diffuse large B-cell lymphoma, esophageal tumors, esophageal squamous cell carcinoma, advanced small cell lung cancer, gastric adenocarcinoma, gastric cancer, gastroesophageal junction cancer, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, lung cancer, melanoma, mesothelioma, metastatic clear cell renal cancer, metastatic melanoma, metastatic noncutaneous melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin 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 carcinoma, renal cell carcinoma This includes carcinoma, SCCHN, pharyngopituitary squamous cell carcinoma, laryngeal squamous cell carcinoma, small cell lung cancer, squamous cell carcinoma of the head and neck, squamous cell lung cancer, TNBC, transitional cell carcinoma, unresectable or metastatic melanoma, urothelial cancer, and urothelial carcinoma.

[0098] In one embodiment, cancers intended for treatment herein include any cancer that expresses GPC3 on the cell surface of cancer cells. In one specific example, cancers intended for treatment herein include hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and squamous cell lung cancer.

[0099] 14. Treatment CAR-modified cells of the present invention, such as CAR T cells, may be administered alone or as part of a pharmaceutical composition containing diluents and / or other components related to cytokines or cell populations. Briefly, a pharmaceutical composition of the present invention may, for example, contain CAR T cells, as described herein, together with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such a composition may include buffers such as neutral buffered saline or buffered saline; sulfates; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; amino acids such as proteins, polypeptides, or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. A pharmaceutical composition of the present invention can be adapted for therapeutic (or preventive) use.

[0100] CAR-modified cells may also be administered in combination with one or more additional therapeutic agents. In one embodiment, anti-cytokine antibodies may be included as additional therapeutic agents. For example, one or more anti-TNFα antibodies may be used to reduce toxicity at high CAR T doses, which may be associated with CRS-like symptoms and weight loss, and to enhance antitumor activity.

[0101] In certain embodiments, the intended treatment regimen may include one or more biological components, such as CAR T cells and anti-cancer antibodies, and / or chemotherapy components. For example, the treatment regimen may further include other cancer immunotherapy (IO) therapies, such as immune checkpoint inhibitors (ICIs) and immune system agonists, such as those targeting the PD-1 / PD-L1 axis (PDX).

[0102] Antibodies intended include anti-PD-L1 antibodies such as durvalumab (MEDI4736), avelumab, atezolizumab, and KNO35; anti-PD-1 antibodies such as nivolumab, pembrolizumab, REGN2810, SHR1210, IBI308, PDR001, anti-PD-1, BGB-A317, BCD-100, and JS001; and anti-CTLA4 antibodies such as tremelimumab or ipilimumab. Further antibodies are also intended herein. Antibody subparts effective for treatment are also intended herein.

[0103] Information regarding durvalumab (or its fragments) used in the methods provided herein can be found in U.S. Patent No. 8,779,108, No. 9,493,565, and No. 10,400,039, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, durvalumab or its antigen-binding fragment used in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H9OPT antibody disclosed in the above U.S. patents.

[0104] Information regarding tremelimumab (or its antigen-binding fragment) used in the methods provided herein can be found in U.S. Patent No. 6,682,736 (in this document, tremelimumab is referred to as 11.2.1), the disclosures of which are incorporated herein by reference in their entirety.

[0105] Further therapeutic agents (chemotherapeutic agents or biologics) intended herein include, but are not limited to, cisplatin / gemcitabine or methotrexate, vinblastine, ADRIAMYCIN® (doxorubicin), cisplatin (MVAC), carboplatin-based regimens, or monotherapy with taxanes or gemcitabine, temozolomide, or dacarbazine, vinflunin, docetaxel, paclitaxel, nab-paclitaxel, vemurafenib, erlotinib, afatinib, cetuximab, bevacizumab, erlotinib, gefitinib, and / or pemetrexed. Further examples include drugs that target the DNA damage repair system, such as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors, as well as therapeutic agents that inhibit WEE1 protein kinase activity, ATR protein kinase activity, ATM protein kinase activity, Aurora B protein kinase activity, and DNA-PK activity.

[0106] The therapeutic compositions or methods intended herein may be combined with one or more of the other therapeutic compositions and methods provided herein.

[0107] In some embodiments, the disclosure provides a method for treating cancer, comprising administering cells containing an anti-GPC3 chimeric antigen receptor (CAR) comprising an effective amount of antigen-binding domain to a subject in need of cancer treatment. The antigen-binding domain may be an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). The VH may comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 37, CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR3 comprising the amino acid sequence of SEQ ID NO: 39. The VL may comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. In some embodiments, the method further inhibits tumor growth, induces tumor regression, and / or extends the survival of the subject.

[0108] In some embodiments, the cells are self-cells. For example, the self-cells may be selected from a group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

[0109] In some embodiments, the cancer treated by this method is a solid tumor. For example, the cancer may be hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer. In certain embodiments, the cancer is hepatocellular carcinoma.

[0110] This disclosure provides a method for treating cancer, comprising administering to a subject in need of cancer treatment cells containing an effective amount of anti-GPC3 chimeric antigen receptor (CAR) and an effective amount of anti-TNFα antibody.

[0111] It should be understood that the specific embodiments described herein are not limited to the specific embodiments presented and can be modified. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting unless specifically defined herein. Furthermore, the specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation, as will be recognized by those skilled in the art. [Examples]

[0112] The following examples illustrate specific embodiments of the Disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the Disclosure. A glossary of terms is provided in Table 1.

[0113] [Table 1]

[0114] Example 1: Expression of GPC3 method GPC3 IHC was performed using the mouse monoclonal anti-human GPC3 antibody GC33 (Ventana). Secondary staining was performed using anti-mouse HRP. Human tissue microarrays (TMA, US Biomax) representing hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), ovarian cancer, or human colonic ganglion tissue were stained for GPC3 expression, and staining intensity and pattern were measured by microscopy.

[0115] result GPC3 overexpression is observed in 80% of HCC, 30% of squamous cell lung cancer, and 47% of ovarian clear cell carcinoma. However, GPC3 is undetectable by immunohistochemistry in normal liver tissue, including cirrhotic and hyperplastic samples, and its expression is low in normal tissue (e.g., lung). See Figures 1A and 1B.

[0116] Example 2: Development and affinity testing of scFv. overview In this example, anti-GPC3 scFv was developed, and their relative affinity for GPC3 was determined.

[0117] method GPC3-1 (SEQ ID NO: 1) and GPC3-2 (SEQ ID NO: 2) are almost identical. H It has domains (sequences 27 and 29), but V L The domains (SEQ ID NOs. 28 and 30; see Figure 2) are different. GPC3-2 has a complete germline framework, while GPC3-1 does not.

[0118] The apparent binding affinity was determined by the cell surface binding of soluble recombinant GPC3 protein to GPC3-1CAR and GPC3-2CAR expressed on the surface of Jurkat cells. The CAR constructs were expressed on the surface of Jurkat cells using a lentiviral vector. The cells were stained with recombinant His-tagged GPC3 protein (R&D systems) at various concentrations. The bound GPC3 was visualized by staining with a fluorescent-conjugated anti-His tag secondary antibody, and the cells were analyzed by flow cytometry. The binding curve was fit to a simple one-site binding model to determine the apparent K D was determined.

[0119] Alternative measurements of binding affinity were obtained using a BIAcore surface plasmon resonance system and GPC3-1 and GPC3-2 scFv-Fc fusion proteins. The purified scFv-Fc fusion molecules of GPC3-1 and GPC3-2 were covalently coupled to an amine-reactive SPR sensor chip (CM5, GE Healthcare). For GPC-1, soluble GPC3 protein (R&D Systems) was flowed over the chip surface at concentrations of 14, 28, 57, 114, and 228 nM and at a rate of 30 μL / min, and the interaction was monitored. For GPC3-2, concentrations of 4, 7, 14, 28, and 57 nM were flowed at the same flow rate. The data were fit using BIAevaluation software (GE Healthcare) and a simple 1:1 Langmuir binding model, and R max The fit was performed globally for k a , k d and K D The fit was performed locally.

[0120] Results In experiments evaluating soluble GPC3 binding to GPC3-1CAR and GPC3-2CAR expressed on the surface of Jurkat cells, K dThe values ​​were approximately 15 nM for GPC3-1 and 5 nM for GPC3-2 (see Figure 3A). Surface plasmon resonance experiments using the same GPC3 protein and purified GPC3-1 / GPC3-2scFv-Fc fusion protein used for cell-based binding showed that the K levels of GPC3-1 and GPC3-2 were high. D The values ​​were approximately 73 nM and 11 nM, respectively. See Table 1 and Figures 3B and 3C.

[0121] [Table 2]

[0122] Table 2 shows the reported Kd values ​​for the four scFvs.

[0123] [Table 3]

[0124] Example 3. Development of a CAR structure and in vitro testing. overview In this example, an anti-GPC3 CAR construct was developed, and its cytokine activity and multifunctionality were tested.

[0125] method CAR structure. All CAR constructs used the CSFR2 signaling peptide (used in many clinical-stage CAR T constructs). The IgG4P (S228P mutation) hinge domain was used as a "spacer" between the scFv and the membrane, and the CD28 transmembrane domain was used. Different costimulatory domains were tested intracellularly, including various combinations of CD28, 4-1BB, and CD3 zeta costimulatory domains. Constructs using costimulatory domains from inducible T cell costimulatory factor (ICOS), OX40, and glucocorticoid-induced TNFR family-related genes (GITR) were also tested. The sequences of the GPC3-1 and GPC3-2 CAR constructs are shown in SEQ ID NOs: 3-10, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 11-18.

[0126] Other known CARs for GPC3 (based on GPC3-3 and GPC3-4 scFv) were constructed for comparison with the GPC3-1 and GPC3-2 CAR constructs. The GPC3-3 CAR contained a short IgG1 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular domain. Another GPC3-3 CAR construct with both CD28 and 4-1BB costimulatory domains was also developed. The GPC3-4 CAR construct contained an IgG4P hinge, a CD28 transmembrane domain, and a 4-1BB costimulatory domain. The sequences of the GPC3-3 and GPC3-4 CARs are shown in SEQ ID NOs: 19-21, and the corresponding nucleic acid sequences are shown in SEQ ID NOs: 22-24.

[0127] Generation of CAR T cells. Purified human T cells are divided into 0.2 × 10⁻⁶ cells. 6 Cells were seeded in AIM-V medium containing 5% human serum and 1% penicillin-streptomycin at a concentration of cells / mL + IL-2 (300 IU / mL). T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Invitrogen), and transduction was performed by spinoculation 24 hours later. Lentiviruses were added to the wells (MOI 100), the plates were centrifuged at 2000 rpm at 37°C for 2 hours, and placed in a 37°C, 5% CO2 incubator. The cell density was approximately 0.5–1 × 10⁶. 6 Cells were divided as needed to maintain the cell / mL concentration. The immunophenotype of CAR-T cells was first analyzed 7 days after transduction, and CAR-T cells were evaluated by in vitro and in vivo functional assays approximately 11 days after transduction.

[0128] Cell line testing. Various cell types were treated with CAR T cells containing different CAR constructs. In all cytokine tests, 5 × 10⁶ cells were used. 4Nine CAR-T cells were co-cultured with target cells in a 1:1 ratio in RPMI 10% FCS. After 24 hours, the supernatant was collected. Cytokines were analyzed using the Meso Scale Discovery 4-plex kit to detect IFN-γ, IL-2, TNF-α, and IL-10. Cytokine concentrations (picograms / milliliter) were measured.

[0129] Cytotoxicity tests were performed using cell impedance monitoring technology (xCELLigence). 3 × 10 4 Target cells were plated, and CAR-T cells were added 24 hours later in effector:target (E:T) ratios of 3, 1, or 0.3. After treatment with various CAR constructs, normalized cell indices were determined for Hep3B, Huh7, and SNU-182 cells. Hep3B expressed high GPC3 (14k / cell), Huh7 expressed medium / low GPC3 (7k / cell), and SNU-182 was GPC3-negative (0 / cell).

[0130] Multifunctionality tests were also conducted on the CAR constructs. Here, GPC3-1 BZ or the indicated CAR-T cells were co-cultured with Hep3B or A375 for 6 hours in the presence of Golgi Stop and a fluorescently labeled antibody against the degranulation marker CD107a. Binding to the target induced CAR-T degranulation, which in turn induced binding of the fluorescently labeled anti-CD107 present in the culture medium. The accumulation of CD107 detected by flow cytometry was directly proportional to the degree of degranulation, indicating target cell lysis. Since the cells were incubated in the presence of Golgi Stop, the production of effector cytokines (IFN-γ, IL-2, TNF-α) could also be evaluated by intracellular staining. Boolean gates combining each function (CD107a, IFN-γ, IL-2, and TNF-α) were generated using Flowtop, and a pie chart of the results was generated using Spice analysis software.

[0131] result The GPC3-2 and GPC3-3 constructs generally produced higher levels of TNFα and IL-2 compared to GPC3-1. Treatment with CAR T cells containing the GPC3-1 and GPC3-2 CAR constructs induced antigen-specific cytokines. On the other hand, the GPC3-4 BZ construct induced cytokines even in GPC3-negative cell types. The GPC3-1 and GPC3-2 constructs did not produce cytokines in the absence of the target. Cytokine production appears to be dependent on antigen density and antigen affinity. See, for example, Figures 4A and 4B.

[0132] The GPC3-1 and GPC3-2 constructs showed cytotoxicity only against GPC3-expressing cells, while the GPC3-4 construct showed cytotoxicity against both GPC3-positive cells (Hep3B and Huh7) and GPC3-negative cells (SNU-182). Low-affinity CARs (GPC3-1 BZ) exhibited cytotoxicity comparable to high-affinity (GPC3-2 BZ) CARs. See Figure 5.

[0133] GPC3-1 BZs exhibited cytotoxicity against HCC cell lines expressing low levels of GPC3. All target cells tested showed high susceptibility to GPC3-1 killing at E:T ratios of 3:1 and 0.3:1, with only one cell line with low GPC3 expression showing reduced death at an E:T ratio of 0.3:1. However, the completely equivalent death rates observed in our isogeneic GPC3-high and low Hep3B cell lines suggest that reduced antigen density is not a key factor limiting CAR-T mediated cytolysis. See Figure 6.

[0134] Both GPC3-2 and GPC3-1 CAR T cells are multifunctional regardless of the intracellular domain used. GPC3-1 BZ CAR T cells were multifunctional, with a higher proportion of cells exhibiting 2+ function. Furthermore, CAR T cells possessing CD28 were less multifunctional in vitro than CAR T cells possessing the 4-1BB intracellular domain. See Figure 7.

[0135] conclusion Treatment with GPC3-1 resulted in the lowest overall cytokine production of the tested CARs. Both GPC3-1 and GPC3-2 were multifunctional and showed specific cytotoxicity against cells expressing GPC3.

[0136] Example 4: In vivo testing of multiple CAR constructs in an animal model of hepatocellular carcinoma overview In this example, anti-GPC3 CAR constructs were tested in vivo, and their effects on body weight, tumor size, and survival were compared.

[0137] method 5 x 10 6 Hep3B cells were transplanted into the flanks of NSG mice (10 mice / group). The average tumor volume was 150 mm². 3 At this stage, mice were administered 4 million GPC3-2 BZ or GPC3-1 BZ. Body weight, tumor volume (2x / week), and survival were monitored. Animals whose body weight decreased to 80-90% were given nutritional supplements, and those whose body weight decreased to less than 80% were euthanized. The survival event (death) occurred at 1500 mm. 3 The determination was made based on tumor size exceeding a certain threshold. Each experiment was performed twice.

[0138] result Weight loss was observed with the use of the high-affinity GPC3-2 construct but not with the low-affinity GPC3-1 construct, indicating that the low-affinity binder is less toxic in vivo. GPC3-2-based CAR T cells were not tolerable at in vivo doses equivalent to those of GPC3-1-based CAR T cells. The greater toxicity of GPC3-2 BZ correlated with extensive infiltration of CAR T cells in the lungs of normal mice. Only a slight level of infiltration was observed in the lungs of mice treated with GPC3-1 BZ. See Figures 8A and 8B.

[0139] GPC3 CAR T induced Hep3B tumor regression in NSG mice. GPC3-1 BZ showed superior antitumor activity compared to GPC3-3 BZ and GPC3-4 BZ. See Figure 9.

[0140] GPC3-1 and GPC3-2 CAR T cells significantly extended the survival time of tumor-bearing NSG mice compared to GPC3-3 or GPC3-4 CAR T cells (p<0.01 vs. GPC3-3 BZ; Kaplan-Meier and Mantelcox-Logrank). See Figure 10. Similarly, it was determined that WPRE deletion did not have a negative functional effect on GPC3-1 BZ cells in vitro or in vivo.

[0141] conclusion Of the CARs tested, GPC3-1 BZ and GPC3-2 BZ showed the greatest antitumor activity and provided the greatest survival benefit. GPC3-1 BZ was less toxic and infiltrated normal tissue less than GPC3-2 BZ.

[0142] Example 5: In vivo comparison of GPC3-1 CAR structures overview In this example, multiple GPC3-1 CAR constructs containing different signaling domains were tested and compared in vivo.

[0143] method Analysis of differentiation and depletion. Differentiation and depletion of multiple GPC3-1 CAR constructs were investigated. Mice with Hep3B tumors were treated with CAR-T cells having different signaling domains (TZ=GPC3-1 TZ; BZ=GPC3-1 BZ; 28Z=GPC3-1 28Z), and the spleen and tumors were analyzed by flow cytometry 7 days after cell injection. Differentiation and depletion were assayed using FACs that detect multiple markers in spleen and tumor cells. The differentiation state of T cells was analyzed by combined expression of CD62L and CD45RO (CD62L+ / CD45RO-=naive; CD62L+ / CD45RO+=central memory; CD62L- / CD45RO+=effector memory; CD62L- / CD45RO-=effector memory cells re-expressing CD45RA (EMRA)). CD3% was used as a measure of persistence and expansion.

[0144] 5x10 on the mouse 6 Injecting individual Hep3B cells resulted in an average size of 150 mm. 3 The tumor was established. 4 million GPC3-1 BZ or GPC3-1 TZ T cells were administered to non-tumor-bearing mice or mice with Hep3B tumors. The effect on body weight in both tumor-bearing and non-tumor-bearing mice was measured up to 35 days after administration. Tumor volume was also assayed twice a week. Blood was collected regularly from the animals after administration for analysis of IFN-γ and TNF-α in the blood. Cytokines in the serum were analyzed 8 days after CAR-T administration. Each experiment was performed twice.

[0145] To investigate the potential peripheral neurotoxicity in GPC3+ tumor-bearing (Hep3B HCC strain) and non-tumor-bearing NSG mice, human anti-GPC3 CAR-T cells were administered to the animals. Histological examinations were performed on tumors and enteric nerve tissue from animals with Hep3B tumors treated with GPC3-1 BZ.

[0146] [Table 4]

[0147] result GPC3 CAR-T cells possessing a 4-1BB / CD3 zeta (BZ) signaling domain exhibit greater central memory and less depletion in vivo than CD28 / CD3 zeta (28Z) cells. The results indicate that splenic GPC3-1 BZ CAR T cells are less differentiated than GPC3-1 28Z CAR T cells, while retaining the ability to fully activate and differentiate in antigen-present tumors. See Figures 11A-11D.

[0148] GPC3-1 BZ showed persistence. Expression of the activation / waste markers LAG3 and PD1 confirmed that GPC3-1 BZ CAR T cells maintained fewer activated / wasted cells in the periphery. See Figure 12.

[0149] GPC3-1 BZ did not induce weight loss in either tumor-bearing or non-tumor-bearing mice at tumor regression doses. See Figure 13.

[0150] Complete tumor regression was observed only in GPC3-1 BZ CAR T cell-treated mice. See Figure 14.

[0151] Very small amounts of systemic cytokines (transient elevations of IFN-γ and TNF-α measured 7 days after the 8-day infusion) were detected. At effective CAR T doses, only very small and transient systemic cytokines were detected, and no weight loss was observed. Only human IFN-γ and TNF-α were transiently detected at elevated serum levels after the regression dose of CAR therapy. Further human or mouse cytokine levels, including hIL-2, mIL-10, mIL-6, mTNFα, and mIFNγ, were below the detection limit (BDL). See Figure 15.

[0152] As the tumor regressed, extensive T-cell infiltration and expansion were observed within the tumor. The tumors shrank due to a decrease in tumor cells, became necrotic, and were infiltrated by mononuclear cells. The mice used lacked lymphocytes, and therefore, any mononuclear cell infiltration is considered to be human CAR-T cells. See Figure 16. The enteric nervous system, which expresses only low levels of GPC3, was unaffected. See Figure 17. Very slight mononuclear cell infiltration was observed in the lungs and liver (data not shown).

[0153] conclusion Compared to constructs with other signaling domains, the GPC3-1 BZ construct is persistent, promotes the central memory response, and shows increased activity against tumors. Furthermore, treatment causes only a transient increase in some cytokines and does not cause weight loss. Treatment leads to T cell infiltration and necrosis in tumors, but normal tissues remain unaffected.

[0154] Example 6: Further characterization of GPC3-1 BZ CAR T cells overview In this example, GPC3-1 BZ CAR T cells were further characterized in treatment with cells of various tumor types and with different GPC3 expression levels. Cytokine responses were analyzed.

[0155] method Cytokine levels in response to treatment with GPC3-1 BZ CAR T cells were investigated in tumor types with various GPC3 expression levels. Immunohistochemical staining was also performed on representative Hep3B and Huh7 tumor xenografts.

[0156] GPC3 expression analysis was also performed on cells within tumor types. Staining intensity was graded on a scale of 1 to 4, with 1 being the lowest intensity and 4 being the highest. A staining intensity of 2 indicates low / medium intensity. Relative GPC3 expression was determined by FACs. GPC3 expression on Hep3B cells was determined by surface staining with a fluorescently labeled anti-GPC3 antibody and subsequent flow cytometry analysis. Based on GPC3 expression, Hep3B cells were gated as low, medium, or high expression cells, and the frequency of GPC3 in each gate was plotted.

[0157] Cytokine levels were measured by ELISA. Cell lines were co-cultured with GPC3-1 BZ CAR T cells in RPMI 10% FCS in a 1:1 ratio. After 24 hours, the supernatant was collected and cytokines were analyzed using the Meso Scale Discovery 4-plex Kit, detecting IFN-γ, IL-2, TNF-α, and IL-10. Cytokine analysis was performed after exposing cells to GPC3-1 BZ T cells for 24 hours. Cytokine levels were tested in different cell types after GPC3-1 BZ treatment.

[0158] result In GPC3-expressing cell lines, GPC3-1 BZ CAR T cell-induced cytokine production was proportional to surface GPC3 expression. See Figures 18-22.

[0159] GPC3-1 BZ CAR T cells did not induce a cytokine response in GPC3-negative or normal tissues. See Figures 23 and 24.

[0160] conclusion GPC3-1 BZ CAR T cells induce cytokine production at levels proportional to the GPC3 expression of the treated cells.

[0161] Example 7: Treatment with GPC3-1 CAR T cell construct and anti-cytokine antibody overview In this example, GPC3-1 CAR T cell therapy was attempted in combination with an anti-cytokine antibody.

[0162] method Tumors were treated with different combinations of GPC3-1 CAR T cell therapy and anti-cytokine antibodies. Hep3B tumor mice (10 mice / group) were administered 5 million GPC3-1 BZ or GPC3-1 TZ-transformed cells (TZ = truncated CD3 zeta, a non-signaling negative control) in or without 100 μg of anti-human TNFα (golimumab, Janssen) or anti-mouse IL-6 (Bio X Cell).

[0163] Using the HCC resistance model Huh7, high doses (1e7-3e7) of GPC3-1 CAR T cells were tested in combination with anti-TNF-α administration at two different time points. Mice with Huh7 tumors (10 mice / group) were administered the indicated doses of GPC3-1 BZ T cells (10 million or 3,000 cells), and 100 μg of anti-TNFα was administered on the same day as CAR T administration (day 0) or two days after the start of treatment (day 2).

[0164] result Blocking TNF-α instead of IL-6 eliminated the effectiveness of GPC3-1 benzoate. See Figure 25.

[0165] In the resistant HCC model Huh7, higher doses of CAR T cells were required to induce tumor growth inhibition, but these higher doses were also associated with CRS-like symptoms and weight loss. Weight loss was reversed by delayed administration of anti-TNFα to achieve tumor growth inhibition. See Figures 26A-26C.

[0166] conclusion The use of anti-TNFα therapy in combination with GPC3-1 benzodiazepine therapy can mitigate the effects of weight loss caused by high-dose CAR T-cell therapy.

[0167] The embodiments described herein may be implemented in the absence of any elements, sets of elements, limitations, or sets of limitations not specifically disclosed herein. These terms and expressions used are for illustrative purposes only and are not intended to be limitations, nor are they intended to exclude any equivalents of the shown and described features or parts thereof, and it should be recognized that various modifications are possible within the scope of the claimed embodiments. Therefore, although this description is specifically disclosed by embodiments, those skilled in the art should understand that any features, modifications, and variations of the concepts disclosed herein may be adopted, and such modifications and variations should be considered within the scope of these embodiments as defined by the description and the accompanying claims. While certain aspects of this disclosure may be identified as particularly advantageous, this disclosure is not intended to be limited to these specific aspects.

[0168] A claim or statement containing "or" between one or more members of a group is deemed satisfied if one, two or more, or all, of the members of that group are present in, used in, or otherwise related to a given product or process, unless the opposite meaning is stated or the context makes a different interpretation apparent. This disclosure includes embodiments in which exactly one of the members of that group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which two or more, or all, of the members of that group are present in, used in, or otherwise related to a given product or process.

[0169] Furthermore, this disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations in other claims dependent on the same base claim. Where elements are presented, for example, as a list in Markush group form, each subgroup of elements is also disclosed, and any element may be excluded from that group.

[0170] In general, whereever the Disclosure or aspects thereof are referred to as including certain elements and / or features, a particular embodiment or aspect of the Disclosure should be understood to consist of, or substantially consist of, such elements and / or features. For brevity, those embodiments are not specifically described herein using these terms.

[0171] All patents and publications described herein are incorporated herein by reference to the same extent as if each individual patent and publication were explicitly and individually directed to be incorporated by reference. Any citation or confirmation of any reference in any section of this application should not be construed as an admission that such reference is available as prior art to the present invention.

[0172] Table 5

[0173] Table 6

[0174] Table 7

[0175] Table 8

[0176] Table 9

[0177] Table 10

[0178] Table 11

[0179] Table 12

[0180] Table 13

[0181] Table 14

[0182] Table 15

[0183]

Table 16

[0184]

Table 17

[0185] Sequence Listing <110> MEDIMMUNE, LLC <120> COMPOSITIONS AND METHODS OF TREATING CANCER WITH CHIMERIC ANTIGEN RECEPTORS TARGETING GLYPICAN 3 <130> PA26-065 <150> US 62 / 951,309 <151> 2019-12-20 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile 145 150 155 160 Gly Ser Asn Thr Val Asn Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Val Tyr Phe Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly 195 200 205 Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp 210 215 220 Asp Ser Leu Asn Ala Pro Val Phe Gly Gly Gly Thr Lys Val Thr Val 225 230 235 240 Leu <210> 2 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr 130 135 140 Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Gly Ser Ser Asp Ile 145 150 155 160 Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro 165 170 175 Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg Pro Ser Gly Val Pro Asp 180 185 190 Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser 195 200 205 Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp 210 215 220 Asp Arg Met Tyr Ser Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val 225 230 235 240 Leu <210> 3 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> 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> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 305 310 315 320 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 325 330 335 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser 340 345 350 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 355 360 365 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 370 375 380 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 385 390 395 400 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 405 410 415 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 420 425 430 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 435 440 445 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 6 <211> 501 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gly Val His Ser Glu Val Gln Leu Leu Glu 20 25 30 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 50 55 60 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser 65 70 75 80 Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu 145 150 155 160 Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr 165 170 175 Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 180 185 190 Trp Phe Arg Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe 195 200 205 Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys 210 215 220 Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp 225 230 235 240 Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro 245 250 255 Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly 260 265 270 Pro Pro Cys Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly 275 280 285 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 290 295 300 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 305 310 315 320 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 325 330 335 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys 340 345 350 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 355 360 365 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 370 375 380 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 385 390 395 400 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 405 410 415 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 420 425 430 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 435 440 445 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 450 455 460 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 465 470 475 480 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 485 490 495 Ala Leu Pro Pro Arg 500 <210> 7 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> 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> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Tyr Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Ala Thr Trp Asp Asp Arg Met Tyr Ser Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser 290 295 300 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 305 310 315 320 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 325 330 335 Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360 365 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 370 375 380 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 385 390 395 400 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 405 410 415 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 420 425 430 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 435 440 445 Met Gln Ala Leu Pro Pro Arg 450 455 <210> 10 <211> 497 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> 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 agggaaggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta ttttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttatt ctctgctggt cacagtggct 900 ttcatcatct tctgggtcaa gcgaggccgg aagaaactgc tgtacatctt caaacagcct 960 tttatgcgcc cagtgcagac aactcaggag gaagacggct gctcttgtcg gttccccgag 1020 gaagaggaag ggggatgtga gctgcgcgtg aagttttctc gaagtgccga tgctcctgca 1080 tatcagcagg gacagaacca gctgtacaac gagctgaatc tgggccggag agaggaatac 1140 gacgtgctgg ataagaggcg cggcagagac ccagaaatgg gcgggaagcc acgacggaaa 1200 aacccccagg aggggctgta taatgaactg cagaaggaca aaatggccga ggcttacagc 1260 gaaatcggga tgaagggaga gagaaggcgc ggaaaaggcc acgatggact gtatcagggc 1320 ctgagcactg ccaccaagga cacctacgat gctctgcaca tgcaggcact gccacccagg 1380 tga 1383 <210> 12 <211> 957 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 12 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaagggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttatt ctctgctggt cacagtggct 900 ttcatcatct tctgggtccg cgtgaagttt tctcgaagtg ccgatgctcc tgcatga 957 <210> 13 <211> 1380 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 13 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaagggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta tttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg gcatgttat ccctgctggt cactgtggcc 900 ttcatcatct tctgggtgcg gagcaagcgg agccggctgc tgcactctga ctacatgaac 960 atgactccac ggagacccgg ccctacccgg aaacattatc agccctacgc cccacccaga 1020 gattttgccg cttataggtc cagggtgaag ttttctcgca gtgcagatgc ccctgcttat 1080 cagcagggac agaatcagct gtacaacgag ctgaatctgg gcaggcgcga ggaatacgac 1140 gtgctggata agcgacgggg cagagacccc gaaatgggag ggaagcccag aaggaaaaac 1200 cctcaggagg ggctgtataa tgaactgcag aaggacaaaa tggcagaggc ctacagtgaa 1260 atcgggatga agggagagcg ccgacgggga aaaggccacg atggactgta tcagggcctg 1320 tctactgcca ccaaggacac ctacgatgcc ctgcacatgc aggctctgcc tccacgctga 1380 <210> 14 <211> 1506 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 14 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctggtg tacactccga ggtgcagctg ttggagtctg ggggaggctt ggtacagcct 120 ggggggtccc tgagactctc ctgtgcagcc tctggattca cctttagcag ctatgccatg 180 agctgggtcc gccaggctcc agggaaggg ctggagtggg tctcagctat tagtggtagt 240 ggtggtagca catactacgc agactccgtg aagggccggt tcaccatctc cagagacaat 300 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ccgaggacac ggccgtgtat 360 tactgtgcga gaggaaagcg atactttgac tactggggcc aggggacaat ggtcaccgtc 420 tcgagtggtg gggggggcag cggtggtgga ggctctggtg gaggagggag ctcctatgag 480 ctgactcagc caccctcagc gtctgggacc cccgggcaga gggtcaccat ctcttgttct 540 ggaggcagct ccaacatcgg aagtaatact gtaaactggt tccggcagct cccaggaacg 600 gcccccaaac tcctcgttta ttttaataat cagcgaccct caggggtccc tgaccgattc 660 tctggctcca agtctggcac ctcggcctcc ctggccatcg gtgggctcca gtctgacgat 720 gaggctgact attactgtgt agcatgggat gactctctga atgctccggt gttcggcgga 780 gggaccaagg tcaccgtcct agagagcaaa tatggaccac catgccctcc atgtcctttt 840 tgggtcctgg tggtcgtggg aggcgtgctg 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 agggacaga tcagctgtac aacgagctga atctgggcag gcgcgagga tacgacgtgc tggataagcg acggggcaga gaccccgaaa tggggaggga gcccagaagg aaaaaccctc aggaggggct gtataatgaa ctgcagaagg acaaaatggc agaggcctac agtgaaatcg ggatgaaggg agagcgccga cggggaaaag gccacgatgg actgtatcag ggcctgtcta ctgccaccaa ggacacctac gatgccctgc acatgcaggc tctgcctcca cgctga 1506 <210> 15 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 15 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 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 gtccttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctgtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaactgctg tacatctca aacagcctttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tctgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaatgggc gggaagccac gacggaaaaa cccccaggag 1200 gggctgtata atgactgca gaaggacaa atggccgagg cttacagcga atcgggatg 1260 aagggagaga gaggcgcgg aaaggccac gatggactgt atcaggggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacccaggtg a 1371 <210> 16 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 16 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc ccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gacacagcca 480 cctagcgcct ccggaacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gacattggga gcaacaccgt gaattggtat cagcagctgc ctggaacagc tccaaagctg 600 ctgatctact ataacaatca gaggccctcc ggcgtccctg atcgcttctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aaaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttactcc ctgctggtca ctgtggcctt catcatcttc 900 tgggtgcggg tgaagttttc tcgcagtgcc gacgctccg catga 945 <210> 17 <211> 1368 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 17 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc ccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 ggcaaaaggt actttgatta ttggggacag ggcactatgg tgaccgtctc tagtggagga 420 ggaggaagcg gaggaggagg atccggcgga ggaggcagtc agtcagtgct gacacagcca 480 cctagcgcct ccggaacccc aggacagcgg gtcacaatct cttgtagtgg gggatcaagc 540 gacattggga gcaacaccgt gaattggtat cagcagctgc ctggaacagc tccaaagctg 600 ctgatctact ataacaatca gaggccctcc ggcgtccctg atcgcttctc aggcagcaaa 660 tccgggactt ctgcaagtct ggccattagt ggcctgcagt cagaggacga agccgattac 720 tattgtgcta cctgggacga taggatgtac tctcccgtgt tcggcggggg aacaaagctg 780 actgtcctgg agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc 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> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 18 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgagg tccagctgct ggagagcgga ggaggactgg tgcagcctgg aggaagtctg 120 cgactgtcat gcgccgctag cggcttcacc ttcagctcct atgcaatgag ctgggtgcga 180 caggcaccag gcaaggggct ggagtgggtc tccgctatct ccggctctgg aggctctact 240 tactatgcag acagtgtgaa ggggcggttc acaatctcca gagataactc taagaacact 300 ctgtacctgc agatgaactc tctgagagct gaggacaccg cagtgtacta ttgcgccaag 360 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> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Put Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110 Tyr Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro 260 265 270 Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 275 280 285 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 290 295 300 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 305 310 315 320 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 325 330 335 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360 365 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 370 375 380 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 385 390 395 400 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 405 410 415 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 420 425 430 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 435 440 445 His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 20 <211> 497 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110 Tyr Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr Cys Pro Pro 260 265 270 Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr 275 280 285 Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys 290 295 300 Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg 305 310 315 320 Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp 325 330 335 Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 340 345 350 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 355 360 365 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 370 375 380 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 385 390 395 400 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 405 410 415 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 420 425 430 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg <210> 21 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 21 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Gln Ser Gly Gly Gly 20 25 30 Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Gly Leu His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Asp Asp 100 105 110 Thr Ala Leu Tyr Phe Cys Ala Arg Gly Trp Phe Val Glu Pro Leu Ser 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln 145 150 155 160 Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys 165 170 175 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln 180 185 190 Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln 195 200 205 Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr 210 215 220 Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp 225 230 235 240 Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly 245 250 255 Thr Gly Thr Lys Leu Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys 260 265 270 Pro Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 275 280 285 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys 290 295 300 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 305 310 315 320 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 325 330 335 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 340 345 350 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 355 360 365 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 370 375 380 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 385 390 395 400 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 405 410 415 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 420 425 430 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 435 440 445 Leu His Met Gln Ala Leu Pro Pro Arg 450 455 <210> 22 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 22 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgatg tcgtgatgac gcagagccct ctctctcttc ccgttacccc tggtgaaccc 120 gcatcaataa gttgccgctc cagtcaatca cttgtacatt caaatcgcaa tacctacctg 180 cactggtatt tgcagaagcc gggacaatcc cctcaattgt tgatatataa ggtatccaat 240 cgcttttctg gagttcctga tagattcagc ggatccgggt ctggtactga tttcactctg 300 aaaatatcca gggtcgaagc tgaggacgta ggcgtatatt attgctctca gaacacgcat 360 gtcccgccga ctttcggcca gggcactaaa cttgagatca agggtggggg gggcagcggt 420 ggtggaggct ctggtggagg agggagccag gtccaactcg ttcaaagtgg cgcagaggtc 480 aaaaagccag gcgcgagcgt taaagtatca tgtaaggcca gcggttatac tttcactgat 540 tatgaaatgc actgggtgcg acaagccccc gggcaaggtc ttgagtggat gggtgcactt 600 gatccaaaaa ctggggatac tgcctatagc cagaaattca aagggcgcgt cacactcact 660 gccgacaaaa gtacgagcac agcttatatg gaattgagtt cactgacgag cgaggatacg 720 gcagtttatt actgtacgcg cttctactct tacacttatt gggggcaagg cactttggtt 780 actgtgtcct ctgacaagac ccatacgtgt ccaccgtgtc ccttctgggt attggttgtg 840 gtcggcggtg tccttgcttg ttacagcctt ctcgtgacag tcgcattcat aattttttgg 900 gtgaaaagag gtcggaaaaa gttgctgtat attttcaaac aaccctttat gagacctgta 960 caaacgactc aggaagga tggttgtagt tgcaggtttc cggaggagga ggaaggtggg tgcgaactgc gggtgaatt taggedc gctgacgcac cagcttacca acaaggacag aaccaattgt acaacgagct taacttgggt aggagggagg aatatgatgt actggacaaa aggcgaggtc gcgatccgga aatgggaggc aagccacagc gccggaaaaa cccgcagga ggcttgtaca acgaacttca gaaagataaa atggcagaag catactccga aatagggatg aaaggtgaac ggcggcgagg caagggccac gacggtctgt accaagggtt gtcaacggca actaaagaca cgtatgatgc acttcatatg caagctctgc cacccaggtg a <210> 23 <211> 1494 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 23 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctgatg tcgtgatgac gcagagccct ctctctcttc ccgttacccc tggtgaaccc 120 gcatcaataa gttgccgctc cagtcaatca cttgtacatt caaatcgcaa tacctacctg 180 cactggtatt tgcagaagcc gggacaatcc cctcaattgt tgatatataa ggtatccaat 240 cgcttttctg gagttcctga tagattcagc ggatccgggt ctggtactga tttcactctg 300 aaaatatcca gggtcgaagc tgaggacgta ggcgtatatt attgctctca gaacacgcat 360 gtcccgccga ctttcggcca gggcactaaa cttgagatca agggtgggg gggcagcggt 420 ggtggaggct ctggtggagg agggagccag gtccaactcg ttcaaagtgg cgcagaggtc 480 aaaaagccag gcgcgagcgt taaagtatca tgtaaggcca gcggttatac tttcactgat 540 tatgaaatgc actgggtgcg acaagcccccc gggcaaggtc ttgagtggat gggtgcactt 600 gatccaaaaa ctggggatac tgcctatagc cagaaattca aagggcgcgt cacactcact 660 gccgacaaaa gtacgagcac agcttatatg gaattgagtt cactgacgag cgaggatacg 720 gcagtttatt actgtacgcg cttctactct tacacttatt gggggcaagg cactttggtt 780 actgtgtcct ctgacaagac ccatacgtgt ccaccgtgtc ccttctgggt attggttgtg 840 gtcggcggtg tccttgcttg ttacagcctt ctcgtgacag tcgcattcat aattttttgg 900 gtgcggagca agcggagccg gctgctgcac tctgactaca tgaacatgac tccacggaga 960 cccggcccta cccggaaaca ttatcagccc tacgccccac ccagagattt tgccgcttat 1020 aggtccaaaa gaggtcggaa aaagttgctg tatattttca aacaaccctt tatgagacct 1080 gtacaaacga ctcaggaaga ggatggttgt agttgcaggt ttccggagga ggaggaaggt 1140 gggtgcgaac tgcgggtgaa atttagtaga agcgctgacg caccagctta ccaacaagga 1200 cagaaccaat tgtacaacga gcttaacttg ggtaggaggg aggaatatga tgtactggac 1260 aaaaggcgag gtcgcgatcc ggaaatggga ggcaagccac agcgccggaa aaacccgcag 1320 gaaggcttgt acaacgaact tcagaaagat aaaatggcag aagcatactc cgaaataggg 1380 atgaaaggtg aacggcggcg aggcaagggc cacgacggtc tgtaccaagg gttgtcaacg 1440 gcaactaaag acacgtatga tgcacttcat atgcaagctc tgccacccag gtga 1494 <210> 24 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 24 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg 60 attcctcagg tccagcttgt gcaaagcgga ggaggagtgg tacagcctgg ccgctctttg 120 agactgtctt gtgcggccag tggattaca ttctcttctt atgggttgca ttgggtcaga 180 caagcaccgg gcaaaggatt ggaatgggtc gcggccatta gctatgatgg ctcaaagaaa 240 tattatgccg attccgtaaa agggaggttg acaataagcc gggataacag caagaacact 300 ttgtatcttc agatgaatag cctccgaccg gacgacacgg cactgtattt ttgcgcacgc 360 gggtggttg tagaacccct gagttgggga caaggtactc ttgtcacggt atcttctggc 420 ggaggtggga gtggtggggg tggcagtggc gggggtgggt cacaaagcgt gcttacacaa 480 cctccttctg cgagcggaac tccgggacaa cgggttacga tttcatgctc cggctcaagt 540 agcaatatag gatcaaatac agtgaattgg tatcaacaac tccctggcac agcgcccaag 600 ctgctgatct actctaataa ccagaggccg agtggtgtgc cagataggtt cagtggctct 660 aaatcaggta ctagcgcgag cctcgccatt tcaggacttc aatcagagga tgaagcggac 720 tactactgtg ccgcgtggga tgattcactt aatggatatg tttcgggac cggaacaaaa 780 ttgacggtat tggagagcaa atatggacca ccatgccctc catgtcctttt ttgggtcctg 840 gtggtcgtgg gaggcgtgct ggcatgttat tctctgctgg tcacagtggc tttcatcatc 900 ttctgggtca agcgaggccg gaagaaactg ctgtacatct tcaaacagcc tttatgcgc 960 ccagtgcaga caactcagga ggaacggc tgctcttgtc ggttccccga ggaagaggaa 1020 gggggatgtg agctgcgcgt gaagttttct cgaagtgccg atgctcctgc atatcagcag 1080 ggacagaacc agctgtacaa cgagctgaat ctgggccgga gagaggaata cgacgtgctg 1140 gataagaggc gcggcagaga cccagaaatg ggcgggaagc cacgacggaa aaacccccag 1200 gaggggctgt ataatgaact gcagaaggac aaaatggccg aggcttacag cgaaatcggg 1260 atgaagggag agagaaggcg cggaaaaggc cacgatggac tgtatcaggg cctgagcact 1320 gccaccaagg acacctacga tgctctgcac atgcaggcac tgccacccag gtga 1374 <210> 25 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 65 70 75 80 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro 145 150 155 160 Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser 165 170 175 Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn Trp Phe Arg Gln 180 185 190 Leu Pro Gly Thr Ala Pro Lys Leu Leu Val Tyr Phe Asn Asn Gln Arg 195 200 205 Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser 210 215 220 Ala Ser Leu Ala Ile Gly Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr 225 230 235 240 Tyr Cys Val Ala Trp Asp Asp Ser Leu Asn Ala Pro Val Phe Gly Gly 245 250 255 Gly Thr Lys Val Thr Val Leu Glu Ser Lys Tyr Gly Pro Pro Cys Pro 260 265 270 Pro Cys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys 275 280 285 Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 290 295 300 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 305 310 315 320 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 325 330 335 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 340 345 350 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 355 360 365 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 370 375 380 Arg Asp Pro Glu 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> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 26 atgctgctgc tggtgacaag cctgctgctg tgcgaactgc cccatcccgc cttcctgctg attcctgagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 120 agactctcct gtgcagcctc tggattcacc tttagcagct atgccatgag ctgggtccgc 180 caggctccag ggaaggggct ggagtgggtc tcagctatta gtggtagtgg tggtagcaca 240 actcgcag actccgtgaa gggccggttc accatctcca gagacaattc caagacacg ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ccgtgtatta ctgtgcgaga ggaaagcgat actttgacta ctggggccag gggacaatgg tcaccgtctc gagtggtggg 420 gggggcagcg gtggtggagg ctctggtgga ggagggagct cctatgagct gactcagcca 480 ccctcagcgt ctgggacccc cgggcagagg gtcaccatct cttgttctgg aggcagctcc 540 aacatcggaa gtaatactgt aaactggttc cggcagctcc caggaacggc ccccaaactc 600 ctcgtttatt ttaataatca gcgaccctca ggggtccctg accgattctc tggctccaag 660 tctggcacct cggcctccct ggccatcggt gggctccagt ctgacgatga ggctgactat 720 tactgtgtag catgggatga ctctctgaat gctccggtgt tcggcggagg gaccaaggtc 780 accgtcctag agagcaaata tggaccacca tgccctccat gtcctttttg ggtcctggtg 840 gtcgtgggag gcgtgctggc atgttattct ctgctggtca cagtggcttt catcatcttc 900 tgggtcaagc gaggccggaa gaaactgctg tacatcttca aacagccttt tatgcgccca 960 gtgcagacaa ctcaggagga agacggctgc tcttgtcggt tccccgagga agaggaaggg 1020 ggatgtgagc tgcgcgtgaa gttttctcga agtgccgatg ctcctgcata tcagcaggga 1080 cagaaccagc tgtacaacga gctgaatctg ggccggag aggaatacga cgtgctggat 1140 aagaggcgcg gcagagaccc agaatgggc gggaagccac gacggaaaaa cccccaggag 1200 gggctgtata atgactgca gaaggacaa atggccgagg cttacagcga atcgggatg 1260 aagggagaga gaaggcgcgg aaaaggccac gatggactgt atcaggggcct gagcactgcc 1320 accaaggaca cctacgatgc tctgcacatg caggcactgc cacccagg 1368 <210> 27 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 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 Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 29 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Lys Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 31 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser 130 135 140 Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Glu 145 150 155 160 Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 165 170 175 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 180 185 190 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 195 200 205 Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220 Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser <210> 32 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Ser Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Gly Trp Phe Val Glu Pro Leu Ser Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly 130 135 140 Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn 145 150 155 160 Ile Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala 165 170 175 Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro 180 185 190 Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile 195 200 205 Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp 210 215 220 Asp Asp Ser Leu Asn Gly Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr 225 230 235 240 Val Leu <210> 33 <211> 723 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 33 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaggaaag 300 cgatactttg actactgggg ccaggggaca atggtcaccg tctcgagtgg tggggggggc 360 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> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 34 gaggtccagc tgctggagag cggaggagga ctggtgcagc ctggaggaag tctgcgactg 60 tcatgcgccg ctagcggctt caccttcagc tcctatgcaa tgagctgggt gcgacaggca 120 ccaggcaagg ggctggagtg ggtctccgct atctccggct ctggaggctc tacttactat 180 gcagacagtg tgaaggggcg gttcacaatc tccagagata actctaagaa cactctgtac 240 ctgcagatga actctctgag agctgaggac accgcagtgt actattgcgc caagggcaaa 300 aggtactttg attattgggg acagggcact atggtgaccg tctctagtgg aggaggagga 360 agcgaggag gaggatccgg cggaggaggc agtcagtcag tgctgacaca gccacctagc 420. gcctccgga ccccaggaca gcgggtcaca atctcttgta gtgggggatc aagcgacatt 480 gggagcaaca ccgtgaattg gtatcagcag ctgcctggaa cagctccaaa gctgctgatc tactataaca atcagaggcc ctccggcgtc cctgatcgct tctcaggcag caaatccgggg acttctgcaa gtctggccat tagtggcctg cagtcagagg acgaagccga ttactattgt gctacctggg acgataggat gtactctccc gtgttcggcg ggggaacaaa gctgactgtc 720 ctg 723 <210> 35 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> 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> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 36 caggtccagc ttgtgcaaag cggaggagga gtggtacagc ctggccgctc tttgagactg 60 tcttgtgcgg ccagtggatt tacattctct tcttatgggt tgcattgggt cagacaagca 120 ccgggcaaag gattggaatg ggtcgcggcc attagctatg atggctcaaa gaaatattat 180 gccgattccg taaaagggag gttgacaata agccgggata acagcaagaa cactttgtat 240 cttcagatga atagcctccg accggacgac acggcactgt atttttgcgc acgcgggtgg 300 360. tttgtagaac ccctgagttg gggacaaggt actcttgtca cggtatcttc tggcggaggt gggagtggtg ggggtggcag tggcgggggt gggtcacaaa gcgtgcttac acaacctcct 420 tctgcgagcg gaactccggg acaacggggtt acgatttcat gctccggctc aagtagcaat attack attack attack ttggtat caactccctg gcacagcgcc caagctgctg atctactcta ataaccagag gccgagtggt gtgccagata ggttcagtgg ctctaatca ggtactagcg cgagcctcgc catttcagga cttcaatcag aggatgaagc ggactactac tgtgccgcgt gggatgattc acttaatgga tatgttttcg ggaccggac aaaattgacg 720 gtattg 726 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptides <400> 37 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial 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> <223> 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> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Ser Gly Gly Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> 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> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Ser Gly Gly Ser Ser Asp Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 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> <223> 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> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(15) <223> This sequence may encompass 1-3 "Gly Gly Gly Gly Ser" repeating units <220> <223> See specification as filed for detailed description of substitutions and preferred embodiments <400> 46 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 sequence encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. Ｄ The CAR construct is an isolated nucleic acid sequence that does not induce cytokine production in GPC3 cells.

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

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

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

34.

5. An isolated nucleic acid sequence according to any one of claims 1 to 4, further encoding a transmembrane domain, a co-stimulatory domain, and a signaling domain.

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

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

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

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

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

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

12. The isolated nucleic acid sequence according to claim 1, wherein the nucleic acid sequence includes 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.

13. An anti-GPC3 chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL); The VH comprises CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The VL is an anti-GPC3 CAR comprising CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO:

45.

14. The anti-GPC3 CAR according to claim 13, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO:

29.

15. The anti-GPC3 CAR according to claim 13, wherein the VL comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO:

30.

16. The anti-GPC3 CAR according to any one of claims 13 to 15, further comprising a transmembrane domain, a co-stimulatory domain, and a signaling domain.

17. The anti-GPC3 CAR according to claim 16, wherein the CAR comprises 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.

18. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises 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.

19. A cell comprising the vector according to claim 18.

20. A cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR includes an antigen-binding domain specific to glypican 3 (GPC3), and the antigen-binding domain has an equilibrium dissociation constant (K) of approximately 100 nanomoles (nM) or less. Ｄ ) and the CAR construct is a cell that does not induce cytokine production in GPC3 cells.

21. The cell according to claim 20, wherein the nucleic acid sequence includes 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.

22. Cells comprising an anti-GPC3 chimeric antigen receptor (CAR) containing an antigen-binding domain, wherein the antigen-binding domain comprises an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The VL is a cell comprising CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO:

45.

23. The cell according to claim 22, wherein the VH comprises the amino acid of SEQ ID NO: 27 or SEQ ID NO:

29.

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

30.

25. The cell according to any one of claims 22 to 24, wherein the CAR further comprises a transmembrane domain, a costimulatory domain, and a signaling domain.

26. The cell according to any one of claims 22 to 25, wherein the CAR comprises 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.

27. The cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells, as described in any one of claims 19 to 26.

28. The cells according to claim 27, wherein the cells exhibit anti-tumor immunity upon contact with tumor cells expressing GPC3.

29. A method of treating cancer, The treatment for cancer involves administering cells containing an anti-GPC3 chimeric antigen receptor (CAR) containing an effective amount of antigen-binding domain to a subject requiring cancer treatment, wherein the antigen-binding domain includes an antibody, Fab, or scFv containing a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises CDR1 containing the amino acid sequence of SEQ ID NO: 37, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 39; and The method wherein the VL includes CDR1 containing the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, CDR2 containing the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and CDR3 containing the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO:

45.

30. The method according to claim 29, further comprising inhibiting tumor growth, inducing tumor regression, and / or extending survival of the target tumor.

31. The method according to claim 29, wherein the cells are the body's own cells.

32. The method according to claim 31, wherein the self-cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

33. The method according to any one of claims 29 to 32, wherein the cancer is a solid tumor.

34. The method according to claim 33, wherein the cancer is hepatocellular carcinoma, non-small cell lung cancer, ovarian cancer, and / or squamous cell lung cancer.

35. The method according to claim 34, wherein the cancer is hepatocellular carcinoma.

36. The method according to any one of claims 29 to 35, further comprising administering an effective amount of anti-TNFα antibody to the subject.

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