Transforming growth factor beta-responsive polypeptide and method of use thereof

Abstract

To provide polypeptides capable of not only neutralizing TGF-β but also specifically triggering T cell activation in the presence of TGF-β when expressed in a cell.SOLUTION: The disclosure provides such a peptide comprising a signal peptide, an antigen binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, a peptide spacer, a transmembrane domain and an endodomain, where the antigen binding domain specifically binds to TGF-β. T-cell activation spurs the immune cell to produce immunostimulatory cytokines and proliferate, thus turning TGF-β from an immunosuppressive signal to an activating stimulus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 248,685, filed on 30 October 2015. The entirety of the disclosures referenced above is incorporated herein by reference without waiver.

[0002] This invention was made with government support, pursuant to grants CA183528 and OD012133 from the National Institutes of Health. The government has certain rights in this invention.

[0003] 1. Field of Invention The present invention generally relates to the fields of biotechnology and medicine. More specifically, the present invention relates to polypeptides useful for stimulating an immune response in the presence of TGF-β, and cells containing said polypeptides. [Background technology]

[0004] 2. Background TGF-β is a pleiotropic cytokine that is found at high levels in various pathogenic conditions, including solid tumors, fibrosis, and dysregulated wounds. In the treatment planning of solid tumors, neutralization of TGF-β in the tumor microenvironment has been a target. Although many anti-TGF-β antibodies exist, antibodies as therapeutic agents have several drawbacks. For example, antibodies can be large compared to other antigen-binding molecules and are multi-chain proteins encoded by multiple genes. Both of these aspects lead to high production costs. Furthermore, while chemical substances that inhibit TGF-β have been identified, they often come with toxicity problems due to metabolic by-products in the liver.

[0005] The use of adoptive T cell therapy with TGF-β insensitive T cells expressing dominant-negative TGF-β receptors is being investigated. However, neutralizing TGF-β signaling alone may not be sufficient, and reversing TGF-β signaling from immunosuppression to immunostimulation may lead to more promising therapeutic strategies.

[0006] Therefore, there is a need in this field for more effective treatments that neutralize the effects of TGF-β and also provide the benefit of cost-effective production. [Overview of the project]

[0007] The polypeptides described herein satisfy the needs of the art by providing polypeptides that, when expressed intracellularly, can not only neutralize TGF-β but also specifically induce T cell activation in the presence of TGF-β. T cell activation encourages immune cells to produce immunostimulatory cytokines and proliferate, thereby changing TGF-β from an immunosuppressive signal to an activating stimulus. Accordingly, aspects of the present disclosure relate to polypeptides comprising a signal peptide, an antigen-binding domain having a heavy-chain variable (VH) region and a light-chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the antigen-binding domain specifically binds to TGF-β.

[0008] In some embodiments, the present disclosure relates to a polypeptide comprising a signal peptide, an antigen-binding domain having a heavy-chain variable (VH) region and a light-chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO: 5 (HCDR1), SEQ ID NO: 6 (HCDR2), and SEQ ID NO: 7 (HCDR3), and the VL region comprises SEQ ID NO: 8 (LCDR1), SEQ ID NO: 9 (LCDR2), and SEQ ID NO: 10 (LCDR3). In some embodiments, VH comprises SEQ ID NO: 1 and VL comprises SEQ ID NO: 2.

[0009] In some embodiments, the present disclosure relates to a polypeptide comprising a signal peptide, an antigen-binding domain having a heavy-chain variable (VH) region and a light-chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO:11(HCDR1), SEQ ID NO:12(HCDR2), and SEQ ID NO:13(HCDR3), and the VL region comprises SEQ ID NO:14(LCDR1), SEQ ID NO:15(LCDR2), and SEQ ID NO:16(LCDR3). In some embodiments, VH comprises SEQ ID NO:3 and VL comprises SEQ ID NO:4.

[0010] In some embodiments, the present disclosure relates to a polypeptide comprising a signal peptide, an antigen-binding domain having a heavy-chain variable (VH) region and a light-chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO:21(HCDR1), SEQ ID NO:22(HCDR2), and SEQ ID NO:23(HCDR3), and the VL region comprises SEQ ID NO:24(LCDR1), SEQ ID NO:25(LCDR2), and SEQ ID NO:26(LCDR3). In some embodiments, the VH comprises SEQ ID NO:19 and the VL comprises SEQ ID NO:20.

[0011] The polypeptides described above and herein are continuous single-strand polypeptides.

[0012] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage of "sequence identity" or "homology" to another sequence (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or any range within that range), meaning that when the two sequences are aligned and compared, that percentage of bases (or amino acids) are the same. This alignment and percentage homology or sequence identity can be calculated using software programs known in the art (e.g., the program described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology).

[0013] The polypeptides of this disclosure may have regions, domains, linkers, spacers, etc., having at least, at most, or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any range obtained therefrom) with respect to all or part of the amino acid sequences described herein. In certain embodiments, the polypeptides described throughout this disclosure are isolated, meaning they are not found in the cellular environment. Polypeptides may also be purified, meaning they are generally purified if they are not completely separated from polypeptides having different amino acid sequences and / or chemical formulas.

[0014] In some embodiments, VH and VL are separated by a peptide linker. The peptide linker is considered capable of separating any domain / region described in the polypeptide of this disclosure. In some embodiments, the peptide linker is a peptide (glycine-serine linker) consisting only of glycine and serine residues. In some embodiments, the peptide linker is at least, as many as, or exactly 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, or 200 amino acids (or any range obtainable therefrom). In some embodiments, the peptide linker is known in the art or is described herein.

[0015] In some embodiments, the polypeptide has the structure SX-PL-Y-PS-TE or SY-PL-X-PS-TE, where S is the signal peptide, X is VH, PL is the peptide linker, Y is VL, PS is the peptide spacer, T is the transmembrane domain, and E is the endodomain. In some embodiments, the polypeptide has the structure SXY-PS-TE or SYX-PS-TE, where S, X, Y, PS, T, and E are defined above. Where peptides and polypeptides are referred to herein, sequences and structures are described and interpreted as N-terminal to C-terminal, which is standard practice in the art.

[0016] In some embodiments, the polypeptide further comprises a co-stimulatory region. In some embodiments, the co-stimulatory region is located between the transmembrane domain and the endodomain. In some embodiments, the polypeptide comprises at least, as many as, or exactly one, two, three, four, five, six, seven, eight, nine, or ten (or any range thereof) co-stimulatory regions. In some embodiments, the co-stimulatory regions are known in the art or are described herein.

[0017] In some embodiments, the transmembrane domain includes a transmembrane domain of CD28. In some embodiments, the transmembrane domain is all or part of transmembrane domains known in the art or described herein.

[0018] In some embodiments, the end domain includes a CD28 signaling domain or a CD3 zeta signaling domain, or both. In some embodiments, the end domain is all or part of an end domain known in the art or described herein. In some embodiments, the end domain is a CD3 zeta signaling domain. In some embodiments, the end domain includes one or more parts of suitable end domains described herein, for example, two, three, four, five, six, seven, eight, nine, or ten parts.

[0019] In some embodiments, the peptide spacer includes a hinge region. In some embodiments, the hinge is the hinge region of an IgG molecule. In some embodiments, the hinge is a hinge region known in the art or described herein. In some embodiments, the peptide spacer includes, or further includes, the CH2CH3 region of an IgG molecule. In some embodiments, the peptide spacer includes one or more of the hinge region, CH1 region, CH2 region, and CH3 region. In some embodiments, the peptide spacer is derived from the hinge region, CH1 region, CH2 region, and / or CH3 region, or other region, of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM from a human, mouse, rat, dog, donkey, goat, or rabbit. In some embodiments, the peptide spacer includes the hinge region and the CH2CH3 region of an IgG molecule. In some embodiments, the CH2CH3 region of the IgG molecule further has the L235E / N297Q or L235D / N297Q mutation to prevent binding to the Fc receptor. In some embodiments, the peptide spacer consists of the hinge region of the IgG molecule. In some embodiments, the peptide spacer consists of fewer than 30, fewer than 20, fewer than 15, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, or fewer than 4 amino acids. In some embodiments, the peptide spacers are 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 20 The number of amino acids is 0, 210, 220, 225, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 450, 500, 550, 600, or 700, or fewer than, more than, or exactly those values ​​(or any range within that range).In some embodiments, the peptide spacer comprises less than 50 amino acids. In some embodiments, the peptide spacer comprises more than 50 amino acids.

[0020] In some embodiments, the polypeptide further comprises a detection peptide. In some embodiments, the detection peptide is the peptide of SEQ ID NO: 17, an HA tag (SEQ ID NO: 94), or a cMyc tag (SEQ ID NO: 95). In some embodiments, the detection peptide is adjacent to a linker. In some embodiments, the linker (e.g., a peptide linker described herein) is located at the amino portion of the detection peptide. In some embodiments, the linker (e.g., a peptide linker described herein) is located at the carboxy portion of the detection peptide. In some embodiments, the linker is located at both the amino portion and the carboxy portion of the detection peptide. In some embodiments, the detection peptide is located at the amino portion of the VH region and the VL region. In some embodiments, the detection peptide is located between the signal peptide and the antigen binding domain.

[0021] In some embodiments, the signal peptide comprises SEQ ID NO: 18. In some embodiments, the signal peptide is known in the art or as described herein.

[0022] In some embodiments, the polypeptide further comprises a cancer molecule-specific antigen binding domain. For example, the polypeptide may be a bispecific chimeric antigen receptor (CAR), and the polypeptide comprises an antigen binding domain against TGF-β and an antigen binding domain against a cancer molecule or a cancer antigen. These antigen binding domains may be separated by a peptide spacer or a linker. In some embodiments, the cancer molecule comprises Her2. In some embodiments, the cancer molecule comprises CD19 or CD20. In some embodiments, the cancer molecule or cancer antigen is known in the art or as described herein.

[0023] In some embodiments, the antigen-binding domain specifically binds to soluble TGF-β. It has not been previously known that a polypeptide similar to the polypeptide of the present disclosure can bind to a soluble antigen and transduce a signal in response to the soluble antigen.

[0024] The polypeptides described herein include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, and 59 of the SEQ ID NO: 1 to 95. , 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces, 88 pieces, 89 pieces, 9 0 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 101 pieces, 102 pieces, 103 pieces, 10 4 pieces, 105 pieces, 106 pieces, 107 pieces, 108 pieces, 109 pieces, 110 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 116 pieces, 117 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces, 123 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces pieces, 129 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 135 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces, 140 pieces , 141 pieces, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces, 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 156 pieces, 157 pieces, 158 pieces, 159 pieces, 160 pieces, 161 pieces, 162 pieces, 163 pieces, 164 pieces, 1 65 pieces, 166 pieces, 167 pieces, 168 pieces, 169 pieces, 170 pieces, 171 pieces, 172 pieces, 173 pieces, 174 pieces, 175 pieces, 176 pieces, 17 7 pieces, 178 pieces, 179 pieces, 180 pieces, 181 pieces, 182 pieces, 183 pieces, 184 pieces, 185 pieces, 186 pieces, 187 pieces, 188 pieces, 189 pieces, 190 pieces, 191 pieces, 192 pieces, 193 pieces, 194 pieces, 195 pieces, 196 pieces, 197 pieces, 198 pieces, 199 pieces, 200 pieces, 201 pieces , 202 pieces, 203 pieces, 204 pieces, 205 pieces, 206 pieces, 207 pieces, 208 pieces, 209 pieces, 210 pieces, 211 pieces, 212 pieces, 213 pieces,214 pieces, 215 pieces, 216 pieces, 217 pieces, 218 pieces, 219 pieces, 220 pieces, 221 pieces, 222 pieces, 223 pieces, 224 pieces, 225 pieces, 226 pieces, 227 pieces, 228 pieces, 229 pieces, 23 0 pieces, 231 pieces, 232 pieces, 233 pieces, 234 pieces, 235 pieces, 236 pieces, 237 pieces, 238 pieces, 239 pieces, 240 pieces, 241 pieces, 242 pieces, 243 pieces, 244 pieces, 245 pieces, 246 pieces, Within a sequence of 247, 248, 249, 250, 300, 400, 500, 550, 1000, or more consecutive amino acids, or within any range obtainable therefrom, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 pieces, 25 pieces, 26 pieces, 27 pieces, 28 pieces, 29 pieces, 30 pieces, 31 pieces, 32 pieces, 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces , 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 It may contain 1, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more mutant amino acids.

[0025] Further embodiments of this disclosure relate to nucleic acids encoding polypeptides described herein. In some embodiments, this disclosure relates to cells comprising one or more polypeptides described herein. In some embodiments, the cells further comprise cancer-specific CARs. In some embodiments, the cancer-specific CAR is a polypeptide different from TGF-β CAR. In some embodiments, the cancer-specific CAR specifically binds to Her2. In some embodiments, the cancer-specific CAR specifically binds to CD19 or CD20. In some embodiments, the cancer-specific CAR specifically binds to cancer molecules or antigens known in the art and / or described herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are progenitor cells or stem cells. In some embodiments, the progenitor cells or stem cells are differentiated into immune cells in vitro. In some embodiments, the cells are T cells. In some embodiments, the cells are CD4+ T cells or CD8+ T cells. In some embodiments, the cells are natural killer cells. In some embodiments, the cells are ex vivo. The term immune cells includes cells of the immune system that are involved in defending the body against both infectious diseases and foreign substances. Examples of immune cells include lymphocytes such as neutrophils, eosinophils, basophils, natural killer cells, B cells, and T cells, as well as monocytes. T cells may include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, γδ T cells, regulatory T cells, suppressor T cells, and natural killer T cells. In certain embodiments, the T cells are regulatory T cells.

[0026] Further embodiments of this disclosure relate to methods for stimulating an immune response, comprising contacting cells of this disclosure (i.e., cells containing antigen-binding polypeptides as described herein) with TGF-β. In some embodiments, stimulating an immune response involves increasing the expression and / or secretion of immunostimulatory cytokines and / or immunostimulatory molecules. In some embodiments, the cytokines and / or molecules are inflammatory cytokines or inflammatory molecules. In some embodiments, the immunostimulatory cytokines and / or immunostimulatory molecules are one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor. In some embodiments, stimulating an immune response involves increasing the proliferation of immune cells. In some embodiments, the immune cells are T cells. In some embodiments, TGF-β is endogenous TGF-β produced in a human subject requiring immune stimulation. In some embodiments, the human subject has cancer, fibrosis, or an open wound. In some embodiments, the human subject has a B-cell malignancy. In some embodiments, the human subject has a solid tumor. A solid tumor is an abnormal mass of tissue that does not typically contain a cyst or fluid area. A solid tumor may be benign (not cancerous) or malignant (cancerous). Each type of solid tumor is named based on the type of cells that form the solid tumor. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. In some embodiments, the method is for treating a person having a certain condition characterized by a pathogenic level of TGF-β expression. In some embodiments, the cells and polypeptides described herein may be used to treat cancer, dysregulated wounds, fibrosis, open wounds, solid tumors, etc., where the pathogenesis of the condition is at least partially based on TGF-β expression. In some embodiments, the cells (i.e., cells of the disclosure containing antigen-binding polypeptides) are present in a human subject requiring immunostimulation. In some embodiments, the method further includes administering cells of the disclosure containing polypeptides or nucleic acids to a human subject.

[0027] Further embodiments of the method relate to a method for detecting TGF-β in solution, comprising contacting cells of the present disclosure and measuring immunostimulation, wherein an increase in immunostimulation indicates the presence of TGF-β, and the absence of an increase in immunostimulation indicates the absence of TGF-β. In some embodiments, immunostimulation includes the expression of immunostimulatory cytokines and / or immunostimulatory molecules. In some embodiments, the immunostimulatory cytokines and / or immunostimulatory molecules are one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factors. In some embodiments, immunostimulation includes an increase in the proliferation of immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the cells are ex vivo.

[0028] The increase in expression or proliferation described herein may be at least, at most, or exactly 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, or 1000-fold increase compared to baseline expression levels of a control (disease-free control, non-TGF-β control, or non-antigen-binding polypeptide control).

[0029] Further aspects of this disclosure relate to methods for producing a polypeptide, including expressing a nucleotide encoding the polypeptide in a cell. Further aspects relate to cultured cells, frozen cells, suspension cells, or adherent cells comprising the polypeptide described herein.

[0030] Aspects of this disclosure relate to methods for treating a disease or pathological condition, including administering the cells of this disclosure to a patient. In some embodiments, the patient is a human patient.

[0031] In some embodiments, the cells are regulatory T cells (i.e., regulatory T cells containing the TGF-β binding polypeptide described herein). In some embodiments, the disease is an autoimmune disease. In some embodiments, the autoimmune disease is rheumatoid arthritis. In some embodiments, the autoimmune disease is one described herein.

[0032] In some embodiments of the method, the method further includes administering TGF-β as the target.

[0033] In some embodiments, the method comprises or further comprises growing T cells in vitro and / or inducing T cell proliferation, the method comprising contacting the in vitro T cells of the Disclosure with a composition comprising TGF-β. In some embodiments, the T cells are regulatory cells. In some embodiments, the T cells are the T cells described herein. In some embodiments, the grown regulatory T cells contain less than 10% non-regulatory T cells. In some embodiments, the proliferated regulatory T cells include less than 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 14%, less than 16%, less than 18%, less than 20%, less than 22%, less than 24%, less than 26%, less than 28%, less than 30%, less than 32%, less than 34%, less than 36%, less than 38%, less than 40%, less than 42%, less than 44%, less than 46%, less than 48%, or less than 50%, or any range obtained therefrom.

[0034] In some embodiments, the composition contains 1 to 50 ng / mL of TGF-β. In some embodiments, the composition contains at least, at most, or about 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 8.5 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL Contains TGF-β (or any range obtainable within that range) in the following concentrations: mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL.

[0035] In some embodiments, the composition further comprises IL-2. In some embodiments, the composition comprises 20 to 400 U / mL of IL-2.In some embodiments, the composition contains at least, at most, or about 5 U / mL, 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 35 U / mL, 40 U / mL, 45 U / mL, 50 U / mL, 55 U / mL, 60 U / mL, 65 U / mL, 70 U / mL, 75 U / mL, 80 U / mL, 85 U / mL, 90 U / mL, 95 U / mL, 100 U / mL, 105 U / mL, 110 U / mL, 115 U / mL, 120 U / mL, 125 U / mL, 130 U / mL, 135 U / mL, 140 U / mL, 145 U / mL, and 150 U / mL. , 155U / mL, 160U / mL, 165U / mL, 170U / mL, 175U / mL, 180U / mL, 185U / mL, 190U / mL, 195U / mL, 200U / mL, 205U / mL, 210U / mL, 215U / mL, 220U / mL, 225U / mL, 230 U / mL, 235U / mL, 240U / mL, 245U / mL, 250U / mL, 255U / mL, 260U / mL, 265U / mL, 270U / mL, 275U / mL, 280U / mL, 285U / mL, 290U / mL, 295U / mL, 300U / mL, 305U / mL, 310U / mL, 315U / mL, 320U / mL, 325U / mL, 330U / mL, 335U / mL, 340U / mL, 345U / mL, 350U / mL, 355U / mL, 360U / mL, 365U / mL, 370U / mL, 375U / mL, 380U / mL, 385U / mL, 390U / mL, 395U / mL, 400U / mL, 405U / mL, 410U / mL, 415U / mL, 420U / mL, 425U / mL, 430U / mL, 435U / mL, 440U / mL, 445U / mL, 450U / mL, 455U / mL, 460U / mL, 4 This includes IL-2 (or any range obtainable within it) of 65 U / mL, 470 U / mL, 475 U / mL, 480 U / mL, 485 U / mL, 490 U / mL, 495 U / mL, 500 U / mL, 505 U / mL, 510 U / mL, 515 U / mL, 520 U / mL, 525 U / mL, 530 U / mL, 535 U / mL, 540 U / mL, 545 U / mL, 550 U / mL, 555 U / mL, 560 U / mL, 565 U / mL, 570 U / mL, 575 U / mL, 580 U / mL, 585 U / mL, 590 U / mL, 595 U / mL, and 600 U / mL.

[0036] In some embodiments, the method further includes bringing cells into contact with feeder cells. In some embodiments, the feeder cells are irradiated. The feeder cells or supporting cells may include, for example, fibroblasts, mouse fetal fibroblasts, JK1 cells, SNL76 / 7 cells, human fetal skin cells, human fibroblasts, and human foreskin fibroblasts.

[0037] In some embodiments, the method omits contacting T cells with feeder cells. The excluded feeder cells may be derived from a different animal species than the T cells.

[0038] In one embodiment of the method described herein, the subject is a human subject. The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and mean mammals, including, but not limited to, rodents (e.g., rats, mice), rabbits (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc.

[0039] Before further description of the present invention, it should be understood that the invention is not limited to the specific embodiments described and is therefore naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them, and the scope of the invention is limited only to the appended claims.

[0040] Where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range (up to one-tenth of the lower limit unless otherwise clearly indicated in the context), and any other specified or intermediate values ​​within that specified range, are included within the scope of the invention. The upper and lower limits of such narrow ranges may be independently included within the narrow range and are included within the scope of the invention, but subject to any limitations specifically excluded within the specified range. If a specified range includes one or both of these limits, the range excluding one or both of the included limits is also included within the invention.

[0041] [Invention 1001] A polypeptide comprising a signal peptide, an antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the antigen-binding domain specifically binds to TGF-β. [Invention 1002] A polypeptide comprising a signal peptide, an antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO:5(HCDR1), SEQ ID NO:6(HCDR2), and SEQ ID NO:7(HCDR3), and the VL region comprises SEQ ID NO:8(LCDR1), SEQ ID NO:9(LCDR2), and SEQ ID NO:10(LCDR3). [Invention 1003] The polypeptide of the present invention 1002, wherein the VH region contains SEQ ID NO:1 and the VL region contains SEQ ID NO:2. [Invention 1004] A polypeptide comprising a signal peptide, an antigen-binding domain having a VH region and a VL region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO:11(HCDR1), SEQ ID NO:12(HCDR2), and SEQ ID NO:13(HCDR3), and the VL region comprises SEQ ID NO:14(LCDR1), SEQ ID NO:15(LCDR2), and SEQ ID NO:16(LCDR3). [Invention 1005] The polypeptide of the present invention 1004, wherein the VH region contains SEQ ID NO:3 and the VL region contains SEQ ID NO:4. [Invention 1006] A polypeptide comprising a signal peptide, an antigen-binding domain having a VH region and a VL region, a peptide spacer, a transmembrane domain, and an endodomain, wherein the VH region comprises SEQ ID NO:21(HCDR1), SEQ ID NO:22(HCDR2), and SEQ ID NO:23(HCDR3), and the VL region comprises SEQ ID NO:24(LCDR1), SEQ ID NO:25(LCDR2), and SEQ ID NO:26(LCDR3). [Invention 1007] The polypeptide of the present invention 1004, wherein the VH region includes SEQ ID NO:19 and the VL region includes SEQ ID NO:20. [Invention 1008] A polypeptide according to any of invention 1001 to 1007, wherein the VH region and VL region are separated by a peptide linker. [Invention 1009] The polypeptide according to any of the inventions 1001 to 1008, wherein the polypeptide has the structure SX-PL-Y-PS-TE or SY-PL-X-SP-TE, where S is the signal peptide, X is VH, PL is the peptide linker, Y is VL, PS is the peptide spacer, T is the transmembrane domain, and E is the end domain. [Invention 1010] A polypeptide according to any one of the present invention 1001 to 1009, further comprising a co-stimulatory region. [Invention 1011] The polypeptide of the present invention 1010, wherein the co-stimulatory region is located between the transmembrane domain and the endodomain. [Invention 1012] A polypeptide according to any one of the present invention 1001 to 1011, wherein the transmembrane domain includes a transmembrane domain of CD28. [Invention 1013] A polypeptide according to any one of the invention 1001 to 1012, wherein the end domain comprises a CD28 signaling domain or a CD3 zeta signaling domain. [Invention 1014] A polypeptide according to any one of the present invention 1008 to 1013, wherein the peptide linker is a glycine-serine linker. [Invention 1015] A polypeptide according to any one of the present invention 1003 to 1014, wherein the peptide linker is at least four amino acids. [Invention 1016] A polypeptide according to any one of the present invention 1001 to 1015, wherein the endodomain is a CD3 zeta signaling domain. [Invention 1017] A polypeptide according to any of the present invention 1001 to 1016, wherein the peptide spacer contains fewer than 50 amino acids. [Invention 1018] A polypeptide according to any of the present invention 1001 to 1016, wherein the peptide spacer contains more than 50 amino acids. [Invention 1019] A polypeptide according to any one of the present invention 1001 to 1018, wherein the peptide spacer includes a hinge region of an IgG molecule. [Invention 1020] A polypeptide according to any of the present invention 1001 to 1019, wherein the peptide spacer includes the hinge region and the CH2CH3 region of an IgG molecule. [Invention 1021] The polypeptide of the present invention 1019, wherein the peptide spacer consists of the hinge region of an IgG molecule. [Invention 1022] A polypeptide according to any of the present invention 1001 to 1021, further comprising the detection peptide. [Invention 1023] The polypeptide of the present invention 1022, wherein the detected peptide is the peptide with SEQ ID NO: 17, 94, or 95. [Invention 1024] A polypeptide according to the present invention 1022 or 1023, wherein the detected peptide is adjacent to the linker. [Invention 1025] A polypeptide according to any of the present invention 1022 to 1024, wherein the detected peptide is at the N terminus with respect to the VH region and the VL region. [Invention 1026] A polypeptide according to any of invention 1022 to 1025, wherein the detection peptide is located between the signal peptide and the antigen-binding domain. [Invention 1027] A polypeptide according to any of the present invention 1001 to 1026, wherein the signal peptide contains SEQ ID NO:18. [Invention 1028] A polypeptide according to any one of the present invention 1001 to 1027, further comprising a cancer molecule-specific antigen-binding domain. [Invention 1029] The polypeptide of the present invention 1028, wherein the aforementioned cancer molecule contains Her2. [Invention 1030] The polypeptide of the present invention 1028, wherein the aforementioned cancer molecule contains CD19 or CD20. [Invention 1031] A polypeptide according to any one of the present invention 1002 to 1030, wherein the antigen-binding domain specifically binds to soluble TGF-β. [Invention 1032] An isolated nucleic acid encoding any polypeptide according to invention 1001 to 1031. [Invention 1033] A cell comprising a polypeptide according to any of Invention 1001 to 1031 or a nucleic acid according to Invention 1032. [Invention 1034] Cells of the present invention 1033, further comprising cancer-specific chimeric antigen receptors (CARs). [Invention 1035] The polypeptide of the present invention 1034, wherein the cancer-specific CAR specifically binds to Her2. [Invention 1036] The polypeptide of the present invention 1034, wherein the cancer-specific CAR specifically binds to CD19 or CD20. [Invention 1037] A cell of the present invention 1036, which is an immune cell. [Invention 1038] A T cell, according to the cell of invention 1037. [Invention 1039] The cells of the present invention 1038, which are CD4+ T cells or CD8+ T cells. [Invention 1040] A natural killer cell, according to the present invention 1037. [Invention 1041] The cell according to the present invention 1038, wherein the T cell is a regulatory T cell. [Invention 1042] Cells of any of the invention 1033 to 1040, present in ex vivo. [Invention 1043] A method for stimulating an immune response, comprising contacting any of the cells described in invention 1033 to 1042 with TGF-β. [Invention 1044] The method of the present invention 1043, comprising stimulating an immune response by increasing the expression and / or secretion of immunostimulatory cytokines and / or immunostimulatory molecules. [Invention 1045] The method of the present invention 1044, wherein the immunostimulatory cytokine and / or immunostimulatory molecule is one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor. [Invention 1046] A method of the present invention 1043, comprising stimulating an immune response to increase the proliferation of immune cells. [Invention 1047] The method of the present invention 1046, wherein the immune cells are T cells. [Invention 1048] The method according to any one of the present invention 1043 to 1047, wherein the cells are in vivo in a target requiring immune stimulation. [Invention 1049] The method of the present invention 1048, wherein the TGF-β is endogenous TGF-β produced in the subject requiring immune stimulation. [Invention 1050] The method of the present invention 1049, wherein the human subject has cancer, fibrosis, or an open wound. [Invention 1051] The method of the present invention 1050, wherein the cancer is melanoma. [Invention 1052] The method of the present invention 1049, wherein the human subject has a B-cell malignant tumor. [Invention 1053] The method of the present invention 1049, wherein the human subject has a solid tumor. [Invention 1054] Any method of the present invention 1043 to 1053, further comprising administering the cells to a human subject. [Invention 1055] Any method of the present invention 1047 to 1054, further comprising administering TGF-β to the subject. [Invention 1056] A method for detecting TGF-β in a solution, comprising contacting any of the cells of the present invention 1033 to 1042 with the solution and measuring immunostimulation, wherein an increase in immunostimulation indicates the presence of TGF-β, and the absence of an increase in immunostimulation indicates the absence of TGF-β. [Invention 1057] The method of the present invention 1056, wherein the immune stimulation comprises the expression of immunostimulatory cytokines and / or immunostimulatory molecules. [Invention 1058] The method of the present invention 1057, wherein the immunostimulatory cytokine and / or immunostimulatory molecule is one or more of TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor. [Invention 1059] The method of the present invention 1056, wherein immune stimulation includes increasing the proliferation of immune cells. [Invention 1060] The method of invention 1059, wherein the immune cells are T cells. [Invention 1061] The method according to any of the present invention 1056 to 1060, wherein the cells are present ex vivo. [Invention 1062] A method for producing any polypeptide according to invention 1001 to 1031, comprising expressing a nucleotide encoding the polypeptide in a cell. [Invention 1063] A method for proliferating T cells in vitro, comprising contacting in vitro T cells according to any of the present invention 1038 to 1041 with a composition containing TGF-β. [Invention 1064] The method of the present invention 1063, wherein the composition contains 1 to 50 ng / mL of TGF-β. [Invention 1065] The method of the present invention 1063 or 1064, wherein the composition further comprises IL-2. [Invention 1066] The method of the present invention 1065, wherein the composition contains 20 to 400 U / mL of IL-2. [Invention 1067] Any method 1063 to 1066 of the present invention, further comprising bringing the cells into contact with feeder cells. [Invention 1068] The method of the present invention 1067, wherein the feeder cells are irradiated with radiation. [Invention 1069] Any method 1063 to 1066 of the present invention, wherein contact between the T cell and the feeder cell is eliminated. [Invention 1070] The method according to any of the present invention 1063 to 1069, wherein the T cells are regulatory T cells. [Invention 1071] The method of the present invention 1070, wherein the proliferated regulatory T cells include less than 10% non-regulatory T cells. [Invention 1072] A method for treating a disease or pathological condition in a patient, comprising administering any of the cells described in items 1038 to 1042 of the present invention to the patient. [Invention 1073] The method of the present invention 1072, wherein the cells are regulatory T cells. [Invention 1074] The method of the present invention 1073, wherein the disease is an autoimmune disease. [Invention 1075] The method of the present invention 1072, wherein the disease is cancer. [Invention 1076] Any method of the present invention 1072 to 1075 further comprises growing the cells in vitro by a method comprising contacting the cells in vitro with a composition comprising TGF-β. [Invention 1077] The method of the present invention 1076, wherein the composition contains 1 to 50 ng / mL of TGF-β. [Invention 1078] The method of the present invention 1076 or 1077, wherein the composition further comprises IL-2. [Invention 1079] The method of the present invention 1078, wherein the composition contains 20 to 400 U / mL of IL-2. [Invention 1080] Any method of the present invention 1076 to 1079, further comprising bringing the cells into contact with feeder cells. [Invention 1081] The method of the present invention 1080, wherein the feeder cells are irradiated with radiation. [Invention 1082] Any method according to 1076 to 1079 of the present invention, wherein contact between the T cell and the feeder cell is eliminated. [Invention 1083] The method according to any of the present invention 1076 to 1082, wherein the T cells are regulatory T cells. [Invention 1084] The method of the present invention 1083, wherein the proliferated regulatory T cells include less than 10% non-regulatory T cells. [Invention 1085] The method according to either Invention 1074 or Invention 1076-1084, wherein the autoimmune disease is rheumatoid arthritis. [Invention 1086] Any method of the present invention 1072 to 1085, further comprising administering TGF-β to the patient. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are provided for illustrative purposes only, illustrating preferred embodiments of the present invention. [Brief explanation of the drawing]

[0042] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed descriptions of specific embodiments presented herein.

[0043] [Figure 1] This study demonstrates that TGF-β scFv neutralizes human TGF-β. Specified amounts of TGF-β and anti-TGF-β scFv were added to HepG2 cell culture for 30 minutes. Neutralization of TGF-β was indicated by a decrease in the phosphorylated SMAD2 response detected by Western blotting. [Figure 2]This shows that TGF-β CAR is expressed on the cell surface. TGF-β CAR was prepared using scFv#2. Surface staining and flow cytometry show that TGF-β CAR is present on the cell surface of CD4+ T cells and CD8+ T cells. The extracellular domain of the receptor contains the FLAG epitope. EGFRt is a cleaved epidermal growth factor receptor, which indicates cell transduction. [Figure 3] Figures 3A and 3B show that TGF-β CARs block endogenous TGF-β signaling in (A) CD8+ T cells and (B) CD4+ T cells. Expression of TGF-β CARs in T cells blocks TGF-β signaling via the SMAD pathway. T cells expressing the specified receptors were incubated with TGF-β for 30 minutes and probed for phosphorylated SMAD2 by Western blotting. 'scFv' represents a CAR without any ligand (antigen)-binding scFv domain, and 'EGFRt' represents a cleaved epidermal growth factor receptor independent of the other components. [Figure 4](A) In Jurcut cells that stably express TGF-β CAR and NFAT reporter (EGFP expressed from an NFAT-inducible promoter), increased activation is observed in response to increasing TGF-β concentration. (B, C) In primary human CD4+ T cells that stably express TGF-β CAR, (B) CD69 expression and (C) Th1 cytokine production are upregulated in response to TGF-β stimulation. Upregulation of CD69 was monitored by surface staining after 1-day incubation with or without TGF-β. After 1-day incubation with or without TGF-β, cytokine production was detected by applying the protein transport inhibitor brefeldin A and performing intracellular staining. "Mock" represents T cells transduced with an unrelated construct. "ScFv-less" represents T cells expressing a CAR that is identical to the TGF-β CAR except that it cannot bind to TGF-β because it lacks an scFv domain. "DNR" is a dominant-negative TGF-β receptor, which is a cleaved TGF-β receptor chain 2 lacking a cytoplasmic signaling domain. The presented values ​​are triplet mean values ​​with error bars indicating ±1 standard deviation (std). *p<0.05, **p<0.005, ***p<0.0005, **** *p≦0.0005. [Figure 5] This demonstrates that dominant-negative TGF-β receptors cannot induce cytokine production. While dominant-negative TGF-β receptors have also been reported to inhibit TGF-β signaling, they do not induce immunostimulatory effects such as TNF-α production. Tβ short and Tβ long are two different types of TGF-β CARs, where Dom-Neg represents a dominant-negative TGF-β receptor, and scFv-less represents a CAR that does not have any ligand-binding scFv domain. Tβ short represents a TGF-β CAR polypeptide having a peptide spacer consisting only of the IgG4 hinge region. Tβ long represents a TGF-β CAR polypeptide having a peptide spacer containing both the IgG4 hinge region and the CH2CH3 region. [Figure 6]This shows that TGF-β CAR-T cells proliferate in response to TGF-β. [Figure 7] This study demonstrates inhibition of mouse TGF-β / SMAD signaling. Specified doses of scFv and mouse TGF-β1 were applied to NIH3T3 fibroblasts for 30 minutes. The cells were lysed and probed for SMAD2 phosphorylation. [Figure 8] In Jurcut cells that stably express TGF-β CAR and an NFAT reporter (EGFP expressed from an NFAT-inducible promoter), increased activation was observed in response to increasing doses of mouse TGF-β1. This indicates that TGF-β CARs engineered to recognize human TGF-β also cross-react with mouse TGF-β. [Figure 9] TGF-β CARs appear on the cell surface more efficiently than dominant-negative TGF-β receptors. [Figure 10] The function of TGF-β CAR can be regulated by selecting the co-stimulatory domain. [Figure 11] TGF-β consistently induces TNF-α production in a dose-dependent manner across cells derived from various donors. [Figure 12] The peptide spacer length of TGF-β CAR regulates the induction threshold. [Figure 13] CAR signaling requires ligand-mediated CAR dimerization, but the ligand or the CAR itself does not need to be pre-existing as a dimer. CD69 surface staining was performed on Jarcut cell lines containing the specified CARs. Both GFP CAR#1 and GFP CAR#3 exist primarily as homodimers, and these two types of CARs can bind to different epitopes on EGFP and simultaneously bind to monomeric EGFP molecules. GFP CAR#1 and GFP CAR#2 bind to the same epitope on EGFP, but CAR#2 exists as a monomer rather than a homodimer. [Figure 14] Soluble dimeric antigen molecules can induce signal transduction by linking receptors on different cells. [Figure 15] TGF-β CAR can be induced by cell-to-cell contact-dependent and cell-independent methods. [Figure 16] TGF-β CAR-T cells can be activated even in the absence of intercellular contact. [Modes for carrying out the invention]

[0044] Description of Exemplary Embodiments Using the polypeptides, cells, and methods described herein, TGF-β can be neutralized and T cell activation can be specifically induced in the presence of TGF-β.

[0045] I. Definition The peptides of this disclosure relate to peptides containing CARs, or chimeric antigen receptors. CARs are engineered receptors that confer arbitrary specificity to immune effector cells. These receptors are commonly used to confer the specificity of monoclonal antibodies to T cells. These receptors are called chimeric because they are composed of parts originating from different sources.

[0046] The terms "protein," "polypeptide," and "peptide" are interchangeable herein when referring to gene products.

[0047] "Homologie," "identity," or "similarity" refers to the degree of sequence similarity between two peptides or two nucleic acid molecules. Identity can be calculated by comparing a position in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules share sequence identity at that position. The degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity or less than 25% identity with any of the sequences in this disclosure.

[0048] The terms "amino moiety," "N-terminus," and "amino terminus," as used herein, refer to the order of regions within a polypeptide. Furthermore, being N-terminus to a region does not necessarily mean it is at the end (or end) of the entire polypeptide, but simply means it is at the end of that region or domain. Similarly, the terms "carboxyl moiety," "C-terminus," and "carboxy terminus," as used herein, refer to the order of regions within a polypeptide, and being C-terminus to a region does not necessarily mean it is at the end (or end) of the entire polypeptide, but simply means it is at the end of that region or domain.

[0049] The terms “polynucleotide,” “nucleic acid,” and “nucleotide” are used interchangeably and refer to polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and nucleic acid primers. Polynucleotides may include modified nucleotides (such as methylated nucleotides and nucleotide analogs). If present, modifications to the nucleotide structure may be made before or after the assembly of the polynucleotide. Non-nucleotide components may be inserted into the nucleotide sequence. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Furthermore, the term refers to both double-stranded molecules and single-stranded molecules. Unless otherwise specified or required, any embodiment of the present invention that is a polynucleotide includes both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0050] The terms “subject,” “individual,” or “patient” are interchangeable herein and mean vertebrates, e.g., primates, mammals, or preferably humans. Mammals include, but are not limited to, horses, dogs, cattle, sheep, mice, rats, monkeys, humans, livestock, sports animals, and pets.

[0051] The terms “Xenofree (XF),” “Animal Component Free (ACF),” or “Animal Free” mean, when used in relation to culture media, extracellular matrices, or culture conditions, culture media, extracellular matrices, or culture conditions that are essentially free from heterologous animal-derived components. When culturing human cells, any protein derived from non-human animals, such as mice, would be considered heterologous components. In certain embodiments, a xenofree matrix is ​​essentially free from any non-human animal-derived components and therefore excludes mouse feeder cells or Matrigel®. Matrigel® is a soluble basement membrane preparation extracted from EHS (Engelbreth-Holm-Swarm) mouse sarcoma, a tumor rich in extracellular matrix proteins, comprising laminin (the main component), collagen IV, heparin sulfate proteoglycan, and entactin / nidogen. In some embodiments, the compositions described herein or the cells of this disclosure are cultured and / or prepared in xenofree media, or animal component-free media, or animal-free media.

[0052] As used herein, cells are "substantially free" of a particular reagent or element, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements, or vector elements, if they make up less than 10% of that element, and are "essentially free" of a particular reagent or element if they make up less than 1% of that element. However, a cell population in which less than 0.5% or less than 0.1% of the total cell population contains exogenous genetic elements or vector elements is even more desirable.

[0053] A culture medium, matrix, or culture medium is "substantially free" of a particular reagent or element (serum, signaling inhibitor, animal component, or feeder cells) if the level of these reagents in the culture medium, matrix, or culture medium is below the level detectable by conventional detection methods known to those skilled in the art, or if these reagents are not added to the culture medium, matrix, or culture medium from an external source. A serum-free medium may not inherently contain serum.

[0054] A gene, polynucleotide, coding region, sequence, segment, fragment, or transgene that codes for a specific protein is a nucleic acid molecule that, under the control of appropriate regulatory sequences, is transcribed in vitro or in vivo and optionally translated into a gene product (e.g., polypeptide). The coding region can exist in cDNA, genomic DNA, or RNA form. When a nucleic acid molecule exists in DNA form, it can be single-stranded (i.e., sense strand) or double-stranded. The boundary of the coding region is determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxy) end. Genes can include, but are not limited to, cDNA derived from prokaryotic mRNA or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic DNA or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically located at the 3' end of the gene sequence.

[0055] The term "cell" is used herein in a broad sense within the art and refers to a living organism that is a structural unit of tissue in a multicellular organism, enclosed by a membrane structure that isolates it from the outside, capable of self-replication, and possessing genetic information and the mechanisms for its expression. The cells used herein may be naturally occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0056] As used herein, the term “stem cell” means a cell that is capable of self-renewal and is pluripotent or multipotent. Typically, stem cells can regenerate injured tissue. Stem cells as used herein may be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells, or tissue stem cells (also referred to as tissue-specific stem cells or somatic stem cells).

[0057] Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. ES cells were first established in 1981 and have been used to create knockout mice since 1989. Human ES cells were established in 1998 and are now available for use in regenerative medicine.

[0058] Unlike ES cells, tissue stem cells have limited differentiation potential. Tissue stem cells reside in specific locations within tissues and possess undifferentiated intracellular structures. Therefore, tissue stem cells generally have low pluripotency. Tissue stem cells have a high nucleus / cytoplasmic ratio and few intracellular organelles. Many tissue stem cells have low pluripotency, a long cell cycle, and proliferative capacity exceeding that of an individual's lifetime. Tissue stem cells are classified into dermal, digestive, myeloid, and nervous systems based on the site of their origin. Dermal tissue stem cells include epidermal stem cells and hair follicle stem cells. Digestive tissue stem cells include pancreatic (common) stem cells and hepatic stem cells. Myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Nervous system tissue stem cells include neural stem cells and retinal stem cells.

[0059] "Induced pluripotent stem cells," commonly abbreviated as iPS cells or iPSCs, refer to a type of pluripotent stem cell that is artificially prepared by introducing specific factors called reprogramming factors into non-pluripotent cells, generally adult somatic cells or terminally differentiated cells (such as fibroblasts, hematopoietic cells, muscle cells, neurons, or epidermal cells).

[0060] "Pluripotent" means a stem cell that has the potential to differentiate into one or more tissues or organs, or in particular into any of the three germ layers: endoderm (endoderm, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), or ectoderm (epidermal tissue and nervous system). As used herein, "pluripotent stem cell" means a cell that can differentiate into a cell derived from any of the three germ layers (e.g., a direct descendant of a totipotent or induced pluripotent cell).

[0061] As used herein, the terms “treatment” and “to treat” mean obtaining a desired pharmacological and / or physiological effect. Such effect may be prophylactic in that it completely or partially prevents a disease or the symptoms of a disease, and / or therapeutic in that it partially or completely cures a disease and / or adverse effects resulting from the disease. As used herein, “treatment” includes any treatment of a disease in a mammal (e.g., human), and “treatment” includes (a) preventing the disease from developing in an object that is susceptible to infection but has not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., preventing its development, and (c) alleviating the disease, i.e., causing its regression.

[0062] In some embodiments, the method is useful for reducing the size and / or cell number of solid tumors. In some embodiments, the method of the present disclosure is useful for inhibiting the growth of tumors, such as solid tumors, in a subject.

[0063] The term “antigen” means any substance to which the immune system produces antibodies or to which T cells respond. In some embodiments, the antigen is a peptide having a length of 5 to 50 amino acids, or at least, at most, or exactly 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or 300 amino acids, or any range obtained therefrom.

[0064] The term "antibody" includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies, and antibody fragments, which may belong to humans, mice, humanized organisms, chimeras, or other species. A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies against a specific antigen site.

[0065] "Antibodies or functional fragments thereof mean immunoglobulin molecules that specifically bind to or immunologically react with a particular antigen or epitope, and include polyclonal and monoclonal antibodies. The term antibody includes genetically modified or altered forms of immunoglobulins, such as intracellular antibodies, peptide bodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heterocoupled antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies). The term functional antibody fragment includes antigen-binding fragments of antibodies, such as Fab' fragments, F(ab')2 fragments, Fab fragments, Fv fragments, rlgG fragments, and scFv fragments. The term scFv means a single-chain Fv antibody in which the heavy-chain variable domain and light-chain variable domain of a conventional double-chain antibody are bound together to form a single chain."

[0066] There is particular interest in the use of single-stranded variable fragments (scFv). scFv are recombinant molecules in which the variable regions of the heavy and light chains of immunoglobulins encoding antigen-binding domains are modified into single-stranded polypeptides. Typically, V H Arrays and V L The sequences are joined by a linker sequence. For example, see Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, which is incorporated herein by reference.

[0067] The "therapeutic dose" or "effective dose" refers to the amount of an agent or a combined amount of two agents that, when administered to a mammal or other subject to treat a disease, is sufficient to have such an effect on treating the disease. The "therapeutic dose" will vary depending on the agent, the disease and its severity, as well as the age and weight of the subject receiving treatment.

[0068] As used herein, "a" or "an" may mean one or more. As used in the claims herein, the words "a" or "an," when used in conjunction with the word "comprising," may mean one or more.

[0069] In the claims, the term “or” is used to mean “and / or” unless it is explicitly indicated that the substitutes are mutually exclusive, but this disclosure supports the definitions of substitutes only and “and / or.” As used herein, “another” may mean at least a second or more.

[0070] Throughout this application, the term “about” is used to indicate that a value includes inherent variations in errors in the apparatus or method used to determine that value, or variations that exist between the objects being tested.

[0071] II. Polypeptides A. Signal peptide A "signal peptide" refers to a peptide sequence that directs the transport and localization of an intracellular protein to a specific organelle (such as the endoplasmic reticulum) and / or the cell surface. Signal peptides guide newly synthesized proteins into the endoplasmic reticulum. This is essential when the receptor needs to be glycosylated and fixed to the cell membrane. It is common to use a signal peptide that is naturally bound to the amino-terminus (for example, in scFv with a light-chain-linker-heavy-chain sequence, the natural signal peptide of the light chain is used). In some embodiments, the signal peptide is SEQ ID NO:18.

[0072] In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., it is a cleavable signal peptide. In some embodiments, the restriction enzyme site is located at the carboxyl terminus of the signal peptide to facilitate cleavage.

[0073] B. Antigen-binding domain The antigen-binding domain is a single-strand variable fragment (scFv) based on the TGF-β antibody. The antibody fragment, either "single-strand Fv" or "scFv", is the V of the antibody. H Domain and V L It includes domains, and these domains are located within a single-chain polypeptide. In some embodiments, the antigen-binding domain is V H Domain and V L A peptide linker is further included between the domain and the scFv, allowing it to form a structure desirable for antigen binding.

[0074] The variable regions of the antigen-binding domains of the polypeptides disclosed herein can be modified by mutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL to improve one or more antibody binding properties (e.g., affinity). The term "CDR" refers to the complementarity-determining region, which is based on the variable chains of immunoglobulins (antibodies) and T cell receptors produced by B cells and T cells, respectively, that enable these molecules to bind to specific antigens. Many sequence divergences related to immunoglobulins and T cell receptors are observed in the CDR, and these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding or other target functional properties can be evaluated by appropriate in vitro or in vivo assays. Preferred conservative modifications are typically introduced, altering one, two, three, four, or five or fewer residues within the CDR region. Mutations may be amino acid substitutions, additions, or deletions.

[0075] By modifying the framework of an antibody, for example, by causing a "reverse mutation" in one or more framework residues to correspond to the germline sequence, immunogenicity can be reduced.

[0076] The antigen-binding domain is also thought to be able to become polyspecific or polyvalent by polymerizing it with VH and VL region pairs that bind to the same antigen (polyvalent) or different antigens (polyspecific).

[0077] As used herein, the term “affinity” means the equilibrium constant of the reversible binding of two active substances and is expressed as the dissociation constant (Kd). Affinity may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater (or any range within that range) than the affinity of an antibody with an unrelated amino acid sequence. As used herein, the term “binding activity” means resistance to the dissociation of a complex of two or more active substances after dilution. The terms “immunoreactivity” and “preferentially binding” are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0078] The term "bond" refers to the direct association between two molecules through interactions such as covalent bonds, electrostatic bonds, hydrophobic bonds, and ionic bonds, as well as / or hydrogen bonds (including interactions such as salt bridges and water bridges).

[0079] C. Peptide Spacer The spacer region connects the antigen-binding domain to the transmembrane domain. This region should be flexible enough to direct the antigen-binding domain in various directions to facilitate antigen recognition. The most basic form is an IgG-derived hinge region. Other examples include the CH2CH3 region of immunoglobulins and a portion of CD3. In some embodiments, the CH2CH3 region may have L235E / N297Q modification or L235D / N297Q modification, or may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity of the CH2CH3 region. For most scFv-type constructs, an IgG hinge is sufficient. However, the best spacer often has to be determined empirically. In some embodiments, the spacer is derived from IgG4.

[0080] As used herein, the term “hinge” means a flexible polypeptide connector region (also referred to as a “hinge region” or “spacer”) that provides structural flexibility and spacing to an adjacent polypeptide region, and can consist of a native polypeptide or a synthetic polypeptide. A “hinge” derived from an immunoglobulin (e.g., IgG1) is commonly defined as the segment from Glu216 to Pro230 in human IgG1 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by positioning the first and last cysteine ​​residues to form an inter-heavy-chain disulfide (SS) bond at the same position. Hinge regions may be spontaneous or non-spontaneous and include, but are not limited to, modified hinge regions described in U.S. Patent No. 5,677,425. The hinge region may include a fully hinged region derived from an antibody of a different class or subclass than the CH1 domain class or subclass. The term “hinge” may also include regions derived from CD8, and regions derived from other receptors that provide similar functions in conferring flexibility and spacing to adjacent regions.

[0081] The peptide spacers are at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 21 The peptide spacer may have a length of 5, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 325, 350, or 400 amino acids (or any range obtained therefrom). In some embodiments, the peptide spacer consists of or includes a hinge region derived from immunoglobulin. The amino acid sequence of the immunoglobulin hinge region is known in the art; see, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res.

[0082] The length of the peptide spacer may affect the response to TGF-β and / or the growth characteristics. In some embodiments, short spacers, such as fewer than 50, 45, 40, 30, 35, 30, 25, 20, 15, or 10 amino acids, may have the advantage of reducing the concentration of TGF-β required for an effective activation response. In some embodiments, at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228 Long spacers, such as amino acid spacers of 1, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids, may have the advantage of increasing growth in vivo or in vitro.

[0083] As a non-limiting example, the immunoglobulin hinge region may contain one of the following amino acid sequences: TIFF0007912329000001.tif48166 In some embodiments, the hinge is SEQ ID NO:98 or SEQ ID NO:99. In some embodiments, the hinge is SEQ ID NO:99.

[0084] The hinge region may contain the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4. The hinge region may contain one or more amino acid substitutions and / or insertions and / or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 in the human IgG1 hinge may be substituted with Tyr, and as a result the hinge region may contain the sequence It will now include TIFF0007912329000002.tif4128.

[0085] The hinge region may contain an amino acid sequence derived from human CD8, for example, the hinge region may contain the following amino acid sequence: TIFF0007912329000003.tif5154 or its variants.

[0086] D. Transmembrane domain The transmembrane domain is a hydrophobic alpha-helix that spans the membrane. Typically, the transmembrane domain is derived from the most proximal component of the endodomain. Different transmembrane domains result in changes to receptor stability.

[0087] The transmembrane domain is inserted between the peptide spacer and the endodomain. In some embodiments, the transmembrane domain is inserted between the peptide spacer and the co-stimulatory region. In some embodiments, a linker is located between the transmembrane domain and the co-stimulatory region or endodomain.

[0088] Suitable applications include any transmembrane domain that inserts polypeptides into the cell membrane of eukaryotic (e.g., mammalian) cells. A non-limiting example is a transmembrane sequence. TIFF0007912329000004.tif4128 may be used. In some embodiments, the transmembrane domain is derived from CD8 beta. TIFF0007912329000005.tif4128, originating from CD4. TIFF0007912329000006.tif4128, derived from CD3 zeta. TIFF0007912329000007.tif4128, originating from CD28 TIFF0007912329000008.tif4128, derived from CD134 (OX40) TIFF0007912329000009.tif4128, or CD7 origin The filename is TIFF0007912329000010.tif4128.

[0089] E. End Domain After recognizing an antigen, the receptors cluster, and signals are transmitted to the cell via the endodomain and / or costimulatory domain. In some embodiments, the costimulatory domain described herein is part of the endodomain. The most commonly used endodomain component is a CD3 zeta containing three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3 zeta alone cannot provide a fully competent activation signal and requires further costimulatory signaling. For example, chimeric CD28 and OX40 can be used with CD3 zeta to transmit proliferation / survival signals, or all three can be used together.

[0090] Further end domains suitable for use in polypeptides of this disclosure include any desired signaling domains that provide a characteristic and detectable signal (e.g., increased production of one or more cytokines by cells, transcriptional changes of target genes, changes in protein activity, changes in cell behavior (e.g., cell death), cell proliferation, cell differentiation, cell survival, regulation of intracellular signaling responses, etc.) in response to activation via binding of an antigen to the antigen-binding domain. In some embodiments, the end domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif described herein. In some embodiments, the end domain includes a DAP10 / CD28 type signaling chain.

[0091] Suitable endodomains for use in the polypeptides of this disclosure include intracellular signaling polypeptides containing an immune receptor tyrosine activation motif (ITAM). The ITAM motif is YX1X2(L / I), where X1 and X2 are independently any amino acids (SEQ ID NO: 64). The endodomain may contain one, two, three, four, or five ITAM motifs. In some cases, the ITAM motif is repeated twice within the endodomain, and the first and second examples of the ITAM motif are separated from each other by 6 to 8 amino acids, e.g., (YX1X2(L / I))(X3). n The formula is (YX1X2(L / I)), where n is an integer between 6 and 8, and each of the 6 to 8 X3 can be any amino acid (SEQ ID NO: 65).

[0092] A preferred end-domain may be an ITAM motif-containing portion derived from an ITAM motif-containing polypeptide. For example, a preferred end-domain may be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Therefore, a preferred end-domain does not need to contain the entire sequence of the entire protein from which it originates. Examples of preferred ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0093] The endodomain may also be derived from DAP12 (also known as TYROBP, TYRO protein tyrosine kinase binding protein, KARAP, PLOSL, DNAX activating protein 12, KAR-related protein, TYRO protein tyrosine kinase binding protein, killer-activated receptor-related protein, etc.). For example, a suitable endodomain polypeptide is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000011.tif70166.

[0094] In some embodiments, a suitable end-domain polypeptide may include an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Therefore, a suitable end-domain polypeptide is, It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000012.tif4128.

[0095] In some embodiments, the endodomain is derived from FCER1G (also known as FCRG, Fc epsilon receptor I gamma chain, Fc receptor gamma chain, fc epsilon Rl gamma, fcR gamma, fceRI gamma, high affinity immunoglobulin epsilon receptor subunit gamma, high affinity gamma chain of immunoglobulin E receptor, etc.). For example, a suitable endodomain polypeptide is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000013.tif12159.

[0096] In some embodiments, a suitable end-domain polypeptide may include an ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Therefore, a suitable end-domain polypeptide is It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000014.tif4128.

[0097] In some embodiments, the endodomain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D, CD3-DELTA, T3D, the delta subunit of the CD3 antigen, CD3 delta, the delta polypeptide of the CD3d antigen (TiT3 complex), the delta chain of OKT3, the T cell receptor T3 delta chain, the T cell surface glycoprotein CD3 delta chain, etc.). For example, a preferred endodomain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to a continuous interval of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 140 aa, about 140 to about 150 aa, or about 150 to about 170 aa from any of the following amino acid sequences (two isoforms): TIFF0007912329000015.tif48160.

[0098] In some embodiments, a suitable end-domain polypeptide may include an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Therefore, a suitable end-domain polypeptide is It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000016.tif4128.

[0099] In some embodiments, the endodomain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). For example, a suitable endo-domain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to a continuous interval of approximately 100 to 110 amino acids (aa), approximately 110 to 115 aa, approximately 115 to 120 aa, approximately 120 to 130 aa, approximately 130 to 140 aa, approximately 140 to 150 aa, or approximately 150 to 205 aa from the following amino acid sequences: TIFF0007912329000017.tif26160.

[0100] In some embodiments, a suitable end-domain polypeptide may contain an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Therefore, a suitable end-domain polypeptide is, It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000018.tif5128.

[0101] In some embodiments, the endodomain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable endodomain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to a continuous interval of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 140 aa, about 140 to about 150 aa, or about 150 to about 180 aa from the following amino acid sequences: TIFF0007912329000019.tif26160.

[0102] In some embodiments, a suitable end-domain polypeptide may include an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. Therefore, a suitable end-domain polypeptide is, It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000020.tif4128.

[0103] In some embodiments, the endodomain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3 zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to a continuous interval of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 140 aa, about 140 to about 150 aa, or about 150 to about 160 aa from any of the following amino acid sequences (two isoforms): TIFF0007912329000021.tif56160.

[0104] In some embodiments, a suitable end-domain polypeptide may contain an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence. Therefore, a suitable end-domain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: TIFF0007912329000022.tif34160.

[0105] In some embodiments, the endodomain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain, CD79a antigen (immunoglobulin-associated alpha), MB-1 membrane glycoprotein, ig alpha, membrane-bound immunoglobulin-associated protein, surface IgM-associated protein, etc.). For example, a preferred endodomain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to a continuous interval of about 100 to about 110 amino acids (aa), about 110 to about 115 aa, about 115 to about 120 aa, about 120 to about 130 aa, about 130 to about 150 aa, about 150 to about 200 aa, or about 200 to about 220 aa from any of the following amino acid sequences (two isoforms): TIFF0007912329000023.tif63161.

[0106] In some embodiments, a suitable end-domain polypeptide may contain an ITAM motif-containing portion of the full-length CD79A amino acid sequence. Therefore, a suitable end-domain polypeptide may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with respect to the following amino acid sequences: TIFF0007912329000024.tif5128.

[0107] In some embodiments, a suitable end-domain may include a DAP10 / CD28 type signaling chain. An example of a DAP10 signaling chain is the amino acid sequence: The filename is TIFF0007912329000025.tif4128. In some embodiments, a preferred end domain is an amino acid sequence. It contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the full length of TIFF0007912329000026.tif4128.

[0108] An example of a CD28 signaling chain is the amino acid sequence The filename is TIFF0007912329000027.tif12158. In some embodiments, a preferred end domain is an amino acid sequence. It contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with respect to the full length of TIFF0007912329000028.tif12158.

[0109] Further end domains suitable for use in the polypeptides of this disclosure include ZAP70 polypeptides, for example, polypeptides containing amino acid sequences having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity for a continuous interval of about 300 to about 400 amino acids, about 400 to about 500 amino acids, or about 500 to about 619 amino acids from the following amino acid sequences: TIFF0007912329000029.tif85160.

[0110] F. Detected peptide Suitable detection peptides include hemagglutinins (HA, for example). TIFF0007912329000030.tif4128), FLAG (for example, TIFF0007912329000031.tif4128), c-myc (for example, This includes TIFF0007912329000032.tif4128). Other suitable detection peptides are known in the art.

[0111] G. Peptide Linker In some embodiments, the polypeptides of this disclosure include peptide linkers (sometimes referred to as linkers). The peptide linkers may separate any of the peptide domains / regions described herein. For example, linkers may be located between a signal peptide and an antigen-binding domain, between the VH and VL regions of an antigen-binding domain, between an antigen-binding domain and a peptide spacer, between a peptide spacer and a transmembrane domain (either adjacent to a costimulatory region or located in the N or C region of a costimulatory region), and / or between a transmembrane domain and an endodomain. Peptide linkers may have any of a variety of amino acid sequences. Domains and regions can generally be linked by flexible peptide linkers, but other chemical bonds are not excluded. Linkers may be peptides of approximately 6 to 40 amino acids in length, or peptides of approximately 6 to 25 amino acids in length. These linkers can be produced using synthetic oligonucleotides encoding the linkers to link proteins.

[0112] A peptide linker with a certain degree of flexibility can be used. The peptide linker can have substantially any amino acid sequence, provided that a suitable peptide linker generally has a sequence that yields a flexible peptide. The use of small amino acids such as glycine and alanine is useful for producing flexible peptides. The creation of such sequences is routine for those skilled in the art.

[0113] A suitable linker can be easily selected and may have any of the following preferred lengths: 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids (e.g., 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids), and may consist of 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0114] As examples of flexible linkers, glycine polymer (G) n , glycine-serine polymer (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 40), and (GGGS) n (SEQ ID NO: 41), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers can be used; since Gly and Ser are relatively unstructured, they can function as neutral tethers between components. Glycine polymers can be used; glycine accesses phi-psi space significantly more than comparable alanine, and is much less restricted than residues with long side chains. Examples of spacers include, but are not limited to, amino acid sequences including, for example, TIFF0007912329000033.tif12144.

[0115] H. Co-stimulatory Domain Non-limiting examples of suitable co-stimulatory domains include, but are not limited to, polypeptides derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0116] The co-stimulatory domain can have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids, or any range of length therebetween. In some embodiments, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9, CD137, 4-1BB, CDwl37, ILA, etc.). For example, a suitable co-stimulatory domain is It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000034.tif4146.

[0117] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000035.tif4155.

[0118] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000036.tif5133.

[0119] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, and TXGP1L). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000037.tif4131.

[0120] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000038.tif19158.

[0121] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000039.tif12160.

[0122] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000040.tif26159.

[0123] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000041.tif12151.

[0124] In some embodiments, the co-stimulatory region is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a preferred co-stimulatory region is: It may contain an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity with TIFF0007912329000042.tif12159.

[0125] I. Additional Modifications The polypeptides of this disclosure may be further chemically modified. The glycosylation of the polypeptide may be modified, for example, by modifying one or more glycosylation sites in the polypeptide sequence, thereby improving the polypeptide's affinity for an antigen (U.S. Patents 5,714,350 and 6,350,861).

[0126] The polypeptides of the present invention can be pegylated to increase their biological half-life by reacting them with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG under conditions that one or more PEG groups are bonded to the polypeptide. The pegylation of polypeptides can be carried out by acylation or alkylation reactions with reactive PEG molecules (or similar reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy polyethylene glycol or aryloxy polyethylene glycol, or polyethylene glycol maleimide. Methods for pegyrating proteins are known in the art and can be applied to the polypeptides of the present invention (European Patents No. 0154316 and 0401384).

[0127] Furthermore, polypeptides can be chemically modified by conjugating or fusing them with serum proteins such as human serum albumin, thereby increasing the half-life of the resulting molecule. Such approaches are described, for example, in European Patent Nos. 0322094 and 0486525.

[0128] The polypeptides of this disclosure can be conjugated into diagnostic or therapeutic agents and used diagnostically to, for example, monitor the onset or progression of a disease and determine the effectiveness of a given treatment regimen. Polypeptides can also be conjugated into therapeutic agents to provide a therapy that combines the immunostimulatory effects of the polypeptides. Examples of diagnostic agents include enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron-emitting metals for various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. Detectable substances can be directly or indirectly linked to or conjugated to polypeptides using techniques known in the art. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent substance is luminol. Examples of bioluminescent substances include luciferase, luciferin, and aequorin. An example of a suitable radioactive substance is, 125 I, 131I, Indium-111, Lutetium-171, Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166, Holmium-166, Examples include rhenium-186, rhenium-188, rhenium-189, lead-212, radium-223, actinium-225, iron-59, selenium-75, arsenic-77, strontium-89, molybdenum-99, rhodium-1105, palladium-109, praseodymium-143, promethium-149, erbium-169, iridium-194, gold-198, gold-199, and lead-211. Chelating agents can be attached via amines (amities) (Meares et al., 1984 Anal. Biochem. 142:68-78), sulfhydral groups of amino acid residues (Koyama 1994 Chem. Abstr. 120:217262t), and carbohydrate groups (Rodwell et al. 1986 PNAS USA 83:2632-2636; Quadri et al. 1993 Nucl. Med. Biol. 20:559-570).

[0129] Polypeptides can also be conjugated to therapeutic agents to provide therapies that combine the immunostimulatory effects of polypeptides.

[0130] Further suitable conjugate molecules include ribonucleases (RNases), DNase I, antisense nucleic acids, repressor RNA molecules (such as siRNA molecules), immunostimulant nucleic acids, aptamers, ribozymes, triple-stranding molecules, and external guide sequences. Aptamers are small nucleic acids ranging from 15 to 50 nucleotides in length that fold into established secondary and tertiary structures (such as stem-loops or G quartets), and can conjugate to small molecules such as ATP (US Patent No. 5,631,146) and theophylline (US Patent No. 5,580,737), as well as larger molecules such as reverse transcriptase (US Patent No. 5,786,462) and thrombin (US Patent No. 5,543,293). Ribozymes are nucleic acid molecules that can catalyze chemical reactions intramolecularly or intermolecularly. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to target substrates. Triple-stranded functional nucleic acid molecules can interact with double-stranded or single-stranded nucleic acids by forming a triple strand, in which the three strands of DNA form a complex based on Watson-Crick and Hoogsteen pairings. The triple-stranded molecule can bind to target regions with high affinity and specificity.

[0131] Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulants of the specific activity of target molecules, or they can possess novel activities independently of any other molecule.

[0132] J. Cancer-specific chimeric antigen receptor In some embodiments, the cell may further contain a cancer-specific chimeric antigen receptor (CAR). In the context of CAR, the term “cancer-specific” means a CAR that has antigen-binding specificity to cancer-specific molecules, such as cancer-specific antigens. In some embodiments, the cancer-specific CAR is located within a cell containing a TGF-β CAR. In some embodiments, the cancer-specific CAR and the TGF-β CAR reside on separate polypeptides. In some embodiments, the CAR is a bispecific CAR that has antigen binding to both cancer-specific molecules and TGF-β. For example, a bispecific CAR may have a signaling peptide, a cancer molecule-specific scFv, optionally a peptide linker / spacer, followed by a TGF-β scFv, and further followed by a spacer, a transmembrane domain, and a costimulatory domain. In some embodiments, the bispecific CAR includes one or more additional peptide segments as described herein.

[0133] In some embodiments, the polypeptides of this disclosure may include CD20 scFv. An example of a CD20 scFv is the following sequence: TIFF0007912329000043.tif34159

[0134] In some embodiments, the polypeptides of this disclosure may include CD19 scFv. An example of a CD19 scFv is the following sequence: TIFF0007912329000044.tif34159

[0135] Other cancer-specific molecules (in addition to CD19 and CD20) may include CAIX, CD33, CD44v7 / 8, CEA, EGP-2, EGP-40, erb-B2, erb-B3, erb-B4, FBP, fetal acetylcholine receptor, GD2, GD3, Her2 / neu, IL-13R-a2, KDR, k-light chain, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen (h5T4), PSCA, PSMA, mAb IgE targeted TAA, TAG-72, and VEGF-R2. In some embodiments, the cancer-specific molecule includes Her2.

[0136] III. Cells Certain embodiments relate to cells comprising polypeptides or nucleic acids of the present disclosure. In some embodiments, the cells are immune cells or T cells. "T cells" include T helper cells (CD4 + cells), cytotoxic T cells (CD8 + This includes all types of CD3-expressing immune cells, including T cells, regulatory T cells (Treg), and gamma delta T cells. "Cytotoxic cells" include CD8 + These include T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses.

[0137] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Cell Culture Preservation Center (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), human fetal kidney (HEK) 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RATI cells, mouse L cells (ATCC number CCLI.3), HLHepG2 cells, Hut-78, Jarcut, HL-60, and NK cell lines (e.g., NKL, NK92, and YTS).

[0138] In some cases, the cells may not be immortalized cell lines, but rather cells obtained from an individual (e.g., primary cells). For example, in some cases, the cells may be immune cells obtained from an individual. One example is T lymphocytes obtained from an individual. In another example, the cells may be cytotoxic cells obtained from an individual. In yet another example, the cells may be stem cells or progenitor cells obtained from an individual.

[0139] IV. Method Aspects of this disclosure relate to methods for stimulating an immune response. Immune response stimulation can be performed in vitro, in vivo, or ex vivo. In some embodiments, the methods relate to cells capable of stimulating an immune response in the presence of TGF-β. The methods typically involve genetically modifying mammalian cells using an expression vector or RNA (e.g., in vitro transcription RNA) containing a nucleotide sequence encoding the polypeptide of this disclosure, or directly transferring the polypeptide into the cells. The cells may be immune cells (e.g., T lymphocytes or NK cells), stem cells, progenitor cells, etc. In some embodiments, the cells are those described herein.

[0140] In some embodiments, the genetic modification is performed ex vivo. For example, T lymphocytes, stem cells, or NK cells (or cells as described herein) are obtained from an organism, and the cells obtained from that organism are genetically modified to express the polypeptides of this disclosure. In some cases, the genetically modified cells are activated ex vivo (i.e., TGF-β is brought into contact with the cells ex vivo). In other cases, the genetically modified cells are introduced into an organism (e.g., the organism from which the cells were collected), and the genetically modified cells are activated in vivo (i.e., by endogenously produced TGF-β).

[0141] In some embodiments, the method further includes the administration of an additional therapeutic agent, such as a bispecific T cell engager (BITE). Such therapeutic agents may be administered to the patient in peptide form or expressed in cells of the Disclosure, such as cells containing TGF-β CAR. BITE may have antigen specificity to cancer antigens / cancer molecules known in the art and / or described herein, and may also have antigen specificity to T cell molecules such as CD3.

[0142] In some embodiments, the method relates to the administration of cells or peptides described herein for treating cancer or for administration to a person having cancer. In some embodiments, cancer is adrenal cancer, anal cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain / CNS tumors in children or adults, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing tumor family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, pharyngeal cancer and hypopharyngeal cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell Lung cancer, pulmonary carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, sarcoma, basal skin cancer, squamous cell carcinoma, melanoma, Merkel cell carcinoma, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, or Wilms' tumor.

[0143] The embodiments can be used to treat or induce remission of many immune-mediated, inflammatory, or autoimmune inflammatory diseases (e.g., hypersensitivity, asthma, diabetes (e.g., type 1 diabetes), graft rejection, etc.). Examples of such diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile-onset systemic rheumatoid arthritis, osteoarthritis, progressive chronic arthritis (arthritis chronica progrediente), osteoarthritis, primary chronic polyarthritis (polyarthritis chronicaprimaria), reactive arthritis, and ankylosing spondylitis, inflammatory hyperproliferative skin diseases, psoriasis (e.g., psoriasis vulgaris, guttate psoriasis, pustular psoriasis, and nail psoriasis), atopic dermatitis (e.g., atopic diseases such as hay fever and Job's syndrome), dermatitis (e.g., contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, 1 Secondary irritant contact dermatitis and atopic dermatitis), X-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria (e.g., chronic allergic urticaria and chronic idiopathic urticaria including chronic autoimmune urticaria), myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis (e.g., systemic sclerosis, multiple sclerosis (MS) (spino-optical type) MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS)), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis), neuromyelitis optica (NMO), inflammation infectious bowel diseases (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis (ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polypoidal colitis, necrotizing enterocolitis, and full-thickness colon) Inflammation, autoimmune inflammatory bowel disease, enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome (e.g., adult or acute respiratory distress syndrome (ARDS)), meningitis, inflammation of the whole or part of the uvea, iritis, choroiditis, autoimmune hematological disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve injury in cases such as meningitis, herpes zoster of pregnancy, bullous pemphigoid of pregnancy, pruritisscroti), autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, IgE-mediated diseases (e.g., anaphylaxis and allergic rhinitis and atopic rhinitis), encephalitis (e.g., Rasmussen's encephalitis and limbic and / or brainstem encephalitis), uveitis (e.g., anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis), glomerulonephritis (GN) with or without nephrotic syndrome (e.g., primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, type I and Chronic or acute glomerulonephritis, including membrane or membranoproliferative GN (MPGN) including type II, and rapidly progressive GN), proliferative glomerulonephritis, autoimmune polyglandular endocrine deficiency, balanitis (e.g., plasma cell-limited balanitis), balanoposthitis, erythema annulare centrifugally, erythema pigmentosum fixed, erythema multiforme, granuloma annulare, lichen glaucoma, lichen sclerosing atrophic, lichen simplex chronic, lichen acanthoid, lichen planus, ichthyosis latifolia, exfoliative hyperkeratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema (e.g., allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and bullous palmoplantar eczema), asthma (e.g., bronchial asthma), bronchial asthmaExamples include asthma, autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood group during pregnancy, chronic inflammatory lung diseases, autoimmune myocarditis, leukocyte adhesion disorders, lupus (e.g., lupus nephritis, lupus encephalitis, lupus inflorescence, lupus in childhood, nonrenal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus erythematosus), juvenile-onset (Type 1) diabetes (e.g., juvenile insulin-dependent diabetes mellitus (IDDM)) and adult-onset (Type 2) diabetes mellitus, as well as autoimmune diabetes. Further considerations include immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, sarcoidosis, granulomatosis (e.g., lymphomatous granulomatosis, Wegener's granulomatosis), agranulocytosis, vasculitis (e.g., vasculitis, large vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis (e.g., systemic necrotizing vasculitis), and ANCA-associated vasculitis (e.g., Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vasculitis)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, and disease. Almond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia (e.g., autoimmune hemolytic anemia (AIHA)), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multi-organ injury syndromes (e.g., secondary to sepsis, trauma, or bleeding), antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid syndromes, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid (e.g., bullous pemphigoid and cutaneous pemphigoid), pemphigus (pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane)Physiological conditions (including pemphigoid and erythematous pemphigus), autoimmune polyglandular endocrine disorders, Reiter's disease or syndrome, burns, pre-eclampsia, immune complex disorders (e.g., immune complex nephritis), antibody-mediated nephritis, polyneuropathy, chronic neuropathy (e.g., IgM polyneuropathy or IgM-mediated neuropathy), autoimmune or immune-mediated thrombocytopenia (e.g., idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP), scleritis (e.g., idiopathic keratoscleritis, episcleritis), autoimmune diseases of the testes and ovaries (e.g., autoimmune orchitis and oophoritis), primary hypothyroidism, hypoparathyroidism, autoimmune endocrine disorders (e.g., autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), Physiitis (or thyroiditis such as subacute thyroiditis), autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polycystic syndromes (e.g., autoimmune polycystic syndrome (or polycystic endocrine disorder syndrome)), paraneoplastic syndromes (e.g., neurological paraneoplastic syndromes such as Lambert-Eaton myasthenia gravis or Eaton-Lambert syndrome, Stiffman or Stiffperson syndrome), encephalomyelitis (e.g., allergic encephalomyelitis or encephalomyelitis-induced allergy, and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis), myasthenia gravis (e.g., (thymoma-associated myasthenia gravis), cerebellar degeneration, neurogenic myotonia, oculoclonus or oculoclonus-myoclonus syndrome (OMS), as well as sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) versus NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient spurt Melting dermatosis, cirrhosis (e.g., primary biliary cirrhosis and pulmonary cirrhosis), autoimmune bowel disease syndrome, celiac or coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases (e.g., autoimmune inner ear disease (AIED), autoimmune hearing loss), polychondritis (e.g., refractory or relapsing or relapsing polychondritis), alveolar proteinosis, Cogan syndrome / non-syphilitic keratoplasty, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, herpes zoster-related pain, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal immunoglobulinemia and monoclonal hypergammaglobulinemia of unknown significance (MGUS)), peripheral neuropathy, paraneoplastic syndromes, channel disorders (e.g., epilepsy, migraine, arrhythmia, muscle disorders, hearing loss, visual loss, periodic paralysis, and CNS channel disorders), autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS),Endocrine eye disorders, uveoretinitis, chorioretinitis, autoimmune hepatopathy, fibromyalgia, polyendocrine insufficiency, Schmidt syndrome, adrenal nephritis, gastric atrophy, presenile dementia, demyelinating diseases (e.g., autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy), Dressler syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorders, kyphosis, and telangiectasia), autoimmune infertility in men and women (e.g., due to anti-sperm antibodies), mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, cardiotomy Postoperative syndrome, Cushing's syndrome, bird vein disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis (e.g., allergic alveolitis and fibrotic alveolitis), interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases (e.g., leishmaniasis, trypanosomiasis, schistosomiasis, aspergillosis, aspergillosis), Sumter syndrome, Kaplan syndrome, dengue fever, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent erythema elevata, fetal erythroblastosis, eosinophilia Myositis, Schulman syndrome, Felty syndrome, filariasis, cyclitis (e.g., chronic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic), or Fuchs' cyclitis), Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroiditis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune Gonadal dysfunction, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritis syndrome, microlesions, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, arthritis, bronchitis, chronic obstructive airway / lung disease, silicosis, aphthous ulcers, aphthous stomatitis, arthrosclerotic disorders, spermatogenesis deficiency (asperniogenese), autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, lens hypersensitivity endophthalmitis, allergic enteritis,Erythema nodosum leprosy, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hanmann-Ricci disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrotic syndrome, sympathetic ophthalmitis, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions with T-cell infiltration, leukocyte adhesion deficiency, and immunological conditions associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes. Diseases with response, leukocyte leakage, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyglandular endocrine disorders, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmitis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insular pancreatitis, polyendocrine insufficiency, polyglandular autoimmune syndrome type 1, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy (e.g., dilated cardiomyopathy), acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis Acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, maxillary sinusitis or sphenoid sinusitis, eosinophil-related disorders (e.g., eosinophilia, pulmonary infiltrative eosinophilia, polymyalgia syndrome with eosinophilia, Löffrel syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia), bronchopneumoniae aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia of infants, Wiscott-Aldrich syndrome, telangiectasia Ataxia tonic syndrome, telangiectasia, autoimmune disorders associated with collagen diseases, rheumatism, neurological disorders, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases with angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of myocardium or other tissues, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, inflammatory disorders of the eye and orbit, granulocyte transfusion-associated syndromes,These include cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intra-arterial hyperplasia, peptic ulcers, valvular heart disease, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis.

[0144] V. Pharmaceutical Compositions This disclosure includes methods for modulating an immune response in subjects requiring an immune response. This disclosure includes cells, which may be in the form of pharmaceutical compositions that can be used to induce or modify an immune response.

[0145] The compositions relating to this disclosure are typically administered via any common route, which includes, but is not limited to, parenteral injection, orthotopic injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intranasal injection, or intravenous injection.

[0146] The compositions of the present invention are typically administered in a therapeutically effective and immunomodulatory amount using a method appropriate to the dosage formulation. The amount to be administered depends on the target being treated. The exact amount of active ingredient required for administration depends on the physician's judgment.

[0147] The application methods can vary widely. Any conventional method of administering a pharmaceutical composition containing cellular components can be applied. The dosage of the pharmaceutical composition depends on the route of administration and varies depending on the size and health condition of the subject.

[0148] In many cases, it is desirable to administer the drug at least three, four, five, six, seven, eight, nine, ten, or more times. Doses may be spaced 2 days to 12 weeks apart, but more commonly 1 to 2 weeks apart. Assays for alloreactive immune responses and T-cell activity may be performed after administration.

[0149] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" mean molecular entities and compositions that, when administered to animals or humans, do not cause adverse reactions, allergic reactions, or other harmful reactions. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, as well as isotonic and absorption retardants. The use of such culture media and activators with pharmaceutically active substances is well known in the art. Unless any commonly used culture media or activator is incompatible with the active ingredient, it is considered appropriate to use a culture media or activator with immunogenic compositions and therapeutic compositions. The pharmaceutical compositions of this disclosure are pharmaceutically acceptable compositions.

[0150] The compositions of this disclosure are formulated for parenteral administration and can be formulated for injection, for example, via intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Such compositions are typically available as injectable preparations, i.e., as liquid solutions or suspensions, and solid forms suitable for preparing solutions or suspensions can also be prepared by adding liquid prior to injection, and the preparations can also be emulsified.

[0151] Suitable drug forms for injection include sterile aqueous solutions or dispersions, and formulations containing sesame oil, peanut oil, or propylene glycol aqueous solutions. Furthermore, these formulations should be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0152] Sterile injection solutions are prepared by mixing the required amount of the active ingredient (i.e., the cells of this disclosure) with various other components listed above as needed in a suitable solvent, and then sterilizing by filtration. Dispersions are prepared by mixing various sterilized active ingredients with a sterile medium, the sterile medium comprising a basic dispersion medium and other necessary components other than those listed above.

[0153] The effective amount of a composition is determined based on the intended objective. The term “unit dose” or “dosage” refers to a physically distinct unit suitable for use in the subject, each unit containing a predetermined amount of the composition, which is calculated to obtain the desired response discussed herein in relation to the administration (i.e., the appropriate route and regimen). The amount administered depends on the desired outcome and / or protection, depending on the number of therapeutic agents and unit doses. The exact amount of the composition is also subject to the physician's judgment and is specific to each individual. Factors influencing the dose include the subject’s physical and clinical condition, the route of administration, the intended therapeutic objective (symptom relief versus cure), and the potency, stability, and toxicity of the particular composition. Once formulated, the solution is administered in a manner suitable for the administration formulation and in an amount that is therapeutically or prophylactically effective. The formulation is readily administered in various forms of administration, such as the injectable solution type described above.

[0154] VI. Arrays The antigen-binding domain of this disclosure includes the following VH (heavy chain variable) and VL (light chain variable) regions. TIFF0007912329000045.tif174160

[0155] The corresponding CDRs for the VH and VL regions of scFv#1 are the following amino acid sequences: TIFF0007912329000046.tif26160.

[0156] The corresponding CDRs for the VH and VL regions of scFv#2 are the following amino acid sequences: TIFF0007912329000047.tif26160.

[0157] The corresponding CDRs for the VH and VL regions of scFv#3 are as follows: TIFF0007912329000048.tif26160.

[0158] Examples of the detection peptides in this disclosure include: TIFF0007912329000049.tif19160 may be included.

[0159] Examples of signal peptides include: This includes TIFF0007912329000050.tif5128. Other exemplary signal peptides include: This includes TIFF0007912329000051.tif12128.

[0160] An example of a peptide spacer hinge region is: This includes TIFF0007912329000052.tif64160.

[0161] Examples of peptide linkers include, for instance, This includes TIFF0007912329000053.tif26163.

[0162] Examples of transmembrane domains include: TIFF0007912329000054.tif4128, derived from CD8 Beta. TIFF0007912329000055.tif4128, originating from CD4. TIFF0007912329000056.tif4128, derived from CD3 zeta. TIFF0007912329000057.tif5128, originating from CD28 TIFF0007912329000058.tif4128, derived from CD134 (OX40) TIFF0007912329000059.tif4128, and CD7-derived This includes TIFF0007912329000060.tif4128.

[0163] Examples of co-stimulatory regions include: This includes TIFF0007912329000061.tif121160.

[0164] In some embodiments, the endodomain contains an ITAM motif. The ITAM motif is YX1X2(L / I), where X1 and X2 are any amino acids independently (SEQ ID NO: 64). In some cases, the ITAM motif is repeated twice within the endodomain, and the first and second examples of the ITAM motif are separated from each other by 6 to 8 amino acids, for example, (YX1X2(L / I))(X3). n The formula is (YX1X2(L / I)), where n is an integer between 6 and 8, and each of the 6 to 8 X3 can be any amino acid (SEQ ID NO: 65).

[0165] An example of an end domain is: TIFF0007912329000062.tif187160TIFF0007912329000063.tif245160TIFF0007912329000064.tif70160 or polypeptides derived from those files. [Examples]

[0166] VII. Examples The following embodiments are included to demonstrate embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments demonstrate that the techniques discovered by the inventors function well in carrying out the present invention and can therefore be considered to constitute a preferred form of implementation. On the other hand, those skilled in the art will understand that, in view of this disclosure, numerous modifications may be made in the specific embodiments disclosed, but similar or comparable results can still be obtained without departing from the spirit and scope of the present invention.

[0167] Example 1 This disclosure describes single-stranded variable fragments (scFv) that neutralize human and mouse TGF-β, and chimeric antigen receptors (CARs) that are responsive to human and mouse TGF-β. The conservation levels of TGF-β across mammals suggest that the described scFv and CARs can bind to most mammalian TGF-β. Two types of scFv are constructed by linking the heavy-chain variable domain (VH) and light-chain variable domain (VL) of an anti-TGF-β antibody using a (G4S)3 linker, ultimately in the order N-terminus-VH-(G4S)3-VL-C-terminus. These scFv can be produced by transtransferring them into eukaryotic cells using DNA sequences encoding the scFv amino acid sequences shown in the table below. TIFF0007912329000065.tif90163

[0168] Each scFv can be tagged with a leader (i.e., signal) peptide derived from the mouse kappa light chain, allowing the secreted scFv to be directly collected from the culture medium of the producing cells. The scFv can also be tagged at its N-terminus (post-leader peptide but anterior to the VH sequence) with markers such as the DYKDDDDK (SEQ ID NO: 17) epitope adjacent to the GGS linker, an HA tag, or a cMyc tag. Each scFv is administered directly to neutralize human or mouse TGF-β. scFv#1 and scFv#2 are superior to scFv#3 (Figures 1 and 7). Furthermore, each scFv can be used to construct a TGF-β responsive CAR. A CAR is a fusion protein composed of an extracellular antigen-binding domain, an extracellular spacer, a transmembrane domain, a costimulatory signaling domain (the number of which varies depending on the specific CAR design), and a CD3 zeta signaling domain / endodomain. TGF-β CARs can be constructed using the aforementioned scFv as the extracellular antigen-binding domain. Immune cells, including T cells and natural killer (NK) cells, can be manipulated to express TGF-β CARs by various methods such as viral transduction, DNA nucleofection, and RNA nucleofection. TGF-β bound to TGF-β CARs activates human T cells, thereby changing TGF-β signaling from an immunosuppressive response to an immunostimulatory response. Furthermore, using TGF-β CARs as co-receptors can counteract immunosuppression and enhance T cell-mediated responses in any adoptive T cell therapy, regardless of whether it is based on tumor-infiltrating lymphocytes, T cell receptor manipulation, or other CARs.

[0169] As shown in Figure 2, TGF-β CARs are efficiently expressed on the surface of primary human T cells. TGF-β CARs were generated using scFv#2. Surface staining and flow cytometry revealed that TGF-β CARs appear on the surface of primary human CD4+ T cells and CD8+ T cells. The extracellular domain of the receptor contains an N-terminal FLAG epitope. EGFRt is a cleaved epidermal growth factor receptor, which indicates cell transduction. We further found that TGF-β CARs appear on the cell surface more efficiently than dominant-negative TGF-β receptors (Figure 9). Dominant-negative TGF-β receptors (DNRs) containing a cleaved TGF-β receptor chain II lacking an intracellular signaling domain have been reported to inhibit TGF-β signaling and improve T cell effector function. However, unlike TGF-β CARs, DNRs are not efficiently expressed on the cell surface. The data shown in Figure 9 were obtained from the transduction of primary human CD4+ T cells using lentiviruses encoding either FLAG-tagged TGF-β CAR or FLAG-tagged DNR. Each receptor is tagged to the cleaved epidermal growth factor receptor (EGFRt) via a T2A cleavage peptide, allowing transduced cells to be identified by EGFRt staining, while receptor surface expression can be identified by FLAG staining. The results in Figure 9 show that TGF-β CAR is expressed on the T cell surface with significantly higher efficiency than DNR.

[0170] Figure 3 shows that TGF-β CARs block endogenous TGF-β signaling. Expression of TGF-β CARs in primary human T cells blocks TGF-β signaling via the SMAD pathway. T cells expressing the specified receptor were incubated with the specified concentration of TGF-β for 30 minutes and probed for phosphorylated SMAD2 by Western blotting. EGFRt represents T cells expressing the truncated epidermal growth factor receptor and acts as a control for "no CAR," while scFv-less represents T cells expressing a CAR that lacks any ligand-binding scFv domain but is otherwise identical in length to the TGF-β CAR. The labels "long" and "short" after TGF-β CAR represent the length of its extracellular peptide spacer. As shown in Figure 6, TGF-β CAR-T cells proliferate in response to TGF-β. T cells expressing TGF-β CARs convert TGF-β from a growth-inhibiting cytokine to a growth-promoting cytokine.

[0171] Furthermore, it was found that TGF-β CARs activate T cells and induce cytokine production (Figure 4). As shown in Figure 4, TGF-β CAR-T cells upregulate the expression of the CD69 activation marker and produce immunostimulatory cytokines IFN-γ and TNF-α in response to TGF-β exposure. After 24-hour incubation with or without TGF-β, the upregulation of CD69 was monitored by surface staining. After 24-hour incubation with or without TGF-β, cytokine production was detected by applying the protein transport inhibitor brefeldin A and performing intracellular staining. "No scFv" represents CARs that do not possess any ligand-binding scFv domains.

[0172] Unlike TGF-β CARs, dominant-negative TGF-β receptors cannot induce cytokine production. Although dominant-negative TGF-β receptors have been reported to inhibit TGF-β signaling, they do not induce immunostimulatory effects such as TNF-α production (Figure 5). "Short Tβ" and "Long Tβ" are two different types of TGF-β CARs, "Dom-Neg" represents a dominant-negative TGF-β receptor, and "no scFv" represents a CAR that does not have any ligand-binding scFv domain.

[0173] We further discovered that the function of TGF-β CARs can be regulated by selecting the co-stimulatory domain. As shown in Figure 10, switching between the CD28 co-stimulatory domain and the 4-1BB co-stimulatory domain alters the TGF-β-responsive cytokine production level. Furthermore, we found that TGF-β consistently induces dose-dependent TNF-α production across cells containing TGF-β CARs from various donors. This suggests that the CARs possess robust performance that is reliable for clinical application (Figure 11).

[0174] Furthermore, we found that the spacer length of the TGF-β CAR modulates the induction threshold. Increasing the length of the extracellular spacer raises the CAR-induced TGF-β threshold (Figure 12). This suggests that CAR responsiveness to application requirements can be customized by altering the binding between the ligand-binding domain and the intracellular signaling domain. The "short spacer" contains the hinge portion of human IgG4, while the "long spacer" contains the hinge-CH2-CH3 of IgG4.

[0175] Next, we found that CAR signaling is induced by soluble ligand-mediated dimerization of CARs. Figure 13 shows Jarcut cell lines possessing the specified CARs. Both GFP CAR#1 and GFP CAR#3 exist primarily in homodimer form, and these two types of CARs can bind to different epitopes on EGFP and simultaneously to individual EGFP molecules. GFP CAR#1 and GFP CAR#2 bind to the same epitope on EGFP, but CAR#2 exists as a monomer rather than a homodimer. The results suggest that CAR signaling can be promoted by ligand-mediated CAR dimerization, but the ligand or the CAR itself does not need to be pre-existing as a dimer (Figure 13). One possible mechanism by which soluble ligands can induce CAR signaling is the formation of an immunological synapse by linking receptors on two different cells. This mechanism is consistent with observations that Jurcut cells expressing a single type of GFP CAR can be activated by dimeric EGFP but not by monomeric EGFP. It is also consistent with observations that a mixture of two Jurcut cell lines expressing different GFP CARs can be activated by both dimeric EGFP and monomeric EGFP. In this example, monomeric EGFP can also induce cell-cell ligation because the CARs in the two Jurcut cell lines bind to two different epitopes on the same EGFP molecule (Figure 14).

[0176] We found that TGF-β CAR can be induced in both cell-to-cell contact-dependent and cell-to-cell contact-independent ways. Cell-to-cell contact is one possible mechanism by which soluble ligands such as EGFP and TGF-β can induce CAR signaling, but CAR-T cell activation can also be induced by soluble ligands even in the absence of cell-to-cell contact. In Figure 15, Jurcut cells stably expressing TGF-β CAR and an EGFP reporter expressed from the NFAT promoter were seeded at various cell densities and incubated in the presence or absence of 5 ng / mL of TGF-β. Even at extremely low densities where cells mainly existed as isolated single cells, EGFP signaling was clearly observed in the presence of TGF-β. Furthermore, no increase in EGFP production in proportion to cell density was observed for cell densities ranging from 10 times higher (500 to 5000 cells / cm²) (Figure 15). These results indicate that soluble TGF-β can induce T cell activation independently of cell-to-cell contact. However, EGFP production significantly increased when the cell density exceeded a threshold, indicating that intercellular contact contributes at high cell density levels. To further evaluate this phenomenon, primary human CD4+ T cells expressing TGF-β CAR were labeled with the calcium indicator Fluo-4-AM and imaged using fluorescence microscopy. The Fluo-4-AM signal observed after the addition of TGF-β in the absence of intercellular contact supports the idea that TGF-β CAR-T cells can be activated by soluble ligands without intercellular junctions (Figure 16).

[0177] TGF-β CARs address the need for immune cells to neutralize the role of TGF-β as an immunosuppressive driver in the tumor microenvironment. While CAR-T cell therapy has shown remarkable clinical results against B-cell malignancies, its efficacy against solid tumors remains significantly limited. Solid tumors are known to create a highly immunosuppressive microenvironment through the overproduction of TGF-β and other cytokines, ultimately leading to T cell inactivation. TGF-β CARs not only confer the ability to counter immunosuppression by reducing signaling via the endogenous TGF-β pathway, but also confer the ability to specifically induce T cell activation in the presence of TGF-β to T cells. T cell activation encourages immune cells to produce and proliferate immunostimulatory cytokines, thereby shifting TGF-β from an immunosuppressive signal to an activating stimulus that activates an anti-tumor immune response.

[0178] Example 2: Design of a multifunctional regulatory T-cell therapy for autoimmune diseases This embodiment describes a method that can be used to manipulate regulatory T cells (Tregs) expressing chimeric antigen receptors (CARs), which can be activated by tumor growth factor beta (TGF-β) to selectively proliferate ex vivo, maintain robust inhibitory function in vivo, and secrete anti-interleukin-6 receptor alpha (IL-6Rα) single-chain variable fragments (scFv) to effectively reduce inflammation in a mouse model of rheumatoid arthritis (RA). It is considered that other autoimmune diseases, such as those known in the art and / or described herein, may also be treated by the methods described in this embodiment and disclosure.

[0179] Adoptive T-cell therapy using conventional T cells expressing chimeric antigen receptors (CARs) (Tconv) has shown remarkable clinical efficacy against refractory cancers, particularly B-cell malignancies. However, the application of CAR-T-cell therapy to the treatment of autoimmune diseases is still in its infancy.

[0180] Treg cells suppress Tconv function through numerous mechanisms, one of which is the secretion of TGF-β, a potent immunosuppressive cytokine that inhibits both effector T cell function and natural killer cell function. The inventors have developed a TGF-β CAR that specifically activates Tconv in the presence of TGF-β (Figure 4), and have confirmed that TGF-β CAR-T cells respond to both soluble and fixed forms of TGF-β. Human CD4+ Tconv and CD8+ Tconv expressing TGF-β CAR induce robust NFAT signaling and produce Th2 cytokines in the presence of TGF-β, despite the fact that TGF-β is normally a highly immunosuppressive agent (Figure 4).

[0181] TGF-β CAR is considered uniquely suitable for Treg therapy for the following reasons: 1) TGF-β is known to promote Treg differentiation, and therefore, TGF-β-mediated proliferation of CAR-expressing T cells presents a method for selectively promoting Treg proliferation while preventing the proliferation of contaminating Tconv. We have confirmed that TGF-β drives robust proliferation of TGF-β CAR-expressing Tconv only in the presence of irradiated feeder cells. In the absence of feeder cells, proliferation of TGF-β CAR-Tconv is specifically inhibited by the presence of TGF-β, and this inhibition is significantly stronger for TGF-β CAR-Tconv compared to unmodified Tconv or Tconv expressing non-TGF-β CAR. Our experimental results suggest that TGF-β, existing as a naturally occurring homodimer, can induce junction between two T cells, both of which express TGF-β CAR. As a result of this intercellular junction, sibling cytotoxicity may occur between Tconv cells. This toxicity can be adequately counteracted by the proliferation response induced by CAR signaling, but only with the assistance of irradiated feeder cells. Unlike Tconv cells, Treg cells do not exhibit granzyme-mediated cytotoxicity and are not suppressed by endogenous TGF-β signaling. Therefore, in the absence of feeder cell assistance, TGF-β CAR-expressing Treg cells are thought to be able to address one of the major obstacles to producing therapeutic Treg cells by being selectively proliferated over contaminating Tconv cells in TGF-β-driven ex vivo proliferation protocols. 2) Activated Treg cells spontaneously produce TGF-β, thus conferring a mechanism for sustained activation of TGF-β CAR-Treg cells in vitro and in vivo. Antigen-specific Treg cells have been shown to be more effective than polyclonal Treg cells in immunosuppression. On the other hand, it has also been shown that once activated, Treg cells can exert suppressor function in an antigen-nonspecific manner. Furthermore, it had been shown that pre-activated polyclonal Tregs inhibit collagen-induced arthritis in mice.In summary, these results suggest that antigen-specific Tregs may be more effective than non-antigen-specific Tregs because they are more likely to be activated, and that specificity to target cells, while perhaps advantageous, is not essential for therapeutic function. Natural antigen-specific Tregs are difficult to isolate and grow to sufficient quantities for therapeutic application. While the introduction of transformed T cell receptors (TCRs) offers an attractive alternative, each disease requires its own specific TCR and the availability of appropriate antigen targets, the latter of which has been recognized as a major bottleneck in the development of T cell therapies. Since TGF-β production is a natural product of Tregs independent of TCR specificity, TGF-β CARs offer a generalized strategy that enables sustained Treg activation and could support Treg-mediated suppression of a wide range of disease targets without requiring disease-specific receptors.

[0182] This example uses rheumatoid arthritis (RA) as an initial disease model to provide a novel methodology and specific Treg products for cellular immunotherapy against autoimmune diseases. The overall objective is to establish a generalized approach for creating therapeutic Tregs with sustained therapeutic function and to elucidate the efficacy of engineered Tregs in a mouse collagen-induced arthritis model.

[0183] A. Development of a TGF-β-mediated ex vivo proliferation protocol for robust Treg proliferation. Primary human Treg cells can be isolated from healthy donor blood samples using the RosetteSep CD4+ T cell enrichment kit, followed by enrichment of CD127- and CD25+ cells based on magnetic beads. The isolated cells can be activated with CD3 / CD28 Dynabeads and cultured for 2 days in complete medium (RPMI + 10% heat-inactivated fetal bovine serum) supplemented with 300 U / ml IL-2 before lentiviral transduction. A lentiviral vector encoding a TGF-β CAR tagged with cleaved epidermal growth factor receptor (EGFRt) (via a T2A cleavage peptide) was pre-constructed and validated by transduction into primary human Tconv cells. CAR-expressing cells can be isolated by sorting EGFRt+ cells based on magnetic beads. This sorting scheme eliminates the need for direct antibody binding to the CAR and reduces the possibility of unproductive T cell activation. The sorted CAR-Treg cells can be grown in 96-well plates under various culture conditions. (a) IL-2 only at 300 U / ml, (b) TGF-β only with a concentration gradient (1-20 ng / ml), (c) gradients of IL-2 (50-300 U / ml) and TGF-β (1-20 ng / ml), (d) IL-2 and TGF-β concentration gradients, plus irradiated feeder cells (TM-LCL) with a T cell:irradiated feeder cell (TM-LCL) ratio of 1:7. The number of viable cells can be quantified by flow cytometry over 3 weeks. Furthermore, Foxp3 expression levels can be quantified by intracellular staining throughout the cell preparation process starting from newly isolated CD4+ / CD25+ / CD127- cells. A "scFv-less" CAR, identical to the TGF-β CAR except for the absence of a TGF-β binding scFv domain, can be constructed and included as a negative control.

[0184] Through the investigations described in this embodiment, the following can be clarified: (1) whether TGF-β CAR expression can produce Treg cells that have the ability to proliferate specifically in response to the addition of TGF-β, and (2) the identification of the optimal combination of IL-2, TGF-β, and / or feeder cells for high-efficiency (defined by high-fold proliferation) and high-purity (defined by the minimum presence of contaminating Tconv) ex vivo Treg proliferation. The inventors developed several TGF-β CARs with different structural properties and observed the individual signaling thresholds of these CARs in response to TGF-β. By evaluating two different TGF-β CARs, one with a short (12 amino acid) extracellular spacer and the other with a long (229 amino acid) extracellular spacer, it is possible to identify the optimal construct for Treg application.

[0185] B. Optimization of the suppressor function of TGF-β CAR-Treg in vitro and in vivo. The suppressor function of Tregs that stably express TGF-β CARs can be further investigated. Tregs are isolated, transduced, and sorted as described above. Both long-spacer and short-spacer TGF-β CARs can be evaluated. As a negative control, a comparison of TGF-β CAR-Tconv to manipulated Tregs can be provided by transducing and sorting a portion of CD4+ T cells (before enrichment of the CD127- / CD25+ phenotype). As a second negative control, Tregs transduced with the scFv-less CAR described above can be included in this study. The sorted CAR-Tregs can be grown in complete medium supplemented with 300 U / ml IL-2, which is an intermediate concentration among those reported in published studies and has been reported to support Treg survival and proliferation ex vivo. The proliferation procedure can be modified to conditions known to support optimal Treg proliferation. Tconv will be grown in complete medium supplemented with 50 U / ml of IL-2 and 1 ng / ml of IL-15.

[0186] The suppressor function of TGF-β CAR-Tregs can be evaluated by co-culture with targeted Tconv cells. Targeted Tconv cells are CD4+ T cells transduced with a lentivirus to express CD19 CAR. To evaluate Treg function, TGF-β CAR-Tregs can be co-incubated with both CFSE-labeled CD19 CAR-Tconv cells and CD19+ Raji lymphoma cells, either in the presence or absence of soluble TGF-β. CD19 CAR-Tconv proliferation can be evaluated by CFSE dilution and by viable cell counting by flow cytometry. To clearly distinguish between Treg and Tconv proliferation in this co-culture setting, a CFSE-based proliferation assay can be performed instead of the more widely used 3H-thymidine uptake assay. CFSE-labeled cells can be accurately quantified from the early stages by flow cytometry, while the CFSE dilution peak represents cell division dynamics over 7 days. As a negative control, TGF-β CAR-Tconv or scFv-less CAR-Treg can be used to replace TGF-β CAR-Treg in co-incubation samples. While scFv-less CAR-Treg exhibits some suppressive function, TGF-β CAR-Treg is expected to have enhanced suppression due to TGF-β production and subsequent TGF-β CAR-mediated autocrine Treg activation. Furthermore, the addition of exogenous TGF-β is expected to further enhance the suppressor function of TGF-β CAR-Treg, resulting in minimal proliferation of CD19 CAR-Tconv despite the presence of CD19+ target cells.

[0187] Verifying the suppressor function of TGF-β CAR-Tregs in vitro allows us to estimate the in vivo suppressor capability of the manipulated Tregs. For animal studies, mouse CD4+ / CD25+ Tregs will be isolated from the lymph nodes and spleen of DBA / 1 mice by cell sorting based on magnetic beads. As previously mentioned for human Tregs, sorted cells can be activated, transduced, proliferated, and sorted with mouse CD3 / CD28 Dynabeads for TGF-β CAR expression. The TGF-β CAR is constructed with scFv targeting human TGF-β, and we have confirmed that this receptor cross-reacts with mouse TGF-β with high efficiency (Figure 8). To induce collagen-induced arthritis (CIA), 100 μl of an emulsion containing a complete Freund's adjuvant—a 1:1 mixture of 2 mg / ml chicken type II collagen dissolved in PBS and 0.1 M acetate—can be injected into the tail base of DBA / 1 mice. One million Tregs can be administered via tail vein injection one day before CIA immunization or two weeks after CIA immunization to evaluate Treg performance at different disease progression stages. TGF-β CAR-Tregs can be compared to scFv-less CAR-Tregs and "Treg-less" controls (i.e., controls not administered Tregs after CIA immunization) in terms of their ability to prevent or alleviate arthritis. Mice can be evaluated for clinical arthritis of the foot based on a 4-point scale, as previously described by Kelchtermans, H. et al. (Arthritis Res Ther 7, R402-415 (2005)). Ten animals can be included in each test condition (40 animals in total). A sample size of n=10 provides 92% power to detect a difference of 1.5 standard deviations in arthritis, scored with α=0.05 in a two-sided t-test. Based on available literature, CAR-Tregs without scFv are expected to achieve significant suppression of arthritis symptoms in the CIA model. On the other hand, the addition of TGF-β CAR is expected to enhance the functionality of Tregs, resulting in further effective inhibition of CIA.Further investigation is needed to determine whether a CAR with a long spacer or a CAR with a short spacer exhibits superior in vivo functionality in the CIA model.

[0188] All methods disclosed and claimed herein can be carried out and implemented without any unnecessary experimentation, taking into consideration this disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and to the steps or sequence of steps of the methods herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted with the agents described herein, while the same or similar results are achieved. All such similar substitutions and modifications that are obvious to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All references, cited documents, patent publications, and any sequence of Genbank accession numbers listed herein are incorporated herein by reference in whole for any purpose.

[0189] References The following references are incorporated herein by reference to the extent that they provide exemplary procedures or other details that supplement those described herein. TIFF0007912329000066.tif19128TIFF0007912329000067.tif241145TIFF0007912329000068.tif240147

[0190] Sequence information SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> TRANSFORMING GROWTH FACTOR-BETA-RESPONSIVE POLYPEPTIDES AND THEIR METHODS FOR USE <150> US 62 / 248,685 <151> 2015-10-30 <160> 101 <170> PatentIn version 3.5 <210> 1 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 1 Glu Val Gln Leu Val 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 Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Ser Ala 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 Ser Gly Gly Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 <210> 2 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala 115 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn 20 25 30 Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Pro Arg Ala Phe Val Leu Asp Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 4 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 4 Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 5 Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Glu 1 5 10 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 6 Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 7 Ser Gly Gly Phe Tyr Phe Asp Tyr 1 5 <210> 8 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 8 Arg Ala Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 9 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 10 His Gln Tyr Leu Ser Ser Asp Thr 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 11 Ser Asn Val Ile Ser 1 5 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 12 Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe Lys 1 5 10 15 Gly <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 13 Pro Arg Ala Phe Val Leu Asp Ala Met Asp Tyr 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 14 Arg Ala Ser Gln Ser Leu Gly Ser Ser Tyr Leu Ala 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 15 Gly Ala Ser Ser Arg Ala Pro 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 16 Gln Gln Tyr Ala Asp Ser Pro Ile Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 17 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 18 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 19 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 19 Glu Val Gln Leu Val Glu 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 Met His Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Ile Lys 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 Thr Gly Glu Tyr Ser Gly Tyr Asp Thr Asp Pro Gln Tyr Ser 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Gly Ile Gly Asp Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile 35 40 45 Tyr Gly Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Ser Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 21 Ser Tyr Gly Met His 1 5 <210> 22 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 22 Val Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 23 Thr Gly Glu Tyr Ser Gly Tyr Asp Thr Asp Pro Gln Tyr Ser 1 5 10 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 24 Arg Ser Ser Gln Gly Ile Gly Asp Asp Leu Gly 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 25 Gly Thr Ser Thr Leu Gln Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 26 Leu Gln Asp Ser Asn Tyr Pro Leu Thr 1 5 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 27 Asp Lys Thr His Thr 1 5 <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 28 Cys Pro Pro Cys 1 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 29 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 1 5 10 15 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 30 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 31 Lys Ser Cys Asp Lys Thr His Thr Cys Pro 1 5 10 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 32 Lys Cys Cys Val Asp Cys Pro 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 33 Lys Tyr Gly Pro Pro Cys Pro 1 5 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 34 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 35 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 36 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 37 Ser Pro Asn Met Val Pro His Ala His His Ala Gln 1 5 10 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 38 Glu Pro Lys Ser Cys Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 39 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 39 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(4) <223> Repeated more than once <400> 40 Gly Ser Gly Gly Ser 1 5 <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (1)..(4) <223> Repeated more than once <400> 41 Gly Gly Gly Ser 1 <210> 42 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 42 Gly Gly Ser Gly 1 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 43 Gly Gly Ser Gly Gly 1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 44 Gly Ser Gly Ser Gly 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 45 Gly Ser Gly Gly Gly 1 5 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 46 Gly Gly Gly Ser Gly 1 5 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 47 Gly Ser Ser Ser Gly 1 5 <210> 48 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 48 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Serum Leu Val Ile Thr Leu Tyr Cys 20 <210> 49 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 49 Leu Gly Leu Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly 1 5 10 15 Val Ala Ile His Leu Cys Cys 20 <210> 50 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 50 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 1 5 10 15 Leu Gly Ile Phe Phe Cys Val Arg Cys 20 25 <210> 51 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 51 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu Arg Val 20 <210> 52 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 52 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 1 5 10 15 Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 53 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptides <400> 53 Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro 1 5 10 15 Leo Ala Ile Leo Leo Ala Leo Tyr Leo Leo 20 25 <210> 54 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 54 Ala Leu Pro Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly 1 5 10 15 Leu Gly Val Ala Cys Val Leu Ala 20 <210> 55 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 55 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 56 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 56 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 1 5 10 15 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 20 25 30 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 57 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 57 Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr 1 5 10 15 Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30 Val Thr Leu 35 <210> 58 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 58 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1 5 10 15 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20 25 30 Thr Leu Ala Lys Ile 35 <210> 59 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 59 Cys Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr 1 5 10 15 Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln 20 25 30 Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr 35 40 45 Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly 50 55 60 Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile 65 70 75 80 Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu 85 90 95 Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val 100 105 110 Arg Ser <210> 60 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 60 His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu 1 5 10 15 Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser 20 25 30 Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser 35 40 45 Pro <210> 61 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 61 Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys Tyr 1 5 10 15 Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg Pro 20 25 30 Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu Pro 35 40 45 Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr Cys 50 55 60 His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp Ala 65 70 75 80 Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro Arg 85 90 95 Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile Met 100 105 110 Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro Glu 115 120 125 Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu Leu 130 135 140 Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro Pro 145 150 155 160 Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly Lys 165 170 175 Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys 180 185 <210> 62 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 62 His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln 1 5 10 15 Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln 20 25 30 Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg 35 40 45 Leu Gly Asp Leu Trp Val 50 <210> 63 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 63 Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val 1 5 10 15 Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile 20 25 30 Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu 35 40 45 Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 50 55 60 <210> 64 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (2)..(3) <223> X is any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> X is leucine or isoleucine <400> 64 Tyr Xaa Xaa Xaa 1 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (2)..(3) <223> X is any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> X is leucine or isoleucine <220> <221> MISC_FEATURE <222> (5)..(12) <223> X is any amino acid or absent <220> <221> MISC_FEATURE <222> (14)..(15) <223> X is any amino acid <220> <221> MISC_FEATURE <222> (16)..(16) <223> X is leucine or isoleucine <400> 65 Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 1 5 10 15 <210> 66 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (82)..(82) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (92)..(92) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (95)..(95) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (97)..(97) <223> X is pyrrolysine <400> 66 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Xaa Arg Ile Thr Glu Thr Glu Ser Pro Tyr Xaa Glu Leu Xaa Gly 85 90 95 Xaa Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys <210> 67 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (81)..(81) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (91)..(91) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (94)..(94) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (96)..(96) <223> X is pyrrolysine <400> 67 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Thr Arg Lys 65 70 75 80 Xaa Arg Ile Thr Glu Thr Glu Ser Pro Tyr Xaa Glu Leu Xaa Gly Xaa 85 90 95 Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 100 105 110 <210> 68 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (71)..(71) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (81)..(81) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (84)..(84) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (86)..(86) <223> X is pyrrolysine <400> 68 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu 20 25 30 Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu 35 40 45 Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 50 55 60 Glu Ala Ala Thr Arg Lys Xaa Arg Ile Thr Glu Thr Glu Ser Pro Tyr 65 70 75 80 Xaa Glu Leu Xaa Gly Xaa Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr 85 90 95 Gln Arg Pro Tyr Tyr Lys 100 <210> 69 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (70)..(70) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (80)..(80) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (83)..(83) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (85)..(85) <223> X is pyrrolysine <400> 69 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu 20 25 30 Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu 35 40 45 Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 50 55 60 Glu Ala Thr Arg Lys Xaa Arg Ile Thr Glu Thr Glu Ser Pro Tyr Xaa 65 70 75 80 Glu Leu Xaa Gly Xaa Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln 85 90 95 Arg Pro Tyr Tyr Lys 100 <210> 70 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (8)..(8) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (10)..(10) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (21)..(21) <223> X is pyrrolysine <400> 70 Glu Ser Pro Tyr Xaa Glu Leu Xaa Gly Xaa Arg Ser Asp Val Tyr Ser 1 5 10 15 Asp Leu Asn Thr Xaa 20 <210> 71 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 71 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 <210> 72 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (12)..(12) <223> X is pyrrolysine <400> 72 Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Xaa Glu Thr Tyr Glu 1 5 10 15 Thr Leu Lys His Glu 20 <210> 73 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (140)..(140) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (147)..(147) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (150)..(150) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (159)..(159) <223> X is pyrrolysine <400> 73 Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg 20 25 30 Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val 35 40 45 Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys 85 90 95 Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val 100 105 110 Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120 125 Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Xaa Ala Leu Leu Arg 130 135 140 Asn Asp Xaa Val Tyr Xaa Pro Leu Arg Asp Arg Asp Asp Ala Xaa Tyr 145 150 155 160 Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys 165 170 <210> 74 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (87)..(87) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (96)..(96) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (103)..(103) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (106)..(106) <223> X is pyrrolysine <400> 74 Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg 20 25 30 Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val 35 40 45 Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Thr Val Xaa Val His Tyr Arg Thr Ala Asp Thr Xaa 85 90 95 Ala Leu Leu Arg Asn Asp Xaa Val Tyr Xaa Pro Leu Arg Asp Arg Asp 100 105 110 Asp Ala Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys 115 120 125 <210> 75 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (2)..(2) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (14)..(14) <223> X is pyrrolysine <400> 75 Asp Xaa Val Tyr Xaa Pro Leu Arg Asp Arg Asp Asp Ala Xaa Tyr Ser 1 5 10 15 His Leu Gly Gly Asn 20 <210> 76 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 76 Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120 125 Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140 Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150 155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190 Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205 <210> 77 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 77 Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser 1 5 10 15 Gly Leu Asn Gln Arg 20 <210> 78 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (139)..(139) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (144)..(144) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (151)..(151) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (158)..(158) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (161)..(161) <223> X is pyrrolysine <400> 78 Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile Ile Leu 1 5 10 15 Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His Leu Val Lys 20 25 30 Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala 35 40 45 Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe 50 55 60 Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp 65 70 75 80 Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro 85 90 95 Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala 100 105 110 Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val 115 120 125 Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Xaa Asp Gly Val Arg Xaa 130 135 140 Ser Arg Ala Ser Asp Lys Xaa Thr Leu Leu Pro Asn Asp Xaa Leu Tyr 145 150 155 160 Xaa Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly 165 170 175 Asn Gln Leu Arg Arg Asn 180 <210> 79 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (2)..(2) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (14)..(14) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (19)..(19) <223> X is pyrrolysine <400> 79 Asp No Tyr No Pro Leu Lys Asp Arg Glu Asp Asp No Tyr Ser 1 5 10 15 His Leu Gly Asn 20 <210> 80 <211> 163 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURES <222> (114)..(114) <223> X is pyrrolysine. <220> <221> MISC_FEATURES <222> (142)..(142) <223> X is pyrrolysine. <400> 80 Met Lys Trp Lys Falcon Phe Thr Falcon Ile Leu Gln Falcon Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Leu Phe Leu Leu Phe Leu Tyr Gly Val Leu Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 100 105 110 Leu Xaa Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 115 120 125 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Xaa Gly Leu 130 135 140 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 145 150 155 160 Pro Pro Arg <210> 81 <211> 164 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (115)..(115) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (143)..(143) <223> X is pyrrolysine <400> 81 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Xaa Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Xaa Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg <210> 82 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (14)..(14) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (17)..(17) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (19)..(19) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (55)..(55) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (63)..(63) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (91)..(91) <223> X is pyrrolysine <400> 82 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Xaa Gln Gly 1 5 10 15 Xaa Asn Xaa Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Xaa Glu Gly Leu Tyr Asn Glu Leu Xaa Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Xaa Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 83 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (2)..(2) <223> X is pyrrolysine <400> 83 Asn Xaa Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 1 5 10 15 Val Leu Asp Lys Arg 20 <210> 84 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 84 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 1 5 10 15 Ser Glu Ile Gly Met Lys 20 <210> 85 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (21)..(21) <223> X is pyrrolysine <400> 85 Asp Gly Leu Tyr Xaa Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 1 5 10 15 Ala Leu His Met Xaa 20 <210> 86 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <220> <221> MISC_FEATURE <222> (170)..(170) <223> X is pyrrolysine <220> <221> MISC_FEATURE <222> (207)..(207) <223> X is pyrrolysine <400> 86 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg Lys Arg Trp Xaa Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Xaa Gly 195 200 205 Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys Pro 225 <210> 87 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 87 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Glu Pro Pro Pro Arg Pro Phe Leu 85 90 95 Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala Glu Gly Ile 100 105 110 Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu Leu Phe Arg 115 120 125 Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu Tyr 130 135 140 Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met 145 150 155 160 Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val Gly 165 170 175 Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro 180 185 <210> 88 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 88 Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met Tyr Glu 1 5 10 15 Asp Ile Ser Arg Gly 20 <210> 89 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 89 Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr Ile Asn Met 1 5 10 15 Pro Gly Arg Gly 20 <210> 90 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 90 Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr Ile Asn Met 1 5 10 15 Pro Gly Arg Gly 20 <210> 91 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 91 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Alas Tyr Arg Dear 65 <210> 92 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 92 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Alas Tyr Arg Dear 65 <210> 93 <211> 619 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 93 Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn 275 280 285 Ser Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys 290 295 300 Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser 305 310 315 320 Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 325 330 335 Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 340 345 350 Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile 355 360 365 Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 370 375 380 Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg 385 390 395 400 Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met 405 410 415 Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 420 425 430 Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly 435 440 445 Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 450 455 460 Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe 465 470 475 480 Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg 485 490 495 Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn 500 505 510 Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 515 520 525 Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys 530 535 540 Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys 545 550 555 560 Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp 565 570 575 Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg 580 585 590 Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 595 600 605 Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 610 615 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 94 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 95 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 96 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 96 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Ala Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 20 25 30 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 35 40 45 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Phe 50 55 60 Thr Asn Tyr Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 65 70 75 80 Glu Trp Val Gly Val Ile Asn Pro Gly Ser Gly Gly Ser Asn Tyr Asn 85 90 95 Glu Lys Phe Lys Gly Arg Ala Thr Ile Ser Ala Asp Asn Ser Lys Asn 100 105 110 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 115 120 125 Tyr Tyr Cys Ala Arg Ser Gly Gly Phe Tyr Phe Asp Tyr Trp Gly Gln 130 135 140 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 165 170 175 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 180 185 190 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Leu Tyr Ser Ser Asn 195 200 205 Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 210 215 220 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Ser 225 230 235 240 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 245 250 255 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Leu 260 265 270 Ser Ser Asp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 275 280 285 Val Ala 290 <210> 97 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 97 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Ala Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 20 25 30 Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 35 40 45 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 50 55 60 Ser Ser Asn Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 65 70 75 80 Glu Trp Met Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala 85 90 95 Gln Arg Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 100 105 110 Thr Thr Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 115 120 125 Tyr Tyr Cys Ala Leu Pro Arg Ala Phe Val Leu Asp Ala Met Asp Tyr 130 135 140 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Val Leu Thr Gln 165 170 175 Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser 180 185 190 Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser Tyr Leu Ala Trp Tyr Gln 195 200 205 Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser 210 215 220 Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val 245 250 255 Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro Ile Thr Phe Gly Gln Gly 260 265 270 Thr Arg Leu Glu Ile Lys 275 <210> 98 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 98 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 1 5 10 <210> 99 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 99 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 1 5 10 <210> 100 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 100 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Asp Trp Tyr Gln Lys Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Gly 100 105 110 Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Val Gln Leu 115 120 125 Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met 130 135 140 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Val 145 150 155 160 Trp Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr 165 170 175 Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala 180 185 190 Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser 195 200 205 Ser Leu Thr Ser Glu Asp Ser Ala Asp Tyr Tyr Cys Ala Arg Ser Asn 210 215 220 Tyr Tyr Gly Ser Ser Tyr Trp Phe Phe Asp Val Trp Gly Ala Gly Thr 225 230 235 240 Thr Val Thr Val Ser Ser 245 <210> 101 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 101 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser 245

Claims

1. A polypeptide comprising a signal peptide, a TGF-β antigen-binding domain having a heavy chain variable (VH) region and a light chain variable (VL) region, a peptide spacer, a transmembrane domain, and an endodomain, further comprising a co-stimulatory region between the transmembrane domain and the endodomain, The VH region includes SEQ ID NO: 11 (HCDR1), SEQ ID NO: 12 (HCDR2), and SEQ ID NO: 13 (HCDR3), and the VL region includes SEQ ID NO: 14 (LCDR1), SEQ ID NO: 15 (LCDR2), and SEQ ID NO: 16 (LCDR3), The peptide spacer comprises the hinge region of an IgG molecule and consists of fewer than 50 amino acids. The aforementioned transmembrane domain includes the transmembrane domain of CD28, The aforementioned endodomain includes a CD3 zeta signaling domain, which transmits an activation signal to T cells after antigen binding. The aforementioned polypeptide.

2. The polypeptide according to claim 1, wherein the TGF-β antigen-binding domain comprises scFv.

3. The polypeptide according to claim 1, wherein the VH region includes SEQ ID NO: 3 and the VL region includes SEQ ID NO:

4.

4. The polypeptide according to any one of claims 1 to 3, wherein the signal peptide comprises SEQ ID NO:

18.

5. The polypeptide according to any one of claims 1 to 4, comprising a CAR, wherein the CAR is a bispecific CAR further comprising a cancer molecule-specific antigen-binding domain.

6. The polypeptide according to claim 5, wherein the cancer molecule-specific antigen-binding domain is specific to GD2 or IL-13R-a2.

7. The polypeptide according to claim 5, wherein the cancer molecule-specific antigen-binding domain is specific to CAIX, CD33, CD44v7 / 8, CEA, EGP-2, EGP-40, erb-B2, erb-B3, erb-B4, FBP, fetal acetylcholine receptor, GD3, Her2 / neu, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen h5T4, PSCA, PSMA, TAG-72, or VEGF-R2.

8. The polypeptide according to claim 1, wherein the aforementioned co-stimulatory region includes a co-stimulatory region derived from 4-1BB or CD28.

9. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 8.

10. A cell comprising a polypeptide according to any one of claims 1 to 8 or a nucleic acid according to claim 9.

11. The cell according to claim 10, further comprising a cancer-specific chimeric antigen receptor (CAR).

12. The cell according to claim 11, wherein the cancer-specific CAR specifically binds to GD2, IL-13R-a2, CAIX, CD33, CD44v7 / 8, CEA, EGP-2, EGP-40, erb-B2, erb-B3, erb-B4, FBP, fetal acetylcholine receptor, GD3, Her2 / neu, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen h5T4, PSCA, PSMA, TAG-72, or VEGF-R2.

13. CD4 ＋ T cells, CD8 ＋ The cell according to claim 12, which is an immune cell that is a T cell, a regulatory T cell, a gamma delta T cell, or a cytotoxic T cell.

14. A pharmaceutical composition for use in a method for stimulating an immune response, comprising cells according to any one of claims 10 to 13, wherein the immune response is stimulated by the cells coming into contact with TGF-β.

15. The pharmaceutical composition according to claim 14, wherein the cells are in vivo of a target requiring immune stimulation.

16. A pharmaceutical composition for use in a method of treating cancer in a subject, comprising cells according to any one of claims 10 to 13, wherein the method comprises administering the cells to the subject having cancer.

17. The pharmaceutical composition according to claim 15, wherein the subject is melanoma, brain tumor, B-cell malignant tumor, or breast cancer.

18. The pharmaceutical composition according to claim 16, wherein the cancer includes melanoma, brain tumor, B-cell malignant tumor, or breast cancer.

19. The pharmaceutical composition according to any one of claims 15 to 18, wherein the subject is a human subject.

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