TGF-beta vaccine

Abstract

To provide a new polypeptide derived from TGFb1 which is a multifunctional cytokine playing important roles in the regulation of immune systems.SOLUTION: Provided is a polypeptide which is an immunogenic fragment of human transforming growth factor 1 (TGFb1) and comprises or consists of a sequence of at least 9 consecutive amino acids of a specific sequence. The invention also relates to uses of the polypeptides, to polynucleotides encoding the polypeptides and their uses, and to compositions comprising the polypeptides and their uses.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel polypeptides derived from transforming growth factor beta 1 (TGFβ1; TGFb1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The present invention also relates to uses and methods of using said polypeptides, polynucleotides, and compositions. [Background technology]

[0002] Background of the Invention TGFb is a multifunctional cytokine that plays an important role in regulating the immune system. It exists in four isoforms, of which isoform 1 (TGFb1) is particularly important in T cell immunity. In the context of cancer, TGFb1 disarms various immune cells, such as cytotoxic T cells (CTLs), tumor-associated neutrophils, and natural killer (NK) cells. This also contributes to tumor angiogenesis and metastasis. Consequently, TGFb1 is a key inhibitory molecule in the tumor microenvironment (TME), contributing to the downregulation of the immune system's antitumor mechanisms and enabling immune evasion by cancer cells.

[0003] In a recent study in a mouse model of metastatic liver cancer, TGFb1 was also found to contribute to the reduced efficacy of immune checkpoint blockade (ICB), such as PD-L1 blockade, as a cancer therapy. Summary of the Invention

[0004] The polypeptide of the present invention is expected to be particularly effective in stimulating beneficial immune responses against TGFb1-expressing cells. The development of novel immunotherapies for cancer requires a thorough understanding of the molecules involved in pathogenesis and the specific proteins recognized by the immune system. In clinical situations, the induction of TGFb1-specific immune responses can directly kill TGFb1-expressing cancer cells, but more importantly, it supports anti-cancer immune responses in general by suppressing the immunosuppressive function of TGFb1. For example, targeting TGFb1 and TGFb1-expressing cells through vaccination with the polypeptide of the present invention is therefore highly synergistic with additional anti-cancer immunotherapies such as immune checkpoint blockers (ICBs).

[0005] TGFb1 is a dimeric cytokine that shares a cysteine ​​knot structure connected together by an intramolecular disulfide bond. TGFb1 is synthesized as a monomeric 390 amino acid precursor protein, which is interchangeably referred to as TGFb1 preprotein; TGFb1 precursor; full-length TGFb1; and prepro-TGFb1. The full-length sequence of TGFb1 preprotein is provided as SEQ ID NO: 1.

[0006] The TGFb1 preprotein monomer has a molecular weight of approximately 25 kDa. As shown in Figure 1E, the TGFb1 protein monomer has three distinct domains: a signal peptide (SP: amino acids 1-29; SEQ ID NO: 2), a latency-associated peptide (LAP: amino acids 30-278; SEQ ID NO: 3), and a mature peptide (mature TGFb1: amino acids 279-390; SEQ ID NO: 4).

[0007] The TGFb1 SP directs the protein to the secretory pathway; the SP is cleaved in the rough endoplasmic reticulum. TGFb1 monomers, including LAP and mature TGFb1, can dimerize in the endoplasmic reticulum through disulfide bridges between cysteine ​​residues in LAP (e.g., Cys223 and Cys225) and the mature TGFb1 peptide (e.g., Cys356) to form a TGFb1 homodimer. This TGFb1 homodimer is called the small latent complex (SLC). SLC can bind to the so-called latent TGF-β binding protein (LTBP) to form a larger complex called the large latent complex (LLC). LLC can be secreted into the extracellular medium (ECM). However, the presence of LAP and LTBP prevents TGFb1 from binding to and activating its extracellular receptor. Active TGFb1 consists of a homodimer of the mature TGFb1 peptide. There are various mechanisms by which mature TGFb1 homodimers are released from LAP and LTBP, including degradation of LAP by proteases, induction of a conformational change in LAP by interaction with thrombospondin, and disruption of the noncovalent bond between LAP and TGFb-1.

[0008] The goal of this study was to develop a T cell-mediated mechanism for depriving the TME of TGFb1. We investigated the presence of spontaneous TGFb1-specific T cell responses in vivo by screening PBMCs from healthy donors and cancer patients. TGFb1-specific T cell populations were then isolated, expanded, and characterized by various assays for HLA restriction, cytokine production, and cytotoxicity.

[0009] The present inventors have identified the most immunogenic regions of human TGFb1. Surprisingly, these immunogenic "hotspot" regions are distributed throughout the human TGFb1 preprotein, including within the SP and LAP domains, as well as within the mature TGFb1 peptide. The present inventors also identified a subregion within human TGFb1 LAP that has a greater frequency of immunogenic peptide sequences, namely, positions 121-160 of SEQ ID NO: 1 (corresponding to the sequence of SEQ ID NO: 65).

[0010] Thus, the present invention provides a polypeptide that is an immunogenic fragment of human TGFb1 (SEQ ID NO: 1) and that comprises or consists of a sequence of at least 9 contiguous amino acids of SEQ ID NO: 1. The sequence of at least 9 contiguous amino acids of SEQ ID NO: 1 may correspond to a sequence of at least 9 contiguous amino acids of SEQ ID NO: 2 or 65. The polypeptide may comprise or consist of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 contiguous amino acids of SEQ ID NO: 1. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 7-9, 43-45, 13-15, 24-26, 29-31, 50-52, 56-58, 64, 65, 2, 66, 67, or 5, and preferably the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67, or 5. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2, and preferably the polypeptide comprises or consists of the amino acid sequence of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63.

[0011] The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids. The C-terminal amino acid of the polypeptide may be replaced with the corresponding amide. The polypeptide may comprise an HLA-A2-restricted epitope. The HLA-A2-restricted epitope may comprise or consist of the amino acid sequence of SEQ ID NO: 66 or 67.

[0012] The present invention further provides a polynucleotide encoding a polypeptide of the present invention. The polynucleotide may be isolated. Also provided by the present invention is a vector comprising the polynucleotide.

[0013] The present invention also provides a composition comprising a polypeptide of the present invention and / or a polynucleotide of the present invention and, optionally, an adjuvant. The composition may further comprise at least one different polypeptide of the present invention; at least one different polynucleotide of the present invention; and / or at least one pharmaceutically acceptable diluent, carrier, or preservative. The adjuvant may be selected from the group consisting of a bacterial DNA-based adjuvant, an oil / surfactant-based adjuvant, a viral dsRNA-based adjuvant, an imidazoquiniline, and a Montanide ISA adjuvant.

[0014] The present invention also provides methods for treating or preventing a disease or condition in a subject, the method comprising administering to the subject a polypeptide of the invention, a polynucleotide of the invention, and / or a composition of the invention. The method may further comprise the simultaneous or sequential administration of an additional cancer therapy, preferably an antibody.

[0015] The present invention also provides a polypeptide of the present invention, a polynucleotide of the present invention, a composition of the present invention, or a combination thereof, for use in the treatment or prevention of a disease or condition. The polypeptide, polynucleotide, composition, or combination thereof may be for use in combination with an additional cancer therapy, preferably an antibody.

[0016] The present invention further provides the use of a polypeptide of the present invention, a polynucleotide of the present invention, a composition of the present invention, or a combination thereof, for the manufacture of a medicament for the treatment or prevention of a disease or condition.

[0017] The disease or condition may be characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb1-expressing cells, and / or the disease or condition is cancer. The disease or condition may be characterized, at least in part, by inappropriate or excessive expression of interleukin-4 (IL-4) and / or interleukin-13 (IL-13). The disease or condition may be cancer. The cancer may be breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), melanoma, leukemia (e.g., acute myeloid leukemia (AML)), or prostate cancer.

[0018] The present invention further provides a method for stimulating TGFb1-specific T cells, said method comprising contacting T cells with a polypeptide of the present invention and / or a composition of the present invention comprising at least one polypeptide of the present invention. The T cells may be present in a sample taken from a healthy subject or a cancer patient, optionally in a tumor sample. [Brief explanation of the drawings]

[0019] [Figure 1-1] Figure 1A-C. Peptide-specific immune responses in PBMCs from six healthy donors were assessed against an array of 38 overlapping 20-mer peptides derived from the TGFb1 preprotein by in vitro IFNγ ELISPOT assays performed in triplicate wells. Each spot represents the average number of IFNγ-secreting cells after subtraction of the respective background signal, and the gray horizontal bar indicates the average value across test donors. Stars indicate peptides that elicited the strongest and most statistically significant DFRx2-based responses and were selected for further screening experiments (summarized in Figure 1D). [Figure 1-2]Figure 1A-C. Peptide-specific immune responses in PBMCs from six healthy donors were assessed against an array of 38 overlapping 20-mer peptides derived from the TGFb1 preprotein by in vitro IFNγ ELISPOT assays performed in triplicate wells. Each spot represents the average number of IFNγ-secreting cells after subtraction of the respective background signal, and the gray horizontal bar indicates the average value across test donors. Stars indicate peptides that elicited the strongest and most statistically significant DFRx2-based responses and were selected for further screening experiments (summarized in Figure 1D). [Figure 1-3] Figure 1A-C. Peptide-specific immune responses in PBMCs from six healthy donors were assessed against an array of 38 overlapping 20-mer peptides derived from the TGFb1 preprotein by in vitro IFNγ ELISPOT assays performed in triplicate wells. Each spot represents the average number of IFNγ-secreting cells after subtraction of the respective background signal, and the gray horizontal bar indicates the average value across test donors. Stars indicate peptides that elicited the strongest and most statistically significant DFRx2-based responses and were selected for further screening experiments (summarized in Figure 1D). [Figure 1-4] Figure 1D. Table summarizing the most immunogenic TGFβ peptides and their respective mean IFNγ ELISPOT counts based on the screening of Figures 1A-1C. The top 8 best-performing peptides were selected for further consideration. [Figure 1-5] Figure 1E. Top: Primary sequence of the TGFb1 preprotein. Highlighted are the amino acid sequences of eight immunogenic TGFb1 peptides selected for further screening. The underlined amino acids 1-29 indicate the location of the signal sequence of the protein (SP), while the underlined amino acids 279-390 indicate the mature TGFb1 monomer protein. Bottom: Schematic representation of the TGFb1 preprotein domains and the location of the eight selected TGFb1-derived peptides. The numbers (1, 29, 279, and 390) indicate the positions of key amino acids that mark the three major domains of the TGFb1 preprotein. [Figure 2-1] Figure 2. A. Peptide-specific immune responses in PBMCs to the eight immunogenic TGFb1-derived peptides identified in Figures 1A-C were validated by evaluating responses in additional healthy donors using an in vitro IFNγ ELISPOT assay. Each spot represents the average number of IFNγ-secreting cells in an individual donor after subtraction of the respective background signal; black horizontal bars indicate the average value across test donors. B. Heatmap showing the amplitude of responses in PBMCs from healthy subjects to the lead epitope (top); representative ELISPOT responses (bottom). [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 3-1] Figure 3. A. We validated peptide-specific immune responses in PBMCs against the eight immunogenic TGFb1 peptides identified in Figures 1A-C, but this time we examined cancer patients by assessing responses with an in vitro IFNγ ELISPOT assay. Each spot represents the average number of IFNγ-secreting cells in an individual cancer patient after subtraction of the respective background signal, and the black horizontal bar indicates the average value across the tested patients. B. Heatmap showing the amplitude of responses in PBMCs from cancer patients against the lead epitope. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1]Figure 4. Intracellular cytokine staining (ICS) analysis was performed to further characterize the functionality of T cells responding to the TGFb1 epitope. In this example, PBMCs from a healthy donor (BC-M-41) were thawed 13 days prior to the assay and stimulated with TGFb-02 (SEQ ID NO: 6). IL-2 was added 1 day after culture (120 U / mL) and 3 days prior to ICS (60 U / mL). In each flow cytometry plot, each cell is represented as a dot, and the functional phenotype of the cell was analyzed based on the simultaneous expression of two markers, each indicated on each axis. Viable cell populations were gated based on the CD3+CD4+ or CD3+CD8+ T cell fraction, and markers for cytokine expression (IFNγ and TNFα) and cytotoxicity (CD107a) were quantified. The percentages of each population are summarized in the hierarchical table on the right. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-2. [Figure 5] Figure 5. A. FACS plot showing CD4+ T cell responses to TGFβ epitopes determined using ICS. B. FACS plot showing CD8+ T cell responses to TGFb1 epitopes determined using ICS. [Figure 6] Figure 6. Bulk cultures specific for several TGFb1-derived epitopes were generated by MACS CD137 enrichment of specific T cells. After enrichment, the enriched cells were expanded and showed variable reactivity to those epitopes. For each of A-D, the top FACS plot shows the amount of specific CD4+ gated cells, and the bottom FACS plot shows the amount of specific CD8+ gated cells for the following epitopes: TGFb-02 (A), TGFb-05 (B), TGFb-26 (C), and TGFb-38 (D). [Figure 7]Figure 7. A. Left: Response amplitude in PBMCs from both cancer patients and healthy subjects measured by ex vivo ELISPOT. PBMCs were rested overnight and then plated directly into ELISPOT wells and stimulated with epitopes in the ELISPOT wells for 48 hours. Right: Examples of ex vivo ELISPOT responses to some of the TGFb1 lead epitopes. B. CD8+ T cell responses identified against the epitope TGFb-15 just 5 hours after stimulation with ICS. [Figure 8] Figure 8. A. PBMCs from a patient with prostate cancer showing CD8+ T cell responses to the TGFb-15 epitope after 18 hours of stimulation with the epitope, with 14 days of prior in vitro stimulation with peptide. B. TGFb15-specific T cells from donor UR1121.14 were enriched twice after stimulation with TGFb-15, restimulated after 14 days of in vitro culture, and then enriched the next day using the MACS CD137 enrichment method. Both CD4+ T cells (top FACS plots for each of A and B) and CD8+ T cells (bottom FACS plots for each of A and B) responded to stimulation with TGFb-15. [Figure 9] Figure 9. FACS plot showing the results of ICS analysis of TGFb-15-specific CD8+ T cell clones stimulated with TGFb-15. [Figure 10-1]Figure 10. TGFb-15-specific CD8+ T cell clones kill target cells in an HLA-restricted manner and kill TGFb1-expressing cancer cell lines. A. TGFb-15-specific CD8+ T cells efficiently lysed T2 cells pulsed with TGFb-15 peptide. B. To confirm that the TGFb-15 response was HLA-A2-restricted, peptide-pulsed HLA-A2+ target cells were lysed, but peptide-pulsed HLA-A3+ target cells were not. C. Stimulation of the clones with the HLA-A2+ cancer cell lines UKE-1 and THP-1 activated the TGFb-15-specific CD8+ T cell clones. Other HLA-A2+ cancer cells did not activate the T cells. D. Both THP-1 and UKE-1 cancer cell lines were readily killed by TGFb-15-specific T cells. E. Activation of TGFb-15-specific T cells was enhanced upon stimulation with cytokine-treated THP-1 cells. F. Stimulation of THP-1 cells with the Th2 cytokines IL-4 or TGFb1 enhanced the amount of THP-1 cells killed by TGFb-15-specific T cells. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11-1] Figure 11. Results of IFN-γ (A) and TNF-α (B) ELISPOT assays used to analyze responses to TGF nonamer library spanning. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12-1]Figure 12. CD8+ T cells specific for an HLA-A2-binding decameric epitope in the TGFb1 signal peptide sequence readily kill TGFb1-expressing cancer cell lines in an HLA-A2-restricted manner. A. Healthy donor PBMCs secreted IFN-γ upon stimulation with the HLA-A2-binding decameric epitope TGFb-A2-01 peptide after 14 days of in vitro culture. B. Intracellular cytokine staining of healthy donor PBMCs demonstrated a CD8+ T cell response to TGFb-A2-01, as stimulated CD8+ cells showed increased expression of both IFN-γ and IFN-α (left), in addition to increased CD107a expression (right), upon stimulation with TGFb-A2-01. C. TGFb-A2-01-specific CD8+ T cells from a healthy donor killed TGFb-A2-01-pulsed HLA-A2+ target cells, but not unpulsed or peptide-pulsed HLA-A3+ target cells. D. CD8+ T cells killed HLA-A2+ TGFb1-expressing UKE-1 target cells, but not MARIMO or WM852 cells. E. HLA-A2+ THP-1 cells were readily killed by TGFb-A2-01-specific T cells, but modulation of TGFb1 expression in THP-1 cells by stimulation with different cytokines 48 hours prior to assay increased the fraction of killed target cells. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-2. [Figure 13] Figure 13. FACS plot showing the results of ICS analysis of TGFb-A2-01-specific T cell clones stimulated with TGFb-A2-01. [Figure 14] Figure 14. Amino acid sequences of 20-mer peptides in the TGFβ library. Overlapping amino acid sequences are underlined.

[0020] A brief description of arrays SEQ ID NO: 1 is the amino acid sequence of the full-length human TGFb1 precursor (also called TGFb1 preprotein). SEQ ID NO: 2 is the amino acid sequence of the signal peptide of human TGFb1. SEQ ID NO: 3 is the amino acid sequence of the LAP peptide of human TGFb1. SEQ ID NO: 4 is the amino acid sequence of mature human TGFb1. SEQ ID NOs: 5 to 64 are each the amino acid sequences of polypeptide fragments derived from human TGFb1. SEQ ID NO: 65 is the amino acid sequence of the LAP subregion containing a frequently immunogenic sequence. SEQ ID NO: 66 is the amino acid sequence of the minimal epitope sequence within the TGFb-15 peptide sequence (SEQ ID NO: 28). SEQ ID NO: 66 is also referred to herein as "TGFb-15short." SEQ ID NO: 67 is the amino acid sequence of TGFb-A2-01.

[0021] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods may be tailored to particular needs in the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0022] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0023] As used herein, the term "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" encompasses short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to any natural and / or unnatural amino acid or synthetic amino acid, including both the D- and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0024] The terms "patient" and "subject" are used interchangeably and generally refer to a human.

[0025] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0026] As used herein, "immunogenic" preferably means that the polypeptide can induce an immune response against TGFb1 protein when the TGFb1 protein is present in or on a cell that expresses the TGFb1 protein. In other words, the polypeptide can be described as being immunogenic to TGFb1. Alternatively, the polypeptide can be described as an immunogenic fragment of TGFb1. The immune response is preferably a T cell response, and the polypeptide can be described as an immunogenic fragment of TGFb1 that contains a T cell epitope. The immune response can be detected in at least one individual (or a sample taken from an individual) after administration of the polypeptide to the individual (or the sample taken from the individual).

[0027] A polypeptide can be identified as immunogenic using any suitable method, including in vitro methods. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics (i) to (iii): i. capable of inducing IFN-γ-producing cells in PBL populations of healthy subjects and / or cancer patients, as determined by ELISPOT assay; and / or ii. CTLs reactive with TGFb1 can be detected in situ in a sample of tumor tissue; and / or iii. It can induce the development of specific T cells in vitro. Suitable methods for determining whether a polypeptide is immunogenic are also described in the Examples section below.

[0028] The polypeptide of the present invention is an immunogenic fragment of human TGFb1 (SEQ ID NO:1) comprising or consisting of a sequence of at least 9 consecutive amino acids of SEQ ID NO:1.

[0029] The sequence of at least 9 consecutive amino acids of SEQ ID NO:1 may correspond to a sequence of at least 9 consecutive amino acids of the SP domain of TGFb1, for example, a sequence of at least 95 consecutive amino acids of SEQ ID NO:2.

[0030] A sequence of at least 9 consecutive amino acids of SEQ ID NO:1 may correspond to a sequence of at least 9 amino acids of the LAP domain of TGFb1, for example, at least 9 consecutive amino acids of SEQ ID NO:3.

[0031] The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may correspond to a sequence of at least 9 consecutive amino acids located within the LAP subregion bounded by amino acid positions 121 and 160 of SEQ ID NO: 1, for example, a sequence of at least 9 consecutive amino acids of SEQ ID NO: 65.

[0032] A sequence of at least 9 consecutive amino acids of SEQ ID NO:1 may correspond to a sequence of at least 9 consecutive amino acids of a mature TGFb1 polypeptide, for example, a sequence of at least 9 consecutive amino acids of SEQ ID NO:4.

[0033] The polypeptide may comprise or consist of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO:1.

[0034] The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 2 and 5-67.

[0035] The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 5, 7-9, 43-45, 13-15, 24-26, 29-31, 50-52, 56-58, 64, 65, 2, 66, 6 or 5. Polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5 are preferred.

[0036] The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2. Polypeptides comprising or consisting of the amino acid sequence of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63 are particularly preferred.

[0037] The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids. The C-terminal amino acid of the polypeptide may be replaced with the corresponding amide. The polypeptide may be isolated.

[0038] Particularly preferred polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, or 63. Particularly preferred polypeptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63. Longer polypeptide fragments of SEQ ID NO: 1 incorporating these sequences are also preferred.

[0039] The polypeptide may comprise an HLA-A2-restricted epitope. Preferably, the HLA-A2-restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO: 66. A preferred peptide comprising an HLA-A2-restricted epitope consisting of the amino acid sequence of SEQ ID NO: 66 is a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 28 to 31, or 65. Alternatively, the HLA-A2-restricted epitope preferably comprises or consists of the amino acid sequence of SEQ ID NO: 67. A preferred peptide comprising an HLA-A2-restricted epitope consisting of the amino acid sequence of SEQ ID NO: 67 is a peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5, 8, 9, or 2.

[0040] In any polypeptide described herein, the amino acid sequence may be modified by one, two, three, four, or five (i.e., up to five) additions, deletions, or substitutions, so long as the polypeptide having the modified sequence exhibits equivalent or increased immunogenicity against TGFb1 compared to the polypeptide having the unmodified sequence. "Equivalent" should be understood to mean that the polypeptide having the modified sequence does not exhibit significantly reduced immunogenicity against TGFb1 compared to the polypeptide having the unmodified sequence. Any comparison of immunogenicity between sequences should be performed using the same assay. Unless otherwise specified, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. In conservative substitutions, an amino acid is replaced with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid it replaces. Alternatively, in conservative substitutions, an aromatic or aliphatic amino acid may be introduced in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 major amino acids defined in Table A1 below. If the amino acids have similar polarity, this can be determined by referring to the hydropathic index for the amino acid side chains in Table A2.

[0041] [Table 1]

[0042] [Table 2]

[0043] In any of the polypeptides disclosed herein, any one or more of the following modifications may be made to improve physiochemical properties (e.g., stability), provided that the polypeptide exhibits equivalent or increased immunogenicity to TGFb1 compared to a polypeptide having an unmodified sequence: substitution of the C-terminal amino acid with the corresponding amide (which can increase resistance to carboxypeptidases); Substitution of the N-terminal amino acid with the corresponding acylated amino acid (which can increase resistance to aminopeptidases); Substitution of one or more amino acids with corresponding methylated amino acids, which can improve resistance to proteolysis; and / or Substitution of one or more amino acids with the corresponding amino acid in the D-configuration, which can improve resistance to proteolysis.

[0044] Any polypeptide disclosed herein may have at least one additional moiety attached to the N- and / or C-terminus to improve solubility, stability, and / or aid in production / isolation, so long as the polypeptide exhibits equivalent or increased immunogenicity to TGFb1 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR may be added to the N- and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0045] The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides may be directly synthesized using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, the polypeptides may be produced by transforming cells, usually bacterial cells, with a nucleic acid molecule or vector encoding the polypeptide. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the present invention. The present invention also provides host cells containing such nucleic acids or vectors.

[0046] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention can be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there may be substantial, but not complete, isolation of the polypeptide from any surrounding medium. Polynucleotides may be mixed with carriers or diluents that do not interfere with their intended use and still be considered substantially isolated. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example, in an expression vector. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences include, but are not limited to, viral cDNA, prokaryotic or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0047] Polynucleotides can be synthesized according to methods well known in the art, as illustrated by examples in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes, which contain regulatory sequences operably linked to an insert sequence, thereby allowing expression of the polypeptides of the present invention in vivo. These expression cassettes are then typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing a polynucleotide of the present invention can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information to allow expression of the polypeptides of the present invention.

[0048] Thus, the present invention includes expression vectors containing such polynucleotide sequences. Such expression vectors are constructed routinely in the art of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, that may be necessary and positioned in the correct orientation to enable expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0049] The present invention also encompasses cells that have been modified to express a polypeptide of the present invention. Such cells typically include prokaryotic cells such as bacterial cells, e.g., E. coli. Such cells may be cultured using conventional methods to produce the polypeptide of the present invention.

[0050] The polypeptide of the present invention can be in a substantially isolated form. The polypeptide of the present invention can be mixed with a carrier, preservative, or diluent, and / or adjuvant that does not interfere with the intended use, and can still be considered substantially isolated. The polypeptide of the present invention can also be in a substantially purified form, in which case the polypeptide of the present invention generally comprises at least 90%, for example, at least 95%, 98%, or 99% of the protein in the preparation.

[0051] Compositions containing polypeptides The present invention provides compositions comprising the polypeptides of the present invention and / or the polynucleotides of the present invention. For example, the present invention provides compositions comprising one or more polypeptides of the present invention and / or one or more polynucleotides of the present invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative, and / or excipient.

[0052] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polypeptides of the invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0053] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polynucleotides of the invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0054] Carriers, preservatives, and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, all ingredients and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may preferably include an adjuvant. An adjuvant is any substance whose incorporation into a composition enhances or otherwise modifies the immune response elicited by the composition. Broadly defined, an adjuvant is a substance that promotes an immune response. Adjuvants may also preferably have a depot effect, in that they also provide a slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986), pp. 61-63.

[0055] Adjuvants include AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, also known as MTP-PE), RIBI in 2% squalene / Tween-80 RTM emulsion (MPL+TDM+CWS), lipopolysaccharides and their various derivatives including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., poly IC and poly AU acid), Mycobacterium tuberculosis (Mycobacterium tuberculosis), The adjuvant may be selected from the group consisting of wax D from B. tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the Brucella genus, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. Nos. 5,876,767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrix or GMDP, interleukin 1, interleukin 2, Montanide ISA-51, and QS-21. Various saponin extracts have also been suggested as useful adjuvants in immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may also be used as an adjuvant.

[0056] Preferred adjuvants for use with the present invention include oil / surfactant-based adjuvants, such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants containing CpG oligonucleotide sequences. Yet other preferred adjuvants are viral dsRNA-based adjuvants, such as poly I:C. GM-CSF and imidazolinone are also examples of preferred adjuvants.

[0057] The adjuvant is most preferably a Montanide ISA adjuvant, preferably Montanide ISA51 or Montanide ISA720.

[0058] Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986), pp. 61-63, also states that coupling to an immunogenic carrier is recommended when the antigen of interest has a low molecular weight or is insufficiently immunogenic. Thus, the polypeptide of the present invention may be coupled to a carrier. The carrier may exist independently of an adjuvant. The function of the carrier may be to increase the molecular weight of the polypeptide fragment, for example, to enhance activity or immunogenicity, to confer stability, to enhance biological activity, or to increase serum half-life. Furthermore, the carrier may aid in presenting the polypeptide or its fragment to T cells. Thus, in the composition, the polypeptide may be associated with a carrier as described below. The carrier may be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell, such as a dendritic cell (DC). Carrier proteins include keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins), or hormones (such as insulin) or palmitic acid. Alternatively, the carrier protein can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be dextran, such as Sepharose. The carrier must be physiologically acceptable and safe for humans.

[0059] If the composition contains an excipient, the excipient must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, and sulfates; and organic acid salts, such as acetates, propionates, malonates, and benzoates. A complete discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0060] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and techniques, all of which are readily available to those skilled in the art. Such compositions can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit dosage form, e.g., in ampoules or in multi-dose containers, optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in a dry (e.g., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can contain, in addition to the active ingredient, additional ingredients such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable formulations may be prepared using non-toxic, parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic saline, and fixed oils such as synthetic mono- or diglycerides. Other useful compositions include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. Alternatively, the active ingredient of the composition may be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368.Other microparticle systems and polymers, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules, can also be used.

[0061] How to use The polypeptides, polynucleotides, or compositions of the present invention, or combinations thereof, may be used in a method for treating or preventing a disease or condition in a subject. The polypeptides, polynucleotides, or compositions of the present invention, or combinations thereof, may be used in the manufacture of a medicament for use in a method for treating or preventing a disease or condition in a subject. The method comprises administering the polypeptide, polynucleotide, composition, or combination to the subject. Administration may be a therapeutically or prophylactically effective amount of the polypeptide, polynucleotide, composition, or combination to a subject in need thereof.

[0062] The disease or condition may be characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb1. The disease or condition may be characterized, at least in part, by inappropriate or excessive expression of IL-4 and / or IL-13. The disease or condition may be cancer, preferably a cancer that expresses TGFb1 and / or is associated with inappropriate or excessive immunosuppressive function of TGFb1 and / or inappropriate or excessive expression of IL-4 and / or IL-13. The cancer may be breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer, or pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), melanoma, leukemia (e.g., acute myeloid leukemia (AML)), or prostate cancer. The cancer may be AML characterized by inappropriate or excessive immunosuppressive function of TGFb1 and / or inappropriate or excessive expression of IL-4 and / or IL-13. The cancer may be AML, which is characterized by inappropriate or excessive immunosuppressive function of TGFb1 and inappropriate or excessive expression of IL-4 and / or IL-13.

[0063] The method may include simultaneous or sequential administration with an additional cancer therapy. The additional cancer therapy may be a dual-specific inhibitor of TGFb (e.g., TGFb1) and PD-L1. The dual-specific inhibitor may be capable of simultaneously binding to and / or inhibiting the activity of TGFb and PD-L1. The dual-specific inhibitor may be a fusion protein comprising an anti-TGFb portion and an anti-PD-L1 portion, optionally wherein the anti-PD-L1 portion comprises or consists of an anti-PD-L1 antibody and / or the anti-TGFb portion comprises or consists of a receptor for TGFb or a portion thereof, e.g., TGFb receptor II or a portion thereof.

[0064] The additional cancer therapy may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiation therapy, immune stimulants, gene therapy, or antibodies.

[0065] Antibodies include abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atlizumab (= tocilizumab), atollimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, and bevacizumab. Zumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, con Cizumab, crenezumab, CR6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab alitox, drozitumab, durigotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, Thalasizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624,Ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, roderucizumab, lorvotuzumab mertansine, lucatumumab, Lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomabutafenatox, namilumab, naptumomab estafenatox, narutumumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obinutuzumab, ocralizumab, ozlimomab , ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placurab, polatuzumab vedotin, ponezumab, priliximab, pritoxakis Cimab, Pritumumab, PRO140, Kirizumab, Racotumomab, Ladletuzumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Lorezumab, Romosozumab, Rontalizumab, Rovelizumab, Rupirizumab, Samalizumab, Sarilumab, Satumomabpendetide, Secukinumab, Seribantumab, Cetoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (= atlizumab), toralizumab, tositumomab, It may be tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, zoralimumab, or zolimomab alitoxin.

[0066] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, mepolizumab, reslizumab, tocilizumab, and ustequitin. These include numab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tositumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab, and vedotin.

[0067] Particularly preferred antibodies that may be used in the methods of the invention include daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab, and ofatumumab.

[0068] The concomitant cancer therapy may be selected from the group consisting of actimid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, revlimid, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0069] The polypeptides of the present invention and / or compositions of the present invention comprising at least one polypeptide of the present invention can be used to induce TGFb1-specific T cells, e.g., CD4 + and CD8 + The cells may also be used in methods for stimulating T cells. The method may be performed ex vivo. The cells may be present in a sample, such as a tumor sample, taken from a healthy subject or a cancer patient.

[0070] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples, both separately and in any combination thereof, may be material for realizing the invention in diverse forms thereof. [Example]

[0071] Example 1 – Materials and Methods Patients and donors Buffy coats from anonymous blood donors were obtained from the blood bank in Rigshospitalet, Copenhagen, Denmark. Buffy coats from cancer patients were obtained from the Department of Oncology, Herlev Hospital, Herlev, Denmark. All participants provided informed consent before study enrollment in accordance with the Declaration of Helsinki. PBMCs were isolated using Lymphoprep (Axis Shield, Oslo, Norway) and frozen in fetal bovine serum containing 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).

[0072] peptide Peptides were provided by Pepscan (Lelystad, The Netherlands) and dissolved in DMSO at a concentration of 10 mM. After identification of the TGFβ1 lead epitope, these peptides were provided with higher purity (>90%) by KJ Ross-Petersen (Klampenborg, Denmark). The sequences of the peptides used in these experiments are shown in the section entitled "Sequences." Peptides are described by SEQ ID NO: 6, name, or by reference to the start and end positions of each peptide sequence within the amino acid sequence of the full-length precursor of human TGFb1. Each designation may be used interchangeably, as shown in the table in the "Sequences" section below. For example, the peptide of SEQ ID NO: 6 may alternatively be designated by the name TGFb-02 (or TGFB02), or alternatively by the name TGFb1. 11-30 (from a start position of 11 and an end position of 30). The intended reference in each case will be clear from the context.

[0073] In vitro ELISPOT assay For in vitro ELISPOT, PBMCs from cancer patients and healthy donors were pulsed in 24-well plates with 20 μM TGFβ-derived peptide (or no peptide as a control) and 120 U / ml IL-2 for 7–10 days before use in ELISPOT assays. Cells were seeded onto 96-well nitrocellulose ELISPOT plates (MultiScreen IP Filter Plate, MSIPN4W50; Millipore) precoated with an IFNγ capture antibody (Mabtech). TGFβ peptide was added to a final concentration of 5 μM, and control stimuli (DMSO, HIV, or scrambled peptide) were added to control wells. The plates were incubated at 37°C for 16–20 h. After incubation, cells were washed, and a secondary biotinylated Ab (Mabtech, catalog no. 3420-6-1000) was added for 2 h at room temperature. Unbound secondary antibody was washed away, and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech, Cat. No. 3310-10) was added for 1 hour at room temperature. The assay was developed by washing away unbound conjugated enzymes and adding BCIP / NBT substrate (Mabtech, Cat. No. 3650-10). Developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6 Ultimate-V Analyzer using Immunospot Software v5.1. Responses were reported as the difference between the mean number of spots in wells stimulated with TGFβ peptide and wells without peptide. Unless otherwise noted, all experiments were performed in vitro using IFN-γ ELISPOT assays, and all experiments were performed in triplicate. Statistical analysis was performed using the distribution-free resampling (DFR) method and the more classical DFR2x method described by Moody et al. (Cancer Immunol Immunother 2010; 59: 1489-1501).

[0074] Ex vivo ELISPOT assay PBMCs from cancer patients or healthy donors were thawed and incubated overnight in 24-well plates in X-VIVO medium (optionally supplemented with 1 μg / ml DNase I). The next day, cells were counted and transferred to 96-well nitrocellulose ELISPOT plates (MultiScreen IP Filter Plate, MSIPN4W50; Millipore) precoated with IFNγ capture antibody (Mabtech). TGFβ peptide was added to a final concentration of 5 μM, and control stimuli (DMSO, HIV, or scrambled peptide) were added to control wells. The plates were then incubated at 37°C for 24–72 hours. The plates were then stained with secondary antibodies and the development protocol was performed according to the in vitro ELISPOT protocol described above.

[0075] Intracellular cytokine staining (ICS) and fluorescence-activated cell sorting (FACS) PBMCs were stimulated with TGFβ-derived peptides in the presence of BD GolgiPlug™ (added after the first hour of peptide stimulation) for 5 hours (or incubated without peptide as a control), followed by intracellular staining of the cell cultures. CD107a-PE (catalog no. 555801, BD Biosciences) antibody was added at the beginning of the incubation. Stimulated cells were stained with fluorescently labeled antibodies for surface markers (CD3, CD4, CD8) and then permeabilized using a mixture of fixation / permeabilization enrichment and diluent (eBioscience, catalog nos. 00-5123-43 and 00-5223-56) according to the manufacturer's instructions. Permeabilized cells were then stained with fluorescently labeled antibodies for IFNγ and TNFα. Flow cytometry analysis was performed using a FACSCanto™ II (BD Biosciences). Antibodies used were: IFNγ-APC (Cat. No. 341117), TNFα-455 BV421 (Cat. No. 562783), CD4-FITC (Cat. No. 347413) or CD4-PerCP (Cat. No. 345770), CD8-PerCP (Cat. No. 345774) or CD8-FITC (Cat. No. 345772), and CD3-APC-H7 (Cat. No. 560275) (all BD Biosciences). Dead cells were stained with Fixable Viability Stain 510 (BD Biosciences, San Jose, CA, USA). As an alternative method to identify activated T cells, T cells were stimulated overnight with either antigen or target cells. Eighteen to 24 hours after stimulation, cells were stained with the surface antigen-specific antibodies and Fixable Viability Stain described above, along with staining with anti-CD107a-PE and anti-CD137-BV421 (BD Biosciences, San Jose, CA, USA). Donor PBMCs were analyzed for HLA-A2 by staining with anti-HLA-A2-FITC (BD Biosciences, San Jose, CA, USA) with appropriate isotype controls.

[0076] Rapid Amplification Protocol In some experiments, T cells were expanded using a rapid expansion protocol (REP) with allogeneic irradiated peripheral blood mononuclear cells (PBMCs) from at least three different healthy donors, 30 ng / mL of anti-CD3 antibody (OKT3, Janssen-Cilag or Miltenyi Biotec), and high-dose IL-2 (6,000 IU / mL IL-2; Novartis Proleukin).

[0077] Live cell FACS To enrich for specific T cells from primary PBMC cultures, we adapted the in vitro culture method for cell culture for analysis in ELISPOT (see above). Cells were then stimulated with antigen overnight and washed twice in FACS buffer the following day. They were then stained for 30 minutes with the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Waltham, MA, USA), anti-CD4-FITC, anti-CD8-PerCP, anti-CD107a-PE, and anti-CD137-BV421 (BD Biosciences, San Jose, CA, USA). Cells were then washed twice and resuspended in FACS buffer. Cells were then sorted using a FACS ARIA flow cytometer with appropriate application settings and compensation controls. Cell sorting was performed using the purity setting. After sorting, cells were divided into two fractions. Half of the enriched cells were amplified using the rapid amplification protocol, and the other half of the cells were cloned using limiting dilution with triplicate seedings per well. The cloned cells were amplified using a rapid amplification protocol.

[0078] Magnetic Activated Cell Sorting (MACS) Antigen-specific T cells were enriched using MACS from both primary and enriched cultures. To enrich specific T cells from primary PBMC cultures, we employed the in vitro culture method for cell cultures for analysis in ELISPOT (see above). Cells were then stimulated overnight with antigen and enriched the following day using the MACS CD137 Enrichment Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's protocol. The enriched cells were expanded using a rapid amplification protocol. Where indicated, some of the enriched cells were cloned by limiting dilution. The cloned cells were expanded using a rapid amplification protocol.

[0079] Chromium 51 cytotoxicity assay and cytokine stimulation of target cells Chromium 51 cytotoxicity assays were used to assess the killing potential of specific T cells as described by Andersen MH et al. (J Immunol 1999;163:3812-3818). To manipulate TGFβ expression in several cancer cell lines, cancer cell lines were stimulated with IL-4 (100 U / mL), IL-13 (20 U / mL), and TGFβ1 (2.5 ng / mL) (all from Peprotech, Rocky Hill, NJ, USA), either alone or in combination, for 48 hours prior to assay.

[0080] Example 2 - In vitro ELISPOT screening of 20mer peptides An array of 38 overlapping 20mer peptides derived from the full-length TGFb1 precursor was designed and generated as described above. Each of the 20mer peptides overlaps by 10 amino acids (see Figure 14).

[0081] Peptide-specific immune responses in PBMCs from six healthy donors were assessed for spontaneous immune responses to an array of 20-mer peptides in triplicate wells using an in vitro IFNγ ELISPOT assay. The results of these assays are shown in Figures 1A-C. Peptides that elicited the strongest and most statistically significant responses were selected for further screening experiments. The identification numbers of the best-performing peptides are summarized in Figure 1D, and the positions of the peptides within the full-length sequence are shown in Figure 1E.

[0082] Surprisingly, immunogenic peptides were not clustered within a single immunogenic "hot spot" or located within the amino acid sequence of the mature TGFb1 peptide, but were found to be located throughout the full-length sequence of the TGFb1 precursor protein. Notably, immunogenic epitope peptides were identified in the signal peptide region of the TGFb1 precursor and in the LAP peptide, which are not present in the mature, active form of TGFb1. Furthermore, a LAP subregion with a high frequency of immunogenic peptides was identified, namely, amino acids 121-160 of SEQ ID NO: 1 (corresponding to SEQ ID NO: 65). This region contains the immunogenic peptides TGFb-13 and TGFb-15.

[0083] The eight most immunogenic peptides, namely, TGFb-02, TGFb-26, TGFb-05, TGFb-13, TGFb-15, TGFb-30, TGFb-33, and TGFb-38 (corresponding to SEQ ID NOs: 6, 42, 12, 23, 28, 49, 55, and 63, respectively), were selected for further study.

[0084] Example 3 – Verification of peptide-specific immune responses Additional in vitro IFNγ ELISPOT assays were performed to verify responses in additional healthy subjects to eight selected epitope peptides identified in the initial screen (see Example 2). The results of these assays are shown in Figure 2. Strong and frequent responses were observed to all of the epitope peptides tested, with TGFb-02, TGFb-26, TGFb-33, and especially TGFb-15 showing strong and frequent responses (Figure 2B).

[0085] Because healthy subjects and cancer patients may exhibit different patterns of immune responses to epitopes, we also examined the immunogenic potential of selected epitopes in cancer patients. Peptide-specific immune responses in PBMCs to eight immunogenic TGFb1-derived peptides were also validated by examining cancer patients, again by assessing responses using in vitro IFNγ ELISPOT assays. The results of these assays are shown in Figure 3. TGFb-02, TGFb-15, TGFb-26, and TGFb-33 were found to be highly immunogenic in patients (Figure 3B).

[0086] Example 4 – Cytokine analysis Intracellular cytokine staining (ICS) analysis was performed to further characterize the functionality of T cells responding to the TGFb1 epitope. In this example, PBMCs from a healthy donor (BC-M-41) were thawed 13 days prior to the assay and stimulated with TGFb-02 (SEQ ID NO: 6). IL-2 was added 1 day after culture (120 U / mL) and 3 days prior to ICS (60 U / mL). FACS analysis was performed to characterize the viable cell population as CD3 + CD4 + T cell fraction or CD3 + CD8 + Gating was based on the T cell fraction. Cytokine expression (IFNγ and TNFα) and markers for cytotoxicity (CD107a) were quantified. FACS plots for cytokine analysis are shown on the left side of Figure 4, while the percentages of each population are summarized in a hierarchy table on the right side of Figure 4.

[0087] CD3 + CD4 + T cell fraction (CD3 + CD8 + The T cell fraction (but not the T cell fraction) was found to be responsive to TGFb-02 (SEQ ID NO: 6) as indicated by secretion of TNFα (either alone or in combination with IFNγ) accompanied by absent / low expression of CD107a.

[0088] ICS was also used to identify epitopes TGFb-05 (SEQ ID NO: 12) and TGFb-26 (SEQ ID NO: 42) that bind to CD4 + T cell responses (Figure 5A) and CD8 + After enrichment of specific cells by magnetic activated cell sorting (MACS), we demonstrated that the antibodies elicited both strong CD4 T cell responses against some of the lead epitopes. + and CD8 + T cell responses were also detected (Fig. 6), indicating the high immunogenic potential of several epitopes in TGFβ.

[0089] Example 5 – Identification of ex vivo responses to TGFβ epitopes Ex vivo responses to several epitopes by PBMCs from both healthy subjects and cancer patients. Cells were thawed, rested overnight before seeding, and then stimulated for 48 hours. Both healthy subject and patient cells released significant amounts of IFN-γ (Figure 7A), indicating that both healthy subject and cancer patient-derived cells harbored large numbers of freely circulating TGFβ-specific T cells. Most surprisingly, CD8 +T cell responses were detected in ex vivo-seeded PBMCs from a patient with prostate cancer just 5 hours after stimulation with the epitope TGFb-15 (Figure 7B). This finding suggested that this patient had a high fraction of circulating TGFb-specific cytotoxic T cells. Given this strong response to TGFb-15, a TGFb-15-specific T cell culture was established using PBMCs from this patient. First, it was established that CD137 can be used as an activation marker to sort specific T cells from this donor. Next, patient PBMCs were stimulated with TGFb-15, and the cells were maintained in culture for 14 days. After that, the PBMCs were restimulated with TGFb-15 for 18 hours and then analyzed for the expression of CD137 and CD107a using fluorescence-activated cell sorting (FACS). From this experiment, CD8 + 16.6% of T cells expressed CD137 after stimulation with peptide + Considering that CD137 was significantly higher than that of T cells, CD137 was shown to be a suitable marker for enriching specific T cells (Fig. 8A).

[0090] TGFb-15-specific T cells were enriched using the MACS CD137 enrichment kit, which was used to identify TGFb-15-specific CD4 + T cells and CD8 + Cultures containing both TGFβ- and TGFβ-specific T cell responses were established (Figure 8B).

[0091] Example 6 – TGFb-15-specific T cells can recognize and kill cancer cells Using limiting dilution, CD8 + TGFb-15-specific T cell clones were constructed from TGFβ as described in Example 5. CD8 + The TGFb-15-specific clones showed high reactivity to TGFb-15 (Figure 9). HLA-A2 + Staining of PBMCs from this patient with specific antibodies revealed that the donor was HLA-A2 +Next, a standard chromium 51 cytotoxicity assay was performed to determine whether specific T cells reacted with peptide-pulsed HLA-A2 + We investigated whether peptide-pulsed HLA-A2 could lyse target cells. + T2 cells were readily lysed by specific T cells, whereas non-pulsed T2 cells were not killed (Fig. 10A).

[0092] T2 cells are HLA-A2 + Because the HLA alleles were not the only HLA alleles expressed, it was possible that the death of these cells was mediated by the matching of other HLA alleles. Therefore, further experiments were performed using K562 cells as targets. Although the original K562 line is HLA-deficient, these experiments were performed using two lines genetically engineered to stably express HLA-A2 or HLA-A3. This ensured that the HLA alleles expressed by each cell were the only HLA alleles. Peptide-pulsed HLA-A2 + Only K562 cells were killed by TGFb-15 specific clones, but not by non-pulsed HLA-A2 + K562 cells and peptide-pulsed HLA-A3 + K562 cells were not recognized (Fig. 10B).

[0093] Almost all cells can secrete TGFβ, which is crucial for creating a tumor-suppressive environment. Therefore, TGFβ-15-specific T cells express HLA-A2 + We investigated whether two HLA-A2 antibodies could recognize cancer cell lines. + Melanoma cell lines (WM852 and FM88) and K562 cells and HLA-A2 +The cell lines UKE-1, SET-2, and THP-1, all derived from patients with acute myeloid leukemia (AML), were used as target cells along with K562 cells. TGFb-15-specific T cells were stimulated overnight with each target cell at an effector:target ratio of 3:1. The specific T cells recognized the two cancer cell lines, THP-1 and UKE-1, but the other cell lines did not activate the T cells (Figure 10C). Furthermore, chromium 51 cytotoxicity experiments revealed that TGFb-15-specific T cells killed both UKE-1 and THP-1 cells (Figure 10D).

[0094] The THP-1 cell line is a relatively undifferentiated line, and treatment with different cytokines can affect gene expression in these cells. Interleukin (IL)-4 is a cytokine fundamental in the development of Th2 responses. Therefore, we speculated that treatment of THP-1 cells with IL-4 might increase TGFβ expression by these cells. Furthermore, because TGFβ generates a positive feedback loop regarding its intracellular production, we speculated that treatment of THP-1 cells with TGFβ would also induce TGFβ expression. Notably, the target epitope TGFb-15 is expressed in the LAP peptide portion of the TGFβ precursor protein but not in the mature, active form of TGFβ (see Figure 1E). Therefore, pretreatment of THP-1 cells with active TGFβ would not confer the recognized epitope to THP-1 cells and would only increase intracellular production of TGFβ.

[0095] THP-1 cells treated with either IL-4 or TGFβ for 48 hours were used to detect TGFβ-specific CD8 + T cells were stimulated for 18 hours. Cytokine-treated THP-1 cells were shown to induce greater activation of TGFβ-15-specific T cells compared to unstimulated THP-1 cells (Figure 10E). Finally, cytokine stimulation of THP-1 cells with IL-4 or TGFβ was shown to increase the number of lysed cells (Figure 10F).

[0096] Example 7 – Identification of the minimal TGFβ epitope sequence Because the TGFb-15 epitope is a 20-mer, it cannot exist in its entirety on HLA-I molecules. Therefore, further experiments were performed to determine the minimal epitope sequence recognized by TGFb-15-specific T cells. Specifically, the TGFb-15 epitope sequence was divided into a 9-mer peptide library with eight overlapping amino acids, generating 12 9-mer peptides. TGFb-15-specific CD8 + T cells from the TGFb-15-specific bulk culture used to generate the T cell clones were seeded in ELISPOT and stimulated with each of the 9-mer peptides. The results showed that the minimal epitope within the TGFb-15 sequence was the sequence VLLSRAELRL (TGFb-15short; SEQ ID NO: 66) (see Figure 11).

[0097] Example 8 - A decameric epitope in the signal peptide of TGFβ is a specific T cell target High frequency of CD8 against several 20-mer epitopes within the TGFβ sequence + Considering the T cell response, we attempted to identify other HLA-A2-restricted decameric epitopes. Using the SYFPEITHI database of MHC ligands and peptide motifs by Rammensee et al. (SYFPEITHI: a database of MHC ligands and peptide motifs; www.syfpeithi.de; accessed October 30, 2014), we searched the entire TGFβ sequence for decameric epitopes with high binding affinity to HLA-A2. The peptide sequence LLLLLPLLWL (TGFβ-A2-01; SEQ ID NO: 67) emerged as the top-ranked binding decameric epitope, with a binding affinity score of 30. Subsequently, we searched for decameric epitopes with high binding affinity to HLA-A2 from healthy subjects. + We examined spontaneous T cell responses to the TGFb-A2-01 epitope by PBMCs. Surprisingly, the majority of PBMCs demonstrated responses to the TGFb-A2-01 epitope (Figure 12A). Using ICS, these responses were CD8 + It was confirmed that it was derived from T cells (Fig. 12B).

[0098] Next, TGFb-A2-01-specific T cells were isolated from a healthy subject (BC363), and a single in vitro stimulation of PBMCs from the subject followed by 14 days of culture demonstrated a robust response to TGFb-A2-01. PBMCs were then stimulated overnight with TGFb-A2-01. CD3 + , CD8 + , CD137 + Specific T cells were enriched using FACS with gating on cells. The enriched cells were expanded as described in Example 1. After 14 days of culture, several cell lines showed high specificity for the TGFb-A2-01 peptide (Figure 13).

[0099] Peptide-pulsed HLA-A2 expression of TGFb-A2-01-specific T cells + The ability to lyse K562 target cells was tested in a standard Cr51 cytotoxicity assay. + K562 cells were lysed but not unpulsed HLA-A2 + and peptide-pulsed HLA-A3 + The target cells were not lysed (Fig. 12C).

[0100] Because the TGFb-15-specific T cells described above killed the AML cell lines UKE-1 and THP-1, we tested whether TGFb-A2-01-specific clones could also kill these target cancer cells. UKE-1 and THP-1 cancer cells were readily killed by TGFb-A2-01-specific T cells (see Figures 12D and 12E). Furthermore, stimulation of THP-1 cells with IL-13, TGFβ, or a combination of both IL-13 and TGFβ increased the fraction of dead target cells (Figure 12E).

[0101] conclusion TGFb1 is a crucial enforcer of immune homeostasis and tolerance, inhibiting the amplification and function of many components of the immune system. Disruptions in TGFb1 signaling underlie inflammatory diseases and promote tumor development. TGFb1 is also central to immunosuppression within the tumor microenvironment, and recent studies have revealed its role in tumor immune evasion and poor response to cancer immunotherapy. Expression of TGFb1 is a key characteristic of both tumor-associated macrophages (TAMs) and bone marrow-derived immunosuppressive cells. TGFb1-expressing cells also play a key role in the development of an immunosuppressive microenvironment by preventing the proliferation of effector lymphocytes at the tumor site. Therefore, activation of TGFb1-specific T cells, for example, by vaccination, should lead to T cell infiltration at the tumor site.

[0102] For the first time, we have found that TGFb1-specific effector T cells can be used to specifically target TGFb1-expressing cells. Specifically, we identified naturally occurring peripheral TGFb1-specific T cells in both cancer patients and healthy donors by screening a peptide library covering the entire amino acid sequence of TGFb1. Interestingly, we discovered that TGFb1 contains multiple epitopes frequently recognized by peripheral T cells, distributed across different regions of the TGFb1 sequence.

[0103] A high frequency of T cell responses to TGFb1 was observed, highlighting the unexpected finding that TGFb1 is highly immunogenic. Given that TGFb1 is so central to immune suppression, it is particularly unexpected that TGFb1 would be highly immunogenic to the extent observed by the inventors. In addition, regions of TGFb1 that can generate particularly strong immune responses have been identified, which would be ideal for use in peptide-based vaccination approaches.

[0104] Our findings are surprising given the role of TGFb1 in suppressing the immune system, e.g., in the TME, but suggest that it may be possible to boost TGFb1-specific immune responses in most patients with solid tumors as well as hematological malignancies.

[0105] Many different therapeutic strategies focus on targeting the immunosuppressive tumor microenvironment (TME) with the goal of depleting or reprogramming immunosuppressive cells or targeting functional mediators secreted by these cells. The unexpected results discussed above suggest that immunomodulatory vaccination targeting TGFb1 may be an effective method for targeting immunosuppressive cells in the TME. In contrast to other clinical strategies, this unique approach combines both the depletion of immunosuppressive cells, including cancer cells (through direct killing by cytotoxic T cells) and the reprogramming of immunosuppressive cell populations (by introducing pro-inflammatory cytokines into the immunosuppressive microenvironment). Because TGFb1 expression is a major contributor to the phenotype of immunosuppressive cells, TGFb1-specific T cells may specifically respond to immunosuppressive cells. A TGFb1 vaccine that rebalances the microenvironment should enhance the efficacy of T cell-potentiating drugs, such as checkpoint blockade drugs like anti-PD1 antibodies. Therefore, combination therapy of a TGFb1 vaccine with a checkpoint blockade antibody should increase the number of patients who can respond to therapy.

[0106] In conclusion, the experimental results discussed above provide a useful approach to directly target a major contributor to the lack of immune response in most patients with cancer: TGFb1.

[0107] Sequence Listing Full-length human TGFb1 preprotein (NP_000651.3) (SEQ ID NO: 1) JPEG2026010046000004.jpg59130

[0108] Unless otherwise indicated, in Table 1 below, "start position" and "end position" refer to positions in the full-length human TGFb1 preprotein (SEQ ID NO: 1). Table 1 [Table 3] JPEG2026010046000006.jpg183169

[0109] SEQUENCE LISTING <110> IO Biotech ApS <120> TGF-BETA VACCINE <130> PA25-466 <150> GB1908012.6 <151> 2019-06-05 <160> 67 <170> PatentIn version 3.5 <210> 1 <211> 390 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(390) <223> Full-length human TGFb1 pre-protein (NP_000651.3) <400> 1 Met Pro Pro Ser Gly Leu Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr 20 25 30 Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala 35 40 45 Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 50 55 60 Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu 65 70 75 80 Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90 95 Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 100 105 110 Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr 115 120 125 His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140 Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu 145 150 155 160 Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn 165 170 175 Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser 180 185 190 Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu 195 200 205 Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser 210 215 220 Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe Thr 225 230 235 240 Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro 245 250 255 Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln 260 265 270 Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser 275 280 285 Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys 290 295 300 Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn 305 310 315 320 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr 325 330 335 Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala 340 345 350 Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 355 360 365 Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val 370 375 380 Arg Ser Cys Lys Cys Ser 385 390 <210> 2 <211> 29 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(29) <223> TGFb1 signal peptide <400> 2 Met Pro Pro Ser Gly Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly 20 25 <210> 3 <211> 249 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(249) <223> TGFb1 LAP <400> 3 Leu Ser Thr Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg 1 5 10 15 Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser 20 25 30 Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val 35 40 45 Leu Ala Leu Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala 50 55 60 Glu Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr 65 70 75 80 Arg Val Leu Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys 85 90 95 Gln Ser Thr His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg 100 105 110 Glu Ala Val Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu 115 120 125 Leu Arg Leu Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys 130 135 140 Tyr Ser Asn Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro 145 150 155 160 Ser Asp Ser Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg 165 170 175 Gln Trp Leu Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala 180 185 190 His Cys Ser Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn 195 200 205 Gly Phe Thr Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met 210 215 220 Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln 225 230 235 240 His Leu Gln Ser Ser Arg His Arg Arg 245 <210> 4 <211> 112 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(112) <223> Mature TGFb1 <400> 4 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60 Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65 70 75 80 Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser 100 105 110 <210> 5 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-01 <400> 5 Met Pro Pro Ser Gly Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Trp Leo Leo Val 20 <210> 6 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-02 <400> 6 Leu Leu Leu Pro Leu Leu Trp Leu Leu Val Leu Thr Pro Gly Arg Pro 1 5 10 15 Ala Ala Gly Leu 20 <210> 7 <211> 25 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(25) <223> TGFb-02.1 <400> 7 Leu Leu Leu Pro Leu Leu Trp Leu Leu Val Leu Thr Pro Gly Arg Pro 1 5 10 15 Ala Ala Gly Leu Ser Thr Cys Lys Thr 20 25 <210> 8 <211> 25 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(25) <223> TGFb-02.2 <400> 8 Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu Trp Leu Leu Val Leu 1 5 10 15 Thr Pro Gly Arg Pro Ala Ala Gly Leu 20 25 <210> 9 <211> 30 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(30) <223> TGFb-02.3 <400> 9 Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu Trp Leu Leu Val Leu 1 5 10 15 Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr Cys Lys Thr 20 25 30 <210> 10 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-03 <400> 10 Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr Cys Lys Thr Ile 1 5 10 15 Asp Met Glu Leu 20 <210> 11 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-04 <400> 11 Ser Thr Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile 1 5 10 15 Glu Ala Ile Arg 20 <210> 12 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-05 <400> 12 Val Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys 1 5 10 15 Leu Arg Leu Ala 20 <210> 13 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-05.1 <400> 13 Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly 1 5 10 15 Gln Ile Leu Ser Lys Leu Arg Leu Ala 20 25 <210> 14 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-05.2 <400> 14 Val Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys 1 5 10 15 Leu Arg Leu Ala Ser Pro Pro Ser Gln 20 25 <210> 15 <211> 30 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(30) <223> TGFb-05.3 <400> 15 Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly 1 5 10 15 Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser Gln 20 25 30 <210> 16 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-06 <400> 16 Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser Gln Gly 1 5 10 15 Glu Val Pro Pro 20 <210> 17 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-07 <400> 17 Ser Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala 1 5 10 15 Leo's Wing Leo's Wing 20 <210> 18 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-08 <400> 18 Gly Pro Leu Pro Glu Ala Val Leu Ala Leu Tyr Asn Ser Thr Arg Asp 1 5 10 15 Arg Val Ala Gly 20 <210> 19 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-09 <400> 19 Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 1 5 10 15 Pro Glu Pro Glu 20 <210> 20 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-10 <400> 20 Glu Ser Ala Glu Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys 1 5 10 15 Glu Val Thr Arg 20 <210> 21 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-11 <400> 21 Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu Met Val Glu Thr 1 5 10 15 His Asn Glu Ile 20 <210> 22 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-12 <400> 22 Val Leu Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln 1 5 10 15 Ser Thr His Ser 20 <210> 23 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-13 <400> 23 Tyr Asp Lys Phe Lys Gln Ser Thr His Ser Ile Tyr Met Phe Phe Asn 1 5 10 15 Thr Ser Glu Leu 20 <210> 24 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-13.1 <400> 24 Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr His Ser Ile 1 5 10 15 Tyr Met Phe Phe Asn Thr Ser Glu Leu 20 25 <210> 25 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-13.2 <400> 25 Tyr Asp Lys Phe Lys Gln Ser Thr His Ser Ile Tyr Met Phe Phe Asn 1 5 10 15 Thr Ser Glu Leu Arg Glu Ala Val Pro 20 25 <210> 26 <211> 30 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(30) <223> TGFb-13.3 <400> 26 Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr His Ser Ile 1 5 10 15 Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val Pro 20 25 30 <210> 27 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-14 <400> 27 Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val Pro Glu 1 5 10 15 Pro Val Leu Leu 20 <210> 28 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-15 <400> 28 Arg Glu Ala Val Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg 1 5 10 15 Leo Leo Arg Leo 20 <210> 29 <211> 25 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(25) <223> TGFb-15.1 <400> 29 Asn Thr Ser Glu Leu Arg Glu Ala Val Pro Glu Pro Val Leu Leu Ser 1 5 10 15 Arg Ala Glu Leu Arg Leu Leu Arg Leu 20 25 <210> 30 <211> 25 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(25) <223> TGFb-15.2 <400> 30 Arg Glu Ala Val Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg 1 5 10 15 Leu Leu Arg Leu Lys Leu Lys Val Glu 20 25 <210> 31 <211> 30 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(30) <223> TGFb-15.3 <400> 31 Asn Thr Ser Glu Leu Arg Glu Ala Val Pro Glu Pro Val Leu Leu Ser 1 5 10 15 Arg Ala Glu Leu Arg Leu Leu Arg Leu Lys Leu Lys Val Glu 20 25 30 <210> 32 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-16 <400> 32 Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu Lys Leu Lys Val Glu Gln 1 5 10 15 His Val Glu Leu 20 <210> 33 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-17 <400> 33 Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn 1 5 10 15 Asn Ser Trp Arg 20 <210> 34 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-18 <400> 34 Tyr Green Light Tyr Ser Donkey Donkey Ser Trp Arg Tyr Leu Ser Donkey Arg Leu 1 5 10 15 Leu Ala Pro Ser 20 <210> 35 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-19 <400> 35 Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser Pro Glu Trp Leu 1 5 10 15 Ser Phe Asp Val 20 <210> 36 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-20 <400> 36 Asp Ser Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln 1 5 10 15 Trp Leu Ser Arg 20 <210> 37 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-21 <400> 37 Thr Gly Val Val Arg Gln Trp Leu Ser Arg Gly Gly Glu Ile Glu Gly 1 5 10 15 Phe Arg Leu Ser 20 <210> 38 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-22 <400> 38 Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser Cys Asp 1 5 10 15 Ser Arg Asp Asn 20 <210> 39 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-23 <400> 39 Ala His Cys Ser Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile 1 5 10 15 Asn Gly Phe Thr 20 <210> 40 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-24 <400> 40 Thr Leu Gln Val Asp Ile Asn Gly Phe Thr Thr Gly Arg Arg Gly Asp 1 5 10 15 Leu Ala Thr Ile 20 <210> 41 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-25 <400> 41 Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro 1 5 10 15 Phe Leu Leu Leu 20 <210> 42 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-26 <400> 42 His Gly Met Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu 1 5 10 15 Arg Ala Gln His 20 <210> 43 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-26.1 <400> 43 Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro Phe Leu Leu Leu Met 1 5 10 15 Ala Thr Pro Leu Glu Arg Ala Gln His 20 25 <210> 44 <211> 25 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(25) <223> TGFb-26.2 <400> 44 His Gly Met Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu 1 5 10 15 Arg Ala Gln His Leu Gln Ser Ser Arg 20 25 <210> 45 <211> 30 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(30) <223> TGFb-26.3 <400> 45 Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro Phe Leu Leu Leu Met 1 5 10 15 Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln Ser Ser Arg 20 25 30 <210> 46 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-27 <400> 46 Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln Ser Ser Arg His 1 5 10 15 Arg Arg Wing Leo 20 <210> 47 <211> 20 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(20) <223> TGFb-28 <400> 47 Leu Gln Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe 1 5 10 15 Ser Ser Thr Glu 20 <210> 48 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-29 <400> 48 Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg 1 5 10 15 Gln Leu Tyr Ile 20 <210> 49 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-30 <400> 49 Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu 1 5 10 15 Gly Trp Lys Trp 20 <210> 50 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-30.1 <400> 50 Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp 1 5 10 15 Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 <210> 51 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-30.2 <400> 51 Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu 1 5 10 15 Gly Trp Lys Trp Ile His Glu Pro Lys 20 25 <210> 52 <211> 30 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(30) <223> TGFb-30.3 <400> 52 Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp 1 5 10 15 Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys 20 25 30 <210> 53 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-31 <400> 53 Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly 1 5 10 15 Tyr His Ala Asn 20 <210> 54 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-32 <400> 54 Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 1 5 10 15 Pro Tyr Ile Trp 20 <210> 55 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-33 <400> 55 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr 1 5 10 15 Ser Lys Val Leu 20 <210> 56 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-33.1 <400> 56 Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser 1 5 10 15 Leu Asp Thr Gln Tyr Ser Lys Val Leu 20 25 <210> 57 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-33.2 <400> 57 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr 1 5 10 15 Ser Lys Val Leu Ala Leu Tyr Asn Gln 20 25 <210> 58 <211> 30 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(30) <223> TGFb-33.3 <400> 58 Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser 1 5 10 15 Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln 20 25 30 <210> 59 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-34 <400> 59 Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His 1 5 10 15 Asn Pro Gly Ala 20 <210> 60 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-35 <400> 60 Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys 1 5 10 15 Val Pro Gln Ala 20 <210> 61 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-36 <400> 61 Ser Ala Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile 1 5 10 15 Val Tyr Tyr Val 20 <210> 62 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-37 <400> 62 Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val 1 5 10 15 Glu Gln Leu Ser 20 <210> 63 <211> 20 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(20) <223> TGFb-38 <400> 63 Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser 1 5 10 15 Cys Lys Cys Ser 20 <210> 64 <211> 25 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(25) <223> TGFb-38.1 <400> 64 Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn 1 5 10 15 Met Ile Val Arg Ser Cys Lys Cys Ser 20 25 <210> 65 <211> 40 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(40) <223> TGFb1 LAP sub-region <400> 65 Tyr Asp Lys Phe Lys Gln Ser Thr His Ser Ile Tyr Met Phe Phe Asn 1 5 10 15 Thr Ser Glu Leu Arg Glu Ala Val Pro Glu Pro Val Leu Leu Ser Arg 20 25 30 Wing Glu Leu Arg Leu Leu Arg Leu 35 40 <210> 66 <211> 10 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(10) <223> TGFb-15short <400> 66 Val Leu Leu Ser Arg Ala Glu Leu Arg Leu 1 5 10 <210> 67 <211> 10 <212> PRT <213> Homo sapiens <220> <221> peptide <222> (1)..(10) <223> TGFb-A2-01 <400> 67 Leo Leo Leo Leo Leo Pro Leo Leo Trp Leo 1 5 10

Claims

1. A polypeptide which is an immunogenic fragment of human transforming growth factor 1 (TGFb1) and which comprises or consists of a sequence of at least 9 consecutive amino acids of SEQ ID NO:

1.

2. 2. The polypeptide of claim 1, comprising or consisting of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO:

1.

3. 3. The polypeptide of claim 1 or 2, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2.

4. 4. The polypeptide of claim 3, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

5. 5. The polypeptide according to any one of claims 1 to 4, having a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 amino acids and / or wherein the C-terminal amino acid is replaced by the corresponding amide.

6. 6. The polypeptide of any one of claims 1 to 5, comprising an HLA-A2 restricted epitope, optionally wherein the HLA-A2 restricted epitope comprises or consists of the amino acid sequence of SEQ ID NO: 66 or 67.

7. A polynucleotide encoding a polypeptide according to any one of claims 1 to 6, optionally contained within a vector.

8. A composition comprising a polypeptide according to any one of claims 1 to 6 and / or a polynucleotide according to claim 7; and optionally an adjuvant.

9. The composition of claim 8, further comprising at least one different polypeptide of any one of claims 1 to 6; at least one different polynucleotide of claim 7; and / or at least one pharmaceutically acceptable diluent, carrier or preservative.

10. 10. The composition of claim 8 or 9, comprising an adjuvant selected from the group consisting of a bacterial DNA-based adjuvant, an oil / surfactant-based adjuvant, a viral dsRNA-based adjuvant, an imidazoquinoline, a Montanide ISA adjuvant.

11. 11. A method of treating or preventing a disease or condition in a subject, said method comprising administering to the subject a polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a composition according to any one of claims 8 to 10.

12. The disease or condition is (i) optionally, the cancer is selected from the group consisting of breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), melanoma, leukemia (e.g., acute myeloid leukemia), or prostate cancer; and / or (ii) characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb1-expressing cells and / or inappropriate or excessive expression of IL-4 and / or IL-13; The method of claim 11.

13. 13. The method of claim 11 or 12, wherein the disease or condition is cancer and the method further comprises the simultaneous or sequential administration of an additional cancer therapy, preferably an antibody.

14. 11. A method for stimulating TGFb1-specific T cells, said method comprising contacting T cells with a polypeptide according to any one of claims 1 to 6 and / or a composition according to any one of claims 8 to 10, said composition comprising at least one polypeptide as defined in any one of claims 1 to 6.

15. 15. The method of claim 14, wherein the T cells are present in a sample taken from a healthy subject or a cancer patient, optionally in a tumor sample.

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