TGF-beta vaccine

Abstract

The present invention relates to novel polypeptides derived from TGFb1. The present invention also relates to uses of the polypeptides, polynucleotides encoding the peptides and uses thereof, and compositions comprising the polypeptides and uses thereof.

Description

TECHNICAL FIELD

[0001] The present invention relates to novel polypeptides derived from transforming growth factor beta 1 (TGFb1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The present invention further relates to uses and methods of using the polypeptides, polynucleotides and compositions. BACKGROUND

[0002] TGFb is a multifunctional cytokine that plays a key role in the regulation of the immune system. There are 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. It also contributes to tumor vascularization and metastasis. Thus, TGFb1 is a key suppressive molecule in the tumor microenvironment (TME), contributing to the downregulation of anti-tumor mechanisms of the immune system and enabling cancer cells to evade immunity.

[0003] In a recent study on a mouse model of metastatic liver cancer, TGFb1 has also been suggested to contribute to the reduced efficiency of cancer treatments with immune checkpoint blockers (ICBs) such as PD-L1 blockers. SUMMARY

[0004] The polypeptides of the present invention are expected to be particularly effective in stimulating a beneficial immune response against cells expressing TGFb1. The development of novel cancer immunotherapies requires a thorough understanding of the molecules involved in the pathogenesis and of specific proteins that are recognized by the immune system. In a clinical setting, the induction of a TGFb1 -specific immune response can directly kill cancer cells expressing TGFb1, but more importantly it generally supports anti-cancer immune responses by suppressing the immunosuppressive function of TGFb1. Targeting TGFb1 and cells expressing TGFb1 will therefore have a high degree of synergy with additional anti-cancer immunotherapies, such as immune checkpoint blockers (ICBs), e.g. by vaccination with the polypeptides of the present invention.

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

[0006] The TGFb1 proprotein monomer has a molecular weight of about 25 kDa. 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), as shown in Figure 1

[0007] The TGFb1 SP targets the protein to the secretory pathway; the SP is cleaved in the rough endoplasmic reticulum. The TGFb1 monomer, comprising the LAP and the mature TGFb1, can dimerize in the endoplasmic reticulum through disulfide bonds between cysteine residues in the LAP (e.g., Cys 223 and Cys 225) and cysteine residues in the mature TGFb1 peptide (e.g., Cys 356), forming a TGFb1 homodimer. This TGFb1 homodimer is referred to as a small latent complex (SLC). The SLC can associate with a so-called Latent TGF-β Binding Protein (LTBP) to form a larger complex, which is referred to as a large latent complex (LLC). The LLC can be secreted into the extracellular medium (ECM). However, the presence of the LAP and the LTBP prevents TGFb1 from binding to and activating its extracellular receptors. Active TGFb1 consists of a homodimer of the mature TGFb1 peptide. There are multiple mechanisms by which the mature TGFb1 homodimer is released from the LAP and the LTBP, including protease degradation of the LAP, induction of a conformational change in the LAP through interaction with thrombospondin, and non-covalent bond breakage between the LAP and TGFβ-1.

[0008] It is an object of the present invention to develop a T cell-mediated mechanism for removing TGFb1 from the TME. The present inventors 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 regarding HLA restriction, cytokine production, and cytotoxicity.

[0009] The present inventors have identified regions of human TGFb1 that are maximally immunogenic. Surprisingly, these immunogenic "hotspot" regions are located throughout the human TGFb1 proprotein, including within the SP and LAP domains and the mature TGFb1 peptide. The present inventors have also identified a sub-region within the human TGFb1 LAP, namely positions 121-160 of SEQ ID NO: 1 (corresponding to the sequence of SEQ ID NO: 65), which contains a higher frequency of immunogenic peptide sequences.

[0010] ​Accordingly, the present application provides a polypeptide which is an immunogenic fragment of human TGFbl (SEQ ID NO: 1) and which 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 can correspond to a sequence of at least 9 contiguous amino acids of SEQ ID NO: 2 or 65. The polypeptide can 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 can 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; 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 can 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; preferably, the polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

[0011] The polypeptide can 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 can be replaced by the corresponding amide. The polypeptide can comprise an HLA-A2 restricted epitope. The HLA-A2 restricted epitope can comprise or consist of the amino acid sequence of SEQ ID NO: 66 or 67.

[0012] The present application also provides a polynucleotide encoding the polypeptide of the present application. The polynucleotide can be isolated. The present application also provides a vector comprising the polynucleotide.

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

[0014] The present application also provides a method of treating or preventing a disease or a disorder in a subject, the method comprising: administering to the subject the polypeptide of the present application, the polynucleotide of the present application, and / or the composition of the present application. The method can further comprise administering simultaneously or sequentially another cancer therapy, preferably an antibody.

[0015] The present application also provides the use of the polypeptide of the present application, the polynucleotide of the present application, the composition of the present application, or a combination thereof, in the treatment or prevention of a disease or a disorder. The polypeptide of the present application, the polynucleotide of the present application, the composition of the present application, or a combination thereof, can be used in combination with another cancer therapy, preferably an antibody.

[0016] The present application also provides the use of the polypeptide of the present application, the polynucleotide of the present application, the composition of the present application, or a combination thereof, in the manufacture of a medicament for the treatment or prevention of a disease or a disorder.

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

[0018] The present application also provides a method of stimulating TGFbl -specific T cells, the method comprising contacting a T cell with a polypeptide of the application and / or a composition of the application comprising at least one polypeptide of the application. The T cell can be present in a sample taken from a healthy subject or a cancer patient, optionally a tumor sample. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A-C. Peptide-specific immune responses in PBMCs from 6 healthy donors were assessed against an array of 38 overlapping 20-mer peptides derived from the TGFbl proprotein by in vitro IFNy ELISPOT assay (set up in triplicate wells). Each dot represents the average number of IFNy-secreting cells after subtraction of the corresponding background signal, and the grey level bar indicates the mean value for the tested donors. Asterisks indicate the peptides leading to the strongest and statistically most significant responses based on DFRx2, and they were selected for further screening experiments (summarized in Figure 1 D).

[0020] Figure 1 D. Table summarizing the most immunogenic TGFβ peptides based on the screening of A-1C and their respective average IFNy ELISPOT counts. The top eight best-performing peptides were selected for further investigation. Figure 1 A-1C. Table summarizing the most immunogenic TGFβ peptides based on the screening of A-1C and their respective average IFNy ELISPOT counts. The top eight best-performing peptides were selected for further investigation.

[0021] Figure 1 E. Top panel: Primary sequence of the TGFbl proprotein. Highlighted are the amino acid sequences of the eight immunogenic TGFbl peptides selected for further screening. Underlined amino acid positions 1-29 indicate the position of the signal sequence of the protein (SP), while underlined amino acid positions 279-390 indicate the mature TGFbl monomeric protein. Bottom panel: Schematic representation of the TGFbl proprotein domains and the position of the eight selected TGFbl -derived peptides. Numbers (1, 29, 279, and 390) indicate key amino acid positions marking the three main domains of the TGFbl proprotein.

[0022] Figure 2A. By assessing the response of another healthy donor through in vitro IFNγELISPOT assay, targeting... Figure 1 Eight immunogenic TGFb1-derived peptides identified in AC validated peptide-specific immune responses in PBMCs. Each dot represents the mean number of IFNγ-secreting cells in an individual donor after subtracting their respective background signal, and the black horizontal bars represent the mean of the tested donors. B. The heatmap depicts the magnitude of the response to the lead epitope in PBMCs from healthy subjects (top); a representative ELISPOT response (bottom).

[0023] Figure 3 A. By assessing the response through in vitro IFNγELISPOT assay, targeting Figure 1 Eight immunogenic TGFb1 peptides identified in AC validated peptide-specific immune responses in PBMCs, but this time the examination was conducted on cancer patients. Each spot represents the mean number of IFNγ-secreting cells in an individual cancer patient after subtracting their respective background signal, and the black horizontal bars represent the mean of the patients tested. B. The heatmap depicts the magnitude of the response to the leader epitope in PBMCs from cancer patients.

[0024] Figure 4 Intracellular cytokine staining (ICS) analysis was established to further characterize the functionality of T cells responding to the TGFb1 epitope. In this embodiment, PBMCs from a healthy donor (BC-M-41) were thawed and stimulated with TGFb-02 (SEQ ID NO:6) 13 days prior to assay. IL-2 was added one day after culture establishment (120 U / mL) and three days before ICS establishment (60 U / mL). In each flow cytometry plot, each cell was represented as a point, and the functional phenotype of the cells was analyzed based on the expression of two markers at a time, one marker per axis. Based on CD3 + CD4 + T cells or CD3 + CD8 + The T cell component gates the live cell population and quantifies the expression of cytokines (IFNγ and TNFα) and the cytotoxic marker (CD107a). The percentages of each population are summarized in the hierarchical table on the right.

[0025] Figure 5 A. FACS plot shows CD4 targeting the TGFβ epitope, determined using ICS. + T cell response. B. FACS plot shows CD8 targeting the TGFb1 epitope as determined by ICS. + T cell response.

[0026] Figure 6MACS CD137 enrichment of specific T cells generated bulk cultures specific for several TGFbl derived epitopes. Enriched cells were expanded after enrichment and showed different reactivity to their epitopes. For each of A-D, FACS plots on top show specific CD4 + gated cells and FACS plots on bottom show specific CD8 + gated cells.

[0027] Figure 7 A. Left panel: Amplitude of responses in PBMCs from cancer patients and healthy subjects measured by ex vivo ELISPOT. PBMCs were rested overnight and then directly plated in ELISPOT wells and stimulated with epitopes in ELISPOT wells for 48 hours. Right panel: Example of ex vivo ELISPOT responses to several TGFbl lead epitopes. B. CD8 + T cell responses identified against epitope TGFb-15 using ICS after only 5 hours of stimulation.

[0028] Figure 8 A. PBMCs from a prostate cancer patient show CD8 + T cell responses against TGFb-15 epitope after 14 days of in vitro stimulation with the peptide and 18 hours of stimulation with TGFb-15. B. TGFb15 specific T cells from donor UR1121.14 were enriched twice after stimulation with TGFb-15, re-stimulated after 14 days of in vitro culture and then enriched using the MACS CD137 enrichment method the next day. CD4 + T cells (FACS plots on top of A and B respectively) and CD8 + T cells (FACS plots on bottom of A and B respectively) both responded to stimulation with TGFb-15.

[0029] Figure 9 FACS plots showing TGFb-15 specific CD8 + T cell clones analyzed by ICS.

[0030] Figure 10 TGFb-15 specific CD8 + T cell clones kill target cells in an HLA restricted manner and kill cancer cell lines expressing TGFbl. A. TGFb-15 specific CD8 +T cells efficiently lyse T2 cells pulsed with TGFb-15 peptide. B. To ensure that the TGFb-15 response is HLA-A2 restricted, it has been demonstrated that TGFb-15 specific CD8 T cells do not lyse HLA-A3 target cells pulsed with the TGFb-15 peptide. + Target cells, but not HLA-A3 + Target cells are lysed. C. HLA-A2 + Cancer cell lines UKE-1 and THP-1 stimulate the activation of TGFb-15 specific CD8 + T cell clones. Other HLA-A2 + Cancer cells do not activate T cells. D. TGFb-15 specific T cells readily kill THP-1 and UKE-1 cancer cell lines. E. Activation of TGFb-15 specific T cells is enhanced after stimulation with cytokine treated THP-1 cells. F. Stimulation of THP-1 cells with the Th2 cytokine IL-4 or TGFbl increases the number of THP-1 cells killed by TGFb-15 specific cells.

[0031] Figure 11 Results of IFN-g (A) and TNF-a (B) ELISPOT assays to analyze responses spanning the TGF nine-mer library.

[0032] Figure 12 CD8 T cells specific for the decamer epitope binding to HLA-A2 in the TGFbl signal peptide sequence. + T cells readily kill TGFbl expressing cancer cell lines in an HLA-A2 restricted manner. A. After 14 days in vitro culture, healthy donor PBMCs show secreted IFN-g after stimulation with the decamer epitope TGFb-A2-01 peptide binding to HLA-A2. B. Intracellular cytokine staining of healthy donor PBMCs shows CD8 T cell responses to TGFb-A2-01, as stimulated cells show increased expression of CD107a (right panel) and increased expression of IFN-g and TNF-a (left panel). + T cell responses, as stimulated CD8 + T cells in addition to showing increased CD107a expression after stimulation with TGFb-A2-01 (right panel), also show increased expression of IFN-g and TNF-a (left panel). C. TGFb-A2-01 specific CD8 T cells from a healthy donor kill TGFb-A2-01 pulsed HLA-A2 + Target cells, but not un-pulsed cells and peptide pulsed HLA-A3 + Target cells, but not un-pulsed cells and peptide pulsed HLA-A3 + Target cells, but not un-pulsed cells and peptide pulsed HLA-A3 + Target cells, but not un-pulsed cells and peptide pulsed HLA-A3 + Target cells, but not un-pulsed cells and peptide pulsed HLA-A3 +THP-1 cells were readily killed by TGFb-A2-01 -specific T cells, and 48 hours prior to assaying for enhanced portions of killed target cells, TGF-1 cell TGFbl expression was modulated by stimulating with different cytokines.

[0033] Figure 13 FACS plots showing results of ICS analysis of TGFb-A2-01 -specific T cell clones stimulated with TGFb-A2-01.

[0034] Figure 14 Amino acid sequences of the deca-peptides in the TGFβ library. Overlapping amino acid sequences are underlined.

[0035] Brief explanation of sequences

[0036] SEQ ID NO: 1 is the amino acid sequence of the full-length precursor of human TGFbl (also referred to as TGFbl proprotein).

[0037] SEQ ID NO: 2 is the amino acid sequence of the signal peptide of human TGFbl.

[0038] SEQ ID NO: 3 is the amino acid sequence of the LAP peptide of human TGFbl.

[0039] SEQ ID NO: 4 is the amino acid sequence of mature human TGFbl.

[0040] SEQ ID NOs: 5-64 are each the amino acid sequence of a polypeptide fragment derived from human TGFbl.

[0041] SEQ ID NO: 65 is the amino acid sequence of a LAP sub-region comprising a high frequency of immunogenic sequences.

[0042] SEQ ID NO: 66 is the amino acid sequence of the smallest epitope sequence in the TGFb-15 peptide sequence (SEQ ID NO: 28). SEQ ID NO: 66 is also referred to herein as “TGFb-15 short”.

[0043] SEQ ID NO: 67 is the amino acid sequence of TGFb-A2-01. DETAILED DESCRIPTION

[0044] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to be limiting.

[0045] Also, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "a plurality of polypeptides" and the like.

[0046] "Polypeptide" is used herein in its broadest form to refer to a compound of two or more subunits amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0047] The terms "patient" and "subject" can be used interchangeably and generally refer to a human.

[0048] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0049] "Immunogenic" refers herein to a polypeptide that is capable of eliciting an immune response to a TGFbl protein, preferably when said protein is present in or on a cell expressing the TGFbl protein. In other words, the polypeptide can be described as immunogenic to TGFbl. Alternatively, the polypeptide can be described as an immunogenic fragment of TGFbl. The immune response is preferably a T cell response, and thus the polypeptide can be described as an immunogenic fragment of TGFbl comprising a T cell epitope. The immune response can be detected in at least one individual (or in a sample taken from an individual) after administration of the polypeptide to said individual (or to said sample).

[0050] 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:

[0051] i. it is capable of eliciting IFN-gamma producing cells in a PBL population of healthy subjects and / or cancer patients, as determined by ELISPOT analysis; and / or

[0052] ii. it is capable of detecting CTLs reacting with TGFbl in situ in tumor tissue samples; and / or

[0053] iii. it is capable of inducing in vitro growth of specific T cells.

[0054] Methods suitable for determining whether a polypeptide is immunogenic are also described in the Examples section below.

[0055] The polypeptide of the application is an immunogenic fragment of human TGFbl (SEQ ID NO: 1) comprising or consisting of a sequence of at least 9 contiguous amino acids of SEQ ID NO: 1.

[0056] The sequence of at least 9 contiguous amino acids of SEQ ID NO: 1 can correspond to a sequence of at least 9 contiguous amino acids of the SP domain of TGFbl, for example a sequence of at least 95 contiguous amino acids of SEQ ID NO: 2.

[0057] The sequence of at least 9 contiguous amino acids of SEQ ID NO: 1 can correspond to a sequence of at least 9 amino acids of the LAP domain of TGFbl, for example a sequence of at least 9 contiguous amino acids of SEQ ID NO: 3.

[0058] The sequence of at least 9 contiguous amino acids of SEQ ID NO: 1 can correspond to a sequence of at least 9 contiguous amino acids within the LAP subregion delimited by amino acid positions 121 and 160 of SEQ ID NO: 1, for example a sequence of at least 9 contiguous amino acids of SEQ ID NO: 65.

[0059] The sequence of at least 9 contiguous amino acids of SEQ ID NO: 1 can correspond to a sequence of at least 9 contiguous amino acids of the mature TGFbl polypeptide, for example a sequence of at least 9 contiguous amino acids of SEQ ID NO: 4.

[0060] The polypeptide can 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.

[0061] The polypeptide can comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 2 and 5-67.

[0062] The polypeptide can 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. 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.

[0063] The polypeptide can 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. Particularly preferably, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63.

[0064] The polypeptide can 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 can be replaced with the corresponding amide. The polypeptide can be isolated.

[0065] Particularly 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, or 63. Particularly preferably, the polypeptide comprises or consists 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 comprising these sequences are also preferred.

[0066] The polypeptide can 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. Preferred peptides comprising an HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO: 66 are peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 28-31 or 65. Alternatively, the HLA-A2 restricted epitope preferably comprises or consists of the amino acid sequence of SEQ ID NO: 67. Preferred peptides comprising an HLA-A2 restricted epitope consisting of the amino acid sequence of SEQ ID NO: 67 are peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5, 8, 9, or 2.

[0067] In any of the polypeptides described herein, the amino acid sequence can be modified by one, two, three, four, or five (up to five) additions, deletions, or substitutions compared to the polypeptide having the unmodified sequence, so long as the polypeptide having the modified sequence exhibits the same or increased immunogenicity to TGFbl compared to the polypeptide having the unmodified sequence. By“same” is understood that the polypeptide having the modified sequence does not exhibit a significant decrease in immunogenicity to TGFbl compared to the polypeptide having the unmodified sequence. Any comparison of immunogenicity between sequences will be performed using the same assay. Unless otherwise specified, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. A conservative substitution replaces an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, a conservative substitution can introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 primary amino acids as defined in Table Al below. When amino acids have similar polarity, this can be determined with reference to the scale of hydrophilicity of amino acid side chains in Table A2.

[0068] Table A1 - Chemical properties of amino acids

[0069] Ala (A) Aliphatic, hydrophobic, neutral Met (M) Hydrophobic, neutral Cys (C) Polar, hydrophobic, neutral Asn (N) Polar, hydrophilic, neutral Asp (D) Polar, hydrophilic, charged (-) Pro (P) Hydrophobic, neutral Glu (E) Polar, hydrophilic, charged (-) Gln (Q) Polar, hydrophilic, neutral Phe (F) Aromatic, hydrophobic, neutral Arg (R) Polar, hydrophilic, charged (+) Gly (G) Aliphatic, neutral Ser (S) Polar, hydrophilic, neutral His (H) Aromatic, polar, hydrophilic, charged (+) Thr (T) Polar, hydrophilic, neutral Ile (I) Aliphatic, hydrophobic, neutral Val (V) Aliphatic, hydrophobic, neutral Lys (K) Polar, hydrophilic, charged (+) Trp (W) Aromatic, hydrophobic, neutral Leu (L) Aliphatic, hydrophobic, neutral Tyr (Y) Aromatic, polar, hydrophobic

[0070] Table A2 - Hydrophilicity scale

[0071]

[0072] In any of the polypeptides disclosed herein, any one or more of the following modifications can be made to improve physicochemical properties (e.g., stability), so long as the polypeptide exhibits the same or increased immunogenicity to TGFbl compared to the polypeptide having the unmodified sequence:

[0073] replacing the C-terminal amino acid with the corresponding amide (may increase resistance to carboxypeptidases);

[0074] replacing the N-terminal amino acid with the corresponding acylated amino acid (may increase resistance to aminopeptidases);

[0075] replacing one or more amino acids with the corresponding methylated amino acid (may increase proteolytic resistance); and / or

[0076] replacing one or more amino acids with the corresponding amino acid in the D configuration (may increase proteolytic resistance).

[0077] Any of the polypeptides disclosed herein can have at least one additional moiety attached at the N- and / or C-terminus to improve solubility, stability, and / or aid in manufacturing / isolation, so long as the polypeptide exhibits the same or increased immunogenicity against TGFbl compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequence KK, KR, or RR can be added at the N- and / or C-terminus. Other suitable moieties include Albumin or PEG (Polyethylene Glycol).

[0078] The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides can be synthesized directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry, or by solution phase peptide synthesis. Alternatively, the polypeptides can be produced by transforming a cell (typically a bacterial cell) with a nucleic acid molecule or vector encoding the polypeptide. The present application provides nucleic acid molecules and vectors encoding the polypeptides of the present application. The present application also provides host cells comprising such nucleic acids or vectors.

[0079] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include a gene, a gene fragment, 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 application can be provided in isolated or substantially isolated form. Substantially isolated means that the polypeptide is essentially separated from any surrounding medium, but not completely isolated. The polynucleotides can be mixed with carriers or diluents that do not interfere with their intended use and still be considered substantially isolated. A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid molecule, which when expressed, i.e., when transcribed and translated in vivo, in vitro, or when placed under the control of appropriate control elements, the product transcribed and translated is a polypeptide. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Such coding sequences can include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0080] A polynucleotide can be synthesized according to methods well known in the art, as described in the examples of Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press. A nucleic acid molecule of the application can be provided in the form of an expression cassette comprising control sequences operably linked to the inserted sequence, allowing expression of a polypeptide of the application in vivo. In turn, these expression cassettes are typically provided in a vehicle (e.g. a plasmid or a recombinant viral vehicle). Such expression cassettes can be administered directly to a host subject. Alternatively, a vehicle comprising a polynucleotide of the application can be administered to a host subject. Preferably, the polynucleotide is prepared and / or administered using a genetic vehicle. A suitable vehicle can be any vehicle capable of carrying sufficient genetic information and allowing expression of a polypeptide of the application.

[0081] The application thus includes expression vehicles comprising such polynucleotide sequences. Such expression vehicles are routinely constructed in the art of molecular biology and can for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements (e.g. polyadenylation signals) which can be necessary and which are positioned in the correct orientation to allow expression of a peptide of the application. Other suitable vehicles will be apparent to those skilled in the art. As a further example in this regard we refer to Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0082] The application also includes cells which have been modified to express a polypeptide of the application. Such cells typically include prokaryotic cells, such as bacterial cells, for example E. coli. Such cells can be cultured using routine methods to produce a polypeptide of the application.

[0083] A polypeptide of the application can be in substantially isolated form. It can be mixed with carriers, preservatives or diluents which do not interfere with the intended use, and / or with adjuvants, and still be regarded as substantially isolated. It can also be in substantially purified form, in which case it typically comprises at least 90%, for example at least 95%, 98% or 99% of the proteins in the preparation.

[0084] Compositions comprising a polypeptide

[0085] The application provides a composition comprising a polypeptide of the application and / or a polynucleotide of the application. For example, the application provides a composition comprising: one or more polypeptides of the application and / or one or more polynucleotides of the application, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0086] The composition can 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 application, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0087] The composition can 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 application, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0088] The carrier, preservative and excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject to whom the composition is administered. In general, all components and the final composition are sterile and pyrogen-free. The composition can be a pharmaceutical composition. The composition can preferably comprise an adjuvant. An adjuvant is any substance added to the composition that increases or otherwise modifies the immune response elicited by the composition. Adjuvants are broadly substances that facilitate an immune response. Adjuvants can also preferably have a depot effect in that they also lead to 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 (2ndEdition, 1986), pages 61-63.

[0089] The adjuvant can be selected from the group consisting of AlK(S04)2, AlNa(S04)2, AlNH4(S04), silicon dioxide, alum, Al(OH)3, Ca3(P04)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 1687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '2'-dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL + TDM + CWS) in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharides and various derivatives thereof including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g. poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis and Brucella members, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21. Various saponin extracts have also been suggested for use as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) can also be used as an adjuvant.

[0090] Preferred adjuvants for use in the present application include oil / surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA based adjuvants such as adjuvants comprising CpG oligonucleotide sequences. Other preferred adjuvants are viral dsRNA based adjuvants such as poly I:C.GM-CSF, and imidazoquinolines are also examples of preferred adjuvants.

[0091] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0092] It is also noted that, when the molecular weight of the antigen of interest is low or poorly immunogenic, it is recommended to couple it to an immunogenic carrier, as described in Goding, Monoclonal Antibodies: Principles & Practice (2nded., 1986), pages 61-63. Thus, the polypeptides of the present application can be coupled to a carrier. The carrier can be present independently of the adjuvant. For example, the function of the carrier can be to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, to confer stability, to increase biological activity or to increase serum half-life. In addition, the carrier can assist in the presentation of its polypeptide or fragment thereof to T cells. Thus, in the composition, the polypeptide can be associated with a carrier as listed below. The carrier can be any suitable carrier known to the skilled person, for example, 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 a dextran, such as agarose. The carrier must be physiologically acceptable and safe for humans.

[0093] If the composition comprises an excipient, it must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not elicit an immune response in the individual receiving the composition, and which can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, salts of mineral acids, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, carriers, and auxiliary substances is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0094] Formulation of suitable compositions can be achieved using standard pharmaceutical formulation chemistries and methods, all of which are readily available to the skilled artisan. 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 in ampoules or in multi-dose containers, for example, with an added preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations in oily or aqueous vehicles. In one embodiment of the composition, the active ingredient is provided in dry (e.g., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oleaginous suspension or solution. This suspension or solution can be formulated according to known techniques using suitable dispersing or wetting agents (so-called "supporting agents") and suspending agents. In addition to the active ingredient, such compositions can further include adjuvants, excipients, and auxiliary substances, as described herein. For example, sterile injectable suspensions or solutions can 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 sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful compositions include those including the active ingredient in microcrystalline form, in liposome preparations, or as a component of biodegradable polymer systems. Compositions for sustained release or implantation can include 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 can be encapsulated, adsorbed onto, or otherwise associated with, microparticulate carriers. Suitable microparticulate carriers include those derived from poly(methyl methacrylate) polymers, as well as PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other microparticulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0095] Methods of use

[0096] The polypeptide, polynucleotide, or composition of the present application, or a combination thereof, can be used in a method of treating or preventing a disease or disorder in a subject. The polypeptide, polynucleotide, or composition of the present application, or a combination thereof, can be used in the preparation of a medicament for use in a method of treating or preventing a disease or disorder in a subject. The method comprises administering to the subject the polypeptide, the polynucleotide, the composition, or the combination. A therapeutically or prophylactically effective amount of the polypeptide, the polynucleotide, the composition, or the combination can be administered to a subject in need thereof.

[0097] The disease or disorder can be characterized at least in part by an inappropriate or excessive immunosuppressive function of TGFbl. The disease or disorder can be characterized at least in part by an inappropriate or excessive expression of IL-4 and / or IL-13. The disease or disorder can be a cancer, preferably a cancer expressing TGFbl and / or associated with an inappropriate or excessive immunosuppressive function of TGFbl and / or an inappropriate or excessive expression of IL-4 and / or IL-13. The cancer can be breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer or pancreatic cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), melanoma, leukemia (such as acute myeloid leukemia (AML)) or prostate cancer. The cancer can be AML characterized by an inappropriate or excessive immunosuppressive function of TGFbl and / or an inappropriate or excessive expression of IL-4 and / or IL-13. The cancer can be AML characterized by an inappropriate or excessive immunosuppressive function of TGFbl and an inappropriate or excessive expression of IL-4 and / or IL-13.

[0098] The method can comprise administering simultaneously or sequentially with a further cancer therapy. The further cancer therapy can be a bispecific inhibitor of TGFb (e.g. TGFbl ) and PD-L1. The bispecific inhibitor can be capable of simultaneously binding to TGFb and PD-L1 and / or inhibiting the activity of TGFb and PD-L1. The bispecific inhibitor can be a fusion protein comprising an anti-TGFb moiety and an anti-PD-L1 moiety, optionally wherein the anti-PD-L1 moiety comprises or consists of an anti-PD-L1 antibody and / or the anti-TGFb moiety comprises or consists of a TGFb receptor or a portion thereof, such as TGFb receptor II or a portion thereof.

[0099] The further cancer therapy can be selected from a cytokine therapy, a T cell therapy, an NK therapy, an immune system checkpoint inhibitor, a chemotherapy, a radiotherapy, an immune stimulatory substance, a gene therapy or an antibody.

[0100] The antibody can be Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (=Tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumab pegol, Cibisatamab, Cibisatamab, Cixutumumab, Clenoliximab, Codritas, Conatumumab, Crizanlizumab, Daratumumab, Denosumab, Detumumab, Dinutuximab, Durvalumab, Efalizumab, Eculizumab, Elsilomab, Eltrombital, Enavatuzumab, Enavatuzumab metuxin, Enokizumab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximabpegol), Cetuximab, Ch. 14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab (Elovalumab), Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab metasesquiox, Epratuzumab, Erasmusumab, Ergozumab, Erilumab, Erizumab, Erlicotuzumab, Erlozotumab, Ervektumab, Erwiniamab, Erzotumab, Esbapezumab, Eseliximab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucab, Eselvucozogamicin), Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecan, Irinotecanozogamicin), Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, NofetumomabMerpentan), Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Prothrombin complex concentrate, Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Koagulationsfaktor VIII), Prothrombin complex concentrate (Ko140、Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Temu- 15 -Aritox), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= Tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (= Atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox).

[0101] Preferred antibodies include Natalizumab, Vedolizumab, Belimumab, Atacicept, Alefacept, Otelixizumab, Teplizumab, Rituximab, Ofatumumab, Ocrelizumab, Epratuzumab, Alemtuzumab, Abatacept, Eculizumab, Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab, Ustekinumab, Briakinumab, Etanercept, Infliximab, Adalimumab, Certolizumab pegol, Golimumab, Trastuzumab, Gemtuzumab, Ozogamicin, Ibritumomab, Tiuxetan, Tostitumomab, Cetuximab, Bevacizumab, Panitumumab, Denosumab, Ipilimumab, Brentuximab, and Vedotin.

[0102] Particularly preferred antibodies that can be used in the methods of the application include daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab, and ofatumumab.

[0103] The additional cancer therapy can be selected from the group consisting of Coenzyme B12 (Actimide), 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.

[0104] The polypeptides of the application and / or the compositions of the application comprising at least one polypeptide of the application can also be used in a method of stimulating TGFb1 -specific T cells (such as CD4 + and / or CD8 + T cells), comprising contacting the cells with said polypeptides and / or said compositions. The method can be performed ex vivo. The cells can be present in a sample taken from a healthy subject or a cancer patient, for example in a tumor sample.

[0105] The present application is further illustrated by the following examples, which should not, however, be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the application in diverse forms thereof.

[0106] Example

[0107] Example 1 - Materials and methods

[0108] Patients and donors

[0109] PBMC from anonymous blood donors were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. PBMC from cancer patients were obtained from the Department of Oncology, Herlev Hospital, Herlev, Denmark. All participants gave informed consent according to the Helsinki declaration prior to entering the study. PBMC were isolated using Lymphoprep (Axis Shield, Oslo, Norway) and frozen in fetal calf serum with 10% dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).

[0110] Peptides

[0111] Peptides were provided by Pepscan (Lelystadt, The Netherlands) and dissolved in DMSO at a concentration of 10 mM. After identification of the TGFβ1 lead epitope, these peptides were provided in higher purity (>90%) by KJ Ross-Petersen (Klampenborg, Denmark). The sequences of the peptides used in these experiments are shown in the section titled "Sequences". The peptides are described by SEQ ID NO, name or by the start and end position within the amino acid sequence of the reference human TGFb1 full-length precursor. Each name can be used interchangeably as indicated in the table listed in the sequence section below. For example, the peptide of SEQ ID NO: 6 can alternatively be referred to as name TGFb-02 (or TGFB02), or alternatively as TGFb1 11-30 (hypothetical start position 11 and end position 30). The intended reference in each case is clear from the context.

[0112] In vitro enzyme-linked immunospot assay (ELISPOT) analysis

[0113] For in vitro ELISPOT, PBMC from cancer patients and healthy donors were pulsed with 20 mM of TGFp-derived peptides (or no peptide as control) and 120 U / ml IL-2 in 24-well plates for 7-10 days before use for ELISPOT analysis. Cells were plated in 96-well nitrocellulose ELISPOT plates (MultiScreen IP filter plates, MSIPN4W50; Millipore) pre-coated with IFNy capture antibody (Mabtech). TGFp peptides were added to a final concentration of 5 mM, control stimulators (DMSO, HIV or scrambled peptide) were added to control wells, and plates were incubated at 37°C for 16-20 hours. After incubation, cells were washed away and secondary biotinylated antibody (Mabtech, cat# 3420-6-1000) was added for 2 hours at room temperature. Unbound secondary antibody was washed away, and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech, cat# 3310-10) was added for 1 hour at room temperature. Unbound conjugated enzyme was washed away, and the assay was developed by adding BCIP / NBT substrate (Mabtech, cat# 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 TGFp peptides and wells with no peptide added. Unless otherwise stated, all experiments were performed using the in vitro IFN-g ELISPOT assay, and all experiments were repeated in triplicate. Statistical analysis was performed using the distribution free resampling (DFR) method and the more conservative DFR 2x method as described by Moodie et al. (Cancer Immunol Immunother 2010; 59: 1489-1501).

[0114] Ex vivo ELISPOT analysis

[0115] PBMC from cancer patients or healthy donors were thawed in X-VIVO medium in 24-well plates and rested overnight. (Optional: addition of 1 pg / ml DNase I). The next day cells were counted and transferred to 96-well nitrocellulose ELISPOT plates (MultiScreen IP filter plates, MSIPN4W50; Millipore) previously coated with IFNy capture antibody (Mabtech). TGFp peptides were added to a final concentration of 5 pM, control stimuli (DMSO, HIV or scrambled peptide) were added to control wells, and plates were incubated at 37°C for 24-72 hours. Plate staining with secondary antibody and chromogenic protocol followed the in vitro ELISPOT protocol described above. Intracellular cytokine staining (ICS) and fluorescence-activated cell sorting (FACS)

[0116] In BD GolgiPlug TMAfter 5 hours of stimulation of PBMC with TGFp-derived peptides (or no incubation with peptides as control), intracellular staining of cell cultures was performed. CD107a-PE (cat. 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) according to the manufacturer's instructions, followed by permeabilization using a mixture of Fixation / Permeabilization Concentrate and Diluent (eBioscience, cat. no. 00-5123-43 and 00-5223-56). Permeabilized cells were then stained with fluorescently labeled antibodies for IFNy and TNFa. Flow cytometry analysis was performed on a FACSCanto™ II (BD Biosciences). Antibodies used: IFNy-APC (cat. no. 341117), TNFa-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), CD3-APC-H7 (cat. no. 560275) (all from BD Biosciences). Dead cells were stained with Fixable Viability Stain 510 (BD Biosciences, San Jose, CA, USA). Another method to identify activated T cells is to stimulate T cells overnight with antigen or target cells. After 18-24 hours of stimulation, cells are stained with the above-mentioned surface antigen-specific antibodies and fixable viability stain, while staining with anti-CD107a-PE and anti-CD137-BV421 (BD Biosciences, San Jose, CA, USA). HLA-A2 of donor PBMCs was analyzed by staining with anti-HLA-A2-FITC (BD Biosciences, San Jose, CA, USA) using appropriate isotype controls.

[0117] Rapid expansion protocol

[0118] 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 anti-CD3 antibody (OKT3, from Janssen-Cilag or Miltenyi Biotec), and a high dose of IL-2 (6,000 IU / mL IL2; Proleukin from Novartis).

[0119] FACS of live cells

[0120] To enrich specific T cells from primary PBMC cultures, the in vitro culturing method for cell cultures analyzed in ELISPOT (see above) was followed. Next, cells were stimulated with antigen overnight and washed twice in FACS buffer the next day before staining for 30 min with LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific, 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. Next, cells were sorted on a FACSARIA flow cytometer with appropriate application settings and compensation controls. Cell sorting was performed using the purity setting. After sorting, cells were split into two parts - half of the enriched cells were expanded using the rapid expansion protocol while the other half of the cells were cloned using limiting dilution, seeding three cells / well. Cloned cells were expanded using the rapid expansion protocol.

[0121] Magnetic activated cell sorting (MACS)

[0122] MACS was used to enrich antigen-specific T cells from primary cultures and from cultures that had already been enriched. Enrichment of specific T cells from primary PBMC cultures followed the in vitro culturing method for cell cultures analyzed in ELISPOT (see above). Next, cells were stimulated with antigen overnight and enriched using the MACS CD137 enrichment kit (Miltenyi Biotech, Bergisch Gladbach, Germany) the next day according to the manufacturer’s protocol. Enriched cells were expanded using the rapid expansion protocol. As described, some of the enriched cells were cloned by limiting dilution. Cloned cells were expanded using the rapid expansion protocol. Chromium-51 cytotoxicity assay with cytokine stimulation of target cells

[0123] Chromium-51 cytotoxicity assay was 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 the expression of TGFp in several cancer cell lines, cancer cell lines were stimulated with IL-4 (100 U / mL), IL-13 (20 U / mL) and TGFpl (2.e5 ng / mL) (all from Peprotech, Rocky Hill, NJ, USA) alone or in combination for 48 hours prior to analysis.

[0124] Example 2 - In vitro ELISPOT screening of 20-mer peptides

[0125] As described above, a batch of 38 overlapping 20-mer peptides derived from the full-length TGFb1 precursor were designed and produced. Each of the 20-mer peptides overlaps with 10 amino acids (see [link to product description]). Figure 14 ).

[0126] Peptide-specific immune responses in PBMCs from six healthy donors were assessed using an in vitro IFNγELISPOT assay (set in three replicate wells) to assess spontaneous immune responses against 20-meric peptide arrays. Results of these assays showed… Figure 1 In AC, peptides that elicited the strongest and most statistically significant responses were selected for further screening experiments. Figure 1 D summarized the characteristics of the best-performing peptides, while Figure 1 E indicates the location of the peptides described in the full-length sequence. Surprisingly, the immunogenic peptides were observed to be located throughout the entire full-length sequence of the TGFb1 precursor protein, rather than clustered within a single immunogenic "hotspot" or located within the amino acid sequence of the mature TGFb1 peptide. Notably, the immunogenic epitope peptides were identified using the signal peptide region and LAP peptide of the TGFb1 precursor, 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.

[0127] Eight of the 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 NO: 6, 42, 12, 23, 28, 49, 55 and 63, respectively), were selected for further research.

[0128] Example 3 - Verification of peptide-specific immune responses

[0129] Additional in vitro IFNγELISPOT assays were established to validate responses in additional healthy subjects to the eight selected epitope peptides identified in the initial screening (see Example 2). The results of these assays were... Figure 2 The results show strong and frequent responses to all tested epitope peptides, with TGFb-02, TGFb-26, TGFb-33, and especially TGFb-15 exhibiting strong and frequent responses. Figure 2B ).

[0130] Healthy subjects and cancer patients can exhibit different modalities of immune response to epitopes; therefore, the immunogenic potential of selected epitopes in cancer patients was also investigated. Peptide-specific immune responses against eight immunogenic TGFb1-derived peptides in PBMCs were also validated by examining cancer patients and then assessing the responses again using an in vitro IFNγELISPOT assay. The results of these assays were... Figure 3 The results show that TGFb-02, TGFb-15, TGFb-26, and TGFb-33 were observed to be highly immunogenic in patients. Figure 3B ).

[0131] Example 4 - Cytokine analysis

[0132] Intracellular cytokine staining (ICS) analysis was performed to further characterize the functionality of T cells responding to the TGFb1 epitope. In this embodiment, PBMCs from a healthy donor (BC-M-41) were thawed and stimulated with TGFb-02 (SEQ ID NO:6) 13 days prior to assay. IL-2 was added one day after culture establishment (at 120 U / mL) and three days before ICS establishment (at 60 U / mL). FACS analysis was performed, based on CD3... + CD4 + T cells or CD3 + CD8 + The T cell component gates the live cell population. Cytokine expression (IFNγ and TNFα) and the expression of cytotoxic markers (CD107a) were quantified. FACS plots of the cytokine analysis were shown... Figure 4 On the left, the percentages of each group are summarized in... Figure 4 The hierarchical structure table on the right.

[0133] CD3 was discovered + CD4 + T cell portion (not CD3) + CD8 + The T cell portion is responsive to TGFb-02 (SEQ ID NO: 6), as indicated by secretion of TNFα (alone or bound to IFNg), and has no / low expression of CD107a.

[0134] ICS was also used to show that epitopes TGFb-05 (SEQ ID NO: 12) and TGFb-26 (SEQ ID NO: 42) triggered CD4. + ( Figure 5 A) and CD8 + T cell response ( Figure 5B). After enriching specific cells by magnetically activated cell sorting (MACS), strong CD4 activity targeting several leader epitopes was also detected. + and CD8 + T cell response ( Figure 6 This demonstrates the high immunogenic potential of several epitopes in TGFβ.

[0135] Example 5 - Identification of ex vivo responses to TGFβ epitopes

[0136] In vitro responses of PBMCs from healthy subjects and cancer patients to several epitopes. Cells were thawed and incubated overnight before plating, followed by stimulation for 48 hours. Both healthy and patient cells released large amounts of IFN-γ (…). Figure 7 A) This study demonstrated that cells from healthy subjects and cancer patients contained a large number of freely circulating TGFβ-specific T cells. Most surprisingly, CD8+ was detected in PBMCs from ex vivo platings of prostate cancer patients only 5 hours after stimulation with the epitope TGFβ-15. + T cell response ( Figure 7 B). This finding indicates that the patient had a high proportion of circulating TGFβ-specific cytotoxic T cells. Given this strong response to TGFβ-15, a TGFβ-specific T cell culture was established using PBMCs from this patient. First, CD137 was identified as an activation marker for sorting specific T cells from this donor. The patient's PBMCs were then stimulated with TGFβ-15 and held in culture for 14 days, followed by restimulation with TGFβ-15 for 18 hours. CD137 and CD107a expression was then analyzed using fluorescence activated cell sorting (FACS). This experiment demonstrated that, considering the 16.6% CD8+ response after peptide stimulation, a high proportion of circulating TGFβ-specific cytotoxic T cells were present. + T cells are CD137 + CD137 is a suitable marker for enriching specific T cells. Figure 8 A).

[0137] TGFb-15-specific T cells were enriched using the MACS CD137 enrichment kit and used to establish CD4+ cells containing TGFb-15-specific T cells. + and CD8 + T cell culture ( Figure 8 B). Use this culture to assess TGFβ-specific T cell responses.

[0138] Example 6 - TGFb-15-specific T cells are able to recognize and kill cancer cells

[0139] CD8 was constructed from TGFβ as described in Example 5 using limiting dilution. + TGFb-15-specific T cell clones. CD8 +TGFb-15 specific clones showed a high reactivity towards TGFb-15 Figure 9 PBMCs from this patient were used to stain for HLA-A2 + Specific antibody staining showed that the donor was HLA-A2 + (data not shown). Subsequently, standard chromium-51 cytotoxicity assays were performed to check whether specific T cells could lyse peptide-pulsed HLA-A2 + target cells. Peptide-pulsed HLA-A2 + T2 cells were readily lysed by specific T cells, while non-pulsed T2 cells were not killed Figure 10 A).

[0140] T2 cells are not only HLA-A2 + So the killing of these cells can be mediated by a match of another HLA allele. For this reason, further experiments were performed using K562 cells as targets. The original K562 line is HLA-deficient, but these experiments were performed with two lines that were genetically modified to stably express HLA-A2 or HLA-A3. This ensures that these are the only HLA alleles expressed by the cells. Only peptide-pulsed HLA-A2 + K562 cells were killed by TGFb-15 specific clones, while non-pulsed HLA-A2 + K562 cells and peptide-pulsed HLA-A3 + K562 cells were not recognized Figure 10 B).

[0141] Almost all cells can secrete TGFβ, which largely contributes to the creation of a tumor suppressive environment. For this reason, it was investigated whether TGFb-15 specific T cells could recognize HLA-A2 + cancer cell lines. Cell lines UKE-1, SET-2 and THP-1, all derived from patients with acute myeloid leukemia (AML), were used together with two HLA-A2 + melanoma cell lines (WM852 and FM88) and K562 and HLA-A2 + K562 cells as target cells. TGFb-15 specific T cells were stimulated overnight with the respective target cells at an effector: target ratio of 3:1. Specific T cells recognized the two cancer cell lines THP-1 and UKE-1, while the other cell lines did not activate T cells Figure 10 C). Further, chromium-51 cytotoxicity experiments showed that TGFb-15 specific T cells killed UKE-1 and THP-1 cells Figure 10 D).

[0142] The THP-1 cell line is a relatively undifferentiated cell line, and treatment with different cytokines will affect gene expression in these cells. Interleukin (IL)-4 is a cornerstone cytokine in the development of Th2 responses. Therefore, it was hypothesized that treatment of THP-1 cells with IL-4 would increase TGFβ expression in these cells. In addition, because TGFβ production feeds into a positive feedback loop for its intracellular production, it was hypothesized 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, and not in the mature active form of TGFβ (see Figure 1 E). Therefore, pre-treatment of THP-1 cells with active TGFβ would not add the recognized epitope to THP-1 cells, but would only increase the production of TGFβ intracellularly.

[0143] THP-1 cells treated with IL-4 or TGFβ for 48 hours were used to stimulate TGFb-15 specific CD8+ T cells for 18 hours. It was demonstrated that cytokine-treated THP-1 cells induced greater activation of TGFb-15 specific T cells compared to unstimulated THP-1 cells Figure 10 E). Finally, it was shown that cytokine stimulation of THP-1 cells with IL-4 or TGFβ increased the number of lysed cells Figure 10 F).

[0144] Example 7 - Identification of minimal TGFβ epitope sequences

[0145] Because the TGFb-15 epitope is a 20-mer, it cannot be presented on an HLA-I molecule in full length form. 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 library of nonamer peptides, with 8 overlapping amino acids, resulting in 12 nonamer peptides. T cells from a TGFb-15 specific CD8 + T cell clone were plated in ELISPOT and stimulated with each of the 9-mer peptides. The results indicated that the minimal epitope within the TGFb-15 sequence was the sequence VLLSRAELRL (TGFb-15 short; SEQ ID NO: 66) (see Figure 11 ).

[0146] Example 8 - Decamer epitope in the signal peptide of TGFβ is a target for specific T cells

[0147] In view of the high frequency of CD8 +T cell responses, the inventors sought to identify additional HLA-A2 restricted decapeptide epitopes. Using the SYFPEITHI database of MHC ligands and peptide motifs, Rammensee et al. (SYFPEITHI: database for MHC ligands and peptide motifs; www.syfpeithi.de.; accessed on October 30, 2014), the entire TGFβ sequence was searched for decapeptide epitopes with high binding affinity to HLA-A2. The peptide sequence LLLLLPLLWL (TGFb-A2-01; SEQ ID NO: 67) emerged as the top binding decapeptide epitope with a binding affinity score of 30. It was then investigated whether the HLA-A2 + PBMC spontaneous T cell responses against the TGFb-A2-01 epitope. Surprisingly, the majority of PBMC showed responses against the TGFb-A2-01 epitope Figure 12 A). Using ICS, it was confirmed that these responses were from CD8 + T cells Figure 12 B).

[0148] TGFb-A2-01 specific T cells were then isolated from a healthy subject (BC363) with a solid response to TGFb-A2-01 by performing a single in vitro stimulation of PBMC from said subject followed by 14 days of culture. Next, PBMC were stimulated overnight with TGFb-A2-01. CD3 + , CD8 + , CD137 + cells were then gated and specific T cells were enriched using FACS. The enriched cells were expanded as described in Example 1. After 14 days of culture, several cell lines showed a high degree of specificity to the TGFb-A2-01 peptide Figure 13 ).

[0149] TGFb-A2-01 specific T cells were then tested for their ability to lyse peptide pulsed HLA-A2 + K562 target cells in a standard Cr51cytotoxicity assay. Peptide pulsed HLA-A2 + K562 cells were lysed, while non-pulsed HLA-A2 + and peptide pulsed HLA-A3 + target cells were not lysed Figure 12 C).

[0150] Since the above TGFb-15 specific T cells killed the AML cell lines UKE-1 and THP-1, it was tested whether the TGFb-A2-01 specific clones were also able to kill these target cancer cells. UKE-1 and THP-1 cancer cells were readily killed by TGFb-A2-01 specific T cells (see Figure 12 D and E). Furthermore, stimulation of THP-1 cells with IL-13, TGF, or both IL-13 and TGF combined increased the proportion of target cells killed Figure 12 E).

[0151] Conclusion

[0152] TGFb1 is a key player in immune homeostasis and tolerance, suppressing expansion and function of many components of the immune system. Perturbation of TGFb1 signaling is causative for inflammatory diseases and contributes to tumor emergence. TGFb1 is also central to immune suppression in the tumor microenvironment, and recent studies have revealed its role in tumor immune evasion and adverse reactions to cancer immunotherapy. TGFb1 expression is a major feature of both tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells. TGFb1 -expressing cells also play an important role in the development of an immunosuppressive microenvironment, as they prevent effector lymphocyte proliferation at the tumor site. Activation of TGFb1 -specific T cells, for example, by vaccination, should therefore lead to T cell infiltration at the tumor site.

[0153] It was discovered for the first time that TGFb1 -expressing cells can be specifically targeted using TGFb1 -specific effector T cells. In particular, the inventors identified peripheral TGFb1 -specific T cells naturally occurring in cancer patients and healthy donors by screening a peptide library covering the entire amino acid sequence of TGFb1. Interestingly, it has been found that TGFb1 contains multiple epitopes that are frequently recognized by peripheral T cells distributed in different regions of the TGFb1 sequence.

[0154] The observation of frequent T cell responses against TGFb1 underscores the surprising finding that TGFb1 is highly immunogenic. It is particularly unexpected that TGF1 b would be as highly immunogenic as the inventors observed, given that TGFb1 is so important for immune suppression. Furthermore, regions of TGFb1 have been identified that are able to generate particularly strong immune responses, and these regions would be ideal candidates for peptide-based vaccination approaches.

[0155] In view of the role of TGFb1 in suppressing the immune system, for example in the TME, the inventors' surprising finding that it is possible to enhance TGFb1 -specific immune responses in most patients with solid tumors and hematological malignancies.

[0156] Many different therapeutic strategies have focused 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 surprising results discussed above suggest that immunomodulatory vaccination targeting TGFbl would be an effective approach to target immunosuppressive cells in the TME. This unique approach would combine the depletion of immunosuppressive cells, including cancer cells (through direct killing by cytotoxic T cells), with the reprogramming of the immunosuppressive cell population (by introducing proinflammatory cytokines into the immunosuppressive microenvironment) compared to other clinical strategies. Since TGFbl expression is a major factor of the immunosuppressive cell phenotype, TGFbl-specific T cells can react specifically with immunosuppressive cells. A TGFbl vaccine to rebalance the microenvironment should increase the effect of T cell enhancing drugs such as checkpoint blockers like anti-PDl antibodies. Thus, combination therapy of a TGFbl vaccine and checkpoint blocking antibodies should increase the number of patients who respond to therapy.

[0157] In summary, the results of the above discussion provide a valuable approach to directly target a major factor in the lack of immune response in most cancer patients: TGFbl.

[0158] Sequences

[0159]

[0160]

[0161] In Table 1 below, unless otherwise indicated, the "start position" and "end position" indicate the position within the full-length human TGFbl proprotein (SEQ ID NO: 1).

[0162] Table 1

[0163]

[0164] SEQUENCE LISTING <110> IO BIOLOGICAL TECHNOLOGIES, INC. <120> TGF- BETA VACCINE <130> N415362WO <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 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 lie 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 Gin His Val Glu Leu Tyr Gin 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 Gin Trp Leu 195 200 205 Ser Arg Gly Gly Glu lie Glu Gly Phe Arg Leu Ser Ala His Cys Ser 210 215 220 Cys Asp Ser Arg Asp Asn Thr Leu Gin Val Asp lie Asn Gly Phe Thr 225 230 235 240 Thr Gly Arg Arg Gly Asp Leu Ala Thr lie His Gly Met Asn Arg Pro 245 250 255 Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gin His Leu Gin 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 Gin Leu Tyr He Asp Phe Arg Lys 290 295 300 Asp Leu Gly Trp Lys Trp He His Glu Pro Lys Gly Tyr His Ala Asn 305 310 315 320 Phe Cys Leu Gly Pro Cys Pro Tyr He Trp Ser Leu Asp Thr Gin Tyr 325 330 335 Ser Lys Val Leu Ala Leu Tyr Asn Gin He Asn Pro Gly Ala Ser Ala 340 345 350 Ala Pro Cys Cys Val Pro Gin Ala Leu Glu Pro Leu Pro He Val Tyr 355 360 365 Tyr Val Gly Arg Lys Pro Lys Val Glu Gin Leu Ser Asn Met He 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> TGFbl 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 Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu 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 Gin His Val Glu Leu Tyr Gin 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 He Glu Gly Phe Arg Leu Ser Ala 180 185 190 His Cys Ser Cys Asp Ser Arg Asp Asn Thr Leu Gin Val Asp He Asn 195 200 205 Gly Phe Thr Thr Gly Arg Arg Gly Asp Leu Ala Thr He His Gly Met 210 215 220 Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gin 225 230 235 240 His Leu Gin Ser Ser Arg His Arg Arg 245 <210> 4 <211> 112 <212> PRT <213> Homo sapiens <220> <221> Peptide <222> (1)..(112) <223> mature TGFbl <400> 4 Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val Arg Gin Leu Tyr He Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 He His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr He Trp Ser Leu Asp Thr Gin Tyr Ser Lys Val Leu Ala Leu 50 55 60 Tyr Asn Gin His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65 70 75 80 Gln Ala Leu Glu Pro Leu Pro He Val Tyr Tyr Val Gly Arg Lys Pro 85 90 95 Lys Val Glu Gin Leu Ser Asn Met He 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 Leu Leu 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 Val Leu Ala Leu 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-1 3.2 <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-1 3.3 <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 Leu Leu Arg Leu 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 Gin 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 Gin His Val Glu Leu Tyr Gin 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 Gin Lys Tyr Ser Asn Asn Ser Trp Arg Tyr Leu Ser Asn 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 lie 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 Gin Val Asp lie 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 Gin Val Asp lie Asn Gly Phe Thr Thr Gly Arg Arg Gly Asp 1 5 10 15 Leu Ala Thr lie 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 Ala Leu 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 Gin Leu Tyr lie 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 Gin Leu Tyr lie 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 Gin Leu Tyr lie Asp Phe Arg Lys Asp Leu 1 5 10 15 Gly Trp Lys Trp lie 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 Gin Leu Tyr He Asp 1 5 10 15 Phe Arg Lys Asp Leu Gly Trp Lys Trp He 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 He 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 Val Pro Gln Ala Leu Glu Pro Leu Pro Ile 1 5 10 15 Val Tyr Tyr Val 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 Val Tyr Tyr Val Gly Arg Lys Pro Lys Val 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 subregion <400> 65 Tyr Asp Lys Phe Lys Gln Ser Thr His Ser lie 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 Ala 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-15 short <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 Leu Leu Leu Leu Leu Pro Leu Leu Trp Leu 1 5 10

Claims

1. A polypeptide, which is an immunogenic fragment of human transforming growth factor 1 (TGFb1), and which consists of up to 20 consecutive amino acids of SEQ ID NO: 1 and contains the amino acid sequence of SEQ ID NO:

66.

2. The polypeptide according to claim 1, wherein it comprises up to 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 consecutive amino acids of SEQ ID NO:

1.

3. The polypeptide according to claim 1, wherein it comprises an amino acid sequence of any one of SEQ ID NO: 66 or 28.

4. The polypeptide according to claim 1, wherein the C-terminal amino acid is replaced by the corresponding amide.

5. The polypeptide according to claim 1, wherein it contains an HLA-A2 restriction epitope.

6. The polypeptide according to claim 5, wherein, The HLA-A2 restriction epitope comprises or consists of the amino acid sequence of SEQ ID NO:

66.

7. A polynucleotide encoding a polypeptide according to any one of claims 1-6.

8. A vector comprising the polynucleotide according to claim 7.

9. A composition comprising: a polypeptide according to any one of claims 1-6 and / or a polynucleotide according to claim 7.

10. The composition according to claim 9 further comprises an adjuvant.

11. The composition according to claim 9 or 10, further comprising: at least one different polypeptide according to any one of claims 1-6; at least one different polynucleotide according to claim 7; and / or at least one pharmaceutically acceptable diluent, carrier, or preservative.

12. The composition of claim 9, comprising an adjuvant selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, and imidazoquinones.

13. The composition according to claim 9, comprising Montanide ISA adjuvant.

14. Use of the polypeptide according to any one of claims 1-6, the polynucleotide according to claim 7, the carrier according to claim 8, and / or the composition according to any one of claims 9-13 in the preparation of a medicament for treating a disease or condition of a subject, wherein, The disease or condition referred to is cancer, selected from the group consisting of melanoma, leukemia, or prostate cancer.

15. The use according to claim 14, wherein, The leukemia mentioned is acute myeloid leukemia.

16. The use of the polypeptide according to any one of claims 1-6, the polynucleotide according to claim 7, the carrier according to claim 8, and / or the composition according to any one of claims 9-13, combined with an antibody, in the preparation of a medicament for treating a disease or condition of a subject, wherein, The disease or condition referred to is cancer, selected from the group consisting of melanoma, leukemia, or prostate cancer.

17. An in vitro method for stimulating TGFb1-specific T cells, the method comprising contacting the T cells with a polypeptide according to any one of claims 1-6 and / or a composition according to any one of claims 9-13, wherein, The composition comprises at least one polypeptide as defined in any one of claims 1-6.

18. The ex vivo method according to claim 17, wherein, The T cells are present in samples taken from healthy subjects or cancer patients.

19. The ex vivo method according to claim 18, wherein, The sample mentioned is a tumor sample from a cancer patient.

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