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

[0001] This application is a divisional application of patent application No. 202080040978.4 filed on June 4, 2020, entitled "TGF-β Vaccine". Technical Field

[0002] This invention relates to novel polypeptides derived from transforming growth factor β1 (TGFβ1; TGFb1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The invention also relates to the use and methods of using the polypeptides, polynucleotides, and compositions. Background Technology

[0003] TGFb is a multifunctional cytokine that plays a crucial role in the regulation of the immune system. There are four subtypes, with subtype 1 (TGFb1) being particularly important in T-cell immunity. In the context of cancer, TGFb1 desensitizes various immune cells, such as cytotoxic T cells (CTLs), tumor-associated neutrophils, and natural killer (NK) cells. It also contributes to tumor angiogenesis and metastasis. Therefore, TGFb1 is a key inhibitory molecule in the tumor microenvironment (TME), helping to downregulate the immune system's anti-tumor mechanisms and enabling cancer cells to evade the immune response.

[0004] In recent studies on mouse models of metastatic liver cancer, TGFb1 has also been shown to help reduce the efficacy of cancer treatment with immune checkpoint blockers (ICBs) such as PD-L1 blockers. Summary of the Invention

[0005] The peptides of this invention are expected to be particularly effective in stimulating beneficial immune responses against cells expressing TGFb1. The development of novel cancer immunotherapies requires a thorough understanding of the molecules involved in pathogenesis and the specific proteins recognized by the immune system. In a clinical setting, the induction of a TGFb1-specific immune response can directly kill TGFb1-expressing cancer cells, but more importantly, it typically supports anti-cancer immune responses by inhibiting the immunosuppressive function of TGFb1. For example, vaccination with the peptides of this invention will thus target TGFb1 and TGFb1-expressing cells, resulting in a high degree of synergy with other anti-cancer immunotherapies, such as immune checkpoint blockers (ICBs).

[0006] TGFb1 is a dimerized cytokine sharing a cysteine ​​knot structure linked together by intramolecular disulfide bonds. 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 TGFb1 pre-protein is provided as SEQ ID NO: 1.

[0007] The TGFb1 preprotein monomer has a molecular weight of approximately 25 kDa. The TGFb1 protein monomer has three distinct domains: a signal peptide (SP: amino acids 1-29; SEQ ID NO: 2), a potential related 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 below. Figure 1 As shown in E.

[0008] TGFb1 SP targets the protein secretion pathway; SP is cleaved in the rough endoplasmic reticulum (ER). TGFb1 monomers containing LAP and mature TGFb1 can dimerize in the ER via disulfide bonds between cysteine ​​residues in LAP (e.g., Cys 223 and Cys 225) and cysteine ​​residues in the mature TGFb1 peptide (e.g., Cys 356), forming TGFb1 homodimers. These TGFb1 homodimers are called small potential complexes (SLCs). SLCs can bind to so-called potential TGF-β binding proteins (LTBPs) to form larger complexes called large potential complexes (LLCs). LLCs can be secreted into the extracellular culture medium (ECM). However, the presence of LAP and LTBPs prevents TGFb1 from binding to and activating its extracellular receptor. Active TGFb1 consists of homodimers of the mature TGFb1 peptide. There are several mechanisms by which mature TGFb1 homodimers are released from LAP and LTBP, including protease degradation of LAP, induction of conformational changes in LAP through interaction with platelet-reactive proteins, and non-covalent bond cleavage between LAP and TGFβ-1.

[0009] The objective of this invention is to develop a T cell-mediated mechanism for removing TGFb1 from the TME. The inventors investigated the presence of spontaneous TGFb1-specific T cell responses in vivo by screening PBMCs from healthy donors and cancer patients. A TGFb1-specific T cell population was then isolated and expanded, and characterized by various assays related to HLA restriction, cytokine production, and cytotoxicity.

[0010] The inventors have identified regions of human TGFb1 with the highest immunogenicity. Surprisingly, these immunogenic "hotspot" regions are located throughout the entire human TGFb1 preprotein, including the SP and LAP domains and the mature TGFb1 peptide. The inventors have also identified a subregion within the human TGFb1 LAP, specifically positions 121-160 of SEQ ID NO: 1 (corresponding to the sequence in SEQ ID NO: 65), which contains a higher frequency of immunogenic peptide sequences.

[0011] Therefore, the present invention provides a polypeptide, which is an immunogenic fragment of human TGFb1 (SEQ ID NO: 1), and which comprises, or is composed of, the sequence of at least 9 consecutive amino acids of SEQ ID NO: 1. The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may correspond to the sequence of at least 9 consecutive amino acids of SEQ ID NO: 2 or 65. The polypeptide may comprise up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO: 1, or may be composed of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO: 1. The polypeptide may comprise any one of the amino acid sequences of SEQ ID NO: 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, or be composed of any one of the amino acid sequences of SEQ ID NO: 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 any one of the amino acid sequences of SEQ ID NO: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5, or be composed of any one of the amino acid sequences of SEQ ID NO: It consists of an amino acid sequence of any one of the following: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67, or 5. The polypeptide may comprise any one of the amino acid sequences of SEQ ID NO: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2, or be composed of any one of the amino acid sequences of SEQ ID NO: 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 any one of the amino acid sequences of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63, or be composed of any one of the amino acid sequences of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

[0012] The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids. The C-terminal amino acid of the polypeptide may be replaced by a corresponding amide. The polypeptide may contain an HLA-A2 restriction epitope. The HLA-A2 restriction epitope may comprise or consist of the amino acid sequence of SEQ ID NO: 66 or 67.

[0013] The present invention also provides a polynucleotide encoding the polypeptide of the present invention. The polynucleotide may be isolated. The present invention also provides a vector containing the polynucleotide.

[0014] The present invention also provides a composition comprising: the polypeptide of the present invention and / or the polynucleotide of the present invention, and optionally an adjuvant. The composition may further comprise: at least one different polypeptide of the present invention; at least one different polynucleotide of the present invention; and / or at least one pharmaceutically acceptable diluent, carrier, or preservative. The adjuvant may be selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazochiniline, and Montanide ISA adjuvants.

[0015] The present invention also provides a method for treating or preventing a disease or condition in a subject, the method comprising: administering to the subject a polypeptide of the present invention, a polynucleotide of the present invention, and / or a composition of the present invention. The method may further comprise the simultaneous or sequential administration of another cancer therapy, preferably an antibody.

[0016] The present invention also provides the use of the polypeptides, polynucleotides, compositions, or combinations thereof of the present invention in the treatment or prevention of diseases or conditions. The polypeptides, polynucleotides, compositions, or combinations thereof of the present invention can be used in combination with other cancer therapies, preferably antibodies.

[0017] The present invention also provides the use of the polypeptides of the present invention, the polynucleotides of the present invention, the compositions of the present invention, or combinations thereof in the preparation of medicaments for treating or preventing diseases or conditions.

[0018] The disease or condition may be characterized at least in part by inappropriate or excessive immunosuppressive function of cells expressing TGFb1, and / or the disease or condition is cancer. The disease or condition may be characterized at least in part by inappropriate or excessive expression of interleukin-4 (IL-4) and / or interleukin-13 (IL-13). The disease or condition may be cancer. The cancer may be breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer, pancreatic cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), melanoma, leukemia (such as acute myeloid leukemia (AML)), or prostate cancer.

[0019] The present invention also provides a method for stimulating TGFb1-specific T cells, the method comprising contacting the T cells with a polypeptide of the present invention and / or a composition of the present invention comprising at least one polypeptide of the present invention. The T cells may be present in a sample taken from a healthy subject or a cancer patient, optionally a tumor sample. Attached Figure Description

[0020] Figure 1 AC. via in vitro IFN ELISPOT assays (set in three replicate wells) assessed peptide-specific immune responses in PBMCs from six healthy donors against an array of 38 overlapping 20-meric peptides derived from TGFb1 proprotein. Each spot represents secreted IFN after subtracting the corresponding background signal. The mean number of cells is shown, and the gray horizontal bars represent the mean of the tested donors. An asterisk indicates the peptide that elicited the strongest and most statistically significant DFRx2-based response, and these were selected for further screening experiments (summarized in...). Figure 1 D (in the middle).

[0021] Figure 1 D. Summarized based on Figure 1 The most immunogenic TGFβ peptides screened by A–1C and their respective average IFN A table of ELISPOT counts. The top eight performing peptides were selected for further investigation.

[0022] Figure 1 E. Top: Primary sequence of TGFb1 preprotein. The amino acid sequences of the eight immunogenic TGFb1 peptides selected for further screening are highlighted. Underlined amino acid positions 1-29 represent the signal sequence of the protein (SP), while underlined amino acid positions 279-390 represent the mature TGFb1 monomeric protein. Bottom: Schematic diagram of the TGFb1 preprotein domains and the positions of the eight selected TGFb1-derived peptides. Numbers (1, 29, 279, and 390) indicate the key amino acid positions that label the three major domains of the TGFb1 preprotein.

[0023] Figure 2.A. In vitro IFN The ELISPOT assay, by evaluating the response of another healthy donor, targets... Figure 1 The identification of eight immunogenic TGFb1-derived peptides in AC validated the peptide-specific immune response in PBMCs. Each spot represents the secreted IFN in an individual donor after subtracting its respective background signal. The average number of cells is shown, and the black horizontal bars represent the mean of the tested donors. B. The heatmap depicts the magnitude of the response to the leader epitope in PBMCs from healthy subjects (top); representative ELISPOT responses (bottom).

[0024] Figure 3.A. In vitro IFN ELISPOT assay, by evaluating response, targets Figure 1 The eight immunogenic TGFb1 peptides identified in AC validated peptide-specific immune responses in PBMCs, but this study examined cancer patients. Each spot represents the amount of IFN secreted in an individual cancer patient after subtracting their respective background signal. The average number of cells is shown, and the black horizontal bars represent the mean of the patients being tested. B. The heatmap depicts the magnitude of the response to the leader epitope in PBMCs from cancer patients.

[0025] 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 + T cells partially gate the live cell population and control cytokine expression (IFN). The expression of TNFα and the cytotoxic marker (CD107a) was quantified. The percentages for each population are summarized in the hierarchical table on the right.

[0026] Figure 5 A. FACS plot showing CD4 targeting the TGFβ epitope, determined using ICS. +T cell response. B. FACS plot showing CD8 targeting the TGFb1 epitope as determined by ICS. + T cell response.

[0027] Figure 6 Large-scale cultures specific to several TGFb1-derived epitopes were generated by MACS CD137 enrichment of specific T cells. The enriched cells expanded post-enrichment and exhibited varying responsiveness to their epitopes. For each of the AD epitopes, specific CD44-derived epitopes were observed at the top of the FACS plot for the following epitopes: TGFb-02 (A), TGFb-05 (B), TGFb-26 (C), and TGFb-38 (D). + The number of gated cells, and the FACS plot at the bottom shows specific CD8. + The number of gated cells.

[0028] Figure 7 A. Left panel: Amplitude of responses in PBMCs from cancer patients and healthy subjects, measured via ex vivo ELISPOT. PBMCs were left to stand overnight, then directly seeded into ELISPOT wells and stimulated with epitopes in the wells for 48 hours. Right panel: Examples of ex vivo ELISPOT responses to several TGFb1 leader epitopes. B. CD8 identified against epitope TGFb-15 only 5 hours after ICS stimulation. + T cell response.

[0029] Figure 8 A. After 14 days of in vitro stimulation with peptides, followed by 18 hours of stimulation with the TGFb-15 epitope, PBMCs from prostate cancer patients showed CD8+ targeting the TGFb-15 epitope. + T cell response. B. TGFb15-specific T cells from donor UR1121.14 were enriched twice after stimulation with TGFb-15, cultured in vitro for 14 days, restimulated, and then enriched using the MACS CD137 enrichment method the following day. CD4 + T cells (FACS images at the top of A and B respectively) and CD8 + T cells (FACS diagrams at the bottom of A and B respectively) both responded to stimulation with TGFb-15.

[0030] Figure 9 FACS images show TGFb-15-specific CD8+ ions stimulated with TGFb-15. + Results of ICS analysis of T cell clones.

[0031] Figure 10 TGFb-15 specific CD8 +T cell clones kill target cells and TGFb1-expressing cancer cell lines in an HLA-restricted manner. A. TGFb-15-specific CD8 + T cells effectively lyse T2 cells using a pulse of TGFb-15 peptide. B. To ensure that the TGFb-15 response is HLA-A2 restricted, it has been demonstrated that HLA-A2 is mediated by a pulse of peptide. + Target cells rather than HLA-A3 + Target cells were lysed. C. Using HLA-A2 + Cancer cell lines UKE-1 and THP-1 stimulated clones to activate TGFb-15-specific CD8. + T cell clones. Other HLA-A2 + A. Cancer cells did not activate T cells. B. TGFb-15-specific T cells readily killed THP-1 and UKE-1 cancer cell lines. C. Stimulation of TGFb-15-specific T cells with cytokines enhanced TGFb-15-specific T cell activation. D. Stimulation of THP-1 cells with Th2 cytokines IL-4 or TGFb1 increased the number of THP-1 cells killed by TGFb-15-specific T cells.

[0032] Figure 11 IFN-γ is used to analyze responses to TGF nonamericium library spanning. (A) and TNF-α (B) results measured by ELISPOT.

[0033] Figure 12 CD8 specific to the HLA-A2 decameric epitope in the TGFb1 signal peptide sequence + T cells readily kill TGFb1-expressing cancer cell lines in an HLA-A2-restricted manner. A. After 14 days of in vitro culture, healthy donor PBMCs showed secretion of IFN-γ upon stimulation with the HLA-A2-binding decameric epitope TGFb-A2-01 peptide. B. Intracellular cytokine staining of healthy donor PBMCs showed CD8+ targeting TGFb-A2-01. + T cell response, due to stimulated CD8 + In addition to showing enhanced CD107a expression after stimulation with TGFb-A2-01 (right figure), the cells also showed enhanced IFN-γ expression. And TFN-α expression (left figure). C. TGFb-A2-01-specific CD8 from healthy donors. + T cells killed the HLA-A2 pulsed by TGFb-A2-01. + Target cells, while non-pulsed cells and peptide pulses of HLA-A3 +The target cells were not killed. D. CD8 + T cells killed HLA-A2 cells expressing TGFb1. + UKE-1 target cells were killed, while MARIMO and WM852 cells were not killed. E. HLA-A2 + THP-1 cells are easily killed by TGFb-A2-01 specific T cells, and TGFb1 expression in TGF-1 cells was regulated by stimulation with different cytokines 48 hours before the enhancement of the killed target cells was measured.

[0034] Figure 13 FACS plots show the results of ICS analysis of TGFb-A2-01-specific T cell clones stimulated with TGFb-A2-01.

[0035] Figure 14 The amino acid sequence of the icosicomeric peptide in the TGFβ library. Overlapping amino acid sequences are underlined.

[0036] Brief description of the sequence

[0037] SEQ ID NO: 1 is the amino acid sequence of the full-length precursor of human TGFb1 (also known as TGFb1 preprotein).

[0038] SEQ ID NO: 2 is the amino acid sequence of the signal peptide of human TGFb1.

[0039] SEQ ID NO: 3 is the amino acid sequence of the LAP peptide of human TGFb1.

[0040] SEQ ID NO: 4 is the amino acid sequence of mature human TGFb1.

[0041] SEQ ID NO: 5-64 are amino acid sequences of polypeptide fragments derived from human TGFb1.

[0042] SEQ ID NO: 65 is the amino acid sequence of the LAP subregion containing a high frequency of immunogenic sequences.

[0043] SEQ ID NO: 66 is the amino acid sequence of the smallest epitope in the TGFb-15 peptide sequence (SEQ ID NO: 28). SEQ ID NO: 66 is also referred to herein as “TGFb-15 short”.

[0044] SEQ ID NO: 67 is the amino acid sequence of TGFb-A2-01. Detailed Implementation

[0045] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in the field. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0046] Furthermore, the singular forms “a,” “an,” and “the” used in this specification and the appended claims include the plural forms unless otherwise expressly stated. Thus, for example, references to “polypeptide” include “multiple polypeptides,” etc.

[0047] The term “polypeptide” is used herein in its broadest sense to refer to a compound consisting of two or more subunit amino acids, amino acid analogs, or other peptide mimics. Therefore, 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 non-natural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptide mimics.

[0048] The terms “patient” and “subject” are used interchangeably and generally refer to humans.

[0049] All publications, patents, and patent applications cited in this article, whether mentioned above or below, are incorporated herein by reference in their entirety.

[0050] "Immunogenicity" herein refers to the ability of a peptide to elicit an immune response to the TGFb1 protein, preferably when said protein is present in or on cells expressing the TGFb1 protein. In other words, the peptide can be described as being immunogenic to TGFb1. Alternatively, the peptide can be described as an immunogenic fragment of TGFb1. The immune response is preferably a T cell response, therefore the peptide can be described as an immunogenic fragment of TGFb1 containing T cell epitopes. After the peptide is administered to the individual (or the sample), the immune response can be detected in at least one individual (or in a sample taken from the individual).

[0051] Peptides 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:

[0052] i. It can induce IFN-γ production in PBL populations of healthy subjects and / or cancer patients. Cells, as determined by ELISPOT analysis; and / or

[0053] ii. It can detect CTLs that respond to TGFb1 in situ in tumor tissue samples; and / or

[0054] iii. It can induce the in vitro growth of specific T cells.

[0055] Methods suitable for determining whether a peptide is immunogenic are also described in the following examples section.

[0056] The polypeptide of the present invention is an immunogenic fragment of human TGFb1 (SEQ ID NO: 1), comprising or consisting of a sequence of at least 9 consecutive amino acids of SEQ ID NO: 1.

[0057] The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may correspond to the sequence of at least 9 consecutive amino acids of the SP domain of TGFb1, such as the sequence of at least 95 consecutive amino acids of SEQ ID NO: 2.

[0058] The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may correspond to the sequence of at least 9 amino acids of the LAP domain of TGFb1, for example, the sequence of at least 9 consecutive amino acids of SEQ ID NO: 3.

[0059] The sequence of at least nine consecutive amino acids of SEQ ID NO: 1 may correspond to the sequence of at least nine consecutive amino acids located in the LAP subregion defined by amino acid positions 121 and 160 of SEQ ID NO: 1, for example, the sequence of at least nine consecutive amino acids of SEQ ID NO: 65.

[0060] The sequence of at least 9 consecutive amino acids of SEQ ID NO: 1 may correspond to the sequence of at least 9 consecutive amino acids of a mature TGFb1 polypeptide, such as the sequence of at least 9 consecutive amino acids of SEQ ID NO: 4.

[0061] The polypeptide may comprise up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO: 1, or may be composed of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 consecutive amino acids of SEQ ID NO: 1.

[0062] The polypeptide may contain or consist of any one of the amino acid sequences in SEQ ID NO: 2 and 5-67.

[0063] The polypeptide may comprise any one of the amino acid sequences of SEQ ID NO: 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, or be composed of any one of the amino acid sequences of SEQ ID NO: 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 an amino acid sequence of any one of SEQ ID NO: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5, or is composed of an amino acid sequence of any one of SEQ ID NO: 6, 42, 12, 23, 28, 49, 55, 63, 66, 67 or 5.

[0064] The polypeptide may comprise any one of the amino acid sequences of SEQ ID NO: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2, or be composed of any one of the amino acid sequences of SEQ ID NO: 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 the amino acid sequence of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63, or be composed of the amino acid sequence of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

[0065] 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 a corresponding amide. The polypeptide can be isolated.

[0066] Particularly preferred is that the polypeptide comprises, or is composed of, any one of the amino acid sequences of SEQ ID NO: 6, 42, 12, 23, 28, 49, 55, or 63. Particularly preferred is that the polypeptide comprises, or is composed of, any one of the amino acid sequences of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49, or 63. Longer polypeptide fragments of SEQ ID NO: 1 containing these sequences are also preferred.

[0067] The polypeptide may contain an HLA-A2 restrictive epitope. Preferably, the HLA-A2 restrictive epitope contains or is composed of the amino acid sequence of SEQ ID NO: 66. A preferred peptide containing an HLA-A2 restrictive epitope composed of the amino acid sequence of SEQ ID NO: 66 is a peptide containing or composed of the amino acid sequences of any one of SEQ ID NO: 28-31 or 65. Alternatively, the HLA-A2 restrictive epitope preferably contains or is composed of the amino acid sequence of SEQ ID NO: 67. A preferred peptide containing an HLA-A2 restrictive epitope composed of the amino acid sequence of SEQ ID NO: 67 is a peptide containing or composed of the amino acid sequences of any one of SEQ ID NO: 5, 8, 9 or 2.

[0068] In any polypeptide described herein, the amino acid sequence may be modified by one, two, three, four, or five (up to five) additions, deletions, or substitutions compared to the polypeptide having the unmodified sequence, provided that the polypeptide having the modified sequence exhibits the same or increased immunogenicity to TGFb1. "Same" should be understood as the polypeptide with the modified sequence not exhibiting significantly reduced immunogenicity to TGFb1 compared to the polypeptide having the unmodified sequence. Any comparison of immunogenicity between sequences will be performed using the same assay. Unless otherwise stated, modification of the polypeptide sequence is preferably a conserved amino acid substitution. A conserved 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 may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, a conserved substitution may introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conserved amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table A1 below. When amino acids have similar polarity, this can be determined by referring to the hydrophilicity level of the amino acid side chains in Table A2.

[0069] Table A1 – Chemical Properties of Amino Acids

[0070]

[0071] Table A2 – Hydrophilicity Level

[0072]

[0073] In any polypeptide disclosed herein, any one or more of the following modifications may be made to improve physicochemical properties (e.g., stability), provided that the polypeptide exhibits the same or increased immunogenicity to TGFb1 compared to a polypeptide having an unmodified sequence:

[0074] Replacing the C-terminal amino acid with the appropriate amide can increase resistance to carboxypeptidase.

[0075] Replacing the N-terminal amino acid with the corresponding acylated amino acid can increase resistance to aminopeptidase.

[0076] Replace one or more amino acids with the corresponding methylated amino acids (this can improve protein hydrolysis resistance); and / or

[0077] Replacing one or more amino acids with the corresponding D-configuration amino acids can improve protein hydrolysis resistance.

[0078] Any polypeptide disclosed herein may be linked to at least one additional moiety at the N-terminus and / or C-terminus to improve solubility, stability, and / or facilitate manufacture / isolation, provided that the polypeptide exhibits the same or increased immunogenicity to TGFb1 compared to a polypeptide lacking that additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR may be added to the N-terminus and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0079] 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 via solution-phase peptide synthesis. Alternatively, the polypeptides can be produced by transforming cells (typically bacterial cells) with a nucleic acid molecule or medium encoding the polypeptide. This invention provides nucleic acid molecules encoding the polypeptides of this invention and a medium. This invention also provides host cells comprising such nucleic acids or mediums.

[0080] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides, or similar molecules) of any length. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of this invention can be provided in a separated or substantially separated form. Substantially separated means that the polypeptide can be substantially separated from any surrounding medium, but not completely separated. Polynucleotides can be mixed with a vector or diluent that does not interfere with their intended use and are still considered substantially separated. The nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that, when placed under the control of a suitable regulatory sequence, such as in an expression medium, is transcribed (in the case of DNA) and translated (in the case of RNA) into a polypeptide in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. For the purposes of this invention, such nucleic acid sequences may include, but are not limited to: cDNA derived from viral, prokaryotic, or eukaryotic mRNA, genomic sequences of viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located at the 3' of the coding sequence.

[0081] Polynucleotides can be synthesized according to methods well-known in the art, as illustrated in the example by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual). Molecular Cloning - a laboratory manual; Cold Spring Harbor Publishing House (Cold Spring Harbor Press). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes, which include control sequences operatively linked to an inserted sequence, thereby allowing the peptides of the present invention to be expressed in vivo. In turn, these expression cassettes are typically provided in a medium (e.g., a plasmid or recombinant viral vector). Such expression cassettes can be administered directly to a host subject. Alternatively, a medium containing the polynucleotide of the present invention can be administered to a host subject. Preferably, the polynucleotide is prepared and / or administered using a genetic medium. A suitable medium can be any medium capable of carrying a sufficient amount of genetic information and allowing the expression of the peptides of the present invention.

[0082] The present invention therefore includes expression media comprising such polynucleotide sequences. Such expression media are conventionally constructed in the field of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements (e.g., polyadenylation signals), which may be necessary and are correctly oriented to allow expression of the peptides of the present invention. Other suitable media 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).

[0083] The present invention also includes cells modified to express the polypeptides of the present invention. Such cells typically include prokaryotic cells, such as bacterial cells, for example, *Escherichia coli* (E. coli). These cells can be cultured using conventional methods to produce the polypeptides of the present invention.

[0084] The polypeptides of the present invention can be in a substantially isolated form. They can be mixed with a carrier, preservative, or diluent that does not interfere with the intended use, and / or with an adjuvant, and are still considered substantially isolated. They can also be in a substantially purified form, in which case they typically constitute at least 90% of the protein in preparation, for example, at least 95%, 98%, or 99%.

[0085] Compositions containing peptides

[0086] This invention provides a composition comprising the polypeptides and / or polynucleotides of the present invention. For example, this invention provides a composition comprising: one or more polypeptides and / or one or more polynucleotides of the present invention, and optionally at least one adjuvant, a pharmaceutically acceptable carrier, a preservative, and / or an excipient.

[0087] The composition may contain at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight different polypeptides of the present invention, and optionally at least one adjuvant, a pharmaceutically acceptable carrier, a preservative, and / or an excipient.

[0088] The composition may contain at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight different polynucleotides of the present invention, and optionally at least one adjuvant, a pharmaceutically acceptable carrier, a preservative, and / or an excipient.

[0089] The carrier, preservative, and excipient must be "acceptable" in the sense of compatibility with the other components of the composition and harmlessness to the recipient of the composition. Typically, all components and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may preferably contain an adjuvant. An adjuvant is any substance added to the composition that increases or otherwise alters the immune response induced by the composition. Adjuvants are broadly defined as substances that promote an immune response. Adjuvants may also preferably have a storage effect, as they also result in a slow and sustained release of the active agent from the site of application. (Goding's Monoclonal Antibodies: Principles & Practice) A general discussion of adjuvants is provided on pages 61-63 of the 2nd edition (1986).

[0090] Adjuvants can be selected from the following groups: AlK SO4)2, AlNa SO42, AlNH4 SO4), silicon dioxide, alum, Al OH)3, Ca3 PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-murayl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normurayl-L-alanyl-D-isoglutamine (CGP 11687, also known as nor-MDP), N-acetylmurayl-L-alanyl-D-isoglutamine-L-alanine-2- 1'2'-Dipalmitoyl-sn-propanetrioxy-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL+TDM+CWS) in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharides and their various derivatives (including lipid A), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g., polyIC and polyAU acid), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5, 554, 372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrix or GMDP, interleukin-1, interleukin-2, Montanide ISA-51 and QS-21. Various saponin extracts have also been suggested as adjuvants in immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0091] The adjuvants preferably used in this invention include oil / surfactant-based adjuvants, such as Montanide adjuvant (available from Seppic in 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 imidazoquinones are also examples of preferred adjuvants.

[0092] The most preferred adjuvant is the Montanide ISA adjuvant. The preferred Montanide ISA adjuvant is Montanide ISA 51 or Montanide ISA 720.

[0093] In Goding's Monoclonal Antibodies: Principles & Practice In the 2nd edition (1986), on pages 61-63, it should also be noted that when the antigen of interest has a low molecular weight or poor immunogenicity, it is recommended to conjugate it with an immunogenic carrier. Therefore, the polypeptides of the present invention can be conjugated to a carrier. The carrier can exist 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, impart stability, increase biological activity, or increase serum half-life. Furthermore, the carrier can assist in the presentation of its polypeptide or fragments to T cells. Therefore, in the composition, the polypeptide can be bound to those carriers listed below. The carrier can be any suitable carrier known to those skilled in the art, such as proteins or antigen-presenting cells, such as dendritic cells (DCs). Carrier proteins include keyhole 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.

[0094] If a composition contains excipients, it must be "pharmaceutically acceptable" in the sense that it is compatible with the other components of the composition and harmless to the recipient. Excipients, such as wetting agents or emulsifiers, pH buffers, etc., may be present in the excipients. These excipients and excipients are generally agents that do not elicit an immune response in the individual receiving the composition and can be administered without excessive 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 may also be included, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. A more detailed discussion of pharmaceutically acceptable excipients, carriers, and excipients is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0095] Suitable compositions can be formulated using standard pharmaceutical chemistry and methods, all of which are readily available to a skilled person. Such compositions can be prepared, packaged, or marketed in forms suitable for bolus or continuous administration. Injectable compositions can be prepared, packaged, or marketed in unit doses, such as in ampoules or in multi-dose containers optionally containing preservatives. Compositions include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations in oily or aqueous carriers. In one embodiment of the composition, the active ingredient is provided in dry (e.g., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile, pyrogen-free water) prior to application of the reconstituted composition. The composition can be prepared, packaged, or marketed as a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and may contain additional components, such as adjuvants, excipients, and excipients as described herein, in addition to the active ingredient. For example, nontoxic, parenteral-acceptable diluents or solvents (e.g., water or 1,3-butanediol) can be used to prepare this sterile injectable formulation. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixative oils such as synthetic monoglycerides or diglycerides. Other useful compositions include those containing the active ingredient in microcrystalline form, in liposome formulations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, microsoluble polymers, or microsoluble salts. Alternatively, the active ingredient of the composition may be encapsulated, adsorbed onto, or bound to a microparticle carrier. Suitable microparticle carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolic acid). See, for example, Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers may also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0096] How to use

[0097] The polypeptides, polynucleotides, or compositions or combinations thereof of the present invention can be used in methods for treating or preventing a disease or condition in a subject. The polypeptides, polynucleotides, or compositions or combinations thereof of the present invention can be used to prepare a medicament for a method of treating or preventing a disease or condition in a subject. The method includes administering the polypeptide, the polynucleotide, the composition, or the combination to the subject. A therapeutically or preventively effective amount of the polypeptide, the polynucleotide, the composition, or the combination can be administered to a subject in need.

[0098] The disease or condition may be characterized at least partially by inappropriate or excessive immunosuppressive function of TGFb1. The disease or condition may be characterized at least partially by inappropriate or excessive expression of IL-4 and / or IL-13. The disease or condition may be cancer, preferably cancer expressing TGFb1 and / or associated with inappropriate or excessive immunosuppressive function of TGFb1 and / or inappropriate or excessive expression of IL-4 and / or IL-13. The cancer may be breast cancer, cervical cancer, gastric cancer, liver cancer, ovarian cancer or pancreatic cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), melanoma, leukemia (such as acute myeloid leukemia (AML)), or prostate cancer. The cancer may be AML characterized by inappropriate or excessive immunosuppressive function of TGFb1 and / or inappropriate or excessive expression of IL-4 and / or IL-13. This cancer can be AML characterized by inappropriate or excessive immunosuppressive function of TGFb1 and inappropriate or excessive expression of IL-4 and / or IL-13.

[0099] This method may include simultaneous or sequential administration with another cancer therapy. The other cancer therapy may be a bispecific inhibitor of TGFb (e.g., TGFb1) and PD-L1. The bispecific inhibitor may be able to simultaneously bind to TGFb and PD-L1 and / or inhibit the activity of both TGFb and PD-L1. The bispecific inhibitor may be a fusion protein comprising an anti-TGFb moiety and an anti-PD-L1 moiety, optionally wherein the anti-PD-L1 moiety comprises or is composed of an anti-PD-L1 antibody, and / or the anti-TGFb moiety comprises or is composed of a TGFb receptor or a portion thereof, such as TGFb receptor II or a portion thereof.

[0100] This additional cancer treatment may be selected from cytokine therapy, T-cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiotherapy, immunostimulants, gene therapy, or antibodies.

[0101] The antibody could be abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, attumomabpentetate, amatuximab, or anatumomab. mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= tocilizumab), Atorlimumab, Bapineuzumab, Basiliximab, Bavixi Monoclonal antibodies (Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab) Mertansine, Blinatumomab, Blosozoumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catuxomab, CC49, Cedelizumab, Certolizumabpegol), Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenzumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dalatumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, drozitumab, duligotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, eddobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epipitumomabcituxetan, eprazuzumab, erlizumab, ertumaxomab, edaracizumab, edaracizumab, edaracizumab, edaracizumab, edaracizumab, exbivirumab, fasolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, fintumumab Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumabozogamicin, Gevokizumab, Girentuximab, and Glembatumumab are all listed. vedotin, golimumab, gomiliximab, GS6624, ibazumab, tiuxetan, icrucumab, igovomab, imciromab, imatrozumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, and innotuzumab.ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lebrikizumab, lemalesomab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lodelcizumab, mozzarella- Lorvotuzumab mertansine, Lucarumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, muromonab-CD3, nacolomabtafenatox, 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, Panobakucumab, Parsatuzumab, Pascol izumab), Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Penditide-Satumomab Satumomabpendetide, Sekunumab, 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, Telimomabaritox, 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 The drugs available include mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox.

[0102] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, offatumumab, ocrelizumab, epratuzumab, alemtuzumab, and abatacept. tacept, Eculizumab, Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab, Ustekinumab, Briakinumab, Etanercept, Infliximab, Adalimumab, Certolizumab pegol, golimumab, trastuzumab, gemtuzumab, oxamicin, ibritumomab, tiuxetan, tostitumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab, and vedotin.

[0103] Particularly preferred antibodies that can be used in the methods of the present invention include: daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab, and ofatumumab.

[0104] Other cancer therapies available include coenzyme B12 (Actimide), azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, and idarubicin. The group consists of Idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, Revlimid, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0105] The polypeptides of the present invention and / or compositions of the present invention comprising at least one polypeptide of the present invention can also be used to stimulate TGFb1-specific T cells (such as CD4+). + and / or CD8 + A method for generating T cells includes contacting the cells with the polypeptide and / or the composition. This method can be performed ex vivo. The cells can be present in samples taken from healthy subjects or cancer patients, such as in tumor samples.

[0106] The invention is further illustrated by the following embodiments; however, these embodiments should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and the following embodiments can be used individually and in any combination thereof as material for implementing the invention in various forms.

[0107] Example

[0108] Example 1 – Materials and Methods

[0109] Patients and donors

[0110] Erythrocyte sedimentation rate (ESR) amber layers from anonymous blood donors were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. ESR amber layers from cancer patients were obtained from the Department of Oncology at Herlev Hospital in Herlev, Denmark. In accordance with Helsinki's statement, all participants provided informed consent before entering the study. PBMCs were isolated using a lymphocyte separation agent (Lymphoprep) (Axis Shield, Oslo, Norway) and frozen in fetal bovine serum containing 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).

[0111] peptides

[0112] The peptides were provided by Pepscan (Lelystadt, Netherlands) and dissolved in DMSO at a concentration of 10 mM. TGF was identified. Following the lead epitope, these peptides were supplied with 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 the start and end positions of each peptide sequence within the amino acid sequence of the SEQ ID NO, name, or reference human TGFb1 full-length precursor. Each name is used interchangeably, as shown in the table listed in the Sequences section below. For example, the peptide of SEQ ID NO: 6 can be alternatively referred to as the name TGFb-02 (or TGFB02), or alternatively as TGFb1. 11-30 (Assume the starting position is 11 and the ending position is 30). The expected reference for each case is clearly visible from the context.

[0113] In vitro enzyme-linked immunospot assay (ELISPOT) analysis

[0114] For in vitro ELISPOT, PBMCs from cancer patients and healthy donors were in 24-well plates with 20 µM TGF. Cells were pulsed with a derivative peptide (or without peptide as a control) and 120 U / ml IL-2 for 7–10 days, followed by ELISPOT analysis. Cells were placed in containers pre-coated with IFN. Capture antibody (Mabtech) in a 96-well nitrocellulose ELISPOT plate (MultiScreen IP filter plate, MSIPN4W50; Millipore). TGF was added. The peptide was added to a final concentration of 5 µM. Control stimulants (DMSO, HIV, or out-of-order peptides) were added to the control wells, and the plate was incubated at 37°C for 16–20 hours. After incubation, cells were washed away, and secondary biotinylated antibody (Mabtech, catalog number 3420-6-1000) was added at room temperature for 2 hours. Unbound secondary antibody was washed away, and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech, catalog number 3310-10) was added at room temperature for 1 hour. Unbound conjugate enzyme was washed away, and the plate was analyzed by adding BCIP / NBT substrate (Mabtech, catalog number 3650-10). The stained ELISPOT plates were analyzed using Immunospot software v5.1 on a CTL ImmunoSpot S6 Ultimate-V analyzer. Response reports were submitted using TGF-β. The difference in the mean number of midpoints between peptide-stimulated pores and pores without peptide addition. Unless otherwise stated, all experiments were performed using in vitro IFN-γ. ELISPOT assays were performed, and all experiments were repeated three times. Statistical analyses were performed using distribution-free resampling (DFR) and the more conservative DFR 2× method, as described by Moodie et al. (Cancer Immunol Immunother 2010; 59: 1489–1501).

[0115] In vitro ELISPOT analysis

[0116] PBMCs from cancer patients or healthy donors were thawed in 24-well plates of X-VIVO medium and allowed to stand overnight. (Optional: Add 1 µg / ml DNase I). The next day, cells were counted and transferred to plates pre-coated with IFN. 96-well nitrocellulose ELISPOT plates (MultiScreen IP filter plates, MSIPN4W50; Millipore) with capture antibodies (Mabtech). TGF added. The peptide was added to a final concentration of 5 µM. A control stimulus (DMSO, HIV, or out-of-order peptide) was added to the control wells, and the plate was incubated at 37°C for 24–72 hours. Plate staining with secondary antibody and chromogenic protocol followed the in vitro ELISPOT protocol described above.

[0117] Intracellular cytokine staining (ICS) and fluorescence-activated cell sorting (FACS)

[0118] In BD GolgiPlug TM (Added one hour after peptide stimulation) in the presence of TGF PBMCs were stimulated with a peptide-derived agent (or used as a control without peptide incubation) for 5 hours, followed by intracellular staining of the cell culture. CD107a-PE (catalog number 555801, BD Biosciences) antibody was added at the start of incubation. Stimulated cells were stained with fluorescently labeled antibodies against surface markers (CD3, CD4, CD8) according to the manufacturer's instructions, and then permeabilized using a mixture of fixation / permeabilization concentrate and diluent (eBioscience, catalog numbers 00-5123-43 and 00-5223-56). The permeabilized cells were then stained with IFN-labeled fluorescent dyes. Staining with TNFα antibody. Flow cytometry analysis was performed on FACSCanto™ II (BD Biosciences). Antibody used: IFN-γ. -APC (catalog number 341117), TNFα-455BV421 (catalog number 562783), CD4-FITC (catalog number 347413) or CD4-PerCP (catalog number 345770), CD8-PerCP (catalog number 345774) or CD8-FITC (catalog number 345772), CD3-APC-H7 (catalog number 560275) (all from BD Biosciences). Dead cells were stained with FixableViability Stain 510 (BD Biosciences, San Jose, CA, USA). Another method for identifying activated T cells is to stimulate T cells overnight with antigens or target cells. After 18-24 hours of stimulation, cells were stained with the above-mentioned surface antigen-specific antibodies and fixable viability stains, and simultaneously stained with anti-CD107a-PE and anti-CD137-BV421 (BD Biosciences, San Jose, CA, USA). Using appropriate isotype controls, HLA-A2 in donor PBMCs was analyzed by staining with anti-HLA-A2-FITC (BD Biosciences, San Jose, CA, USA).

[0119] Rapid expansion protocol

[0120] In some trials, T cells were expanded using a rapid expansion protocol (REP) with allogeneic 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 high doses of IL-2 (6,000 IU / mL IL2; from Novartis' Proleukin).

[0121] FACS for live cells

[0122] To enrich specific T cells from primary PBMC cultures, the in vitro culture method used for cell cultures analyzed in ELISPOT was followed (see above). Cells were then stimulated with the antigen overnight and washed twice the next day in FACS buffer, followed by staining for 30 minutes with the following substances: LIVE / DEADFixable Near-IR Dead Cell Stain Kit (Waltham, MA, USA), anti-CD4-FITC, anti-CD8-PerCP, anti-CD107a-PE, and anti-CD137-BV421 (BD Biosciences, San Jose, CA, USA). Cells were then washed twice and resuspended in FACS buffer. Cells were then sorted on a FACSARIA flow cytometer with appropriate application settings and compensation controls. Cell sorting was performed using purity settings. After sorting, cells were divided into two halves—one half of the enriched cells were expanded using a rapid expansion protocol, while the other half were cloned using limiting dilutions, seeded at three cells / well. The cloned cells were expanded using a rapid expansion protocol.

[0123] Magnetic Activated Cell Sorting (MACS)

[0124] MACS is used to enrich antigen-specific T cells from primary cultures and pre-enriched cultures. Enrichment of specific T cells from primary PBMC cultures followed the in vitro culture method used for cell cultures analyzed in ELISPOT (see above). Cells were then stimulated with the antigen overnight according to the manufacturer's protocol and enriched the following day using the MACSCD137 Enrichment Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The enriched cells were amplified using a rapid amplification protocol. As described, some enriched cells were cloned through limiting dilution. The cloned cells were amplified using a rapid amplification protocol.

[0125] Chromium-51 cytotoxicity assay and cytokine stimulation of target cells

[0126] As described by Andersen MH et al. (J Immunol 1999; 163: 3812–3818), the chromium-51 cytotoxicity assay was used to assess the killing potential of specific T cells. To manipulate TGFβ expression in several cancer cell lines, the cancer cell lines were stimulated for 48 hours prior to analysis with IL-4 (100 U / mL), IL-13 (20 U / mL), and TGFβ1 (2.e5 ng / mL) (all from Peprotech, Rocky Hill, NJ, USA), alone or in combination.

[0127] Example 2 – In vitro ELISPOT screening of 20-meric peptides

[0128] 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 ).

[0129] Using in vitro IFN The ELISPOT assay (set in three replicate wells) assessed the peptide-specific immune response in PBMCs from six healthy donors against the spontaneous immune response to the 20-meric peptide array. 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.

[0130] 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.

[0131] Example 3 – Validation of peptide-specific immune response

[0132] Establish additional in vitro IFN ELISPOT assays were performed to validate responses in additional healthy subjects to eight selected epitope peptides identified in the initial screening (see Example 2). The results of these assays are shown in Figure 2. Strong and frequent responses were observed for all tested epitope peptides, with TGFb-02, TGFb-26, TGFb-33, and especially TGFb-15 exhibiting strong and frequent responses. Figure 2B ).

[0133] 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 examined in cancer patients, and again using in vitro IFN assays. The response was assessed and validated using ELISPOT assays. The results of these assays are shown in Figure 3. TGFb-02, TGFb-15, TGFb-26, and TGFb-33 were observed to be highly immunogenic in the patients. Figure 3B ).

[0134] Example 4 – Cytokine Analysis

[0135] 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 + T cells partially gate the live cell population. This includes the expression of cytokines (IFN-γ). The expression of TNFα and the cytotoxic marker CD107a was quantified. FACS plots of cytokine analysis were shown... Figure 4On the left, the percentages of each group are summarized in... Figure 4 The hierarchical structure table on the right.

[0136] 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.

[0137] 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 5 B). 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 ), proved TGF The high immunogenicity potential of several epitopes in the middle.

[0138] Example 5 – Identification of in vitro responses to TGFβ epitopes

[0139] 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 significant amounts of IFN-γ. ( Figure 7 A) demonstrates that cells from healthy subjects and cancer patients contain a large amount of freely circulating TGF. Specific T cells. Most surprisingly, CD8 was detected in PBMCs from ex vivo plates of prostate cancer patients only 5 hours after stimulation with epitope TGFb-15. + T cell response ( Figure 7 B). This finding indicates that the patient has a high proportion of circulating TGF. Specific cytotoxic T cells. Given this strong response to TGFb-15, a TGFb-15-specific T cell culture was established using PBMCs from this patient. First, CD137 was identified as an activation marker for sorting specific T cells from this donor. Then, the patient's PBMCs were stimulated with TGFb-15 and held in culture for 14 days, followed by restimulation with TGFb-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, specific cytotoxic T cells were effectively induced. + T cells are CD137 + CD137 is a suitable marker for enriching specific T cells ( Figure 8 A).

[0140] 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 evaluate TGF. Specific T cell response.

[0141] Example 6 – TGFb-15-specific T cells can recognize and kill cancer cells

[0142] 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 to TGFb-15. Figure 9 ). HLA-A2 from the patient's PBMCs. + Specific antibody staining showed that the donor was HLA-A2. + (Data not shown). Next, a standard chromium-51 cytotoxicity assay was performed to examine whether specific T cells could cleave the HLA-A2 of the peptide pulse. + Target cells. HLA-A2 of peptide pulses. + T2 cells are easily lysed by specific T cells, while non-pulsed T2 cells are not killed. Figure 10 A).

[0143] T2 cells are not only HLA-A2 +Therefore, the killing of these cells may be mediated by a matching of another HLA allele. For this reason, K562 cells were used as targets for further experiments. The original K562 line was HLA-deficient, but these experiments were performed using two genetically modified lines to stably express either HLA-A2 or HLA-A3. This ensured that these were the only HLA alleles expressed in the respective cells. Only HLA-A2 with peptide pulses was expressed. + K562 cells were killed by a TGFb-15-specific clone, while unpulsed HLA-A2 cells were killed. + HLA-A3 in K562 cells and peptide pulses + K562 cells were not identified. Figure 10 B).

[0144] Almost all cells can secrete TGFβ, which is largely involved in creating a tumor-suppressive environment. For this reason, we investigated whether TGFβ-15-specific T cells can recognize HLA-A2. + Cancer cell lines. All derived from UKE-1, SET-2, and THP-1 cell lines from patients with acute myeloid leukemia (AML), along with two HLA-A2 cell lines. + Melanoma cell lines (WM852 and FM88) as well as K562 and HLA-A2 + K562 cells were used together as target cells. TGFb-15 specific T cells were stimulated overnight with their respective target cells at an effector:target ratio of 3:1. Specific T cells recognized two cancer cell lines, THP-1 and UKE-1, while other cell lines did not activate T cells. Figure 10 C). Furthermore, chromium-51 cytotoxicity assays showed that TGFb-15-specific T cells killed UKE-1 and THP-1 cells (C). Figure 10 D).

[0145] The THP-1 cell line is a relatively undifferentiated cell line, and treatment with different cytokines can affect gene expression in these cells. Interleukin (IL)-4 is a cornerstone cytokine in the development of the Th2 response. Therefore, it is hypothesized that treatment of THP-1 cells with IL-4 may increase TGFβ expression in these cells. Furthermore, because TGFβ generates a positive feedback loop for its intracellular production, it is hypothesized that treatment of THP-1 cells with TGFβ may also induce TGFβ expression. Notably, the target epitope TGFb-15 is expressed in the LAP peptide portion of the TGFβ precursor protein, rather than in the mature active form of TGFβ (see...). Figure 1 E). Therefore, pretreatment of THP-1 cells with active TGFβ does not add the recognized epitopes to THP-1 cells, but only increases the production of intracellular TGFβ.

[0146] 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. This demonstrated that, compared to unstimulated THP-1 cells, cytokine-treated THP-1 cells induced greater activation of TGFb-15-specific T cells. Figure 10 E). Ultimately, it was shown that cytokine stimulation of THP-1 cells with IL-4 or TGFβ increased the number of lysed cells (E). Figure 10 F).

[0147] Example 7 – Identification of the Minimal TGFβ Epitope Sequence

[0148] Because the TGFb-15 epitope is a 20-mer, it cannot be presented on the HLA-I molecule in its full-length form. Therefore, further experiments were conducted to determine the minimal epitope sequence recognized by TGFb-15-specific T cells. Specifically, the TGFb-15 epitope sequence was fragmented into a nonameric peptide library with 8 overlapping amino acids, resulting in 12 nonameric peptides. The TGFb-15 epitope sequence was derived from CD8+ cells used to generate TGFb-15-specific T cells. + T cell plating of a large number of TGFb-15-specific cultures of T cell clones was performed in ELISPOT and stimulated with each of the nine-peptides. Results showed that the smallest epitope within the TGFb-15 sequence was the sequence VLLSRAELRL (TGFb-15 short; SEQ ID NO: 66) (see...). Figure 11 ).

[0149] Example 8 – The decameric epitope in the TGFβ signal peptide is a target for specific T cells.

[0150] Given the high frequency of CD8 targeting several 20-mer epitopes in the TGFβ sequence. + T cell response: The inventors sought to identify other HLA-A2-restricted decameric 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 October 30, 2014), they searched the entire TGFβ sequence for decameric epitopes with high binding affinity to HLA-A2. The peptide sequence LLLLPLLWL (TGFb-A2-01; SEQ ID NO: 67) appeared as the leading binding decameric epitope with a binding affinity score of 30. HLA-A2 from healthy subjects was then investigated. + Spontaneous T cell responses of PBMCs to the TGFb-A2-01 epitope. Surprisingly, most PBMCs showed a response to the TGFb-A2-01 epitope ( Figure 12A). Using ICS, it was confirmed that these responses originated from CD8. + T cells ( Figure 12 B).

[0151] Then, TGFb-A2-01-specific T cells were isolated from healthy subjects (BC363) who had a solid response to TGFb-A2-01 by performing a single in vitro stimulation on PBMCs from the subjects and then culturing them for 14 days. Next, the PBMCs were stimulated overnight with TGFb-A2-01. Then, CD3+... + CD8 + CD137 + Cells were 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 high specificity for the TGFb-A2-01 peptide (…). Figure 13 ).

[0152] HLA-A2 was tested for TGFb-A2-01-specific T cell cleavage peptide pulses in a standard Cr51 cytotoxicity assay. + K562's ability to target cells. HLA-A2 peptide pulses. + K562 cells were lysed, while non-pulsed HLA-A2 cells were lysed. + HLA-A3 and peptide pulses + The target cells were not lysed. Figure 12 C).

[0153] Because the aforementioned TGFb-15-specific T cells killed the AML cell lines UKE-1 and THP-1, the test was conducted to determine whether the TGFb-A2-01-specific clone could also kill these target cancer cells. UKE-1 and THP-1 cancer cells are easily killed by TGFb-A2-01-specific T cells (see...). Figure 12 (D and E). Furthermore, stimulation of THP-1 cells with IL-13, TGFβ, or a combination of IL-13 and TGFβ increased the proportion of target cells killed (D and E). Figure 12 E).

[0154] in conclusion

[0155] TGFb1 is a key executor of immune homeostasis and tolerance, suppressing the expansion and function of many components of the immune system. Perturbations in TGFb1 signaling are the cause of inflammatory diseases and contribute to tumorigenesis. TGFb1 is also central to immunosuppression in the tumor microenvironment, and recent studies have revealed its role in tumor immune evasion and adverse responses to cancer immunotherapy. TGFb1 expression is a major characteristic of both tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells. Cells expressing TGFb1 also play an important role in the development of the immunosuppressive microenvironment because they prevent the proliferation of effector lymphocytes at the tumor site. For example, activation of TGFb1-specific T cells through vaccination should therefore lead to T cell infiltration at the tumor site.

[0156] For the first time, cells expressing TGFb1 have been discovered that can be specifically targeted by TGFb1-specific effector T cells. Specifically, the inventors identified peripheral TGFb1-specific T cells naturally present in cancer patients and healthy donors by screening a peptide library covering the entire amino acid sequence of TGFb1. Interestingly, TGFb1 has been found to contain multiple epitopes, which are frequently recognized by peripheral T cells distributed in different regions of the TGFb1 sequence.

[0157] The observation of frequent T-cell responses to TGFb1 underscores the surprising finding that TGFb1 possesses high immunogenicity. Particularly unexpected is the level of immunogenicity observed by the inventors, given TGFb1's importance in immunosuppression. Furthermore, regions of TGFb1 capable of generating particularly strong immune responses have been identified, and these regions would be ideal for peptide-based vaccination methods.

[0158] Given the role of TGFb1 in suppressing the immune system (e.g., in the TME), the inventors’ surprising findings suggest that it is possible to enhance TGFb1-specific immune responses in most patients with solid tumors and hematologic malignancies.

[0159] Many different therapeutic strategies focus on targeting the immunosuppressive tumor microenvironment (TME), aiming to deplete or reprogram immunosuppressive cells or target functional mediators secreted by these cells. The surprising results discussed above suggest that immunomodulatory vaccination targeting TGFb1 could be an effective approach to targeting immunosuppressive cells within the TME. This unique approach combines 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 pro-inflammatory cytokines into the immunosuppressive microenvironment) compared to other clinical strategies. Since TGFb1 expression is a major factor in the immunosuppressive cell phenotype, TGFb1-specific T cells can specifically respond to immunosuppressive cells. A TGFb1 vaccine that rebalances the microenvironment should enhance the effectiveness of T-cell-enhancing drugs, such as checkpoint blockers like anti-PD1 antibodies. Therefore, combination therapy with TGFb1 vaccines and checkpoint blocking antibodies should increase the number of patients responding to treatment.

[0160] In summary, the results of the above discussion provide a valuable approach for directly targeting TGFb1, a key factor in the lack of immune response in most cancer patients.

[0161] sequence

[0162] Full-length human TGFb1 precursor protein (NP_000651.3) (SEQ ID NO: 1): 10 20 30 40 50

[0164] MPPSGLRLLL LLLPLLWLLV LTPGRPAAGL STCKTIDMEL VKRKRIEAIR 60 70 80 90 100

[0166] GQILSKLRLA SPPSQGEVPP GPLPEAVLAL YNSTRDRVAG ESAEPEPPEPE 110 120 130 140 150

[0168] ADYYAKEVTR VLMVETHNEI YDKFKQSTHS IYMFFNTSEL REAVPEPVLL 160 170 180 190 200

[0170] SRAERLLLRL KLKVEQHVEL YQKYSNNSWR YLSNRLLAPS DSPEWLSFDV 210 220 230 240 250

[0172] TGVVRQWLSR GGEIEGFRLS AHCSCDSRDN TLQVDINGFT TGRRGDLATI 260 270 280 290 300

[0174] HGMNRPFLLL MATPLERAQH LQSSRHRRAL DTNYCFSSTE KNCCVRQLYI 310 320 330 340 350

[0176] DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA 360 370 380 390

[0178] SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS

[0179] In Table 1 below, unless otherwise stated, “start position” and “end position” indicate the position within the full-length human TGFb1 preprotein (SEQ ID NO: 1).

[0180] Table 1

[0181]

[0182]

[0183]

Claims

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

1.

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

1.

3. The polypeptide according to claim 1 or 2, comprising an amino acid sequence of any one of SEQ ID NO: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2, or composed of an amino acid sequence of any one of SEQ ID NO: 66, 28-31, 67, 5-9, 42-45, 12-15, 55-58, 23-26, 49-52, 63, 64, 65 or 2.

4. The polypeptide according to claim 3, comprising an amino acid sequence of any one of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63, or composed of an amino acid sequence of any one of SEQ ID NO: 66, 28, 67, 5, 6, 42, 12, 55, 23, 49 or 63.

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

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

7. A polynucleotide encoding a polypeptide according to any one of claims 1-6, optionally contained in a medium.

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

9. The composition of claim 8 further comprises: 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.

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

11. A method for treating or preventing a disease or ailment of a subject, the method comprising: The subject is administered the polypeptide according to any one of claims 1-6, the polynucleotide according to claim 7, and / or the composition according to any one of claims 8-10.

12. The method according to claim 11, wherein, The disease or symptom described is: (i) Cancer, optionally selected from the group consisting of 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), or prostate cancer; and / or (ii) Characterized at least in part by inappropriate or excessive immunosuppressive function of cells expressing TGFb1 and / or inappropriate or excessive expression of IL-4 and / or IL-13.

13. The method according to claim 11 or 12, wherein, The disease or condition is cancer, and the method further includes the simultaneous or sequential administration of other cancer therapies, preferably antibodies.

14. A 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 8-10, wherein, The composition comprises at least one polypeptide as defined in any one of claims 1-6.

15. The method according to claim 14, wherein, The T cells are present in samples taken from healthy subjects or cancer patients, optionally tumor samples.

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