TGF-β1 vaccine

Abstract

The present invention relates to novel polypeptides derived from transforming growth factor β1 (TGFβ1; TGFb-1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The invention further relates to methods for enhancing the selectivity of an immune response to TGFb-1. The invention also relates to uses and methods of using the polypeptides, polynucleotides, and compositions.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel polypeptides derived from transforming growth factor β1 (TGFβ1; TGFb-1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The invention further relates to methods for enhancing the selectivity of an immune response to TGFb-1. The invention also relates to uses and methods of using the polypeptides, polynucleotides, and compositions. [Background technology]

[0002] Background of the Invention TGFb is a multifunctional cytokine that plays an important role in regulating the immune system. It has three isoforms, of which isoform 1 (TGFb-1) is particularly important in T cell immunity. In cancer, TGFb-1 neutralizes 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. In conclusion, TGFb1 is a key inhibitory molecule in the tumor microenvironment (TME), contributing to the downregulation of the immune system's antitumor mechanisms and enabling immune evasion by cancer cells.

[0003] Recent clinical results (Kjeldsen, JW et al., Nat. Med. 27(12): 2212-2223 (2021)) provide a rationale for cancer immunotherapy based on the activation of "anti-regulatory" T cells, which recognize antigens commonly expressed by immunosuppressive cells and thereby target inflammatory signals to the tumor microenvironment (Andersen, MH, Semin. Immunopathol (2022)). Therapeutic agents based on small molecule inhibitors targeting the TGFb receptor and soluble TGFb receptor-based trap ligands target all three isoforms of TGFb (TGFb-1, TGFb-2, and TGFb-3), and targeting other TGFb isoforms (TGFb-2, TGFb-3) has been suggested to have adverse clinical effects (Tauriello, DVF, E. Sancho, and E. Batlle, Nat. Rev. Cancer, 22(1): 25-44 (2022)).

[0004] Activating TGFb-1-selective T cells (which lack cross-reactivity to TGFb-2 and TGFb-3) may allow for targeting of inflammatory immune responses to TGFb-1-expressing tumors while avoiding the toxicity associated with pan-TGFb blockade. TGFb-1-selective T cells are frequently detected in humans (Holmstrom, MO, et al., Cell Mol. Immunol. 18(2): 415-426 (2021)). Therefore, there remains a need to enhance the selectivity of anti-TGFb-1 immune responses to mitigate potential off-target toxicity. Summary of the Invention

[0005] The present inventors have previously identified immunogenic polypeptide fragments of TGFb-1. These polypeptide fragments are disclosed in WO2020 / 245264, which is incorporated herein by reference. The present inventors have now identified new immunogenic polypeptide fragments derived from TGFb-1.

[0006] Surprisingly, the polypeptides disclosed herein stimulate an immune response selective for TGFb-1. In other words, the polypeptides disclosed herein stimulate TGFb-1-selective T cells that exhibit low cross-reactivity to TGFb-2 and TGFb-3. Thus, the polypeptides of the present invention are believed to be particularly effective in stimulating beneficial, selective immune responses against TGFb-1-expressing cells. In particular, the polypeptides of the present invention are believed to enhance the selectivity of immune responses against TGFb-1-expressing cells without enhancing immune responses against TGFb-2-expressing cells or TGFb-3-expressing cells. Thus, the polypeptides of the present invention are believed to exhibit low off-target toxicity. Longer polypeptides of the present invention, such as those set forth in SEQ ID NOS: 7 and 10, may contain more epitopes than shorter polypeptide fragments of TGFb-1 and are therefore believed to be particularly immunogenic. Such peptides are also believed to have improved properties in terms of ease of production and formulation.

[0007] TGFb-1 is a dimeric cytokine that shares a cysteine ​​knot structure linked by an intramolecular disulfide bond. TGFb-1 is synthesized as a 390 amino acid monomeric precursor protein, which is interchangeably referred to as TGFb-1 preprotein; TGFb-1 precursor; full-length TGFb-1; and preproTGFb-1. The full-length sequence of the TGFb-1 preprotein is shown as SEQ ID NO: 1.

[0008] The molecular weight of the TGFb-1 preprotein monomer is approximately 25 kDa. The TGFb-1 protein monomer has three distinct domains: a signal peptide (SP: amino acids 1-29; SEQ ID NO: 4), a latency associated peptide (LAP: amino acids 30-278; SEQ ID NO: 5), and a mature peptide (mature TGFb-1: amino acids 279-390; SEQ ID NO: 6).

[0009] The SP of TGFb-1 targets the protein to the secretory pathway, where it is cleaved in the rough endoplasmic reticulum. TGFb-1 monomers, including LAP and mature TGFb-1, dimerize in the endoplasmic reticulum via disulfide bonds between cysteine ​​residues in LAP (e.g., Cys223 and Cys225) and the mature TGFb-1 peptide (e.g., Cys356) to form TGFb-1 homodimers. This TGFb-1 homodimer is called the small latent complex (SLC). SLC can bind to the so-called latent TGF-β-binding protein (LTBP) to form a larger complex called the large latent complex (LLC). LLC can be secreted into the extracellular medium (ECM). However, the presence of LAP and LTBP prevents TGFb-1 from binding to and activating extracellular receptors. Active TGFb-1 consists of a homodimer of the mature TGFb-1 peptide. There are various mechanisms by which mature TGFb-1 homodimers are released from LAP and LTBP, including protease-mediated degradation of LAP, induction of a conformational change in LAP by interaction with thrombospondin, and cleavage of the noncovalent bond between LAP and TGFb-1.

[0010] An object of the present invention is to develop an immunogenic polypeptide that induces a selective immune response against TGFb-1. Another object of the present invention is to selectively target immunosuppressive cells in the TME. Because TGFb-1 is highly expressed by immunosuppressive cells in the TME, selective targeting of TGFb-1 on such cells makes it possible to deplete immunosuppressive cells in the TME.

[0011] Thus, the present invention provides a polypeptide that is an immunogenic fragment of TGFb-1, comprising or consisting of a sequence of at least 8 consecutive amino acids of SEQ ID NO:5. Preferably, the polypeptide does not contain any cysteine ​​residues. The polypeptide may have low homology to the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. The polypeptide may have low sequence identity to the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. TGFb-2 may have the amino acid sequence of SEQ ID NO:2. TGFb-3 may have the amino acid sequence of SEQ ID NO:3. The polypeptide fragment may have less than about 80%, 70%, 60%, 50%, 40%, 30%, 25%, or 20% sequence identity to the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. Preferably, the polypeptide has less than about 40% sequence identity to the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. The polypeptide may comprise or consist of up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 consecutive amino acids of SEQ ID NO:5. The polypeptide may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 contiguous amino acids of SEQ ID NO: 5, or may consist of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 contiguous amino acids of SEQ ID NO: 5. Preferably, the polypeptide may comprise at least 25, 26, 27, 28, 29, or 30 contiguous amino acids of SEQ ID NO: 5, or may consist of at least 25, 26, 27, 28, 29, or 30 contiguous amino acids of SEQ ID NO: 5.More preferably, the polypeptide may comprise or consist of at least 30 contiguous amino acids of SEQ ID NO:5. The polypeptide may comprise the amino acid sequence of SEQ ID NO:32. The polypeptide may comprise the amino acid sequence of any one of SEQ ID NOs:7, 10, or 11. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs:7, 10, 32, 11, 14, or 15. Preferably, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:7. Preferably, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:10. In one embodiment, the polypeptide does not comprise or consist of the amino acid sequence of SEQ ID NOs:11, 14, and / or 15. In one embodiment, the polypeptide does not consist of the amino acid sequence of SEQ ID NOs:11, 14, and / or 15.

[0012] The polypeptide can stimulate TGFb-1 selective T cells. TGFb-1 selective T cells may have low cross-reactivity to cells expressing and / or presenting TGFb-2 and / or TGFb-3 polypeptides. Cells expressing and / or presenting TGFb-2 and / or TGFb-3 polypeptides may be cells in the tumor microenvironment (TME). These cells may be tumor cells. Preferably, these cells are immunosuppressive cells. These cells include cancer-associated fibroblasts (CAFs), CD8 + T cells, CD4 + T cells, regulatory CD4 + T cells, exhausted CD8 + It may include T cells, M1 tumor-associated macrophages (M1_TAM), M2 tumor-associated macrophages (M2_TAM), myeloid antigen-presenting cells (APCmye) and / or other cells.

[0013] Measuring cross-reactivity can involve comparing the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting TGFb-1 polypeptides with the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting corresponding polypeptides derived from TGFb-2 or TGFb-3. The reactivity of TGFb-1-selective T cells to cells expressing and / or presenting corresponding polypeptides derived from TGFb-2 or TGFb-3 can be less than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the reactivity of TGFb-1-selective T cells to TGFb-1 polypeptides. Cross-reactivity can be measured by IFNγ ELISPOT assay.

[0014] The present invention further provides a polynucleotide encoding a polypeptide of the present invention. The polynucleotide may be isolated. The polynucleotide may be contained in a vector. The polynucleotide may be messenger RNA (mRNA). The mRNA may be (a) an open reading frame (ORF) encoding at least one polypeptide of the invention; (b) 5′-end cap at the 5′ end; (c) 5′ untranslated region (UTR) contained 5′ of the ORF; (d) the 3'UTR contained 3' of the ORF; and (e) 3' tailing sequence at the 3' end may include:

[0015] The present invention also provides compositions comprising a polypeptide and / or a polynucleotide of the present invention and, optionally, an adjuvant. The compositions 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 adjuvants, oil / surfactant adjuvants, viral dsRNA adjuvants, imidazoquinolines, and montanide ISA adjuvants. Preferably, the compositions may be TGFb-1-selective vaccine compositions. When the compositions comprise a polynucleotide, and the polynucleotide is mRNA, the compositions may be formulated in a lipid nanoparticle composition. The lipid nanoparticle composition may have an average diameter of 50 to 200 nm.

[0016] 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 an additional cancer therapy, preferably an antibody. When the disease or condition is cancer, the method may further comprise stimulating a selective immune response against TGFb-1-expressing cancer cells.

[0017] The present invention also provides a polypeptide of the present invention, a polynucleotide of the present invention, a composition of the present invention, or a combination thereof for use in the treatment or prevention of a disease or condition. The polypeptide, polynucleotide, composition, or combination thereof can be used in combination with an additional cancer therapy, preferably an antibody. When the disease or condition is cancer, the polypeptide, polynucleotide, composition, or combination thereof can stimulate a selective immune response against TGFb-1-expressing cancer cells.

[0018] The present invention further provides use of the polypeptide of the present invention, the polynucleotide of the present invention, the composition of the present invention, or a combination thereof for the manufacture of a medicament for treating or preventing a disease or condition. When the disease or condition is cancer, the use of the polypeptide, polynucleotide, composition, or a combination thereof can be for the manufacture of a medicament for stimulating a selective immune response against TGFb-1-expressing cancer cells.

[0019] The disease or condition may be characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb1-expressing cells. Preferably, the disease or condition is cancer. The cancer may be esophageal cancer or urothelial cancer. The cancer may be colorectal cancer, gastric cancer, head and neck cancer, melanoma, non-small cell lung cancer (NSCLC), or ovarian cancer. The cancer may be breast cancer, cervical cancer, liver cancer, or pancreatic cancer.

[0020] The disease or condition may be a tumor, and the polypeptide, polynucleotide, composition, or combination thereof may modulate the tumor microenvironment (TME). The methods and uses of the present invention may include modulating the TME. This modulation may include promoting the infiltration of T cells into the TME. Preferably, the T cells are CD4 + Administration of the polypeptides, polynucleotides, compositions, or combinations thereof can stimulate a selective immune response against TGFb-1-expressing cells in the TME.

[0021] The present invention also provides a method for stimulating TGFb-1-selective T cells, the method comprising contacting T cells with a polypeptide of the present invention, a polynucleotide of the present invention, a composition of the present invention, or a combination thereof.

[0022] The present invention also provides an ex vivo method for stimulating TGFb-1-selective T cells, the method comprising contacting T cells with a polypeptide of the invention, a polynucleotide of the invention, a composition of the invention, or a combination thereof.

[0023] The present invention further provides the use of the polypeptide of the present invention, the polynucleotide of the present invention, the composition of the present invention, or a combination thereof for stimulating TGFb-1-selective T cells. This use may include contacting T cells with the polypeptide of the present invention, the polynucleotide of the present invention, the composition of the present invention, or a combination thereof. This use may be non-therapeutic and / or ex vivo.

[0024] TGFb-1-selective T cells stimulated with the peptides, polynucleotides, and / or compositions of the present invention may have low cross-reactivity with cells expressing and / or presenting TGFb-2 and / or TGFb-3 polypeptides. Measuring cross-reactivity may involve comparing the reactivity of TGFb-1-selective T cells with cells expressing and / or presenting TGFb-1 polypeptides with the reactivity of TGFb-1-selective T cells with cells expressing and / or presenting corresponding polypeptides derived from TGFb-2 or TGFb-3. The reactivity of TGFb-1-selective T cells with cells expressing and / or presenting corresponding polypeptides derived from TGFb-2 or TGFb-3 may be less than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the reactivity of TGFb-1-selective T cells with TGFb-1 polypeptides. Cross-reactivity may be measured by IFNγ ELISPOT assay. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1A-B: Representative images showing TGFB-1 staining for tumor cells in esophageal cancer (A) and non-small cell lung cancer (NSCLC) (B). [Figure 2]TGFb-1 is expressed by a substantial proportion of cells in the tumor and tumor microenvironment (TME). Box plots and data points show the frequency of TGFb-1 expression in tumor cells and the tumor microenvironment (TME, non-tumor cells) for the indicated cancer types. For each cancer type, the box plot on the left corresponds to TGFb-1 expression in the TME, and the box plot on the right corresponds to TGFb-1 expression in tumor cells. The percentage of TGFb-1-positive cells in each individual region of interest (ROI) is shown as a single data point. [Figure 3] TGFb-1 is expressed by various cell types in the TME. The vertical bars show the percentage of TGFb-1-positive cells for each cell type in the TME (excluding tumor cells) for the cancer types indicated. Some categories are non-exclusive, so the sum may exceed 1.0. The order of cell types listed in the graph legend is the same as the order of the cell types in each bar. [Figure 4] TGFb-1-expressing cells are not limited to TME populations expressing other immunosuppressive markers. Columns indicate the percentage of cells expressing the indicated immunosuppressive antigens. "Combination" indicates cells expressing any combination of two or more antigens. The melanoma samples analyzed here were 70% uveal and 30% cutaneous; no metastatic melanomas were analyzed. The order of immunosuppressive markers listed in the graph legend corresponds to the order of the immunosuppressive markers in each bar. [Figure 5] TGFb sequence alignment. Clustal Omega sequence alignment of human TGFb-1, TGFb-2, and TGFb-3 proteins. The Pep01-Pep12 designations in Figure 5 indicate the positions of peptides disclosed herein mapped to the TGFb-1, TGFb-2, and TGFb-3 sequences. For example, the sequence of Pep01-1 can be found in the TGFb-1 sequence under "Pep01" in Figure 5, the sequence of Pep01-2 can be found in the TGFb-2 sequence under "Pep01" in the figure, etc. [Figure 6-1]IFNγ ELISPOT identifies strong and frequent immune responses. Immune responses to TGFb-1 peptides were identified in PBMCs from 14 healthy donors. PBMCs were stimulated with the indicated TGFb-1 peptides, and responses were analyzed by IFNγ ELISPOT 7 days later. Significant responses (*) were defined by a Fisher's exact P value of <0.01, a peptide / control spot ratio >2, and background-subtracted spots >25. Peptides that elicited responses in the greatest number of donors were then tested for the specificity of the response to TGFb-1. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7A] IFNγ ELISPOT to identify TGFb-1-selective immune responses. Donor PBMCs were stimulated with the indicated TGFb-1 peptides (Pep01-1, Pep04-1, Pep05-1, Pep08-1, Pep09-1, and Pep12-1). Seven days later, recall responses were examined by IFNγ ELISPOT assays using the same TGFb-1 peptides, or homologous TGFb-2 peptides (i.e., Pep01-2, Pep04-2, Pep05-2, Pep08-2, Pep09-2, or Pep12-2), or TGFb-3 peptides (i.e., Pep01-3, Pep04-3, Pep05-3, Pep08-3, Pep09-3, or Pep12-3). The highly homologous peptides Pep12-1, Pep12-2, and Pep12-3 were included as positive controls to detect cross-reactive immune responses to TGFb-2 and TGFb-3. In Figure 7A, for each peptide tested, the left boxplot corresponds to the response with the same TGFb-1 peptide, the middle boxplot corresponds to the response with the homologous TGFb-2 peptide, and the right boxplot corresponds to the response with the homologous TGFb-3 peptide. Figure 7B shows a representative ELISPOT assay set up in triplicate wells. [Figure 7B]IFNγ ELISPOT to identify TGFb-1-selective immune responses. Donor PBMCs were stimulated with the indicated TGFb-1 peptides (Pep01-1, Pep04-1, Pep05-1, Pep08-1, Pep09-1, and Pep12-1). Seven days later, recall responses were examined by IFNγ ELISPOT assays using the same TGFb-1 peptides, or homologous TGFb-2 peptides (i.e., Pep01-2, Pep04-2, Pep05-2, Pep08-2, Pep09-2, or Pep12-2), or TGFb-3 peptides (i.e., Pep01-3, Pep04-3, Pep05-3, Pep08-3, Pep09-3, or Pep12-3). The highly homologous peptides Pep12-1, Pep12-2, and Pep12-3 were included as positive controls to detect cross-reactive immune responses to TGFb-2 and TGFb-3. In Figure 7A, for each peptide tested, the left boxplot corresponds to the response with the same TGFb-1 peptide, the middle boxplot corresponds to the response with the homologous TGFb-2 peptide, and the right boxplot corresponds to the response with the homologous TGFb-3 peptide. Figure 7B shows a representative ELISPOT assay set up in triplicate wells. [Figure 8-1] IFNγ ELISPOT to identify TGFb-1-selective immune responses. The same data shown in Figure 7 were plotted to show responses for individual peptides and donors. Significant cross-reactive responses to the homologous TGFb-2 and TGFb-3 peptides were observed only once (Pep01-2); no cross-reactivity was observed in the other seven donors tested with this peptide combination. The highly homologous peptide Pep12 showed cross-reactivity in all donors that showed significant responses to the TGFb-1 homolog (Pep12-1). Significant responses (*) were defined by a Fisher's exact P value of <0.01, a peptide / control spot ratio >2, and >25 background-subtracted spots. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9]TGFb-1 vaccine induces robust immune responses. Mice were inoculated with two different synthetic long peptides (SLPs), and the immune response was analyzed by IFNγ ELISPOT assay. ELISPOT responses of individual mice are shown. Results for the control peptide are clearly indicated. [Figure 10] Figure 10A-D: TGFb-1 vaccine induces tolerogenic changes in the TME. Tumor weights were compared between groups at the end of the experiment (day 21). TGFB-1 expression was assayed by latency-associated peptide (LAP) staining. CD4+ T cell infiltration was significantly increased in SLP2-vaccinated animals, whereas CD8+ T cell infiltration was unchanged. [Figure 11-1] TGFb-1 vaccines promote targeted in vivo cell killing. An in vivo cytotoxicity assay was used to compare cell killing in mice vaccinated with SLP1 and the class I epitope SLP1_Ib (SIYMFFNT). Vaccinated mice (in duplicate) were injected with differentially labeled splenocytes loaded with the assay peptide (SLP1_Ib) or a control peptide. Cell killing was determined by comparing splenocyte recovery with splenocytes loaded with the control peptide as an internal control for cell recovery. OVA peptide was included as a positive control. [Figure 11-2] This is a continuation of Figure 11-1. DETAILED DESCRIPTION OF THE INVENTION

[0026] A brief description of arrays SEQ ID NO: 1 is the amino acid sequence of the full-length precursor of human TGFb-1 (also called TGFb-1 preprotein).

[0027] SEQ ID NO: 2 is the amino acid sequence of the full-length precursor of human TGFb-2 (also called TGFb-2 preprotein).

[0028] SEQ ID NO: 3 is the amino acid sequence of the full-length precursor of human TGFb-3 (also called TGFb-3 preprotein).

[0029] SEQ ID NO: 4 is the signal peptide of human TGFb-1.

[0030] SEQ ID NO: 5 is the amino acid sequence of the human TGFb-1 LAP (latency associated peptide) domain.

[0031] SEQ ID NO: 6 is the amino acid sequence of mature human TGFb-1.

[0032] SEQ ID NOs: 7 to 17 are the amino acid sequences of polypeptide fragments derived from human TGFb-1.

[0033] SEQ ID NOs: 18 to 22 are the amino acid sequences of polypeptide fragments derived from human TGFb-2.

[0034] SEQ ID NOs: 23 to 27 are the amino acid sequences of polypeptide fragments derived from human TGFb-2.

[0035] SEQ ID NO:28 is the amino acid sequence of a polypeptide fragment derived from human TGFb-1 that has sequence homology to human TGFb-2 and TGFb-3.

[0036] SEQ ID NO:29 is the amino acid sequence of a polypeptide fragment derived from human TGFb-2 that has sequence homology to human TGFb-1 and TGFb-3.

[0037] SEQ ID NO:30 is the amino acid sequence of a polypeptide fragment derived from human TGFb-3 that has sequence homology to human TGFb-1 and TGFb-2.

[0038] SEQ ID NOs: 31 and 34 are synthetic long peptides (SLPs) containing putative MHC class I and class II epitopes.

[0039] SEQ ID NOs: 32, 33, 35, and 36 are the minimal peptides corresponding to the MHC class I and class II epitopes of SLPs.

[0040] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods may be tailored to particular needs in the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0041] definition Unless otherwise defined, the technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.For the purpose of interpreting this specification, the following term explanations shall apply, and whenever applicable, terms used in the singular form ("a", "an", and "the") shall also include the plural form, and vice versa, unless the context clearly indicates otherwise.Thus, for example, when referring to a "polypeptide", it shall include "multiple polypeptides", etc.If the explanation of the terms provided contradicts with the documents incorporated herein by reference, the explanation of the terms provided below shall prevail.

[0042] Where the terms "comprising" and "comprises" are used, it is also provided that "consisting essentially of" or "consisting of" what is set forth.

[0043] As used herein, the term "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics.Thus, the term "polypeptide" includes short peptide sequences and longer polypeptides and proteins.As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

[0044] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0045] The terms "patient" and "subject" are used interchangeably and generally refer to a human.

[0046] As used herein, the term "immunogenic" means that a polypeptide is capable of eliciting an immune response against TGFb protein, particularly TGFb-1 protein, usually when the protein is present in or on cells that express TGFb-1 protein. In other words, the polypeptide can be said to be immunogenic against TGFb. The polypeptide can alternatively be referred to as an immunogenic fragment of TGFb. The immune response can also refer to a T cell response, and thus the polypeptide can be referred to as an immunogenic fragment of TGFb containing a T cell epitope. The immune response can be detected in at least one individual (or a sample taken from the individual) after administration of the polypeptide to the individual (or a sample taken from the individual).

[0047] A polypeptide can be identified as immunogenic using any suitable method, including in vitro methods. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics: i. capable of eliciting IFN-γ-producing cells in PBL populations of healthy subjects and / or cancer patients, as determined by an ELISPOT assay; and / or ii. CTLs reactive with TGFb-1 can be detected in situ in a sample of tumor tissue; and / or iii. It can induce the proliferation of specific T cells in vitro.

[0048] Suitable methods for determining whether a polypeptide is immunogenic are also described in the Examples section below.

[0049] The polypeptides disclosed herein are capable of stimulating a "selective" immune response to TGFb-1, e.g., a selective T cell response to TGFb-1. In this context, a selective immune response to TGFb-1 is considered to be greater than an immune response to TGFb-2 or TGFb-3. For example, a polypeptide is considered to be capable of stimulating a selective T cell response to TGFb-1 so long as the polypeptide does not stimulate a T cell response to TGFb-2 and / or TGFb-3. Similarly, a polypeptide is considered to be capable of stimulating a selective T cell response to TGFb-1 so long as the polypeptide stimulates a T cell response to TGFb-1 that is at least about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or 10,000-fold greater than the T cell response to TGFb-2 and / or TGFb-3 that the polypeptide is capable of stimulating.

[0050] Suitable assays for measuring a selective immune response to TGFb-1 will be apparent to those skilled in the art. An exemplary assay that can be used for this purpose is the IFNγ ELISPOT assay. For example, a polypeptide can be specifically identified as being capable of stimulating a selective immune response to TGFb-1 if: i. capable of eliciting IFN-γ-producing cells in the peripheral blood leukocyte (PBL) population of healthy subjects and / or cancer patients, as determined by an ELISPOT assay; and / or ii. When IFNγ-producing cells are contacted with the corresponding polypeptide of TGFb-2 or TGFb-3, there is less IFNγ produced as determined by an ELISPOT assay.

[0051] References herein to "TGFb," "TGF-b," "T-GF-β," etc., are equivalent to references to TGF-β. However, to avoid use of the Greek symbols and to aid in reproducibility of the present content, the previous nomenclature is used.

[0052] Polypeptides In any of the polypeptides described herein, the amino acid sequence may be modified by one, two, three, four, or five (i.e., up to five) additions, deletions, or substitutions, so long as the polypeptide with the modified sequence exhibits the same or increased immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence. "Identical" means that the polypeptide with the modified sequence does not exhibit significantly reduced immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence. Comparison of immunogenicity between sequences is performed using the same assay. Unless otherwise specified, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have the same polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid it replaces. Alternatively, conservative substitutions may introduce another amino acid that is aromatic or aliphatic in place of an 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 major amino acids as defined in Table A1 below. If the amino acids have similar polarities, this can be determined by reference to the hydropathy scale of amino acid side chains in Table A2.

[0053] TIFF2025538981000001.tif67148

[0054] TIFF2025538981000002.tif120139

[0055] The polypeptides disclosed herein may be modified as long as the polypeptide exhibits the same immunogenicity or increased immunogenicity against TGFb1 compared to a polypeptide having an unmodified sequence. To improve physicochemical properties (e.g., stability), any one or more of the following modifications can be made: Substitution of the C-terminal amino acid with the corresponding amide (which may increase resistance to carboxypeptidases); Substitution of the N-terminal amino acid with the corresponding acylated amino acid (which may increase resistance to aminopeptidases); Substitution of one or more amino acids with corresponding methylated amino acids (which may improve resistance to proteolysis); and / or One or more amino acids with the corresponding D-configuration amino acid (which may improve resistance to proteolysis).

[0056] The polypeptides disclosed herein can be conjugated to at least one additional moiety at the N-terminus and / or C-terminus to improve solubility, stability, and / or aid in production / isolation, as long as the polypeptide exhibits the same or increased immunogenicity against TGFb1 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR can be added to the N-terminus and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0057] Polypeptides as disclosed herein can be produced by any suitable means. For example, the polypeptides can be directly synthesized using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, polypeptides can be produced by transforming cells with a nucleic acid molecule or vector encoding the polypeptide. Such cells generally include prokaryotic cells, such as bacterial cells, e.g., E. coli. Such cells can be cultured using conventional methods to produce the polypeptides of the invention. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the invention. The present invention also provides host cells containing such nucleic acids or vectors.

[0058] The polypeptide of the present invention can be in a substantially isolated form. The polypeptide can be mixed with a carrier, preservative, or diluent that does not interfere with the intended use and / or adjuvant, and still be considered substantially isolated. The polypeptide can also be in a substantially purified form, in which case the polypeptide generally accounts for at least 90%, for example, at least 95%, 98%, or 99% of the protein in the preparation.

[0059] For the purposes of the present invention, to determine the percent identity of two sequences (such as two polypeptide sequences), the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the first sequence for optimal alignment with the second sequence). Then, the nucleotides at each position are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the nucleotides at that position are identical. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical positions / total number of positions in the reference sequence × 100).

[0060] Typically, sequence comparison is performed over the length of the reference sequence. For example, if a user wants to determine whether a given ("test") sequence is less than 80% identical to SEQ ID NO: 18, SEQ ID NO: 18 is the reference sequence. To assess whether a sequence is less than 80% identical to SEQ ID NO: 18 (an example of a reference sequence), one skilled in the art aligns the length of SEQ ID NO: 18 and identifies how many positions in the test sequence are identical to the positions in SEQ ID NO: 18. If less than 80% of the positions are identical, the test sequence is less than 80% identical to SEQ ID NO: 18. If the sequence is shorter than SEQ ID NO: 18, gaps or deletion positions should be considered non-identical positions.

[0061] Those skilled in the art are familiar with various computer programs that can be used to determine the homology or identity between two sequences.For example, the comparison of sequences and the determination of the percent identity between two sequences can be achieved using mathematical algorithms.In one embodiment, the percent identity between two amino acid sequences or nucleic acid sequences is determined by using the Needleman and Wunsch (1970) algorithm, which is incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either Blosum62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0062] Polynucleotides Non-limiting examples of polynucleotides of the present invention 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. Polynucleotides of the present invention can be provided in isolated or substantially isolated form. By substantially isolated, it is meant that the polypeptide may be substantially, but not completely, isolated from the surrounding medium. A polynucleotide may be mixed with a carrier or diluent that does not interfere with its intended use and still be considered substantially isolated. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that can be transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example, in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA derived from viral, prokaryotic, or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0063] Polynucleotides can be synthesized according to methods well known in the art, for example, as described in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes containing regulatory sequences operably linked to an insert sequence, thus allowing expression of the polypeptides of the present invention in vivo. These expression cassettes are generally provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing a polynucleotide of the present invention can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing expression of a polypeptide of the present invention.

[0064] Thus, the present invention includes expression vectors containing such polynucleotide sequences. Such expression vectors are constructed routinely in the field of molecular biology and may, for example, include the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals that may be required, arranged in the proper orientation to enable expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0065] In one embodiment, the polynucleotide is an mRNA. (a) an open reading frame (ORF) encoding at least one immunogenic polypeptide of the present invention; (b) 5′-end cap at the 5′ end; (c) 5′ untranslated region (UTR) contained 5′ of the ORF; (d) the 3'UTR contained 3' of the ORF; and (e) 3' tailing sequence at the 3' end may include:

[0066] The mRNA sequence encoding the immunogenic polypeptide may be interrupted by cleavage-sensitive sites.

[0067] The ORF may comprise multiple copies of each sequence encoding different immunogenic polypeptides, optionally at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 15 copies of each said sequence, preferably the ORF encodes at least 2, 3, 4, 5, 10 or more different immunogenic polypeptides.

[0068] mRNA may be referred to as mRNA vaccine for cancer or mRNA cancer vaccine. mRNA vaccine is described in International Patent Application No. WO2015 / 164674, the entirety of which is incorporated herein by reference. The mRNA cancer vaccine of the present invention can be a composition, including pharmaceutical compositions. The present invention also encompasses the method for preparing, manufacturing, formulating and / or using mRNA cancer vaccine.

[0069] The fact that immunogenic polypeptides are expressed from RNA as intracellular peptides may be advantageous over delivery as exogenous peptides. RNA is delivered intracellularly and expresses epitopes in close proximity to the appropriate cellular machinery for processing, allowing them to be recognized by the appropriate immune cells. Furthermore, targeting sequences can deliver peptide epitopes more specifically. For example, C-terminal ubiquitin ligase targeting proteins (FBox proteins) can be used to target polypeptide processing to the proteasome, more closely mimicking MHC processing. The constructs of the present invention can also include linkers, such as APC-optimized proteolytic cleavage sites. These proteolytic sites are advantageous for enhancing peptide processing in APCs. Once mRNA cancer vaccines are delivered to cells, the intracellular machinery processes the mRNA into polypeptides, which can then be processed into immunogenic polypeptides capable of stimulating the desired immune response.

[0070] In some embodiments, the mRNA cancer vaccine encodes multiple immunogenic polypeptides. This can be referred to as a polyepitope mRNA vaccine because each encoded immunogenic polypeptide contains at least one epitope. The RNA sequence encoding the immunogenic polypeptides may be interrupted by a sequence encoding an amino acid sequence recognized by a protease. Thus, in some embodiments, the mRNA cancer vaccine is an mRNA having an open reading frame encoding a propeptide, since the encoded polypeptide sequence contains multiple immunogenic polypeptides linked directly or via a linker such as a cleavage-sensitive site. An exemplary propeptide has the following peptide sequence: Tm-Yo-(X1-Yo-X2-Yo-…..Xn)-Yo-Tm where T is a targeting sequence and m = 0 to 1. The targeting sequence may be included at the N-terminus, C-terminus, or both termini of the central peptide region. When a polypeptide has two or more targeting sequences, the sequences may be the same or different.

[0071] X1, X2, etc. are each independently an immunogenic polypeptide sequence, where n = 0 to 1000. Each immunogenic polypeptide sequence designated by X may represent a unique immunogenic polypeptide sequence in the propeptide or may refer to copies of an immunogenic polypeptide sequence. Thus, the propeptide encoded by the mRNA may be composed of multiple immunogenic polypeptide sequences, each unique, and / or may contain two or more copies of each unique immunogenic polypeptide sequence. In some embodiments, the propeptide may have at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or more copies of each unique immunogenic polypeptide sequence. Preferably, the propeptide has at least 2, 3, 4, 5, 10, or more different immunogenic polypeptide sequences.

[0072] Y is a linker sequence, preferably a cleavage-sensitive sequence, and o = 0 to 5. Each immunogenic polypeptide sequence may optionally have one or more linkers, optionally a cleavage-sensitive site adjacent to the N-terminus and / or C-terminus. In a multi-epitope design, two or more immunogenic polypeptide sequences may have a cleavage-sensitive site between them. Alternatively, two or more immunogenic polypeptide sequences may be directly linked to each other or linked via a linker that is not a cleavage-sensitive site. A targeting sequence may also be linked to an immunogenic polypeptide sequence via a cleavage-sensitive site, or directly linked to an immunogenic polypeptide sequence via a linker that is not a cleavage-sensitive site.

[0073] The mRNA may encode one or more targeting sequences. This may be an endosomal targeting sequence, such as a portion of the transmembrane domain of lysosome-associated membrane protein-1 (LAMP-1) or a portion of the transmembrane domain of the invariant chain (Ii). The targeting sequence may be a ubiquitination signal attached to either or both ends of the encoded polypeptide. In other embodiments, the targeting sequence is a ubiquitination signal attached to an internal site and / or either end of the encoded polypeptide. Thus, the RNA may contain a nucleic acid sequence encoding a ubiquitination signal at either or both ends of the nucleotides encoding the immunogenic polypeptide.

[0074] Ubiquitination, a post-translational modification, is the process of attaching ubiquitin to target proteins. Ubiquitination signals are peptide sequences that enable targeting and processing of peptides to one or more proteasomes. By targeting and processing peptides using ubiquitination signals, intracellular processing of peptides can more closely mimic antigen processing in antigen-presenting cells (APCs). The efficiency of the processing step can be increased by increasing the number of ubiquitin molecules attached to an antigen. For example, in polyubiquitination, the first ubiquitin molecule is attached to a peptide, followed by the addition of additional ubiquitin molecules. The resulting ubiquitin chain is created by the attachment of a glycine residue of a ubiquitin molecule to a lysine residue of the ubiquitin attached to the peptide. Each ubiquitin molecule contains seven lysine residues and an N-terminus that can function as a site of ubiquitination. When four or more ubiquitin molecules are attached to lysine residues on a peptide antigen, the 26S proteasome recognizes the complex, internalizes it, and degrades the protein into small peptides.

[0075] In some embodiments, the immunogenic polypeptide sequence may be linked by a cleavage-sensitive site. A cleavage-sensitive site is a peptide that is susceptible to cleavage by an enzyme or protease. These sites are also referred to as protease cleavage sites. Preferably, the protease is an intracellular enzyme. The protease may be a serine protease, a threonine protease, a cysteine ​​protease, an aspartic acid protease, a glutamic acid protease, or a metalloprotease. In some preferred embodiments, the protease is a protease found in antigen-presenting cells (APCs). Thus, the protease cleavage site corresponds to a protease that is highly abundant (highly expressed) in APCs. A cleavage-sensitive site that is sensitive to an APC enzyme is referred to as an APC cleavage-sensitive site. Proteases expressed in APCs include, but are not limited to, cysteine ​​proteases such as cathepsin B, cathepsin H, cathepsin L, cathepsin S, cathepsin F, cathepsin Z, cathepsin V, cathepsin O, cathepsin C, and cathepsin K, and aspartic acid proteases such as cathepsin D, cathepsin E, and asparaginyl endopeptidase.

[0076] The cleavage-sensitive site may preferably be a cathepsin B or S-sensitive site. Exemplary cathepsin B-sensitive sites include, but are not limited to, those described in WO2017 / 020026 (incorporated herein by reference; see SEQ ID NOS: 12-407 of WO2017 / 020026). Exemplary cathepsin-sensitive sites include, but are not limited to, those described in WO2017 / 020026 (see SEQ ID NOS: 3-5, 408-1122 of WO2017 / 020026). Other cathepsin-sensitive sites are known in the art or can be readily determined experimentally in digestion assays using only routine experimentation.

[0077] An mRNA cancer vaccine may comprise one or more polynucleotides encoding one or more immunogenic polypeptide sequences of the present invention. Exemplary polynucleotides may comprise at least one chemical modification. Polynucleotides may comprise various substitutions and / or insertions. As used herein in reference to polynucleotides, the term "chemical modification" or, where applicable, "chemically modified" refers to modifications of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in one or more of their positions, patterns, percentages, or populations.

[0078] Modified polynucleotides, when introduced into a cell or organism, may exhibit reduced degradation in the cell or organism compared to unmodified polynucleotides. Modified polynucleotides, when introduced into a cell or organism, may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism. Modifications of polynucleotides are well known in the art and include, for example, those listed in WO2017 / 020026. In general, the modifications described in this section are not intended to refer to ribonucleotide modifications in the native 5'-terminal mRNA cap moiety.

[0079] The polynucleotide may contain natural, non-natural modifications, or both natural and non-natural modifications. The polynucleotide of the mRNA cancer vaccine of the present invention may contain any useful modification to the sugar, nucleobase, or internucleoside linkage (e.g., bridged phosphate / phosphodiester linkage / phosphodiester backbone), etc. One or more atoms of the pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, the modification (e.g., one or more modifications) is present in the sugar and internucleoside linkage. The modification according to the present invention can be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Further modifications are described herein. Non-natural modified nucleotides can be introduced into polynucleotides, such as mRNA cancer vaccines, or nucleic acids during or after the synthesis of the chain to achieve desired functions or properties. Modifications can be to internucleotide groups, purine bases, pyrimidine bases, or sugars. Modifications can be introduced at the end of the chain using chemical synthesis or polymerase enzymes, or anywhere else in the chain. Any region of a polynucleotide can be chemically modified.

[0080] The present disclosure provides modified nucleosides and nucleotides. As described herein, a "nucleoside" is defined as a compound comprising a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "dibasic base"). As described herein, a "nucleotide" is defined as a nucleoside comprising a phosphate group. Modified nucleotides can be synthesized by any useful method, such as those described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or unnatural nucleosides). A polynucleotide can comprise one or more regions of linked nucleosides. Such regions can have various backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide comprises multiple nucleotide regions.

[0081] Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or between modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard and standard bases, or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of the present invention.

[0082] The mRNA may have at least one chemical modification, preferably selected from pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-0-methyluridine.

[0083] As used herein, "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded peptide or polypeptide of interest. Basic components of an mRNA molecule include at least a coding region, a 5' UTR, a 3' UTR, a 5' cap, and a 3' tailing sequence. The mRNA of the present invention generally contains all of these features.

[0084] The "5' untranslated region (UTR)" is the region of an mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome) that does not encode a protein or peptide.

[0085] The "3' untranslated region (UTR)" is the region of an mRNA immediately downstream (i.e., 3') of a stop codon (i.e., a codon in an mRNA transcript that signals the end of translation) that does not encode a protein or peptide.

[0086] An "open reading frame" is a contiguous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) that encodes a protein or peptide.

[0087] A 5' end cap is a specially altered nucleotide at the 5' end of a primary transcript, such as messenger RNA, that promotes stability and translation. A 5' end cap typically consists of a guanine nucleotide attached to mRNA via a specific 5'-5' triphosphate bond. This guanosine is directly methylated at position 7 after capping by methyltransferases in vivo. Therefore, it is sometimes called a 7-methylguanylate cap, abbreviated as m7G. A preferred 5' end cap is m7G(5')ppp(5')NlmpNp.

[0088] The 3' tailing sequence is a poly-A tail, a poly-AG quartet, and / or a stem-loop sequence. The 3' tailing sequence is generally 40 to 200 nucleotides in length. In some embodiments, the 3' tailing sequence is a poly-A tail. A "poly-A tail" is a region of an mRNA that is downstream, e.g., immediately downstream (i.e., 3'), of the 3' UTR and contains multiple consecutive adenosine monophosphates. A poly-A tail may contain 10 to 300 adenosine monophosphates. For example, the poly(A) tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the poly(A) tail contains 50 to 250 adenosine monophosphates. In relevant biological environments (e.g., intracellular, in vivo, etc.), the poly(A) tail functions, for example, to protect the mRNA from enzymatic degradation in the cytoplasm and assists in transcription termination, transport of the mRNA from the nucleus, and translation.

[0089] In some embodiments, the polynucleotide comprises about 200 to about 3,000 nucleotides (e.g., 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, and 2,000 to 3,000).

[0090] The polynucleotides of the present invention can function as mRNA but are distinct from wild-type mRNA in functional and / or structural characteristics. The mRNA cancer vaccines of the present invention can be encoded by in vitro translation (IVT) polynucleotides. As used herein, "in vitro transcription template (IVT)" refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction to produce messenger RNA (mRNA). In some embodiments, the IVT template encodes a 5' untranslated region, contains an open reading frame, and encodes a 3' untranslated region and a polyA tail. The specific nucleotide sequence composition and length of the IVT template depend on the mRNA of interest that the template encodes.

[0091] mRNA can be prepared by any suitable technique known in the art and any suitable synthetic route. The IVT method is preferred. In vitro transcription (IVT) allows for template-directed synthesis of RNA molecules of almost any sequence. The size of RNA molecules that can be synthesized using the IVT method ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. The IVT method allows for the synthesis of large amounts of RNA transcripts (e.g., microgram to milligram amounts) (Beckert et al., Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703:29-41(2011); Rio et al., RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.; Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington DC: ASM Press, 2007, 262-299). Generally, IVT utilizes a DNA template characterized by a promoter sequence upstream of the sequence of interest. The promoter sequence is most commonly derived from a bacteriophage (e.g., a T7, T3, or SP6 promoter sequence), but many other promoter sequences, including those designed de novo, are acceptable. Transcription of the DNA template is usually best achieved by using an RNA polymerase that corresponds to a specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase. IVT generally initiates with dsDNA, but can also proceed with a single strand. Suitable methods include, for example, those described in WO2017 / 020026 (incorporated herein by reference).

[0092] The mRNA disclosed herein can be fully or partially codon-optimized for human expression and / or to reduce immune recognition. Codon optimization methods are known in the art and can be useful in efforts to achieve various results, such as matching the codon frequency in target organisms and host organisms to ensure proper folding, biasing the GC content or reducing secondary structure to increase mRNA stability, minimizing tandem repeat codons or base runs that may impair gene assembly or expression, customizing transcriptional and translational control regions, inserting or removing protein transport sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins, removing or shuffling protein domains, inserting or deleting restriction sites, modifying ribosomal binding sites and mRNA degradation sites, adjusting the translation rate to allow various protein domains to fold properly, or reducing or removing problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. Preferably, the ORF sequence is optimized using an optimization algorithm.

[0093] A codon-optimized sequence can have less than 95%, less than 90%, less than 85%, less than 80%, or less than 75% sequence identity with a native or wild-type sequence (e.g., a native or wild-type mRNA sequence encoding a polypeptide or protein of interest). A codon-optimized sequence can have 65%-85% sequence identity with a native or wild-type sequence (e.g., a native or wild-type mRNA sequence encoding a polypeptide or protein of interest).

[0094] An exemplary codon-optimized RNA may have an increased level of G / C. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 (incorporated herein by reference) discloses a pharmaceutical composition comprising an mRNA stabilized by sequence modification of the translation region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with codons that promote greater RNA stability without changing the resulting amino acids. This approach is limited to the coding region of the RNA.

[0095] Compositions, Formulations, and Encapsulation The present invention provides compositions comprising the polypeptides of the invention and / or the polynucleotides of the invention. For example, the present invention provides compositions comprising one or more polypeptides of the invention and / or one or more polynucleotides of the invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative, and / or excipient.

[0096] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polypeptides of the invention and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0097] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polynucleotides of the invention and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0098] Carriers, preservatives and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, all ingredients and the final composition are sterile and pyrogen-free.

[0099] The composition may be a pharmaceutical composition.

[0100] The composition may be a vaccine composition, preferably a TGFb-1 selective vaccine composition.

[0101] The composition may preferably contain an adjuvant. An adjuvant is any substance that, when incorporated into a composition, enhances or otherwise modulates the immune response elicited by the composition. Broadly defined, an adjuvant is a substance that promotes an immune response. The adjuvant may also preferably have a depot effect, in that it also provides a slow and sustained release of the active agent from the administration site. A general discussion of adjuvants is provided on pages 61-63 of Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986).

[0102] Adjuvants include AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 11687). 19835A, also known as MTP-PE), RIBI in 2% squalene / Tween-80.RTM emulsion (MPL+TDM+CWS), lipopolysaccharides and various derivatives thereof, such as lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., poly IC and poly AU acid), wax D from mycobacteria, substances found in tuberculosis, Corynebacterium parvum, Bordetella pertussis, and members of the Brucella genus, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. No. 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 to be useful as adjuvants for immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0103] Preferred adjuvants for use in the present invention include oil / surfactant-based adjuvants such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants include bacterial DNA-based adjuvants, such as adjuvants containing CpG oligonucleotide sequences. Still other preferred adjuvants include viral dsRNA-based adjuvants, such as poly I:C. GM-CSF and imidazoquinolines are also examples of preferred adjuvants.

[0104] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0105] It should also be noted that, on pages 61-63 of Goding's Monoclonal Antibodies: Principles & Practice (2nd ed., 1986), it is recommended to conjugate the antigen of interest to an immunogenic carrier if the antigen has a low molecular weight or poor immunogenicity. Thus, the polypeptide of the present invention can be conjugated to a carrier. The carrier may exist independently of an adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, to confer stability, to increase biological activity, or to extend serum half-life. Furthermore, the carrier can aid in presenting the polypeptide or its fragment to T cells. Thus, in the present composition, the polypeptide can be conjugated to a carrier as described below. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell, such as a dendritic cell (DC). Carrier proteins include serum proteins such as keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. Alternatively, the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be a dextran such as Sepharose. The carrier must be physiologically acceptable and safe for humans.

[0106] If a composition contains an excipient, it must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and that may be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included therein, including, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, and sulfates; and organic acid salts such as acetates, propionates, malonates, and benzoates. A detailed discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0107] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and methodology, all of which are readily available to those reasonably skilled in the art. Such compositions can be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers, optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in a dry (e.g., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution can be formulated according to known techniques and may contain, in addition to the active ingredient, additional components such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable formulations 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 containing the active ingredient in microcrystalline form in a liposomal preparation or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may contain pharmaceutically acceptable polymers 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, or associated with particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368Jeffery et al. (1993) Pharm. Res. 10:362-368.Other particle systems and polymers can also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0108] Formulations of the compositions described herein can be prepared by any method known in the art or hereafter developed. Generally, such preparation methods include bringing the active ingredient(s) into association with an excipient(s) and / or one or more other accessory ingredients, and then, as necessary and / or desirable, dividing, shaping, and / or packaging the product into desired single- or multi-dose units. The relative amounts of the active ingredient(s), pharmaceutically acceptable excipient(s), and / or optional additional ingredients in pharmaceutical compositions according to the present invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition will be administered. By way of example, the composition may contain 0.1% to 100%, e.g., 0.5 to 50%, 1 to 30%, 5 to 80%, or at least 80% (w / w) of the active ingredient(s).

[0109] mRNA cancer vaccines can be formulated with one or more excipients to (1) enhance stability, (2) increase cell transfection, (3) enable sustained or delayed release (e.g., from depot formulations), (4) alter biodistribution (e.g., target specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) alter the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients such as any solvent, dispersion medium, diluent, or other liquid vehicle, dispersing or suspending aid, surfactant, tonicity agent, thickener or emulsifier, preservative, etc., excipients of the present invention include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with mRNA cancer vaccines (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0110] The mRNA and / or compositions disclosed herein may contain stabilizing elements. Natural eukaryotic mRNA molecules have been found to contain stabilizing elements, including, but not limited to, 5' and 3' UTRs, 5' caps, and 3' tails, as discussed elsewhere herein. Other stabilizing elements that may be included in mRNAs disclosed herein include, for example, histone stem loops. In some embodiments, histone stem loops are generally derived from histone genes and contain two adjacent, partially or fully reverse-complementary sequences separated by a spacer consisting of a short sequence that forms a structural loop. The mRNA may have one or more AU-rich sequences removed. Such sequences may be destabilizing. RNA vaccines may or may not contain enhancer and / or promoter sequences, may be modified or unmodified, and may be activated or inactivated.

[0111] The mRNA cancer vaccines disclosed herein have a diameter of about 10 to about 200 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, Approximately 30 to approximately 100 nm, approximately 40 to approximately 50 nm, approximately 40 to approximately 60 nm, approximately 40 to approximately 70 nm, approximately 40 to approximately 80 nm, approximately 40 to approximately 90 nm, approximately 40 to approximately 100 nm, approximately 50 to approximately 60 nm, approximately 50 to approximately 70 nm The lipid nanoparticles may be formulated into lipid nanoparticles of about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 50 to about 150 nm, about 50 to about 200 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 60 to about 150 nm, about 60 to about 200 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 70 to about 150 nm, about 70 to about 200 nm, about 80 to about 90 nm, about 80 to about 100 nm, about 80 to about 150 nm, about 80 to about 200 nm, about 90 to about 100 nm, about 90 to about 150 nm, and / or about 90 to about 200 nm.

[0112] The lipid nanoparticles can be about 10 to 500 nm in diameter, hi one embodiment, the lipid nanoparticles can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0113] Lipid nanoparticles can be limit-sized lipid nanoparticles as described in International Patent Publication No. WO2013 / 059922 (the entire contents of which are incorporated herein by reference).Limit-sized lipid nanoparticles can comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core, where the lipid bilayer can comprise phospholipids such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacylphosphatidylcholine, and l-palmitoyl-2-oleylphosphatidylcholine (POPC).In another embodiment, limit-sized lipid nanoparticles can comprise polyethylene glycol-lipids such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0114] The RNA vaccine can be delivered, localized, and / or concentrated to a specific location using the delivery method described in International Patent Publication No. WO2013 / 063530 (the entire contents of which are incorporated herein by reference).As a non-limiting example, empty polymer particles can be administered before, simultaneously with, or after the RNA vaccine is delivered to the subject.When the empty polymer particles come into contact with the subject, they undergo a volume change and remain, become embedded, fixed, or encapsulated in a specific location in the subject.

[0115] The lipid nanoparticle composition may comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid in a molar ratio of approximately 20-60% cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0116] How to use The polypeptides, polynucleotides, or compositions of the present invention, or combinations thereof, can be used in methods for treating or preventing a disease or condition in a subject. The polypeptides, polynucleotides, or compositions of the present invention, or combinations thereof, can be used in the manufacture of a medicament for use in a method for treating or preventing a disease or condition in a subject. The method can include administering the polypeptide, polynucleotide, composition, or combination to the subject. A therapeutically or prophylactically effective amount of the polypeptide, polynucleotide, composition, or combination can be administered to a subject in need thereof.

[0117] The disease or condition may be characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb-1. The disease or condition may be cancer, preferably a cancer that expresses TGFb-1 and / or is associated with inappropriate or excessive immunosuppressive function of TGFb-1. The cancer may be esophageal cancer or urothelial cancer. The cancer may be colorectal cancer, gastric cancer, head and neck cancer, melanoma, non-small cell lung cancer (NSCLC), or ovarian cancer. The cancer may be breast cancer, cervical cancer, liver cancer, or pancreatic cancer. The cancer may be a tumor.

[0118] The method may include the simultaneous or sequential administration of an additional cancer therapy. The additional cancer therapy may be a dual-specific inhibitor of TGFb (e.g., TGFb-1) and PD-L1. The dual-specific inhibitor is capable of simultaneously binding to and / or inhibiting the activity of TGFb and PD-L1. The dual-specific inhibitor comprises an anti-TGFb portion and an anti-PD-L1 portion, the anti-PD-L1 portion optionally comprising or consisting of an anti-PD-L1 antibody, and / or the anti-TGFb portion may be a fusion protein comprising or consisting of a TGFb receptor or a portion thereof (e.g., TGFb receptor II or a portion thereof).

[0119] The additional cancer therapy may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiation therapy, immunostimulants, gene therapy, or antibodies.

[0120] The antibodies include Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, and Anatumomab mafenatoxin. Mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= Tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab-mertansine mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab vedotinravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Crivatuzumab tetraxetan Tetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab Aritox, Drozitumab, Durigotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab-SituxetanCituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab-ozogamicin ozogamicin, Gevokizumab, Direntuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicinozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lebrikizumab, lemaresomab emalesomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, rodelcizumab, lorvotuzumab-mertansine Mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentanemerpentan, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaradumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, 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 ab), Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab Pendetide 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, Telimomabaritox, tenatumomab, teneliximab, teplizumab, teprotumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab Tocilizumab (= atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab Celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Bepalimomab, Besencumab, Visilizumab, Volociximab, Vorsetuzumab It may be cefotaxime, cefotaxime (C2000), ...

[0121] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicep, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, meplizumab, respirulina, and the like. These include rituximab, tocilizumab, ustekinumab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tositumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab, and vedotin.

[0122] Particularly preferred antibodies that can be used in the methods of the invention include daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab, and atumumab.

[0123] The additional cancer therapy may be selected from the group consisting of actimid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, revirimide, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0124] The polypeptides of the invention, the polynucleotides of the invention and / or the compositions of the invention may also be used to stimulate TGFb-1-selective T cells, e.g., CD4 + and / or CD8 + The present invention can be used in a method for stimulating T cells, comprising contacting cells with the polypeptide and / or composition. The method can be performed ex vivo. These cells can be present in a sample collected from a healthy subject or a cancer patient, such as a tumor sample. TGFb-1-selective T cells can exhibit low cross-reactivity to TGFb-2 and TGFb-3. The reactivity of TGFb-1-selective T cells can be compared with the reactivity of TGFb-1-specific T cells contacted with corresponding polypeptides derived from TGFb-2 or TGFb-3. The reactivity of TGFb-1-selective T cells to cells expressing and / or presenting TGFb-1 polypeptides can be compared with the reactivity of TGFb-1-specific T cells to cells expressing and / or presenting corresponding polypeptides derived from TGFb-2 or TGFb-3. The reactivity of TGFb-1-selective T cells can be measured by methods known to those skilled in the art, such as an IFNγ ELISPOT assay.

[0125] The polypeptide of the present invention, the polynucleotide of the present invention, and / or the composition of the present invention can also be used in a method for modulating the tumor microenvironment (TME) of a subject. TGFb-1 may be highly expressed in the TME of most cancer types, such as colorectal cancer, esophageal squamous cell carcinoma, gastric cancer, head and neck cancer, melanoma, NSCLC, ovarian cancer, and urothelial carcinoma. In particular, TGFb-1 is expressed in the TME by, for example, cancer-associated fibroblasts (CAFs), CD8 + T cells, CD4 + T cells, regulatory CD4 + T cells, exhausted CD8 +It may be expressed by various cell types, such as T cells, M1 tumor-associated macrophages (M1_TAM), M2 tumor-associated macrophages (M2_TAM), myeloid antigen-presenting cells (APCmye), and other cells. The method includes administering the polypeptide, polynucleotide, composition, or combination to a subject. The polypeptides of the present invention can elicit a TGFb-1-selective T cell response, and therefore, administration of the polypeptides of the present invention and / or compositions comprising at least one polypeptide of the present invention can be used to modulate the TME in a subject suffering from cancer. Modulation of the TME can result in promotion of T cell infiltration in the TME, for example, CD4 expression in the TME. + This may include promoting T cell infiltration.

[0126] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof. [Example]

[0127] Example 1 - Materials and Methods peptide Peptides were synthesized by standard methods and provided in DMSO to a stock concentration of 5 or 10 mM. The sequences of the peptides used in these experiments are provided in the section entitled "Sequence Listing." Peptides are described by SEQ ID NO: 7, name, or by reference to the start and end positions of each peptide sequence within the amino acid sequence of the full-length precursor of human TGFb. As indicated in the table in the Sequence Listing section below, the respective designations can be used interchangeably. For example, the peptide of SEQ ID NO: 7 is sometimes referred to as Pep01-1 and is also referred to as TGFb-1. 112-151 (start position 112, end position 151). The intended reference in each case will be clear from the context.

[0128] In vitro ELISPOT assay For in vitro ELISPOT assays, peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with 20 μM TGFβ-derived peptide and 20 U / ml IL-2 for 7 days in a 24-well plate. The cells were then placed in a 96-well nitrocellulose ELISPOT plate (MultiScreen IP Filter Plate, MSIPN4W50; Millipore) precoated with an interferon-gamma (IFNγ) capture antibody (Mabtech). TGFβ peptide was added to a final concentration of 5 μM, and a control stimulus (DMSO) was added to control wells. The plate was then incubated at 37°C for 16–20 hours. After incubation, the cells were washed, and a biotinylated secondary antibody (Mabtech) was added for 2 hours at room temperature. Unbound secondary antibody was washed away, and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech) was added for 1 hour at room temperature. Unbound bound enzyme was washed away, and the assay was developed by adding BCIP / NBT substrate (Mabtech). Developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6 Ultimate-V analyzer using Immunospot software v5.1.

[0129] Mouse tumor assay For the MC38 tumor study, female C56 / BL6 mice (Tacomic) were inoculated with 100 μg of peptide (SEQ ID NO: 31 or SEQ ID NO: 34) emulsified in DMSO, diluted with water to a total volume of 50 μl (per injection), and then mixed with an equal volume of Montanide ISA51 VG ST adjuvant. The vaccine formulation was administered subcutaneously (sc) at the base of the tail on days 0, 7, and 14. MC38 tumor cells (2e5 cells per injection) were administered sc in the flank on day 0. Tumors were measured every 3-4 days with Vernier calipers, and tumor volume was calculated using the formula: V = L × W / 2, where V is tumor volume, L is tumor length (major axis), and W is tumor width (minor axis).

[0130] Analysis of the Tumor Microenvironment (TME) by Flow Cytometry Freshly isolated tumors were dissociated by collagenase digestion to obtain single-cell suspensions for flow cytometry analysis. Approximately 1 million cells were stained with the following antibodies: anti-mouse LAP BV421 (BD, 565638), anti-mouse CD4 BV605 (BD, 743156), and anti-mouse CD8 BV786 (BD, 563332) for flow cytometric analysis using a Symphony A1 flow cytometer (BD, Becton Dickinson). Data analysis was performed using FlowJo software.

[0131] In vivo cytotoxicity assay In vivo cytotoxicity assays were performed by injecting peptide-loaded splenocytes from untreated donor mice into vaccinated mice. Freshly isolated splenocytes were incubated with 5 μM peptide in culture medium (RPMI, 10% fetal bovine serum) at 37°C for 1 hour. Splenocytes were loaded with the assay peptide (SLP1_Ib) or control peptide (P53, AIYKKSQHM), respectively. Splenocytes were then washed twice in 5 ml PBS + 0.5% BSA. Cells loaded with the assay peptide were then labeled with CellTrace Far Red (ThermoFisher) at 1 / 100 the recommended concentration for 20 minutes at 37°C, while cells loaded with the control peptide were labeled with CellTrace Violet (ThermoFisher). After washing twice in PBS, the labeled cells were combined in equal numbers with PBS, approximately 8 million cells per 200 μl injection volume. The cells in PBS were injected intravenously. Eighteen hours later, splenocytes were isolated from injected mice and cell killing was analyzed by flow cytometry comparing the recovery of FarRed- and Violet-labeled cells. Specific killing of assay-pulsed splenocytes was calculated as follows: (1 - [(FarRed / Violet) inoculated × (Violet / FarRed) injected]) × 100%.

[0132] Example 2 - Characterization of TGFb-1 in the tumor microenvironment of multiple solid cancers Neogenomics MultiOmyx™ technology was used to assess the expression of 18 biomarkers: ARG1, CD3, CD4, CD8, CD11b, CD68, CD163, FAP, FoxP3, HLADR, IDO1, LAG-3, PanCK, PD-1, PD-L1, SOX-10, TGFb-1, TIGIT, and a panel of tumor markers PanCK and SOX10. A proprietary deep learning algorithm was used to classify positive cells for each marker. Data presented here focused on the frequency of positively classified cells and the overlap between the various markers. More than 30 regions of interest (ROIs) were analyzed for each cancer type.

[0133] [Table 1]

[0134] [Table 2]

[0135] As shown in Figures 1-4, TGFb-1 was found to be highly expressed in tumor cells in esophageal and urothelial cancers, but in most cancer types, TGFb-1 was expressed in the TME. TGFb-1-expressing cells comprised tumors at a proportion comparable to that of IDO1 and PD-L1, the antigens targeted by IO Biotech's lead therapeutics, IO102 and IO103.

[0136] Example 3 - Identification and characterization of TGFb-1 specific peptide antigens To identify peptides with high specificity for the TGFb-1 protein sequence, Clustal Omega (www.ebi.ac.uk / Tools / msa / clustalo / ) sequence alignments were performed using the UniProt reference sequences (P01137, P61812, and P10600 for TGFb-1, TGFb-2, and TGFb-3, respectively) shown in Figure 5. Sequence alignments were visualized using Jalview (www.jalview.org / ). Five peptides (Pep01-1 to Pep05-1) were selected from low homology region 1 (amino acids 112 to 151 of SEQ ID NO: 1), three peptides (Pep06-1 to Pep08-1) were selected from low homology region 2 (amino acids 226 to 260 of SEQ ID NO: 1), and three peptides (Pep09-1 to Pep11-1) were selected to encompass the sequence disclosed in WO2020 / 245264 (SEQ ID NO: 205, also known as TGFb-15) with intermediate homology. Peptides were selected based on the following criteria: 1) length greater than 20 amino acids, 2) avoidance of consecutive stretches of eight or more identical or highly similar amino acids, and 3) avoidance of cysteine ​​residues. One peptide (Pep12-1) was selected based on the high homology between TGFb-1, TGFb-2, and TGFb-3 and was used exclusively for TGFb-1 selectivity studies. Selected TGFb-1 peptides are shown in Table 3 , including the percentage of sequence identity with the corresponding peptides in TGFb-2 and TGFb-3 (see Table 5 for details of TGFb-3 and the corresponding peptides in TGFb-3).

[0137] [Table 3]

[0138] Example 4 - Screening of immune responses to TGFb-1 peptides To determine whether the low-homology peptides selected based on sequence alignment could elicit immune responses in humans, we used an IFNγ ELISPOT assay. PBMCs from a total of 14 healthy donors were used for immune response screening. PBMCs were individually exposed to each peptide to induce peptide-specific immune responses and peptide-specific T cell proliferation. Seven days later, the frequency of peptide-specific T cells was assayed by IFNγ ELISPOT. IFNγ ELISPOT identified several peptides that elicited strong immune responses (number of spots) and high frequency of immune responses (number of donors), as shown in Figure 6. Based on this, five peptides were selected (see Table 4). These five peptides were then assayed for TGFb-1 selectivity by assaying cross-reactive immune responses to TGFb-2 and TGFb-3 peptides in IFNγ ELISPOT assays, as shown in Figures 7 and 8. These data indicate that the IFNγ ELISPOT immune response to the low-homology selected TGFb-1 peptide did not cross-react with the homologous TGFb-2 and TGFb-3 peptides. In contrast, the highly homologous selected peptide (Pep12-1) induced immune responses that cross-reacted with the homologous TGFb-2 and TGFb-3 peptides (Pep12-2 and Pep12-3, respectively), reflecting the magnitude of the TGFb-1-specific response.

[0139] [Table 4]

[0140] Taken together, these data identified five peptides (Pep01-1, Pep04-1, Pep05-1, Pep08-1, and Pep09-1) that were selective for TGFb-1 and induced strong and frequent immune responses in healthy donors.

[0141] Example 5 - Functionality of TGFb-1 peptide vaccine in mouse tumor models We developed a murine TGFb-1 vaccine based on synthetic long peptides (SLPs) encoding putative MHC class I and class II epitopes (SEQ ID NOs: 31-36 in Table 1 below). Preclinical studies of anti-TGFb therapies often show no effect on tumor growth when administered as single agents. Therefore, our goals here are primarily to: 1) target CD4 + T cells and CD8 + The study focused on: 1) developing a vaccine that elicits the most potent immune response consisting of both T cells and T cells; 2) determining its effect on tumor growth; 3) determining its effect on the TME; and 4) developing assays to further characterize the vaccine-induced T cell response.

[0142] As shown in Figure 9, both SLPs elicited strong immune responses in IFNγ ELISPOT assays. SLP1 vaccination recognized minimal peptides encoding class I and class II epitopes. In contrast, SLP2 induced primarily class II responses (although a class I response cannot be ruled out, given that class I epitopes were not tested). CD4 + T cell infiltration was significantly enhanced in SLP2-inoculated animals, whereas CD8 + T cell infiltration was unchanged (Figures 10C and 10D). In vivo cytotoxicity assays demonstrated that vaccination with SLP1 resulted in cytotoxic activity against cells loaded with the class I peptide antigen SLP1_Ib, as shown in Figure 11. Notably, cytotoxic activity was higher in animals vaccinated with SLP1_Ib, suggesting superior induction of cytotoxic T cells by the minimal epitope antigen SLP1_Ib compared with SLP1 (Figure 11). A similar assay is currently being developed to evaluate TGFb-1 vaccines in mice.

[0143] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but that it is intended to cover modifications within the spirit and scope of the invention as defined herein.

[0144] Various publications, articles, and patents are cited or described throughout the Background and Specification, and each of these references is incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any of these items form part of the prior art with respect to the invention disclosed or claimed.

[0145] Sequence Listing In Table 5 below, "start" and "end" indicate positions within the full-length human TGFβ preprotein (SEQ ID NO: 1, 2 or 3) unless otherwise specified.

[0146] [Table 5] TIFF2025538981000008.tif202166

Claims

1. A polypeptide which is an immunogenic fragment of human transforming growth factor 1 (TGFb-1) and comprises or consists of a sequence of at least 8 consecutive amino acids of SEQ ID NO:

5.

2. The polypeptide of claim 1 which does not contain any cysteine ​​residues.

3. have low homology and / or low sequence identity to the corresponding polypeptide sequences of human transforming growth factor 2 (TGFb-2) and / or 3 (TGFb-3), and optionally (a) TGFb-2 has the amino acid sequence of SEQ ID NO:2, and / or TGFb-3 has the amino acid sequence of SEQ ID NO:3; and / or (b) the polypeptide fragment has less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 25%, or less than about 20% sequence identity with the corresponding polypeptide sequence of TGFb-2 and / or TGFb-3, preferably, the polypeptide has less than about 40% sequence identity with the corresponding polypeptide sequence of TGFb-2 and / or TGFb-3; A polypeptide according to claim 1 or 2.

4. (a) comprising, or consisting of, up to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 contiguous amino acids of SEQ ID NO:5; or (b) comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 consecutive amino acids of SEQ ID NO:5, or at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 consecutive amino acids of SEQ ID NO:5 Preferably, the polypeptide comprises at least 25, 26, 27, 28, 29, or 30 consecutive amino acids of SEQ ID NO:5, or consists of at least 25, 26, 27, 28, 29, or 30 consecutive amino acids of SEQ ID NO:5, more preferably, the polypeptide comprises at least 30 consecutive amino acids of SEQ ID NO:5, or consists of at least 30 consecutive amino acids of SEQ ID NO:

5. A polypeptide according to any one of claims 1 to 3.

5. 5. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 32, and optionally the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 7, 10, or 11.

6. A polypeptide according to any one of claims 1 to 5, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 7, 10, 32, 11, 14, or 15, preferably SEQ ID NO: 7 or SEQ ID NO:

10.

7. The polypeptide according to any one of claims 1 to 6, which is capable of stimulating TGFb-1 selective T cells.

8. The polypeptide of claim 7, wherein the TGFb-1 selective T cells have low cross-reactivity with cells that express and / or present TGFb-2 and / or TGFb-3 polypeptides.

9. measuring cross-reactivity comprises comparing the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting a polypeptide of TGFb-1 with the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting a corresponding polypeptide from TGFb-2 or TGFb-3, and optionally (a) the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting the corresponding polypeptide from TGFb-2 or TGFb-3 is less than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the reactivity of TGFb-1-selective T cells to TGFb-1 polypeptides; and / or (b) Cross-reactivity is measured by IFNγ ELISPOT assay; The polypeptide of claim 8.

10. A polynucleotide encoding a polypeptide according to any one of claims 1 to 9, optionally contained within a vector.

11. (a) an open reading frame (ORF) encoding at least one polypeptide according to any one of claims 1 to 9; (b) a 5′-end cap at the 5′-terminus; (c) a 5′ untranslated region (UTR) contained 5′ of the ORF; (d) a 3'UTR contained 3' of the ORF; and (e) a 3' tailing sequence at the 3' end The polynucleotide of claim 10, which is an mRNA comprising:

12. A composition comprising a polypeptide according to any one of claims 1 to 9; and / or a polynucleotide according to claim 10 or 11; and optionally an adjuvant.

13. The composition of claim 12, further comprising at least one different polypeptide of any one of claims 1 to 9; at least one different polynucleotide of claim 10 or 11; and / or at least one pharmaceutically acceptable diluent, carrier or preservative.

14. 14. The composition of claim 12 or 13, comprising an adjuvant selected from the group consisting of a bacterial DNA-based adjuvant, an oil / surfactant-based adjuvant, a viral dsRNA-based adjuvant, an imidazoquinoline, and a montanide ISA adjuvant.

15. 14. The composition of claim 12 or 13, formulated in a lipid nanoparticle composition and comprising a polynucleotide, wherein the polynucleotide is mRNA, and optionally the lipid nanoparticles have an average diameter of 50 to 200 nm.

16. The composition according to any one of claims 12 to 15, which is a TGFb-1 selective vaccine composition.

17. 19. A method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a polypeptide according to any one of claims 1 to 9, a polynucleotide according to claim 10 or 11, and / or a composition according to any one of claims 12 to 16.

18. The disease or condition is (a) a cancer optionally selected from the group consisting of esophageal cancer and urothelial cancer, and optionally selected from the group consisting of colorectal cancer, gastric cancer, head and neck cancer, melanoma, non-small cell lung cancer (NSCLC) or ovarian cancer; and / or (b) characterized, at least in part, by inappropriate or excessive immunosuppressive function of TGFb-1-expressing cells; 18. The method of claim 17.

19. 19. The method of claim 17 or 18, wherein the disease or condition is cancer, and further comprising the simultaneous or sequential administration of an additional cancer therapy, preferably an antibody.

20. 20. The method of any one of claims 17 to 19, wherein the disease or condition is cancer and the administration of the polypeptide, polynucleotide and / or composition stimulates a selective immune response in TGFb-1 expressing cancer cells.

21. the disease or condition is a tumor; (a) the polypeptides, polynucleotides, and / or compositions are capable of modulating the tumor microenvironment (TME), optionally wherein said modulation comprises promoting infiltration of T cells into the TME, preferably wherein said T cells are CD4 ＋ is a T cell; (b) the method comprises modulating the TME, and optionally, the modulating comprises promoting infiltration of T cells into the TME, preferably the T cells are CD4 ＋ are T cells; and / or (c) administration of said polypeptides, polynucleotides and / or compositions stimulates a selective immune response against TGFb-1 expressing cells in the TME; The method according to any one of claims 17 to 20.

22. 17. A method for stimulating TGFb-1-selective T cells, the method comprising contacting T cells with a polypeptide according to any one of claims 1 to 9, a polynucleotide according to claim 10 or 11, and / or a composition according to any one of claims 12 to 16.

23. 23. The method of claim 22, wherein the TGFb-1-selective T cells have low cross-reactivity to cells that express and / or present TGFb-2 and / or TGFb-3 polypeptides.

24. measuring cross-reactivity comprises comparing the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting a polypeptide of TGFb-1 with the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting a corresponding polypeptide from TGFb-2 or TGFb-3; and optionally (a) the reactivity of TGFb-1-selective T cells to cells expressing and / or presenting the corresponding polypeptide from TGFb-2 or TGFb-3 is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the reactivity of TGFb-1-selective T cells to TGFb-1 polypeptides; and / or (b) Cross-reactivity is measured by IFNγ ELISPOT assay; 24. The method of claim 23.

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