TGF-beta1 vaccine

Abstract

The present invention relates to a protein derived from transforming growth factor beta1 (TGF beta1; tGFb1), as well as polynucleotides encoding such polypeptides and compositions comprising such peptides. The invention also relates to a method of increasing the selectivity of an immune response to TGFb-1. The invention also relates to uses and methods of use of the polypeptides, polynucleotides and compositions.

Description

Technical Field

[0001] The present invention relates to novel polypeptides derived from transforming growth factor β1 (TGFβ1; TGFb-1), polynucleotides encoding such polypeptides, and compositions comprising such peptides. The present invention also relates to methods for increasing the selectivity of the immune response to TGFb-1. The present invention also relates to the uses and methods of use of said polypeptides, polynucleotides and compositions. Background Art

[0002] TGFb is a multifunctional cytokine that plays a key role in the regulation of the immune system. There are three isoforms, with subtype 1 (TGFb-1) being particularly important in T cell immunity. In the case of cancer, TGFb-1 disarms various immune cells, such as cytotoxic T cells (CTLs), tumor-associated neutrophils, and natural killer (NK) cells. It also contributes to tumor vascularization and metastasis. Thus, TGFb1 is a key inhibitory molecule in the tumor microenvironment (TME), contributing to the downregulation of the anti-tumor mechanisms of the immune system and enabling cancer cells to evade immunity.

[0003] Recent clinical results (Kjeldsen, J.W. et al., Nat. Med. 27(12):2212-2223(2021)) provide the rationale for cancer immunotherapy based on the activation of "counter-regulatory" T cells. Counter-regulatory T cells recognize antigens that are typically expressed by immunosuppressive cells, thereby targeting pro-inflammatory signals to the tumor microenvironment (Andersen, M.H., Semin. Immunopathol(2022)). Therapeutic agents based on small molecule inhibitors targeting the TGFb receptor and capture ligands based on soluble TGFb receptors target all three isoforms of TGFb (TGFb-1, TGFb-2, and TGFb-3), and targeting additional TGFb isoforms (TGFb-2, TGFb-3) is thought to result in adverse clinical effects (Tauriello, D.V.F., E. Sancho, and E. Batlle, Nat. Rev. Cancer, 22(1):25-44(2022)).

[0004] Activating TGFb-1 selective T cells (non-cross-reactive with TGFb-2 and TGFb-3) can target pro-inflammatory immune responses to tumors expressing TGFb-1 while avoiding toxicity associated with pan-TGFb inhibition. TGFb-1 selective T cells are frequently detected in humans (Holmstrom, M.O., et al., Cell Mol. Immunol. 18(2):415-426 (2021)). Thus, there is a current need to enhance the selectivity of anti-TGFb-1 immune responses to mitigate potential off-target toxicity. Summary of the Invention

[0005] The inventors have previously identified polypeptide fragments of TGFb-1 that are immunogenic. These polypeptide fragments are disclosed in WO2020 / 245264, which is incorporated herein by reference. The inventors have now identified new immunogenic polypeptide fragments 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 with TGFb-2 and TGFb-3. Thus, the polypeptides of the present invention are expected to be particularly effective in stimulating a beneficial selective immune response against cells expressing TGFb-1. In particular, the polypeptides of the present invention are expected to enhance the selectivity of the immune response against cells expressing TGFb-1 without enhancing the immune response against cells expressing TGFb-2 or cells expressing TGFb-3. Thus, the polypeptides of the present invention are expected to exhibit low off-target toxicity. The longer polypeptides of the present invention, such as those listed in SEQ ID NO:7 and 10, are also expected to have particular immunogenicity as they may contain more epitopes than shorter TGFb-1 polypeptide fragments. Such peptides are also expected to have improved properties in terms of ease of manufacture and formulation.

[0007] TGFb-1 is a dimeric cytokine that has cysteine structures linked together by intermolecular disulfide bonds. TGFb-1 is synthesized as a monomeric 390 amino acid precursor protein, which can be interchangeably referred to as: TGFb-1 proprotein; TGFb-1 precursor; full-length TGFb-1; pre-pro-TGFb-1. The full-length sequence of the TGFb-1 proprotein is provided as SEQ ID NO:1.

[0008] The TGFb1 proprotein monomer has a molecular weight of 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 TGFb-1 SP targets the protein to the secretory pathway; the SP is cleaved in the rough endoplasmic reticulum. The TGFb-1 monomer containing LAP and mature TGFb-1 can dimerize in the endoplasmic reticulum via disulfide bonds between cysteine residues in LAP (e.g., Cys 223 and Cys 225) and the mature TGFb-1 peptide (e.g., Cys 356) to form a TGFb-1 homodimer. This TGFb-1 homodimer is called the small latent complex (SLC). The SLC may be bound by the so-called latent TGF-β-binding protein (LTBP) to form a larger complex, which is called the large latent complex (LLC). The LLC can be secreted into the extracellular matrix (ECM). However, the presence of LAP and LTBP prevents TGFb-1 from binding and activating its extracellular receptors. Active TGFb-1 consists of a homodimer of the mature TGFb-1 peptide. There are multiple mechanisms for the release of the mature TGFb-1 homodimer from LAP and LTBP, including proteolytic degradation of LAP, induction of conformational changes in LAP by interaction with thrombospondin, and cleavage of the non-covalent bond between LAP and TGFb-1.

[0010] One object of the present invention is to develop an immunogenic polypeptide that generates a selective immune response against TGFb-1. Another object of the present invention is to selectively target immunosuppressive cells in the TME. Considering that immunosuppressive cells in the TME highly express TGFb-1, selectively targeting TGFb-1 on these cells can deplete immunosuppressive cells in the TME.

[0011] Accordingly, the present invention provides a polypeptide, which is an immunogenic fragment of TGFb-1 and which comprises or consists of a sequence of at least 8 consecutive amino acids of SEQ ID NO:5. Preferably, the polypeptide does not contain cysteine residues. The polypeptide may have low homology with the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. The polypeptide may have low sequence identity with 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 a sequence identity of less than about 80%, 70%, 60%, 50%, 40%, 30%, 25% or 20% with the corresponding polypeptide sequences of TGFb-2 and / or TGFb-3. Preferably, the polypeptide has a sequence identity of less than about 40% with 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 or 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 consecutive amino acids of SEQ ID NO:5. Preferably, the polypeptide may comprise or consist of at least 25, 26, 27, 28, 29 or 30 consecutive amino acids of SEQ ID NO:5. More preferably, the polypeptide may comprise or consist of at least 30 consecutive 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 NO:7, 10 or 11. The polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NO: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 the amino acid sequence of SEQ ID NO: 11, 14, and / or 15 or is not composed of the amino acid sequence of SEQ ID NO: 11, 14, and / or 15. In one embodiment, the polypeptide is not composed of the amino acid sequence of SEQ ID NO: 11, 14, and / or 15.

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

[0013] Measurement of cross-reactivity may include comparing the reactivity of the TGFb-1 selective T cells to cells expressing and / or presenting a polypeptide of TGFb-1 with the reactivity of the TGFb-1 selective T cells to cells expressing and / or presenting a corresponding polypeptide from TGFb-2 or TGFb-3. The reactivity of the TGFb-1 selective T cells to cells expressing and / or presenting a corresponding polypeptide 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 the TGFb-1 selective T cells to a polypeptide of TGFb-1. Cross-reactivity may be determined by IFNγ ELISPOT assay.

[0014] The present invention further provides a polynucleotide encoding the 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 comprise:

[0015] (a) an open reading frame (ORF) encoding at least one polypeptide of the present invention;

[0016] (b) a 5' cap at the 5' end;

[0017] (c) a 5' untranslated region (UTR) contained at the 5' of the ORF;

[0018] (d) A 3' UTR contained in the 3' of the ORF; and

[0019] (e) A polyadenylation sequence at the 3' end.

[0020] The present invention also provides a composition comprising the polypeptide and / or polynucleotide of the present invention and an optional adjuvant. The composition may further comprise: at least one different polypeptide of the present invention; at least one different polynucleotide of the present invention; and / or at least one pharmaceutically acceptable diluent, carrier or preservative. The adjuvant may be selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazoquinolines and Montanide ISA adjuvants. Preferably, the composition may be a TGFb-1 selective vaccine composition. When the composition comprises a polynucleotide and the polynucleotide is mRNA, the composition may be formulated in a lipid nanoparticle composition. The lipid nanoparticle composition may have an average diameter of 50 - 200 nm.

[0021] The present invention also provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject the polypeptide of the present invention, the polynucleotide of the present invention and / or the composition of the present invention. The method may further comprise: administering an additional cancer therapy, preferably an antibody, simultaneously or sequentially. When the disease or disorder is cancer, the method may further comprise stimulating a selective immune response against cancer cells expressing TGFb-1.

[0022] The present invention also provides the polypeptide of the present invention, the polynucleotide of the present invention, the composition of the present invention or a combination thereof for treating or preventing a disease or disorder. The polypeptide, polynucleotide, composition or combination thereof may be used in combination with an additional cancer therapy (preferably an antibody). When the disease or disorder is cancer, the polypeptide, polynucleotide, composition or combination thereof may stimulate a selective immune response against cancer cells expressing TGFb-1.

[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 in the preparation of a drug for treating or preventing a disease or disorder. When the disease or disorder is cancer, the use of the polypeptide, polynucleotide, composition or combination thereof may be for the preparation of a drug for stimulating a selective immune response against cancer cells expressing TGFb-1.

[0024] The disease or disorder can be characterized, at least in part, by inappropriate or excessive immunosuppressive function of cells expressing TGFb1. Preferably, the disease or disorder is cancer. The cancer can be esophageal cancer or urothelial cancer. The cancer can be colorectal cancer, gastric cancer, head and neck cancer, melanoma, non-small cell lung cancer (NSCLC), or ovarian cancer. The cancer can be breast cancer, cervical cancer, liver cancer, or pancreatic cancer.

[0025] The disease or disorder can be a tumor, and the polypeptide, polynucleotide, composition, or combination thereof can modulate the tumor microenvironment (TME). The methods and uses of the present invention can include modulating the TME. Modulating can include enhancing the infiltration of T cells into the TME. Preferably, the T cell is a CD4 + T cell. Administering the polypeptide, polynucleotide, composition, or combination thereof can stimulate a selective immune response against cells expressing TGFb-1 in the TME.

[0026] The present invention also provides a method for stimulating TGFb-1 selective T cells, the method comprising: 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.

[0027] The present invention also provides an ex vivo method for stimulating TGFb-1 selective T cells, the method comprising: 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.

[0028] 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. The use can 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. The use can be non-therapeutic and / or ex vivo.

[0029] TGFb-1 selective T cells stimulated by the peptide, polynucleotide, and / or composition of the present invention can have low cross-reactivity against cells expressing and / or presenting polypeptides of TGFb-2 and / or TGFb-3. Measuring cross-reactivity can include comparing the reactivity of the TGFb-1 selective T cells against cells expressing and / or presenting a polypeptide of TGFb-1 with the reactivity of the TGFb-1 selective T cells against cells expressing and / or presenting a corresponding polypeptide from TGFb-2 or TGFb-3. The reactivity of the TGFb-1 selective T cells against cells expressing and / or presenting a corresponding polypeptide 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 the TGFb-1 selective T cells against a polypeptide of TGFb-1. Cross-reactivity can be determined by IFNγ ELISPOT assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A -B. Representative images showing TGFB-1 staining on tumor cells in esophageal cancer (A) and non-small cell lung cancer (NSCLC) (B).

[0031] Figure 2 . TGFb-1 is expressed by most cells in the tumor and the tumor microenvironment (TME). Box plots and data points show the frequency of TGFb-1 expression in tumor cells and the tumor microenvironment (TME, not tumor cells) of the indicated cancer types. For each cancer type, the box plot on the left corresponds to TGFb-1 expression in the TME, while the box plot on the right corresponds to TGFb-1 expression in tumor cells. The proportion of TGFb-1 positive cells in each individual region of interest (ROI) is shown as an individual data point.

[0032] Figure 3 . TGFb-1 is expressed by various cell types in the TME. The bar graph shows the proportion of TGFb-1 positive cells of each cell type in the TME (excluding tumor cells) of the indicated cancer types. Some categories are non-exclusive, so the sum can exceed 1.0. The order of cell types shown in the legend is the same as the order of cell types in each column.

[0033] Figure 4 . Cells expressing TGFb-1 are not limited to TME populations expressing other immunosuppressive markers. The bar graph shows the proportion of cells expressing the indicated immunosuppressive antigens. "Combination" refers to cells expressing any combination of two or more antigens. The melanoma samples analyzed here were 70% uveal and 30% cutaneous, and metastatic melanoma was not analyzed. The order of immunosuppressive markers presented in the legend is the same as the order of immunosuppressive markers in each column.

[0034] Figure 5 . TGFb sequence alignment. Clustal Omega sequence alignment of human TGFb-1, TGFb-2, and TGFb-3 proteins. Figure 5 The labels Pep01 to Pep12 in indicate the positions where the peptides disclosed herein map to the TGFb-1, TGFb-2, and TGFb-3 sequences. For example, the sequence of Pep01-1 can be found in the sequence of TGFb-1 below "Pep01" in Figure 5 and the sequence of Pep01-2 can be found in the sequence of TGFb-2 below "Pep01" in the figure, and so on.

[0035] Figure 6. 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 the responses were analyzed by IFNγ ELISPOT 7 days later. Significant responses (*) were defined by Fisher's exact P value < 0.01, peptide-to-control spot ratio > 2, and background-subtracted spots > 25. Subsequently, the specificity of the peptides that elicited responses in most donors to TGFb-1 responses was tested.

[0036] Figure 7A -B. IFNγ ELISPOT for identifying 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), and the recall response was tested 7 days later by IFNγ ELISPOT assay using the same TGFb-1 peptide or homologous TGFb-2 peptides (i.e., Pep01-2, Pep04-2, Pep05-2, Pep08-2, or Pep12-2) or TGFb-3 peptides (i.e., Pep01-3, Pep04-3, Pep05-3, Pep08-3, Pep08-3, Pep09-3, or Pep12-3). Highly homologous peptides Pep12-1, Pep12-2, and Pep12-3 were included as positive controls for detecting cross-reactive immune responses to TGFb-2 and TGFb-3. In Figure 7A , for each tested peptide, the left box plot corresponds to the response using the same TGFb-1 peptide, the middle box plot corresponds to the response using the homologous TGFb-2 peptide, and the right box plot corresponds to the response using the homologous TGFb-3 peptide. Figure 7B A representative ELISPOT assay is shown, set up in triplicate wells.

[0037] Figure 8 . IFNγ ELISPOT for identifying TGFb-1 selective immune responses. The same data shown in Figure 7 were plotted to show the responses of individual peptides and donors. Significant cross-reactive responses to homologous TGFb-2 and TGFb-3 peptides (Pep01-2) were observed only once, while no cross-reactivity was observed in 7 other 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 homologue (Pep12-1). Significant responses (*) were defined by Fisher's exact P value < 0.01, peptide-to-control spot ratio > 2, and background-subtracted spots > 25.

[0038] Figure 9 . The TGFb-1 vaccine induces a robust immune response. Mice were immunized with two different synthetic long peptides (SLP), and the immune response was analyzed by IFNγ ELISPOT assay. The ELISPOT responses of individual mice are shown. The results of the control peptide are shown clearly.

[0039] Figure 10 A-D. The TGFb-1 vaccine drives changes in immune permissiveness derived from the TME. Tumor weights were compared between groups at the end of the experiment (day 21). The expression of TGFB-1 was detected by latency-associated peptide (LAP) staining. In animals inoculated with SLP2, CD4 + T cell infiltration was significantly enhanced, while CD8 + T cell infiltration remained unchanged.

[0040] Figure 11 . The TGFb-1 vaccine promotes targeted in vivo cell killing. An in vivo cytotoxicity assay was used to compare cell killing in mice inoculated with SLP1 and the class I epitope SLP1_Ib (SIYMFFNT). Inoculated mice were (repeatedly) injected with differentially labeled splenocytes loaded with the assay peptide (SLP1_Ib) or a control peptide. Cell killing was determined by comparing the recovery of splenocytes using splenocytes loaded with the control peptide as an internal control for cell recovery. OVA peptide was included as a positive control.

[0041] Brief Description of the Sequences

[0042] SEQ ID NO:1 is the amino acid sequence of the full-length precursor of human TGFb-1 (also known as TGFb-1 proprotein).

[0043] SEQ ID NO:2 is the amino acid sequence of the full-length precursor of human TGFb-2 (also known as TGFb-2 proprotein).

[0044] SEQ ID NO:3 is the amino acid sequence of the full-length precursor of human TGFb-3 (also known as TGFb-3 proprotein).

[0045] SEQ ID NO:4 is the signal peptide of human TGFb-1.

[0046] SEQ ID NO:5 is the amino acid sequence of the latency-associated peptide (LAP) domain of human TGFb-1.

[0047] SEQ ID NO:6 is the amino acid sequence of mature human TGFb-1.

[0048] SEQ ID NOs: 7-17 are the amino acid sequences of polypeptide fragments derived from human TGFb-1, respectively.

[0049] SEQ ID NO: 18 - 22 are the amino acid sequences of polypeptide fragments derived from human TGFb - 2, respectively.

[0050] SEQ ID NO: 23 - 27 are the amino acid sequences of polypeptide fragments derived from human TGFb - 2, respectively.

[0051] SEQ ID NO: 28 is the amino acid sequence of a polypeptide fragment derived from human TGFb - 1, which has sequence homology with human TGFb - 2 and TGFb - 3.

[0052] SEQ ID NO: 29 is the amino acid sequence of a polypeptide fragment derived from human TGFb - 2, which has sequence homology with human TGFb - 1 and TGFb - 3.

[0053] SEQ ID NO: 30 is the amino acid sequence of a polypeptide fragment derived from human TGFb - 3, which has sequence homology with human TGFb - 1 and TGFb - 2.

[0054] SEQ ID NO: 31 and 34 are synthetic long peptides (SLP), which contain predicted MHC class I and class II epitopes.

[0055] SEQ ID NO: 32, 33, 35 and 36 are the minimal peptides corresponding to the MHC class I and class II epitopes of SLP. Detailed implementation mode

[0056] It should be understood that different applications of the disclosed products and methods can be customized according to the specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing specific embodiments of the present invention only and are not intended to be limiting.

[0057] Definition

[0058] Unless otherwise defined herein, the scientific and technical terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following descriptions of the terms will apply, and where appropriate, terms used in the singular ("a", "an" and "the") will also include the plural, and vice versa, unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "multiple polypeptides" and the like. If any description of the terms conflicts with any document incorporated herein by reference, the following description of the terms shall prevail.

[0059] In cases where the terms "comprising" and "comprise" are used, there is also provided what is stated by "consisting essentially of" or "consisting of".

[0060] "Polypeptide" as used herein in its broadest sense refers 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 also includes longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including D or L optical isomers, amino acid analogs, and peptidomimetics.

[0061] The terms "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0062] The terms "patient" and "subject" are used interchangeably and generally refer to humans.

[0063] As used herein, "immunogenicity" means that a polypeptide is capable of eliciting an immune response against the TGFb protein, particularly the TGFb-1 protein, typically when the protein is present intracellularly or on the surface of cells expressing the TGFb-1 protein. In other words, the polypeptide can be described as being immunogenic to TGFb. The polypeptide can alternatively be described as an immunogenic fragment of TGFb. The immune response can refer to a T cell response, and thus the polypeptide can be described as an immunogenic fragment of TGFb containing a T cell epitope. After administration of the polypeptide to the individual (or the sample), an immune response can be detected in at least one individual (or a sample taken from the individual).

[0064] Any suitable method can be used, including in vitro methods, to identify a polypeptide as immunogenic. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics:

[0065] i. It is capable of eliciting IFN-γ-producing cells in the PBL population of healthy subjects and / or cancer patients, as determined by ELISPOT assay; and / or

[0066] ii. It is capable of detecting CTLs reactive with TGFb-1 in situ in tumor tissue samples; and / or iii. It is capable of inducing the in vitro growth of specific T cells.

[0067] Methods suitable for determining whether a polypeptide is immunogenic are also described in the Examples section below.

[0068] The polypeptides disclosed herein are capable of stimulating a "selective" immune response to TGFβ-1, such as a selective T cell response to TGFβ-1. In this context, a selective immune response to TGFβ-1 is considered to be greater than the immune response to TGFβ-2 or TGFβ-3. For example, if the polypeptide does not stimulate a T cell response to TGFβ-2 and / or TGFβ-3, then the polypeptide can be considered capable of stimulating a selective T cell response to TGFβ-1. Similarly, a polypeptide can be considered capable of stimulating a selective T cell response to TGFβ-1 provided that the polypeptide stimulates a T cell response to TGFβ-1 that is at least about 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold higher than the T cell response to TGFβ-2 and / or TGFβ-3 that the polypeptide is capable of stimulating.

[0069] To those skilled in the art, suitable assays for measuring a selective immune response to TGFβ-1 will be apparent. An exemplary assay useful for this purpose is the IFNγ ELISPOT assay. For example, a polypeptide can be identified as being capable of stimulating a selective immune response to TGFβ-1 if:

[0070] i. it is capable of eliciting cells that produce IFNγ in a population of peripheral blood leukocytes (PBL) from healthy subjects and / or cancer patients, as determined by an ELISPOT assay; and

[0071] ii. the cells that produce IFNγ produce less IFNγ when contacted with the corresponding polypeptide from TGFβ-2 or TGFβ-3, as determined by an ELISPOT assay.

[0072] References herein to "TGFβ", "TGF-β", "T-GF-β", etc. correspond to references to TGF-β. However, for the sake of avoiding the use of Greek symbols and to facilitate the reproducibility of the text, the previous nomenclature has been used.

[0073] Polypeptide

[0074] In any polypeptide described herein, the amino acid sequence can be modified by one, two, three, four, or five (at most five) additions, deletions, or substitutions compared to the polypeptide with the unmodified sequence, provided that the polypeptide with the modified sequence exhibits the same or increased immunogenicity against TGFb1. "Same" should be understood to mean that the polypeptide with the modified sequence does not exhibit a significantly reduced immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence. Any comparison of immunogenicity between sequences will be performed using the same assay. Unless otherwise stated, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acid can have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge similar to that of the amino acid it replaces. Alternatively, a conservative substitution can introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table A1 below. In cases where the amino acids have similar polarities, this can be determined by reference to the hydrophilicity scale of amino acid side chains in Table A2.

[0075] Table A1 - Chemical Properties of Amino Acids

[0076]

[0077] Table A2 - Hydrophilicity Scale

[0078]

[0079]

[0080] In any polypeptide disclosed herein, any one or more of the following modifications can be made to improve physicochemical properties (such as stability), provided that the polypeptide exhibits the same or increased immunogenicity against TGFb1 compared to the polypeptide with the unmodified sequence:

[0081] Replace the C-terminal amino acid with the corresponding amide (which can increase resistance to carboxypeptidase);

[0082] Replace the N-terminal amino acid with the corresponding acylated amino acid (which can increase resistance to aminopeptidase);

[0083] Replace one or more amino acids with the corresponding methylated amino acids (which can increase proteolytic resistance); and / or

[0084] Replace one or more amino acids with the corresponding D-configured amino acids (which can increase proteolytic resistance).

[0085] Any polypeptide disclosed herein may be linked at the N-terminus and / or C-terminus to at least one additional moiety to enhance solubility, stability, and / or facilitate manufacture / isolation, provided that the polypeptide exhibits the same or increased immunogenicity to TGFb1 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR may be added at the N-terminus and / or C-terminus. Other suitable moieties include Albumin or PEG (Polyethylene Glycol).

[0086] The polypeptides disclosed herein may be produced by any suitable means. For example, the polypeptides may be synthesized directly using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or by solution-phase peptide synthesis. Alternatively, the polypeptides may 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., Escherichia coli. Such cells may be cultured using conventional methods to produce the polypeptides of the present invention. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the present invention. The present invention also provides host cells comprising such nucleic acids or vectors.

[0087] The polypeptides of the present invention may be in a substantially isolated form. It may be mixed with carriers, preservatives, or diluents that do not interfere with the intended use, and / or with adjuvants, and still be considered substantially isolated. It may also be in a substantially purified form, in which case it generally comprises at least 90% of the protein in the preparation, e.g., at least 95%, 98%, or 99%.

[0088] For the purposes of the present invention, to determine the percent identity between two sequences (such as two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first sequence for optimal alignment with the second sequence). Then the nucleotides at each position are compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the nucleotides at that position are identical. The percent identity between the two sequences is a function of the number of positions shared by the sequences (i.e., identity% = number of identical positions / total number of positions in the reference sequence × 100).

[0089] Typically, sequence comparisons are performed over the length of the reference sequence. For example, if a user wishes to determine whether the identity of a given (“test”) sequence to SEQ ID NO:18 is less than 80%, then SEQ ID NO:18 would be the reference sequence. To assess whether the identity of a sequence to SEQ ID NO:18 (an example of a reference sequence) is less than 80%, one of ordinary skill in the art would align over the length of SEQ ID NO:18 and determine how many positions in the test sequence are the same as those in SEQ ID NO:18. If less than 80% of the positions are the same, then the identity of the test sequence to SEQ ID NO:18 is less than 80%. If the sequence is shorter than SEQ ID NO:18, then gaps or missing positions should be considered non-identical positions.

[0090] One of ordinary skill in the art is aware of different computer programs that can be used to determine homology or identity between two sequences. For example, mathematical algorithms can be used to perform sequence comparisons and determine the percent identity between two sequences. In an embodiment, the Needleman and Wunsch (1970) algorithm of the GAP program, which has been incorporated into the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), is used with a Blosum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid or nucleic acid sequences.

[0091] polynucleotide

[0092] Non-limiting examples of the 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. The polynucleotides of the present invention can be provided in isolated or substantially isolated form. Substantially isolated means that the polypeptide can be substantially but not completely separated from any surrounding medium. The polynucleotide can 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 that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences (e.g., in an expression vector), is transcribed (in the case of DNA) and translated (in the case of mRNA) in vivo into the polypeptide. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. For the purposes of the present invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. Transcription termination sequences can be located 3' of the coding sequence.

[0093] The polynucleotides can be synthesized according to methods well known in the art, as exemplified 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 an expression cassette that includes control sequences operably linked to the inserted sequence to permit expression of the polypeptide of the present invention in vivo. In turn, these expression cassettes are typically provided in a vector (e.g., a plasmid or a recombinant viral vector). Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing the polynucleotide of the present invention can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. Suitable vectors can be any vector capable of carrying a sufficient amount of genetic information and permitting expression of the polypeptide of the present invention.

[0094] Accordingly, the present invention includes expression vectors containing such polynucleotide sequences. Such expression vectors are routinely constructed in the field of molecular biology and can, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals that may be required and positioned in the correct orientation to permit expression of the peptide of the present invention. Other suitable vectors will be apparent to those skilled in the art. As a further example in this regard, we refer to the literature of Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0095] In one embodiment, the polynucleotide is mRNA. The mRNA can comprise:

[0096] a) an open reading frame (ORF) encoding at least one immunogenic polypeptide of the present invention;

[0097] b) a 5' cap at the 5' end;

[0098] c) a 5' untranslated region (UTR) included at the 5' of the ORF;

[0099] d) a 3' UTR included at the 3' of the ORF; and

[0100] e) a 3' polyadenylation sequence at the 3' end.

[0101] In the mRNA sequences encoding immunogenic polypeptides, each can be interspersed with cleavage-sensitive sites.

[0102] The ORF can include 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, and preferably wherein the ORF encodes at least 2, 3, 4, 5, 10 or more different immunogenic polypeptides.

[0103] The mRNA can be described as an mRNA vaccine against cancer or an mRNA cancer vaccine. mRNA vaccines are described in International Patent Application No. WO2015 / 164674, which is incorporated herein by reference in its entirety. The mRNA cancer vaccine of the present invention can be a composition, including a pharmaceutical composition. The present invention also includes methods of preparing, manufacturing, formulating and / or using the mRNA cancer vaccine.

[0104] The fact that the immunogenic polypeptide is expressed as an intracellular peptide from the RNA can provide advantages over delivery as an exogenous peptide. The RNA is delivered intracellularly and expresses the epitope near the appropriate cellular machinery for processing the epitope such that they will be recognized by the appropriate immune cells. In addition, the targeting sequence can have more specificity in the delivery of the peptide epitope. For example, C-terminal ubiquitin ligase targeting proteins (FBox proteins) can be used to target polypeptide processing to the proteasome and more closely mimic MHC processing. The constructs of the present invention can also include linkers, such as proteolytic cleavage sites optimized for APCs. These proteolytic sites provide an advantage because they enhance the processing of the peptide in APCs. When the mRNA cancer vaccine is delivered to a cell, the mRNA will be processed by the intracellular machinery into a polypeptide, and then the intracellular machinery will process the polypeptide into an immunogenic polypeptide capable of stimulating the desired immune response.

[0105] In some embodiments, the mRNA cancer vaccine encodes multiple immunogenic polypeptides. This can be described as a multi-epitope mRNA vaccine because each encoded immunogenic polypeptide contains at least one epitope. The RNA sequence encoding the immunogenic polypeptide can be interspersed with sequences encoding amino acid sequences recognized by proteolytic enzymes. Thus, in some embodiments, the mRNA cancer vaccine is an mRNA having an open reading frame encoding a propeptide because the encoded polypeptide sequence comprises multiple immunogenic polypeptides linked together directly or via a linker such as a cleavage-sensitive site. Exemplary propeptides have the following peptide sequences:

[0106] Tm–Yo-(X1-Yo-X2-Yo-…Xn)-Yo-Tm

[0107] Where: T is a targeting sequence, and m = 0-1. The targeting sequence can be included at the N-terminus, C-terminus, or both termini of the central peptide region. If the polypeptide has more than one targeting sequence, then these sequences can be the same or different.

[0108] X1, 2, etc. are each independently an immunogenic polypeptide sequence, and n = 0-1000. Each immunogenic polypeptide sequence represented by X can represent a unique immunogenic polypeptide sequence in the propeptide, or it can refer to a copy of the immunogenic polypeptide sequence. Thus, the propeptide encoded by the mRNA can be composed of multiple immunogenic polypeptide sequences, each sequence being unique and / or it can include more than 1 copy of each unique immunogenic polypeptide sequence. In some embodiments, the propeptide can 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.

[0109] Y is a linker sequence, preferably a cleavage-sensitive sequence, and o = 0-5. Each immunogenic polypeptide sequence can optionally have one or more linkers, optionally adjacent to the cleavage-sensitive site at the N-terminus and / or C-terminus. In a multi-epitope design, there can be cleavage-sensitive sites between two or more immunogenic polypeptide sequences. Alternatively, two or more immunogenic polypeptide sequences can be directly linked to each other or linked via a linker that is not a cleavage-sensitive site. The targeting sequence can also be linked to the immunogenic polypeptide sequence via a cleavage-sensitive site, or it can be directly linked to the immunogenic polypeptide sequence via a linker that is not a cleavage-sensitive site.

[0110] mRNA can encode one or more targeting sequences. This can be a targeting sequence for endosomes, such as a part of the transmembrane domain of lysosome-associated membrane protein (LAMP-1) or a part of the transmembrane domain of invariant chain (Ii). The targeting sequence can be a ubiquitination signal, which is attached to either or both ends of the encoded polypeptide. In other embodiments, the targeting sequence is a ubiquitination signal, which is attached to an internal site and / or either end of the encoded polypeptide. Thus, the RNA can contain a nucleic acid sequence encoding a ubiquitination signal at one or both ends of the nucleotides encoding the immunogenic polypeptide.

[0111] Ubiquitination is a post-translational modification, which is the process of attaching ubiquitin to a substrate target protein. The ubiquitination signal is a peptide sequence, which can target and process a peptide to one or more proteasomes. By using the ubiquitination signal to target and process a peptide, the intracellular processing of the peptide can more closely reproduce the antigen processing in antigen-presenting cells (APCs). The number of ubiquitins added to the antigen can enhance the efficacy of the processing step. For example, in polyubiquitination, after the first ubiquitin molecule attaches to the peptide, additional ubiquitin molecules are added. The ubiquitin molecules are linked by connecting the glycine residue of one ubiquitin to the lysine of the ubiquitin bound to the peptide to produce a ubiquitin chain. Each ubiquitin contains seven lysine residues and an N-terminus, which can serve as sites for ubiquitination. When four or more ubiquitin molecules are linked to the lysine residue on the peptide antigen, the 26S proteasome recognition complex internalizes it and degrades the protein into small peptides.

[0112] In some embodiments, the immunogenic polypeptide sequences can be linked by cleavage-sensitive sites. A cleavage-sensitive site is a peptide that is easily cleaved by an enzyme or protease. These sites are also called protease cleavage sites. Preferably, the protease is an intracellular enzyme. The protease can be a serine protease, threonine protease, cysteine protease, aspartic protease, glutamic protease or 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 with high abundance (high expression) in APCs. A cleavage-sensitive site sensitive to APC enzymes is called 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 proteases such as cathepsin D, cathepsin E and asparaginyl endopeptidase.

[0113] The cleavage-sensitive site can preferably be a cathepsin B or S-sensitive site. Exemplary cathepsin B-sensitive sites include but are not limited to those described in WO 2017 / 020026 (which is incorporated herein by reference; see WO

[0114] (SEQ ID NOs: 12 to 407 of WO 2017 / 020026). Exemplary cathepsin S sensitive sites include, but are not limited to, those described in WO 2017 / 020026 (see SEQ ID NOs: 3 to 5, 408 to 1122 of WO 2017 / 020026). Other cathepsin sensitive sites are known in the art or can be readily determined experimentally using digestion assays with routine experimentation only.

[0115] The mRNA cancer vaccine can comprise one or more polynucleotides encoding one or more immunogenic polypeptide sequences of the present invention. Exemplary polynucleotides can include at least one chemical modification. The polynucleotides can include various substitutions and / or insertions. As used herein with respect to polynucleotides, the term "chemical modification" or suitably "chemically modified" refers to a modification of one or more positions, patterns, percentages or amounts of ribose or deoxyribose nucleosides with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C).

[0116] Compared to unmodified polynucleotides, the modified polynucleotides exhibit reduced degradation in a cell or organism when introduced into the cell or organism. The modified polynucleotides can exhibit reduced immunogenicity (e.g., reduced innate response) in a cell or organism when introduced into the cell or organism. Modifications of polynucleotides are well known in the art and include, for example, those listed in WO 2017 / 020026. Generally, the modifications discussed in this section are not intended to refer to ribonucleotide modifications in the naturally occurring 5' end mRNA cap portion.

[0117] The polynucleotide can contain naturally occurring, non-naturally occurring modifications, or the polynucleotide can contain both natural and non-naturally occurring modifications. The polynucleotide of the mRNA cancer vaccine of the present invention can include any useful modifications, such as modifications to the sugar, nucleobase, or internucleoside linkage (e.g., modifications to the linked phosphate / phosphodiester bond / phosphodiester backbone). One or more atoms of the pyrimidine nucleobase can be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol group, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine). In certain embodiments, the modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modifications according to the present invention can be the modification of ribonucleic acid (RNA) into deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein. During the synthesis or post-synthesis process of the strand, non-naturally modified nucleotides can be introduced into the polynucleotide (e.g., the polynucleotide of the mRNA cancer vaccine) or nucleic acid to achieve the desired function or property. The modification can be an internucleotide lineage, a purine or pyrimidine base, or a sugar. The modification can be introduced at the end of the strand or anywhere else in the strand; by chemical synthesis or polymerase. Any region of the polynucleotide can be chemically modified.

[0118] The present disclosure provides modified nucleosides and nucleotides. As described herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method described herein (e.g., chemical methods, enzymatic methods, or recombinant methods to include one or more modified or non-natural nucleosides). A polynucleotide can contain one or more linked nucleoside regions. These regions can have variable backbone linkages. The linkage can be a standard phosphodiester bond, in which case the polynucleotide would contain nucleotide regions.

[0119] Modified nucleobase pairs include not only the standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows for the formation of hydrogen bonds between non-standard bases and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is the 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.

[0120] The mRNA may have at least one chemical modification, which is preferably selected from: pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydropseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methylpseudouridine, 4-thio-pseudouridine (4-thio-seudouridine), 5-azauridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0121] As used herein, "messenger RNA (mRNA)" refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and is capable of being translated in vitro, in vivo, in situ or ex vivo to produce the encoded peptide or polypeptide of interest. The basic composition of an mRNA molecule includes at least a coding region, a 5' UTR, a 3' UTR, a 5' cap and a 3' polyadenylation sequence. The mRNAs of the present invention generally include all these features.

[0122] The "5' untranslated region (UTR)" is the region of the mRNA that is directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome), and this region does not encode a protein or peptide.

[0123] The "3' untranslated region (UTR)" is the region of the mRNA that is directly downstream (i.e., 3') of the stop codon (i.e., the codon of the mRNA transcript that indicates the termination of translation), and this region does not encode a protein or peptide.

[0124] An "open reading frame" is a continuous segment of DNA that starts with a start codon (such as methionine (ATG)), ends with a stop codon (such as TAA, TAG or TGA), and encodes a protein or peptide.

[0125] The 5' cap is a nucleotide that is specifically modified at the 5' end of some primary transcripts (such as messenger RNA), which promotes stability and translation. It is usually composed of a guanine nucleotide linked to the mRNA by an unusual 5′ to 5′ triphosphate bond. This guanosine is directly methylated at the 7 position after capping by a methyltransferase in vivo. Therefore, it can be called a 7-methylguanylate cap, abbreviated as m7G. The preferred 5' cap is m7G(5')ppp(5')NlmpNp.

[0126] The 3'-tail sequence is a polyA tail, a polyA-G tetraloop, and / or a stem-loop sequence. The length of the 3'-tail sequence is typically between 40 and 200 nucleotides. In some embodiments, the 3'-tail sequence is a polyadenylate (polyA) tail. A "polyA tail" is a region of an mRNA that is located downstream of the 3'UTR, such as immediately downstream (i.e., 3'), and that contains multiple consecutive adenosine monophosphates. The polyA tail can contain 10 to 300 adenosine monophosphates. For example, the polyA 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 polyA tail contains 50 to 250 adenosine monophosphates. In the relevant biological context (e.g., in cells, in vivo, etc.), the function of the poly(A) tail is to protect the mRNA from enzymatic degradation, e.g., in the cytoplasm, and to contribute to transcription termination, export of the mRNA from the nucleus, and translation.

[0127] In some embodiments, the polynucleotide comprises from about 200 to about 3000 nucleotides (e.g., 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, and 2000 to 3000).

[0128] The polynucleotides of the invention can function as mRNA, but differ from wild-type mRNA in functional and / or structural characteristics. The mRNA cancer vaccines of the invention can be encoded by in vitro transcribed (IVT) polynucleotides. As used herein, an "in vitro transcription template (IVT)" refers to deoxyribonucleic acid (DNA) that is 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 will depend on the target mRNA encoded by the template.

[0129] mRNA can be prepared via any suitable synthetic route by any suitable technique known in the art. IVT methods are preferred. In vitro transcription (IVT) methods allow for template-directed synthesis of RNA molecules of almost any sequence. The size of RNA molecules that can be synthesized using IVT methods ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods allow for the synthesis of large amounts of RNA transcripts (e.g., amounts ranging from micrograms to milligrams) (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 D.C.: ASM Press, 2007. 262-299). Generally, IVT utilizes a DNA template that is characterized by a promoter sequence upstream of the sequence of interest. The promoter sequence is typically phage-derived (e.g., T7, T3, or SP6 promoter sequences), but many other promoter sequences are acceptable, including those that have been redesigned. Transcription of the DNA template is typically best achieved by using an RNA polymerase corresponding to a particular phage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT typically begins with dsDNA, but can also be performed on single strands. Suitable methods include, for example, those listed in WO2017 / 020026 (which is incorporated herein by reference).

[0130] The mRNA disclosed herein can be wholly or partially codon-optimized for human expression and / or reduced immune recognition. Codon optimization methods are known in the art and can be used to achieve various results, such as matching codon frequencies in the target and host organisms to ensure proper folding, biasing the GC content to increase mRNA stability or reduce secondary structure, minimizing tandem repeat codons or runs of bases that can impair gene construction or expression, customizing transcriptional and translational control regions, inserting or removing protein trafficking sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, adding, removing or shuffling protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites to regulate the translation rate to allow proper folding of the various domains of the protein, or reducing or eliminating problematic secondary structure within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples including services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. Preferably, the ORF sequence is optimized using an optimization algorithm.

[0131] The codon-optimized sequence can have less than 95%, 90%, 85%, 80% or 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest). The codon-optimized sequence can have 65% to 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest).

[0132] Exemplary codon-optimized RNAs can be RNAs with increased G / C levels. The G / C content of a nucleic acid molecule can affect the stability of the RNA. RNAs with an increased number of guanine (G) and / or cytosine (C) residues can be functionally more stable than nucleic acids containing a large number of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 (incorporated herein by reference) discloses a pharmaceutical composition containing an mRNA stabilized by sequence modification in the translation region. Due to the degeneracy of the genetic code, the modification is carried out by replacing those codons that promote greater RNA stability with existing codons without changing the resulting amino acid. The method is limited to the coding region of the RNA.

[0133] Compositions, formulations, encapsulation

[0134] The present invention provides a composition, which comprises the polypeptide and / or polynucleotide of the present invention. For example, the present invention provides a composition comprising: one or more polypeptides and / or one or more polynucleotides of the present invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0135] 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 present invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0136] 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 present invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0137] The carrier, preservative and excipient must be "acceptable" in the sense of being compatible with the other components of the composition and harmless to the subject to which the composition is administered. Generally, all components and the final composition are sterile and pyrogen-free.

[0138] The composition may be a pharmaceutical composition.

[0139] The composition may be a vaccine composition, preferably a TGFb-1 selective vaccine composition.

[0140] The composition may preferably comprise an adjuvant. An adjuvant is any substance added to the composition to enhance or otherwise alter the immune response elicited by the composition. Adjuvants are substances that generally promote an immune response. Adjuvants may also preferably have a depot effect, as they also cause slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants is provided on pages 61-63 of Goding's Monoclonal Antibodies: Principles & Practice (2nd Edition, 1986).

[0141] Adjuvants can be selected from the group consisting of: AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL+TDM+CWS) in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharide and its various derivatives (including lipid A), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (such as polyIC and polyAU acid), wax D from Mycobacterium, substances found in Corynebacterium parvum, Bordetella pertussis and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 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 proposed as adjuvants in immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0142] Preferred adjuvants used in the present invention include oil / surfactant-based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA-based adjuvants such as adjuvants comprising CpG oligonucleotide sequences. Other preferred adjuvants are virus dsRNA-based adjuvants such as poly I:C. GM-CSF and imidazoquinoline are also examples of preferred adjuvants.

[0143] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0144] On pages 61 - 63 of Goding's Monoclonal Antibodies: Principles & Practice (2nd Edition, 1986), it should also be noted that when the molecular weight of the antigen of interest is low or its immunogenicity is poor, it is recommended to conjugate it to an immunogenic carrier. Thus, the polypeptide of the present invention can be conjugated to a carrier. The carrier can exist independently of the adjuvant. For example, the function of the carrier can be to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, confer stability, increase biological activity or increase the serum half - life. In addition, the carrier can assist in presenting its polypeptide or its fragment to T cells. Thus, in the composition, the polypeptide can be bound to carriers such as those listed below. The carrier can be any suitable carrier known to those skilled in the art, for example, a protein or an antigen - presenting cell such as a dendritic cell (DC). Carrier proteins include keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin), immunoglobulins or hormones (such as insulin or palmitic acid). Alternatively, the carrier protein can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be a dextran such as agarose. The carrier must be physiologically acceptable and safe for humans.

[0145] If the composition contains excipients, then it must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and harmless to its recipient. Auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances, etc. can be present in the excipients. These excipients and auxiliary substances are generally agents that do not cause an immune response in the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include but are not limited to liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids such as acetate, propionate, malonate, benzoate, etc. An in - depth discussion of pharmaceutically acceptable excipients, carriers and auxiliary substances is provided in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0146] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and methods, all of which are readily available to those skilled in the art. Such compositions can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit dosage form, such as in an ampoule or in a multi-dose container optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations in an oily or aqueous carrier. In one embodiment of the composition, the active ingredient is provided in a dry (e.g., powder or granule) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and, in addition to containing the active ingredient, can also contain additional ingredients such as adjuvants, excipients, and auxiliary substances as described herein. For example, a sterile injectable preparation can be prepared using a non-toxic parenterally acceptable diluent or solvent (e.g., 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 monoglycerides or diglycerides of glycerol. Other useful compositions include those containing the active ingredient in microcrystalline form, in liposomal formulations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can contain pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts. Alternatively, the active ingredient of the composition can be encapsulated, adsorbed onto, or bound to a particulate carrier. Suitable particulate carriers include those derived from polymethylmethacrylate polymers, as well as PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, for example, Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, such as polymers like polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0147] Formulations of the compositions described herein can be prepared by any method known in the art or developed hereafter. Generally, such a preparation method includes the step of combining the active ingredient with excipients and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into the desired single-dose or multi-dose units. In the pharmaceutical compositions according to the present invention, the relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients will vary depending on the identity, size, and / or condition of the subject to be treated and further depending on the route of administration of the composition. By way of example, the composition can contain from 0.1% to 100%, such as from 0.5% to 50%, 1% to 30%, 5% to 80%, at least 80% (w / w) of the active ingredient.

[0148] One or more excipients can be used to formulate the mRNA cancer vaccine to: (1) increase stability; (2) increase cell transfection; (3) allow sustained release or delayed release (e.g., from a depot formulation); (4) alter biodistribution (e.g., target specific tissues or cell types); (5) increase the 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 and all solvents, dispersion media, diluents, or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives), the excipients of the present invention can include, but are not limited to: lipidoids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with the mRNA cancer vaccine (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0149] The mRNA and / or compositions disclosed herein can include stabilizing elements. It has been found that naturally occurring eukaryotic mRNA molecules contain stabilizing elements, including but not limited to the 5' and 3' UTRs, 5' cap, and 3' tail discussed elsewhere herein. Other stabilizing elements that can be included in the mRNA disclosed herein can include, for example, histone stem-loops. In some embodiments, the histone stem-loop is generally derived from a histone gene and includes intramolecular base pairing of two adjacent partially or fully reverse-complementary sequences separated by a spacer consisting of a short sequence that forms a structural loop. One or more AU-rich sequences can be removed from the mRNA. Such sequences may disrupt stability. The RNA vaccine can contain or not contain enhancer and / or promoter sequences, which can be modified or unmodified, or can be activated or inactivated.

[0150] The mRNA cancer vaccines disclosed herein can be formulated into lipid nanoparticles with a diameter of about 10 nm to about 200 nm, such as but not limited to about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm, about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm to about 100 nm, about 50 nm to about 150 nm, about 50 nm to about 200 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 60 nm to about 150 nm, about 60 nm to about 200 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 70 nm to about 150 nm, about 70 nm to about 200 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm, about 80 nm to about 150 nm, about 80 nm to about 200 nm, about 90 nm to about 100 nm, about 90 nm to about 150 nm, and / or about 90 nm to about 200 nm.

[0151] The diameter of the lipid nanoparticles can be about 10 nm to 500 nm.

[0152] In 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.

[0153] The lipid nanoparticles can be the limited-size lipid nanoparticles as described in International Patent Publication No. WO 2013 / 059922, the content of which is incorporated herein by reference in its entirety. The limited-size lipid nanoparticles can include a lipid bilayer surrounding an aqueous core or a hydrophobic core; wherein, the lipid bilayer can contain phospholipids, such as but not limited to: diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacyl phosphatidylcholine, and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In another aspect, the limited-size lipid nanoparticles can contain polyethylene glycol lipids, such as but not limited to DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0154] The RNA vaccine can be delivered, localized, and / or concentrated at a specific location using the delivery method described in International Patent Publication No. WO 2013 / 063530, the content of which is incorporated herein by reference in its entirety. As a non-limiting example, empty polymer particles can be administered to a subject before, simultaneously with, or after the RNA vaccine is administered to the subject. Once in contact with the subject, the empty polymer particles undergo a volume change and remain, embed, fix, or intercept at a specific location in the subject's body.

[0155] The lipid nanoparticle composition can contain cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. The lipid nanoparticle composition can contain a cationic lipid in a molar ratio of about 20-60%: 5-25% non-cationic lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid.

[0156] Method of Use

[0157] The polypeptide, polynucleotide, or composition of the present invention, or a combination thereof, can be used in a method for treating or preventing a disease or disorder in a subject. The polypeptide, polynucleotide, or composition of the present invention, or a combination thereof, can be used to prepare a medicament in a method for treating or preventing a disease or disorder in a subject. The method can include: administering the polypeptide, the polynucleotide, the composition, or the combination to the subject. A therapeutically or prophylactically effective amount of the polypeptide, the polynucleotide, the composition, or the combination can be administered to a subject in need thereof.

[0158] The disease or disorder is at least partially characterized by an inappropriate or excessive immunosuppressive function of TGFb1. The disease or disorder can be cancer, preferably cancer that expresses TGFb-1 and / or is associated with an inappropriate or excessive immunosuppressive function of TGFb-1. The cancer can be esophageal cancer or urothelial cancer. The cancer can be colorectal cancer, gastric cancer, head and neck cancer, melanoma, non-small cell lung cancer (NSCLC), or ovarian cancer. The cancer can be breast cancer, cervical cancer, liver cancer, or pancreatic cancer. The cancer can be a tumor.

[0159] The method can include administering simultaneously or sequentially with an additional cancer therapy. The additional cancer therapy can be a bispecific inhibitor of TGFb (such as TGFb-1) and PD-L1. The bispecific inhibitor may be capable of binding simultaneously to TGFb and PD-L1 and / or inhibiting the activities of TGFb and PD-L1. The bispecific inhibitor can be a fusion protein comprising an anti-TGFb moiety and an anti-PD-L1 moiety, optionally, wherein the anti-PD-L1 moiety comprises an anti-PD-L1 antibody or consists of an anti-PD-L1 antibody, and / or the anti-TGFb moiety comprises a TGFb receptor or a part thereof or consists of a TGFb receptor or a part thereof, such as TGFb receptor II or a part thereof.

[0160] The additional cancer therapy can be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiotherapy, immunostimulatory substances, gene therapy, or antibodies.

[0161] The antibody can be Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (=tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumabpegol), Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan), Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin), Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomabmerpentan), Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomabaritox), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (= atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox.

[0162] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, mepolizumab, reslizumab, tocilizumab, ustekinumab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tositumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab and vedotin.

[0163] Particularly preferred antibodies for use in the methods of the present invention include: daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab and ofatumumab.

[0164] The additional cancer therapy can be selected from the group consisting of coenzyme B12 (Actimide), azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dauno-rubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, lenalidomide (Revlimid), temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine and vinorelbine.

[0165] The polypeptide, polynucleotide and / or composition of the present invention can also be used to stimulate TGFb-1 selective T cells (such as CD4 + and / or CD8 +In the method of (T cells), the method includes contacting the cells with the polypeptide and / or the composition. The method can be carried out in vitro. The cells can be present in a sample taken 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 contacting the corresponding polypeptides 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 the corresponding polypeptides from TGFb-2 or TGFb-3. The reactivity of TGFb-1 selective T cells can be measured by methods obvious to those skilled in the art, such as the IFNγ ELISPOT assay.

[0166] 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 the method of regulating the tumor microenvironment (TME) of a subject. TGFb-1 can 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 cancer. Specifically, TGFb-1 can be expressed by various cell types in the TME, such as cancer-associated fibroblasts (CAF), CD8 + T cells, CD4 + T cells, regulatory CD4 + T cells, exhausted CD8 + 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, the polynucleotide, the composition, or the combination to the subject. The polypeptide of the present invention can trigger a TGFb-1 selective T cell response. Therefore, administering the polypeptide of the present invention and / or the composition of the present invention containing at least one polypeptide of the present invention can be used to regulate the TME of a subject with cancer. Regulating the TME can include enhancing T cell infiltration in the TME, such as enhancing CD4 + T cell infiltration in the TME.

[0167] The present invention is further illustrated by the following examples. However, these examples should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and the following examples, whether alone or in any combination, are important for implementing the present invention in its different forms.

[0168] Examples

[0169] Example 1 – Materials and Methods

[0170] Peptide

[0171] Peptides were synthesized by standard methods and dissolved in DMSO to obtain a stock concentration of 5 mM or 10 mM. The sequences of the peptides used in these experiments are shown in the section entitled "Sequences". Peptides are described by SEQ ID NO, name, or the starting and ending positions of each peptide sequence in the amino acid sequence of the full-length precursor of human TGFb. Each name can be used interchangeably, as shown in the table listed in the following sequence section. For example, the peptide of SEQ ID NO:7 can alternatively be called Pep01-1, or alternatively called TGFb-1 112-151 (given starting position 112 and ending position 151). The intended reference in each case is clear from the context.

[0172] In vitro ELISPOT assay

[0173] For in vitro ELISPOT, peripheral blood mononuclear cells (PBMCs) from healthy donors were pulsed with 20 μM TGFb-derived peptides and 20 U / ml IL-2 in 24-well plates for 7 days before being used for the ELISPOT assay. The cells were placed in 96-well nitrocellulose ELISPOT plates (MultiScreen IP FilterPlate, MSIPN4W50; Millipore) pre-coated with interferon γ (IFNγ) capture antibody (Mabtech). TGFb peptides were added to a final concentration of 5 μM, and control stimulation (DMSO) was added to the control wells and the plates were incubated at 37 °C for 16 - 20 hours. After incubation, the cells were washed, and biotinylated secondary antibody (Mabtech) was added and kept at room temperature for 2 hours. Unbound secondary antibody was washed off, and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech) was added and kept at room temperature for 1 hour. Unbound conjugated enzyme was washed off, and the assay was developed by adding BCIP / NBT substrate (Mabtech). The developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6 Ultimate-V analyzer using ImmunoSpot software v5.1.

[0174] Mouse tumor studies

[0175] 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), which was formulated in DMSO and then diluted with water to a total volume of 50 μl (per injection volume), and then mixed with an equal volume of Montanide ISA51 VG ST adjuvant to form an emulsion. On days 0, 7, and 14, the vaccine preparation was injected subcutaneously (s.c.) at the base of the tail. On day 0, MC38 tumor cells (2e5 per injection) were injected subcutaneously into the flank. Every 3 to 4 days, the tumors were measured with vernier calipers, and the tumor volume was calculated using the following formula: V = L × W2 / 2, where V is the tumor volume, L is the tumor length (major axis), and W is the tumor width (minor axis). Flow cytometry was used to analyze the tumor microenvironment (TME).

[0176] Freshly isolated tumors were dissociated by collagenase digestion to generate a single-cell suspension for flow cytometry analysis. Approximately 1 million cells were stained using a Symphony A1 flow cytometer (BD, Becton Dickinson) for flow cytometry analysis with the following antibodies: anti-mouse LAP BV421 (BD, 565638), anti-mouse CD4 BV605 (BD, 743156), and anti-mouse CD8 BV786 (BD, 563332). Data analysis was performed using FlowJo software.

[0177] In vivo cytotoxicity assay

[0178] The in vivo cytotoxicity assay was performed by injecting peptide-loaded splenocytes from untreated donor mice into inoculated mice. Freshly isolated splenocytes were incubated with 5 μM of peptide in medium (RPMI containing 10% fetal bovine serum) at 37 °C for 1 hour. The splenocytes were loaded with the assay peptide (SLP1_Ib) or a control peptide (P53, AIYKKSQHM) respectively. The splenocytes were then washed twice in 5 ml PBS + 0.5% BSA and then the cells loaded with the assay peptide were labeled with CellTrace Far Red (ThermoFisher), and the cells loaded with the control peptide were labeled with CellTrace Violet (ThermoFisher), using a recommended concentration of 1 / 100, for 20 minutes at 37 °C. The labeled cells were washed twice in PBS and then the same number of cells were mixed in PBS at a volume of about 8 million cells per 200 μl injection volume. The cells in PBS were injected intravenously. After 18 hours, cell killing was analyzed by isolating splenocytes from the injected mice and comparing the recovery of FarRed- and Violet-labeled cells by flow cytometry. Specific killing of the pulsed splenocytes was calculated as follows: (1 - [(FarRed / Violet)vaccinated × (Violet / FarRed)injected]) × 100%.

[0179] Example 2 – TGFb-1 Characterization in the Tumor Microenvironment of Multiple Solid Cancer Indications

[0180] Neogenomics MultiOmyx TM The Neogenomics MultiOmyx

[0181] Table 1 – Cancer types analyzed

[0182] Abbreviations Cancer Types CRC Colorectal Esophageal Esophageal Squamous Cell Carcinoma Gastric Gastric HNN Head and Neck Cancer Melanoma Melanoma (70% uveal, 30% cutaneous) NSCLC Non-Small Cell Lung Cancer (Adenocarcinoma) Ovarian Ovarian Cancer (Serous Papillary Adenocarcinoma) Urothelial Urothelial Carcinoma (Bladder Cancer)

[0183] Table 2 – Cell types and markers used to define them

[0184]

[0185]

[0186] As shown in Figures 1-4, it was found that TGFb-1 was highly expressed in tumor cells of esophageal cancer and urothelial cancer, while TGFb-1 was expressed in the TME of most cancer types. The proportion of cells expressing TGFb-1 in tumors was comparable to that of IDO1 and PD-L1, which are the antigens targeted by IO Biotech's lead therapies IO102 and IO103.

[0187] Example 3 – Identification and Characterization of TGFb-1 Specific Peptide Antigens

[0188] To identify peptides with high specificity for the TGFb-1 protein sequence, Clustal Omega ( www.ebi.ac.uk / Tools / msa / clustalo / ) sequence alignment was performed using the reference sequences from UniProt (P01137, P61812, and P10600 for TGFb-1, TGFb-2, and TGFb-3 respectively), as Figure 5 shown. The sequence alignment was visualized using Jalview ( www.jalview.org / ). Five peptides (Pep01-1 to Pep05-1) were selected from the low homology region 1 (amino acids 112-151 of SEQ ID NO:1), three peptides (Pep06-1 to Pep08-1) were selected from the low homology region 2 (amino acids 226-260 of SEQ ID NO:1), and three peptides (Pep09-1 to Pep11-1) were selected to include the sequence with intermediate homology (SEQ ID NO:205, also known as TGFb-15) disclosed in WO 2020 / 245264. Peptides were selected based on the following criteria: 1) length equal to or greater than 20 amino acids, 2) avoidance of any continuous fragment of 8 or more identical or highly similar amino acids, and 3) avoidance of cysteine residues. A single peptide (Pep12-1) was selected based on the high homology between TGFb-1, TGFb-2, and TGFb-3 and was only used for the study of TGFb-1 selectivity. Table 3 shows the selected TGFb-1 peptides, including the percentage of sequence identity with the corresponding peptides from TGFb-2 and TGFb-3 (details of the corresponding peptides from TGFb-3 and TGFb-3 are shown in Table 5).

[0189] Table 3 – Sequence Identity

[0190]

[0191]

[0192] Example 4 – Screening for Immune Responses to TGFb-1 Peptides

[0193] To determine whether low-homology peptides selected based on sequence alignment can elicit immune responses in humans, the IFNγ ELISPOT assay was used. PBMC from a total of 14 healthy donors were used to screen for immune responses. The PBMC were exposed to each peptide separately to induce peptide-specific immune responses and the proliferation of peptide-specific T cells. After 7 days, the frequency of peptide-specific T cells was determined using the IFNγ ELISPOT assay. As Figure 6 shown, the IFNγ ELISPOT assay identified several peptides that elicited strong (number of spots) and frequent (number of donors) immune responses. Five peptides were selected based on this (see Table 4). As shown in Figures 7 and Figure 8 shown, these five peptides were then analyzed for TGFb-1 selectivity by analyzing cross-reactive immune responses to TGFb-2 and TGFb-3 peptides in the IFNγ ELISPOT assay. These data indicate that the IFNγ ELISPOT immune responses to TGFb-1 peptides selected for low homology did not cross-react with homologous TGFb-2 and TGFb-3 peptides. In contrast, selection of a highly homologous peptide (Pep12-1) induced an immune response that cross-reacted with homologous TGFb-2 and TGFb-3 peptides (Pep12-2 and Pep12-3, respectively), to reflect the extent of the TGFb-1-specific response.

[0194] Table 4 – Summary of IFNγ ELISPOT screening

[0195]

[0196] Median spot = median of the mean spots minus background for each donor among all donors. Positive donors = number of donors (out of 14) showing a significant response (Fisher's test < 0.01, peptide:control ratio > 2, and mean spots (background subtracted) > 25). The selected peptides were used to test the specificity of the response to the TGFb-1 peptide.

[0197] Collectively, these data identified five peptides (Pep01-1, Pep04-1, Pep05-1, Pep08-1, and Pep09-1) that induced strong and frequent immune responses selective for TGFb-1 in healthy donors.

[0198] Example 5 – Function of TGFb-1 Peptide Vaccine in a Mouse Tumor Model

[0199] Based on synthetic long peptides (SLPs) encoding predicted MHC class I and II epitopes (SEQ ID NO: 31-36, see Table 1 below), a murine TGFb-1 vaccine was developed. When administered as a single therapy, preclinical studies of anti-TGFb therapy have generally failed to show any effect on tumor growth. Therefore, the goals here were mainly focused on: 1) developing an induction by CD4+ and CD8 + 1) a vaccine that elicits the strongest immune response consisting of CD4 and CD8 T cells; 2) determine any effect on tumor growth; 3) determine any effect on the TME; and 4) develop an assay to further characterize the vaccine-induced T cell response.

[0200] As Figure 9 shown, both SLP elicited a strong immune response by IFNγ ELISPOT assay. Immunization with SLP1 led to the recognition of minimal peptides encoding class I and class II epitopes. In contrast, SLP2 mainly induced class II responses (although class I responses cannot be excluded considering the untested class I epitopes). In animals immunized with SLP2, CD4 + T cell infiltration was significantly enhanced, while CD8 + T cell infiltration remained unchanged ( Figure 10 C and 10D). As Figure 11 shown, in vivo cytotoxicity assays demonstrated that immunization with SLP1 led to cytotoxic activity against cells loaded with the class I peptide antigen SLP1_Ib. Notably, the cytotoxic activity was higher in animals immunized with SLP1_Ib, indicating that the minimal epitope antigen SLP1_Ib induced better cytotoxic T cells compared to SLP1 ( Figure 11 ). Similar assays are being developed to evaluate the TGFb-1 vaccine in mice.

[0201] *****

[0202] Those skilled in the art will understand that the above-described embodiments can be changed without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined in this specification.

[0203] Various publications, articles, and patents are cited or described in the background art and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of the documents, acts, materials, devices, articles, etc. included in this specification is provided to provide the background of the present invention. This discussion does not admit that any or all of these matters constitute a part of the prior art of any invention disclosed or claimed.

[0204] Sequence Listing

[0205] In Table 5 below, unless otherwise stated, "start" and "stop" refer to positions in the full-length human TGFb preprotein (SEQ ID NO: 1, 2, or 3).

[0206] Table 5

[0207]

[0208]

[0209]

Claims

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

5.

2. The polypeptide according to claim 1, which does not contain cysteine residues.

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

4. The polypeptide according to any one of claims 1 to 3, which comprises or consists of: (a) 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) 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 wherein the polypeptide comprises or consists of at least 25, 26, 27, 28, 29 or 30 contiguous amino acids of SEQ ID NO:5, more preferably wherein the polypeptide comprises or consists of at least 30 contiguous amino acids of SEQ ID NO:

5.

5. The polypeptide according to any one of claims 1 to 4, which comprises the amino acid sequence of SEQ ID NO:32, optionally wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO:7, 10 or 11.

6. The polypeptide according to any one of claims 1 to 5, which comprises the amino acid sequence of any one of SEQ ID NO:7, 10, 32, 11, 14 or 15, preferably SEQ ID NO:7 or SEQ ID NO:10; or consists of the amino acid sequence of any one of SEQ ID NO: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 according to claim 7, wherein, The TGFb-1 selective T cells have low cross-reactivity to cells expressing and / or presenting polypeptides of TGFb-2 and / or TGFb-3.

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

10. A polynucleotide encoding the polypeptide according to any one of claims 1-9, which is optionally contained in a carrier.

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

12. A composition comprising the polypeptide according to any one of claims 1-9 and / or the polynucleotide according to claim 10 or 11 and an optional adjuvant.

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

14. The composition according to claim 12 or 13, which comprises an adjuvant selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, virus dsRNA-based adjuvants, imidazoquinolines and Montanide ISA adjuvants.

15. The composition according to claim 12 or 13, which is formulated in a lipid nanoparticle composition, wherein the composition comprises a polynucleotide and the polynucleotide is mRNA, optionally, wherein the lipid nanoparticle has an average diameter of 50-200 nm.

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

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

18. The method according to claim 17, wherein the disease or disorder is: (a) cancer, optionally selected from the group consisting of esophageal cancer and urothelial cancer, further 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 an inappropriate or excessive immunosuppressive function of cells expressing TGFb-1.

19. The method according to claim 17 or 18, wherein The disease or disorder is cancer, and the method further comprises: administering an additional cancer therapy, preferably an antibody, simultaneously or sequentially.

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

21. The method according to any one of claims 17 - 20, wherein, The disease or disorder is a tumor, and wherein: (a) The polypeptide, polynucleotide, and / or composition can modulate the tumor microenvironment (TME). Optionally, the modulation includes enhancing the infiltration of T cells into the TME. Preferably, the T cells are CD4 + T cells; (b) The method includes modulating the TME, optionally, wherein the modulation includes enhancing the infiltration of T cells into the TME, preferably, wherein the T cells are CD4 + T cells; and / or (c) administration of the polypeptide, the polynucleotide, and / or the composition stimulates a selective immune response against cells expressing TGFb-1 in the TME.

22. A method of stimulating TGFb-1 selective T cells, the method comprising: Contacting the T cells with a polypeptide as defined in any one of claims 1-9, a polynucleotide as defined in claim 10 or 11, and / or a composition as defined in any one of claims 12-16.

23. The method according to claim 22, wherein, The TGFb-1-selective T cells have low cross-reactivity to cells expressing and / or presenting polypeptides of TGFb-2 and / or TGFb-3.

24. The method according to claim 23, wherein, Measurement of cross-reactivity comprises comparing the reactivity of the TGFb-1-selective T cells to cells expressing and / or presenting a polypeptide of TGFb-1 with the reactivity of the TGFb-1-selective T cells to cells expressing and / or presenting a corresponding polypeptide from TGFb-2 or TGFb-3, optionally, wherein: (a) the reactivity of the TGFb-1-selective T cells to cells expressing and / or presenting a 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 the TGFb-1-selective T cells to a polypeptide of TGFb-1; and / or (b) cross-reactivity is determined by IFNγ ELISPOT assay.

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