Peptide and conjugates thereof

Abstract

A product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein: (i) the peptide is linked to: (a) human serum albumin (HSA), (b) a polyethylene glycol (PEG), or (c) an Fc domain; and / or (ii) the peptide comprises the amino acid sequence of SEQ ID NO: 4; and wherein the peptide is less than or equal to 50 amino acids in length.

Description

[0001]P128268PCT PEPTIDE AND CONJUGATES THEREOF FIELD OF THE INVENTION The present invention relates to peptides and peptide conjugates, and their use in the treatment of disease, for example cancer. 5 BACKGROUND TO THE INVENTION The αvβ6 and αvβ8 integrins are members of the human integrin superfamily. αvβ6 and αvβ8 are heterodimers formed from the αv chain and either the β6 or β8 chain. Integrins αvβ6 and αvβ8 are highly expressed in processes such as tissue modelling and wound healing. A growing body of evidence suggests that the αvβ6- and αvβ8-integrins are upregulated in many 10 tumors, and represent potential targets for tumor imaging and therapy. The expression level of αvβ6 and αvβ8 is correlated with poor prognosis in cancer patients. Integrin αvβ6 is overexpressed in pancreatic ductal adenocarcinoma (PDAC), head and neck squamous cell carcinoma, colon, liver, breast, ovarian, and other types of cancer and has important prognostic implications. Integrin αvβ8 overexpression is seen in various carcinoma cells and15by tumor-infiltrating regulatory T cells (Treg). Compounds capable of targeting these integrinscould be used, in principle, for delivering imaging and therapeutic agents to tumors. Furthermore, considering that both αvβ6 and αvβ8 have high affinity for the RGD sequence of the TGF-β1 latency associated peptide (LAP) and participate in the activation of TGFβ (a potent immunosuppressive cytokine), compounds capable of interfering with such interaction 20 may unleash anti-tumor activities. αvβ6 and αvβ8 are overexpressed in both primary and metastatic pancreatic ductal adenocarcinoma (PDAC), effective treatment of which is an urgent, unmet clinical need. PDAC represents the fourth leading cause of cancer-related death with a 5-year survival rate of about 10%. Novel chemotherapy regimens, such as FOLFIRINOX (fluorouracil, leucovorin, 25 irinotecan, and oxaliplatin) and gemcitabine with nab-paclitaxel (nanoparticle albumin-bound paclitaxel) have reported only incremental increases in overall survival compared with standard gemcitabine chemotherapy, thus underlying the need for more efficacious therapeutic strategies. Furthermore, the upregulation of αvβ6- and / or αvβ8-mediated TGFβ activation is a driver of other diseases, including idiopathic pulmonary fibrosis and primary 30 sclerosing cholangitis. 1 P128268PCT SUMMARY OF THE INVENTION The present inventors have identified peptides that target αvβ6 and / or αvβ8 integrins and that may mediate anti-tumor effects, and also reshape the tumour microenvironment. Without wishing to be bound by theory, peptides of the present invention may accumulate in αvβ6- or 5 αvβ8-positive tumors, and inhibit TGFβ activation by cancer cells. The present inventors have further identified peptides and products comprising peptides that have increased affinity and / or avidity for target integrins and integrin-expressing cells, as well as having increased plasma half-life. In one aspect, the invention provides a product comprising a peptide, wherein the peptide 10 comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to four amino acid substitutions, additions or deletions, wherein: (i) the peptide is linked to: (a) human serum albumin (HSA), (b) a polyethylene glycol (PEG), or 15 (c) an Fc domain; and / or (ii) the peptide comprises the amino acid sequence of SEQ ID NO: 4. In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three 20 amino acid substitutions, additions or deletions, wherein: (i) the peptide is linked to: (a) human serum albumin (HSA), (b) a polyethylene glycol (PEG), or (c) an Fc domain; 25 and / or (ii) the peptide comprises the amino acid sequence of SEQ ID NO: 4. 2 P128268PCT In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the peptide is linked to: (a) human serum albumin (HSA), (b) a polyethylene glycol (PEG), or (c) an Fc domain. 5 In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the peptide is linked to human serum albumin (HSA). In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having 10 up to three amino acid substitutions, additions or deletions, wherein the peptide is linked to a polyethylene glycol (PEG). In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the peptide is linked to an Fc domain. 15 In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the peptide is linked to human serum albumin (HSA). In some embodiments, the peptide is linked to a polyethylene glycol (PEG). In some embodiments, the peptide is linked to an Fc domain. 20 In some embodiments, the peptide is less than or equal to 50 amino acids in length. In some embodiments, the peptide is less than or equal to 45, 40, 35 or 30 amino acids in length. Preferably, the peptide is less than or equal to 30 amino acids in length. Suitably, the peptide is less than or equal to 29 amino acids in length. Suitably, the peptide is less than or equal to 28 amino acids in length. Suitably, the peptide is less than or equal to 27 amino acids in length. 25 More preferably, the peptide is less than or equal to 26 amino acids in length. For example, the peptide may be 26, 25, 24 or 23 amino acids in length. In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein: 30 (i) the peptide is linked to: (a) human serum albumin (HSA), 3 P128268PCT (b) a polyethylene glycol (PEG), or (c) an Fc domain; and / or (ii) the peptide comprises the amino acid sequence of SEQ ID NO: 4; 5 and wherein the peptide is less than or equal to 50 amino acids in length. The product may be, for example, a peptide or a peptide conjugate (e.g. in which the peptide is linked to another agent, such as HSA, a PEG or an Fc domain). In some embodiments, the product does not comprise more than 100 contiguous amino acids of SEQ ID NO: 19. 10 In some embodiments, the product does not comprise more than 80, 79, 78, 77 or 76 (preferably more than 76) contiguous amino acids of SEQ ID NO: 19. In some embodiments, the product does not comprise more than 75 contiguous amino acids of SEQ ID NO: 19. In some embodiments, the product does not comprise more than 50 contiguous amino acids of SEQ ID NO: 19. 15 In some embodiments, the product does not comprise more than 24 contiguous amino acids of SEQ ID NO: 19. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists of an amino acid sequence selected from the group consisting of: (i) SEQ ID NO: 2, (ii) SEQ ID NO: 3, and (iii) SEQ ID NO: 4, or a variant thereof having up to 20 three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide 25 comprises or consists of the amino acid sequence of SEQ ID NO: 3, or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 4, or a variant thereof having up to three amino acid substitutions, additions or deletions. 4 P128268PCT The variant may have, for example, up to two amino acid substitutions, additions or deletions. The variant may have, for example, one amino acid substitution, addition or deletion. In some embodiments, the peptide has a modified C-terminus. Suitably, the peptide may have an amidated C-terminus. In some embodiments, the peptide comprises or consists of the 5 amino acid sequence of SEQ ID NO: 20, or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists 10 of the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide is linked to HSA, a PEG or an Fc domain and the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, the peptide is linked to the HSA, PEG or Fc domain by a chemical crosslinker of a peptide linker. 15 In some embodiments, the HSA comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least 80% sequence identity thereto, for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the PEG is selected from the group consisting of PEG100, PEG200, PEG500, PEG1K, PEG2K, PEG5K, PEG10K, PEG20K, PEG50K and PEG100K. In some 20 embodiments, the PEG is PEG5K or PEG10K. In preferred embodiments, the PEG is PEG10K. In some embodiments, the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least 80% sequence identity thereto, for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some 25 embodiments, the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least 80% sequence identity thereto, for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, and the peptide is linked to HSA. 30 In some embodiments, the peptide is CFETLRGDLRILSILRX1QNLX2KELQD (SEQ ID NO: 2), wherein X1is propargylglycine and X2is azidolysine, and the peptide is linked to HSA. 5 P128268PCT In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, and the peptide is linked to a PEG. In some embodiments, the peptide is CFETLRGDLRILSILRX1QNLX2KELQD (SEQ ID NO: 2), wherein X1is propargylglycine and X2is azidolysine, and the peptide is linked to a PEG. In 5 some embodiments, the peptide is CFETLRGDLRILSILRX1QNLX2KELQD (SEQ ID NO: 2), wherein X1is propargylglycine and X2is azidolysine, and the peptide is linked to PEG10K. In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 3, and the peptide is linked to an Fc domain. In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ 10 ID NO: 3, and the peptide is linked to HSA. In some embodiments, the peptide is linked to a signal peptide, optionally wherein the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least 90% sequence identity thereto, for example at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. 15 In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 4. In one aspect, the invention provides a polynucleotide encoding the product of the invention. In one aspect, the invention provides a vector comprising the polynucleotide of the invention. In one aspect, the invention provides a pharmaceutical composition comprising the product, 20 polynucleotide or vector of the invention, and a pharmaceutically-acceptable carrier, diluent or excipient. In one aspect, the invention provides the product, polynucleotide or vector of the invention for use in therapy. In one aspect, the invention provides the product, polynucleotide or vector of the invention for 25 use in the treatment or prevention of cancer. In some embodiments, the cancer is ανβ6 integrin positive and / or ανβ8 integrin positive. In some embodiments, the cancer is selected from the group consisting of: pancreatic cancer, breast cancer, prostate cancer, fibrosarcoma, oral or skin squamous cell carcinoma, head cancer, neck cancer, ovarian cancer, lung cancer, kidney cancer, cervix cancer, colorectal 6 P128268PCT cancer, gastric cancer, liver cancer, melanoma, and brain tumor (e.g. glioblastoma, astrocytoma or metastatic brain tumors). In some embodiments, the cancer is a pancreatic ductal adenocarcinoma (PDAC). In some embodiments, the cancer is a liver metastasis of PDAC. 5 In some embodiments, the cancer is a mammary adenocarcinoma. In some embodiments, the cancer is a prostate adenocarcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a secondary tumor, wherein the secondary tumor is a metastasis of a primary tumor. In one aspect, the invention provides the product, polynucleotide or vector of the invention for 10 use in the treatment or prevention of fibrosis. In some embodiments, the fibrosis is related to ανβ6 integrin and / or ανβ8 integrin function. In some embodiments, the fibrosis is selected from the group consisting of: pulmonary fibrosis, liver fibrosis, kidney fibrosis, pancreatic fibrosis, and fibrotic posterior capsular opacification (PCO, also known as capsular fibrosis). 15 In some embodiments, the product, polynucleotide or vector is administered to a subject systemically. In some embodiments, the product, polynucleotide or vector is administered to a subject locally. In some embodiments, the product, polynucleotide or vector is administered intravenously. In some embodiments, the product, polynucleotide or vector is administered within the site of primary occurrence of a disease (e.g. intrapancreatically for PDAC, or 20 intrahepatically for liver fibrosis). In some embodiments, the product, polynucleotide or vector is administered within the site of metastasis dissemination (e.g. intrahepatic for PDAC). In some embodiments, the product, polynucleotide or vector is administered to a subject in combination with one or more further therapeutic agent. In some embodiments, the further therapeutic agent is a therapeutic agent used in the 25 treatment or prevention of cancer. In some embodiments, the further therapeutic agent is selected from an immunotherapy, chemotherapy, targeted therapy, hormone therapy or radiotherapy. In some embodiments, the further therapeutic agent is an immunotherapy. In some embodiments, the further therapeutic agent is a therapeutic agent used in the treatment or prevention of fibrosis. 7 P128268PCT In some embodiments, the invention provides the product, polynucleotide or vector in combination with one or further therapeutic agents for use in the treatment or prevention of cancer, wherein the further therapeutic agent is a therapeutic agent used in the treatment or prevention of cancer. 5 DESCRIPITON OF THE FIGURES Figure 1 – Biochemical characterization of 5a-HSA (Lot #A) A) SDS-PAGE analysis of 5a-HSA, HSA, and linker-HSA (*HSA) under reducing (+βMe) and non-reducing conditions (−βMe). B) MALDI-TOF mass spectra of HSA, *HSA, and 5a-HSA (lot #A). The average masses of the principal components and the estimated number of 10 peptides coupled to HSA molecules are indicated. C) Analytical gel-filtration chromatography of 5a-HSA, HSA, and peptide 5a on a Superdex 75 HR column. Void volume (Vo), total volume (Vt), and elution volume of molecular markers (158, 44, 17, 1.35 kDa) are indicated. Figure 2 – Biochemical characterization of 5a-HSA (Lot #B) MALDI-TOF mass spectra of HSA, *HSA, and 5a-HSA (Lot #B). The average masses of the 15 principal components and the estimated number of peptides coupled to HSA molecules are indicated. Figure 3 – Binding of 5a-HSA and peptide 5a to recombinant human αvβ6 and αvβ8 (competitive and direct binding assays) Schematic representation of the assays (left panels) and dose-response curves (middle-right 20 panels). A) Direct binding of 5a-HSA to αvβ6 or αvβ8 coated-microtiter plates as detected with an anti-HSA rabbit polyclonal antibody and HRP-labelled goat anti-rabbit antibody. B) Competitive binding of 5a-HSA and isoDGR-HRP conjugate to αvβ6 or αvβ8 coated-microtiter plates. C) Competitive binding of peptide 5a (upper panels) or 5a-HSA (lower panels) and biotinylated-αvβ6 or -αvβ8 integrins to Latent TGFβ1-coated microtiter plates, as detected with 25 HRP-labelled streptavidin (STV-HRP). Dots, mean±SE of technical duplicates. Inhibition constant (Ki) or dissociation constant (Kd) are also indicated in each panel. Figure 4 – Flow cytometry analysis of αvβ6 and αvβ8 expression in TS / A, WEHI-164, TRAMP- C2, 5M7101 / GFP / CEA, K8484 / GFP / CEA, BxPC-3 and 5637 cells Expression of αvβ6 and αvβ8 by the indicated murine (A) and human (B) cell lines. Flow 30 cytometry was performed as described in Nardelli et al. (2019) Chem Commun 55: 14777-80 using the indicated monoclonal antibodies, followed by an Alexa Fluor 488-goat anti-mouse, 8 P128268PCT or Alexa Fluor 488 goat anti-rabbit IgG polyclonal antibody, or an Alexa Fluor 647-goat anti- mouse polyclonal antibody (only for 5M7101 / GFP / CEA and K8484 / GFP / CEA). Figure 5 – 5a-HSA promotes cell adhesion of αvβ6 / αvβ8 single- or double-positive TRAMP- C2, BxPC-3, and 5637 cell lines 5 Ninety-six-well PVC microplates were coated with 5a-HSA or with *HSA, a control conjugate without peptide 5a, and seeded with the indicated cell lines (0.5-1x105cells / well). After 2-3 h of incubation, non-adherent cells were removed by washing, and the adherent cells were stained with crystal violet and quantified by spectrophotometric analysis. A) Representative photomicrographs of wells coated with 5a-HSA or *HSA; quantification of cell adhesion is also 10 shown (mean±SE, n=3-4 wells). B) Effect of peptides 5a and 2a, i.e., a control peptide with the RGE sequence instead of RGD, on the adhesion of TRAMP-C2 cells to microtiter plates coated with 5a-HSA or *HSA. Figure 6 – Peptide 5a and 5a-HSA inhibit the production of active TGFβ by cancer cells Effect of 5a, 2a, *HSA and 5a-HSA on TGFβ production by WEHI-164, TS / A, TRAMP-C, 15 5M7101 / GFP / CEA and 5637 cells. Cells were seeded on cell culture microplates, left to adhere for 2 h and treated with the indicated compounds for 48-72 h. The amount of active TGFβ in the supernatant was quantified using a bioassay based on HEK-Blue™ TGFβ cells. The results of one representative experiment per each cell line are shown (mean±SE, n=2-4 wells). **, P<0.01; ****P <0.0001 by two-tail t-test. 20 Figure 7 – Peptide 5a binds to human iTregs and inhibits the production of active TGFβ by iTregs A) Characterization of conventional T cells (Tconvs) and induced T-regulatory cells (iTregs) by FACS. Representative overlaid two-parameter FACS plots of Tconvs (blue contour plot) and iTregs (red contour plot) populations stained with the indicated antibodies. The gating25strategy used to identify the cell subset is shown (dashed rectangle with the arrow). B-C)Binding of 5a-IRDye680 and Cys-IRDye680 to peripheral blood mononuclear cells (PBMC), Tconvs and iTregs cell types. B) Representative overlaid two-parameter FACS plots of Tconvs (blue contour plot) and iTregs (red contour plot) incubated with Cys-IRDye680 or 5a-IRDye680 (200 nM) in 25 mM HEPES buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM30magnesium chloride, 1 mM manganese chloride and 2% w / v BSA for 1 h. The dashedrectangle delineates a subset of iTregs, which could bind 5a-IRDye680. C) PBMC (peripheral blood mononuclear cells), Tconvs or Tregs were incubated with 5a-IRDye680 or Cys- IRDye680. Specific binding is depicted as the ratio of the mean fluorescence intensity of 5a- 9 P128268PCT IRDye680 over Cys-IRDye680 (left panel) and the percentage of IRDye680 positive cells (right panel). Cumulative results of 1-3 experiments are shown, performed with the cells isolated from 1-4 donors (dots), Bar, mean±SE. *, P<0.05; **, P<0.01; by two-way ANOVA with post- hoc Tukey's multiple comparisons test. D) Effect of peptides 5a and 2a on active TGFβ 5production by Tconvs and iTregs. Cells were cultured for 25 days before the assay; on theday of the assay, they were seeded in a 96-well microplate and treated with the indicated peptides for 48 h. The amount of active TGFβ in the supernatant was quantified using a bioassay based on HEK-Blue™ TGFβ cells. The results of one experiment are shown (mean±SE, n=2-4 wells). The dotted line indicates the lower limit of detection of the assay. ***,10P<0.001 by two-way ANOVA with post-hoc Tukey's multiple comparisons test.Figure 8 – Pharmacokinetics of 5a-HSA in mice A) Scheme of 5a-HSA administration and blood sampling in healthy mice. BALB / c and C57BL / 6 mice were used for intraperitoneal (i.p.) or intravenous (i.v) pharmacokinetic studies, respectively. B) Plasma levels of 5a-HSA. Plasma levels of 5a-HSA were measured after i.p. 15 or i.v. administration using a homemade sandwich-ELISA for human serum albumin and 5a- HSA as the reference standard. The dashed lines indicate the Kd of 5a-HSA for αvβ6 and αvβ8 integrins. The plasma half-life of 5a-HSA was calculated using a one-phase exponential decay equation. Figure 9 – 5a-HSA inhibits TGFβ signaling in K8484 / GFP / CEA liver metastases 20 A-C) Characterization of K8484 / GFP / CEA liver metastases. A) Binding of 5a-IRDye680 and Cys-IRDye680 to K8484 / GFP / CEA cells as detected by flow cytometry. Dots, mean±SD of technical duplicates. ***, P<0.001 by unpaired two-tailed t-test analysis of the area under the curve for each peptide-IRDye680 binding curve calculated with GraphPad Prism software. B) Expression of integrin αvβ6 in mouse liver with K8484 / GFP / CEA metastases. C57BL / 6N male 25 mice were injected in the portal vein with 1.0x105K8484 / GFP / CEA cells, and liver tumor colonies were left to grow. On day 32, the liver was excised, embedded in paraffin, and processed for immunostaining with a rabbit anti-β6 polyclonal antibody, followed by a secondary HRP-labeled goat anti-rabbit polyclonal antibody and DAB substrate. A representative image of liver tissue with metastases (outlined by the dashed line) and 30 quantification of αvβ6 expression are shown (n=4 mice / group, >3 mets per liver were quantified). ****, P<0.0001 by unpaired two-tailed t-test analysis. C) Representative ex vivo images of the liver with K8484 / GFP / CEA metastases in mice injected with 1.2 pmol of 5a- IRDye800. Arrows, liver metastases; green signal, GFP-positive cells; red signal, 5a- IRDye800-positive cells. D-H) Effect of 5a-HSA on tumor growth and TGFβ signaling in mice 10 P128268PCT bearing K8484 / GFP / CEA liver mets, as detected by microscopy analysis and immunohistochemical quantification of p-SMAD2 / 3. D) Experimental scheme. K8484 / GFP / CEA cells were injected into the portal vein. Seventeen days later, the mice were randomized into two experimental groups: one group (n=5) received 5a-HSA, while the other 5 group (n=5) remained untreated (None). On day 21, the mice were sacrificed, and their livers were explanted for necroscopic and microscopic examination, followed by quantification of p- SMAD2 / 3. E) Representative microphotographs of liver tissue sections counterstained with H&E from untreated and 5a-HSA-treated mice. Red lines delineate liver metastases. F) Quantification of the number of PDAC liver mets found in the tissue sections. Bars, mean ± 10 SE, n=5 mice per group. Note that 3 out of 5 mice treated with 5a-HSA showed no countable liver metastases (i.e., a complete response, CR), as assessed by visual inspection of the tissue sections by bright-field microscopy. G) Immunohistochemical analysis of the expression of p-SMAD2 / 3 in mice bearing K8484 / GFP / CEA liver mets and treated with or without 5a-HSA. Representative microphotographs (acquired at 200x magnification and electronically zoomed- 15 in areas delineated by black dashed rectangles) of p-SMAD2 / 3 staining of liver metastases in untreated or 5a-HSA treated mice are shown. Red arrows indicate positive nuclei. H) Quantification of nuclear p-SMAD2 / 3 positive cells in liver metastases or adjacent healthy liver is shown. The number of positive nuclei was quantified using the QuPath software and is shown as box-plots with 5-95 percentile, median, and min-to-max values (n=2-5 mice per 20 group, ≥1 mets per liver were quantified). *, p<0.05, **p<0.01 by unpaired two-tailed t-test analysis. Figure 10 – 5a-HSA inhibits TGFβ signaling in subcutaneous TS / A tumors. A) Experimental scheme. TS / A cells were subcutaneously injected into 8 mice. On day 13, after cell injection, the mice were randomized into two experimental groups: one group (n=4) 25 received 5a-HSA (four treatments), while the other group (n=4) received the diluent (Vehicle). On day 17, the mice were sacrificed, and the tumors were removed, lysed, and processed for western blot analysis with anti-phospho- or total-SMAD antibody (p-SMAD3 and t-SMAD3, respectively). B) TS / A tumor growth curves. Mean±SE, n=4 mice. C) One representative western blot analysis, out of four performed, of tumor lysates with antibodies against p- 30 SMAD3, t-SMAD3 and β-actin (as loading control) is shown. D-E) Quantification of the ratio of normalized p-SMAD3 / t-SMAD3 in 4 independent western blots with the results reported separately for each mouse (D) or as a cumulative result of the 4 blots per mouse (E). Boxplots with median, interquartile and 5–95 percentile values. **p < 0.01 by t test. Figure 11 – Antitumor effect of 5a-HSA in mice bearing K8484 / GFP / CEA liver metastases 11 P128268PCT A) Experimental scheme. K8484 / GFP / CEA cells were injected in the portal vein. On day 14, mice were randomized into 2 experimental groups. One group (n=9) was treated with 2 weekly cycles of 5a-HSA (50 µg / mouse, i.p., for 4 consecutive days per week), while the other group (n=5) was left untreated (None). B) Quantification of the volume of liver mets by MRI at day 5 21, 33 and 47. Dots, total volume of liver mets per mouse. Bar, mean±SE. *, P< 0.05; by unpaired two-tailed t-test analysis. Mice were euthanized when the tumor burden reached 2000 mm3or when other signs of distress were observed. C) Kaplan-Meier survival curves. ***, P< 0.01, by long rank Mantel-Cox test. D) Cured mice (n=3) were re-challenged subcutaneously with a tumorigenic dose of K8484 / GFP / CEA cells (1x106cells / mouse) at day 120 post-intraportal 10 injection. In parallel, naïve mice (n=3) were challenged subcutaneously with the same cell suspension and served as controls. Tumor growth was monitored by measuring tumor volume using a caliper. Single tumor volume (thin lines) and mean tumor volume (thick lines) are shown. **, P< 0.05, by unpaired two-tailed t-test analysis of the area under the curve (AUC) for each tumor. Figure 12 – Pharmacological and toxicological effects of 5a-HSA in mice bearing different 15 tumors implanted subcutaneously Effect of 5a-HSA on i) the growth of TS / A mammary adenocarcinomas, WEHI-164 fibrosarcomas, or TRAMP-C2 prostate carcinomas, implanted s.c. in mice (panels A and B), ii) animal survival (panels C) and iii) body weight (panels D). Tumor-bearing mice were treated at the indicated times (arrows) with 40 or 50 µg of 5a-HSA (i.p.). For the WEHI-164 20 fibrosarcoma model, cumulative results of two independent experiments (each performed with groups of 6 mice) are shown. A) Graphs of the tumor growth (5-12 mice per group, as indicated, mean ± SE). *, P < 0.05 by unpaired two-tailed t-test analysis of the area under the curve for each tumor growth; B) waterfall plots of percentage change in tumor volume for individual mice over the indicated time; C) Kaplan-Meier survival curves (*, P<0.05 by long 25 rank Mantel-Cox test); D) change in body weight (mean±SE, 5-12 mice per group). See also Figure 13. Figure 13 – Pharmacological and toxicological effects of 5a-HSA in tumor-bearing mice Individual tumor growth curves and individual changes in body weights of the mice reported in Figure 12 are shown. Mice were euthanized when tumors reached 1000-1200 mm3or when 30 extensive tumor ulceration or other suffering symptoms were observed. Figure 14 – Effect of an anti-CD8α monoclonal antibody on the antitumor activity of 5a-HSA A) Scheme of the experiment. WEHI-164 tumor-bearing mice (6 mice / group) were injected (i.p.) with 200 µg (day 5 and 9) or with 100 µg (day 13) of an anti-CD8α monoclonal antibody. 12 P128268PCT Ten minutes later mice were injected with the dose of 5a-HSA (i.p.). The following days (days 6, 7, 8, 12, 14, 15) mice were treated with 5a-HSA. B) Individual tumor growth curves. C) Tumor volume (mean±SE). *, P< 0.05; ***, P< 0.001, ****, P< 0.0001 by two-way ANOVA with post-hoc Tukey's multiple comparisons test. D) Waterfall plots of percentage change in tumor 5 volume for individual mice over the indicated time. E) Kaplan-Meier survival curves. Mice were euthanized when tumors reached >800 mm3or when extensive tumor ulceration or other signs of distress were observed. The cumulative data of the (5a-HSA) and (5a-HSA + isotype control mAb) groups are reported. *P < 0.05; by log-rank (Mantel-Cox) test. Figure 15 – PD-L1 expression by TS / A, WEHI-164, TRAMPC-2, and 5M7101 / GFP / CEA cells 10 FACS analysis of cells was performed using the anti-PD-L1 mAb 10F.9G2 (5 µg / ml) followed by a goat anti-rat Alexa Fluor 488-labeled secondary antibodies (5 µg / ml). The binding of an isotype control antibody (clone RTK-4530) is also shown. Figure 16 – 5a-HSA synergizes with anti-PD-L1 mAb to decrease WEHI-164 tumor size and improve survival of tumor-bearing mice 15 A) Experimental scheme. WEHI-164 fibrosarcoma cells were subcutaneously injected into 24 mice. On day 5 post-implantation, the mice were randomized and grouped into four experimental groups (n=6 each). Mice were injected intraperitoneally with diluent (Vehicle) or with the indicated dose of 5a-HSA (at days 5, 7, 9, 12, and 14) or anti-PD-L1 mAb (on days 5 and 12) or the combination of 5a-HSA and anti-PD-L1 mAb (with anti-PD-L1 mAb administered 20 10 min before 5a-HSA). B-E) Effect of the indicated compounds on WEHI-164 tumor growth. B) Growth curves of tumors (n=6). Mean±SE. *, P< 0.05; **, P< 0.01, by unpaired two-tailed t-test analysis of the area under the curve for each tumor volume calculated with GraphPad Prism software. C) Waterfall plots of percentage change in tumor volume for individual mice over the indicated time. D) Individual tumor growth curves. E) Kaplan-Meier survival curves. 25 *, P< 0.05; **, P< 0.01, by long rank Mantel-Cox test. Mice were euthanized when tumors reached 1000-1200 mm3or when extensive tumor ulceration or other signs of distress were observed. F) Body weight changes in mice bearing WEHI-164 tumors after treatment with the indicated compounds (6 mice / group, mean±SE). Figure 17 – Combined treatment with 5a-HSA and anti-PD-L1 mAb induces long-term anti- 30 tumor immunity in the WEHI-164 fibrosarcoma model Tumor re-challenge of mice that survived 60 days in the WEHI-164 model after treatment with 5a-HSA in combination with anti-PD-L1 mAb (see Figure 15). Control mice (naïve) were not exposed previously to tumor cells. Tumor growth in re-challenged mice was monitored for 30 13 P128268PCT days, and then they were sacrificed, while control mice were monitored until day 12. Challenged mice were then used in further experiments. Figure 18_– Synergistic effect of 5a-HSA with anti-PD-L1 mAb on large TS / A tumors. TS / A mammary adenocarcinoma cells were subcutaneously injected into 53 mice. On day 10 post- 5 implantation, the mice were randomized and grouped into 8 experimental groups (n=5-6 mice). Mice were injected intraperitoneally with diluent (Vehicle) or with the indicated dose of 5a-HSA (8 treatments), anti-PD-L1 mAb (2 treatments) or their combination at the indicated times (arrows). The mAb was administered 10 min before 5a-HSA. A) Growth curves of each tumor. B-D) Tumor volumes of each group (mean±SE, n=5-6 mice / group). *, P< 0.05; **, P< 0.01, ***, P< 0.001 by10 unpaired two-tailed t-test analysis of the area under the curve (AUC) for each tumor. E-F) Kaplan- Meier survival curves of pooled results of the groups treated with 50 µg or 5 µg 5a-HSA, the groups treated with anti-PD-L1 mAb, or their combination. Mice were euthanized when tumors reached 800 mm3or when extensive tumor ulceration or other signs of distress were observed. *, P<0.05; **, P< 0.01, ****, P< 0.0001 by long rank Mantel-Cox test. 15 Figure 19 – Effect of anti-PD-L1 mAb alone and in combination with 5a-HSA in mice with 5M7101 / GFP / CEA liver metastases A) 5a-IRDye800 specifically targeted 5M7101 / GFP / CEA liver metastases. C57BL / 6N mice received (+) or not (-) an intraportal injection of 5M7101 / CEA / GFP cells (1x105cells / mouse). After 21 days, the mice underwent MRI to assess the presence of liver metastases. On day 20 32, the mice were injected i.v. with 5a-IRDye800 (1.2 nmol), and after 24 h, their livers were explanted and analyzed by optical imaging using the indicated filter sets. Control mice, which were not surgically manipulated, were injected with the vehicle or 5a-IRDye800 and used as a reference for quantifying autofluorescence and nonspecific uptake in the liver; Upper panel: representative MRI images of one liver injected (+) or not (–) with 5M7101 / CEA / GFP cells. 25 The boundaries of liver metastases are marked in red. Lower left panels, representative ex vivo fluorescence images of the livers of mice shown in Panel A and injected with 5a- IRDye800. Arrow, liver metastasis; green signal, GFP-positive cells; red signal, IRDye800- positive cells. Quantification of 5a-IRDye800 uptake in the normal liver or liver with metastasis 24 h post-injection. The dotted line corresponds to the autofluorescence signal in normal livers. 30 The livers were imaged on both sides, and the resulting radiance efficiency was averaged. Bar, mean±SE (n=3-4 liver). *, P<0.05; **, P< 0.01, by unpaired two-tailed t-test. B) Experimental scheme. The 5M7101 / GFP / CEA cells were injected as described above. Fourteen days later, mice were randomized into 3 groups (n=5 mice / group) and treated with anti-PD-L1 alone, with the combination of anti-PD-L1 and 5a-HSA or left untreated. Tumor 35 growth was monitored by MRI. C) Effect of the indicated treatments on liver metastases at day 14 P128268PCT 21 as assessed by MRI. The total metastasis volume of each mouse and the % of complete responses (CR) observed are shown. Dots, volume of the tumor burden of each mouse (n=5); bar, median. Figure 20 – Effect of 5a-HSA and murine S-NGR-TNF, alone and in combination, on the 5 growth of subcutaneous WEHI-164 tumors A) Experimental scheme. WEHI-164 cells were subcutaneously injected into 12 mice. On day 5 post-implantation, the mice were randomized into four experimental groups. Mice were injected intraperitoneally with diluent (Vehicle) or with the indicated doses of 5a-HSA and murine S-NGR-TNF, alone or in combination (at days 5, 7, 9, 12, 14, 16). Cumulative results 10 of 2 independent experiments are shown (each conducted with 6 mice / group). B) Individual tumor growth curves; C) waterfall plots showing the percentage change in tumor volume for individual mice over the indicated time; D) Tumor volume (n=12, mean±SE; *, P< 0.05; **, P< 0.01, by unpaired two-tailed t-test analysis of the area under the curve for each tumor volume calculated with GraphPad Prism software); E) Kaplan-Meier survival curves (*, P< 0.05; **, P<150.01, by long rank Mantel-Cox test). Mice were euthanized when tumors reached 800 mm3orwhen extensive tumor ulceration or other signs of distress were observed. Figure 21 – Effect of 5a-HSA, murine S-NGR-TNF, and anti-PD-L1 antibody, alone and in combination, on the growth of subcutaneous WEHI-164 tumors A) Experimental scheme. WEHI-164 cells were subcutaneously injected into 6 mice. On day 20 5 post-implantation, the mice were injected intraperitoneally with diluent (Vehicle) or with the indicated doses of 5a-HSA, murine S-NGR-TNF, or an anti-PD-L1 antibody alone or in combination. B) Tumor volumes (mean+SE); C) Waterfall plots showing the percentage change in tumor volume for individual mice over the indicated time. D) Kaplan-Meier survival curves (*, P< 0.05, by long rank Mantel-Cox test). Mice were euthanized when tumors reached 25 ≥800 mm3or when extensive tumor ulceration or other signs of distress were observed. Figure 22 – Schematic representation and characterization of peptibody 4∆-mFc1. A) N-terminal sequence of peptibody 4∆-mFc1 consisting of: a Signal secretion sequence (derived from a human heavy chain IgG1), Peptide 4∆ (reported in bold-red), 4-glycine spacer fused to a murine IgG1-Fc domain, consisting of hinge plus CH2 and CH3 domains (grey box). 30 Amino acids are reported with the single letter code. Arrow indicates the expected cleavage site of the signal secretion peptide. B) Schematic representation of the peptibody 4∆-mFc1. 15 P128268PCT C) SDS-PAGE analysis of affinity-purified peptibody 4∆-mFc1 under reducing (βMe +) and non-reducing (βMe –) conditions. Two micrograms of peptibody per lane was loaded. MW, molecular weight marker. Figure 23 – Direct and competitive integrin binding assays of peptibody 4∆-mFc1 to purified 5 human αvβ1, αvβ3, αvβ6, αvβ8, and α5β1 A) Schematic representation of the direct integrin binding Assay 1 (left); binding curves of 4∆- mFc1 to microtiter plates coated with the indicated integrin, as detected using an HRP-labeled rat anti-mFc polyclonal antibody (Anti-Fc pAb) (right). The binding of 4∆-mFc1 was determined by subtracting the unspecific binding of 4∆-mFc1 to BSA-coated microtiter wells. 10 B) Schematic representation of the competitive integrin binding Assay 2 (left); binding curves of 4∆-mFc1 to microtiter plates coated with the indicated integrin, as detected using an isoDGR-HRP conjugate. Mean ± SE of technical duplicates or triplicates. The dissociation and inhibition constants values (Kd and Ki) were calculated using the GraphPad Prism software. Figure 24 – Binding of 4∆-mFc1 to human T3M-4 pancreatic ductal adenocarcinoma cells 15 A) Left panel: binding of 4∆-mFc1 to αvβ6 / αvβ8-positive T3M-4 cells at 4°C, as detected by FACS analysis using a goat anti-mouse Alexa Fluor 488-labeled secondary antibody. The binding of a control isotype antibody (murine IgG1, clone MOPC-31C) is also shown. Right panel: quantification of protein binding. The dots represent the mean ± SE of duplicates. The effective concentration 50 (EC50) of the peptibody is shown. B) Effect of the temperature on 20 the binding of 4∆-mFc1 to T3M-4 cells as determined by FACS analysis. The cells were incubated with various amounts of 4∆-mFc1 and then incubated at 4°C or 37°C for 1 h. After cell washing, the bound 4∆-mFc1 to the cell surface was detected as described above. Figure 25 – 4∆-mFc1 inhibits TGFβ activation mediated by 5M701 / GFP / CEA and 5637 cancer cells 25 Cells were seeded in cell culture microtiter plates, allowed to adhere for 2 h, and treated with the indicated proteins and peptides for 24-48 h. The amount of active TGFβ in the supernatant was quantified using a bioassay based on HEK-Blue™ TGFβ cells. Two independent experiments have been performed, and the results of one representative experiment are shown in the upper panels (mean±SE, n=2-4 wells). The EC50of each compound, as 30 calculated from the results of the two experiments, is shown in the lower panel. 16 P128268PCT Figure 26 – Binding of the 4∆-mFc1 to human PDAC tissue sections Binding of the 4∆-mFc1 (left) or control isotype murine IgG1(clone MOPC-31C) (right) (5 µg / ml) to fresh frozen PDAC sections. Binding was detected by immunohistochemical analysis using an HRP-labelled goat anti-mouse antibody and DAB substrate. 5 Figure 27 – Binding of 4∆-mFc1 / IRDye800 to ανβ6 and plasma half-life of this conjugate in mice A) Binding of 4∆-mFc1 / IRDye800 and 5a-IRDye800 to αvβ6-coated microtiter plates. B) Plasma pharmacokinetics of 4∆-mFc1 / IRDye800 in mice. C57BL / 6 (n=4) were injected i.v. with 4∆-mFc1-IRDye800 (50 µg / mouse). Their blood was then collected into heparinized tubes 10 at various time points. Plasma samples (20 µl) were diluted with PBS (50 µl, final volume) and transferred to a black 96-well microtiter plate. The amount of fluorescence in each sample was then quantified using a Li-Cor Odyssey scanner (dots: mean ± SE of 5 mice). Plasma half-life of 4∆-mFc1-IRDye800 was calculated using a one-phase exponential decay equation (GraphPad Prism software, San Diego California). 15 Figure 28 – 4∆-mFc1 inhibits the growth of subcutaneous lesions of WEHI-164 fibrosarcomas in mice A) Experimental scheme. WEHI-164 tumor-bearing mice (6 mice / group) were treated at the indicated times (arrows) with 35 µg of 4∆-mFc1 or vehicle (i.p.). B) Tumor volumes (mean ± SE). *, P < 0.05; **, P<0.01 by two-way ANOVA with post-hoc Sídák's multiple comparisons 20 test. C) Waterfall plots of percentage change in tumor volume for individual mice over the indicated time. D) Single tumor volume growth. E) Kaplan-Meier survival curves. Mice were euthanized when tumors reached ≥800 mm3or when extensive tumor ulceration or other signs of distress were observed. F) Animal weight plot (mean ± SE, n=6 mice). Figure 29 – Biochemical characterization of 5a-PEG5K and 5a-PEG10K 25 A) RP-HPLC of 5a-PEG5K and 5a-PEG10K. B) Analytical gel filtration chromatography of 5a- PEG5K and 5a-PEG10K (Superdex 75 HR column). Void volume (Vo), total volume (Vt). The elution volumes of molecular markers are indicated. Figure 30 – Direct and competitive binding of PEGylate peptide 5a to purified human αvβ6 and αvβ8 17 P128268PCT A) Schematic representation of the direct integrin binding assay (Assay 1) (left); binding curves obtained with the indicated compound to microtiter plates coated with the indicated integrin, as detected using an HRP-labeled mouse anti-PEG antibody (Anti-PEG mAb) (right). B) Schematic representation of the competitive integrin binding assay (Assay 2) (left); binding of 5 isoDGR-HRP conjugate to integrins, as obtained in the presence of various concentrations of the indicated competitors. Each point represents the mean ± SE of technical duplicates or triplicates. Ki and Kd values were calculated using the GraphPad Prism software. Figure 31 – Binding of PEGylated peptide 5a to human T3M-4 pancreatic ductal adenocarcinoma cells 10 A) Binding of 5a-PEG5K, 5a-PEG10K, and PEG8K to αvβ6 / αvβ8-double positive T3M-4 cells, as detected by FACS using an anti-PEG mAb, followed by a FITC-labeled secondary antibody. B) Quantification of the binding. Each point represents the mean ± SE of technical duplicates. ****, p<0.0001, by two-way ANOVA. Figure 32 – Plasma half-life of 5a-PEG5K and 5a-PEG10K in mice 15 RAG2− / −γc− / −mice were divided into 2 groups (4 mice / group) and injected i.v. with 5a-PEG5K or 5a-PEG10K (50 µg / mouse). Their blood was then collected into heparinized tubes at 5, 30, 120, 240, 1440, 2880, and 5760 min (group 1) or after 15, 60, 180, 360, 1440, 2880, and 5760 min (group 2). Plasma samples were diluted with PBS, and plasma levels of the conjugates were determined using a competitive PEG-ELISA (Polyethylene Glycol Backbone ELISA Kit,20 Life Diagnostics, Inc.). Each point represents mean ± SE (2-4 mice / group); the estimated half- life of both compounds is shown. Figure 33 – 5a-PEG10K inhibits the growth of subcutaneous lesions of WEHI-164 fibrosarcomas in mice A) Scheme of the experiment. WEHI-164 tumor-bearing mice (six mice per group) were 25 injected (i.p.) with or without the indicated doses of 5a-PEG10K. B) Tumor volumes (n=6 mice / group, mean±SE). *, P< 0.05; **, P< 0.01, by two-way ANOVA with post-hoc Tukey's multiple comparisons test. C) Waterfall plots of percentage change in tumor volume for individual mice over the indicated time. D) Individual tumor growth curves. Figure 34 – αvβ6 and αvβ8 inhibitor constant (Ki) values of CgA-derived peptides 30 A) A mini library of peptides derived from the indicated peptide 4a, in which residues of the R47I48L49sequence were modified as indicated, was screened by competitive αvβ6 and αvβ8 18 P128268PCT integrin binding assays using an isoDGR-HRP conjugate as a probe. Ki values determined in independent assays for a given peptide and for control peptides (4a, 4, 5a, and 5) are represented by blue and red dots. Box-plots with median, interquartile and 5–95 percentile values are shown. The dotted line represents the average Ki of peptide 5a. ***, P < 0.01, ****, 5 P < 0.0001 by one way ANOVA. From this study, the peptide with A48(CFETLRGDLRA48LSILRHQNLLKELQD, called peptide 7a, SEQ ID NO: 4) was selected for further optimization. B) A mini library of C-terminal amidated peptides derived from peptide 7a, in which R47was modified as indicated, and screened as reported above. Note that none of these peptides were 10 significantly more potent than the parental peptide 7a. Figure 35 – Biochemical characterization of 5a-, 2a-, CG-A20FMDV2-, and Cys-pHsDye conjugates. A) Schematic representation of the pH-sensitive dye (pHsDye, from Promega) coupled to thiol- containing peptides via maleimide chemistry. B) RP-HPLC analysis of peptides and reaction 15 mixtures 16 h after conjugation with pHsDye-maleimide (showing unreacted materials and products). The amount of each compound loaded on the C18 column is indicated in parentheses. Peptide 5a, unlike the others, formed a partially insoluble product upon reaction with pHsDye. Following incubation, the mixture was centrifuged, the pellet was dissolved in water containing 16% DMSO and 5% acetonitrile, and the supernatant (SN) was recovered. 20 The dissolved pellet was then pooled with the SN. Asterisks denote a peak likely corresponding to DMSO in the 5a-pHsDye sample. The red and black dashed lines mark the retention times of unreacted pHsDye and free peptide 5a, respectively. C) Expected and observed monoisotopic masses of peptide-pHsDye conjugates as determined by mass spectrometry analysis of reaction mixtures (not-purified). D) Competitive binding of 5a-HRP to 25 αvβ6-coated microtiter plates in the presence of increasing concentrations of peptide-pHsDye conjugates, as assessed by αvβ6-competitive integrin binding assay. Mean ± SEM (n=4 technical replicates). Figure 36 – Expression of αvβ6 and αvβ8 by BxPC-3 and LN-229 cells, as assessed by FACS analysis. 30 FACS analysis was carried out using the indicated monoclonal antibodies (mAb), followed by AlexaFluor 488-goat anti-mouse or anti-rabbit IgG polyclonal antibodies. These experiments were performed using a new batch of BxPC-3 cells obtained from ATCC, which also expressed 19 P128268PCT the αvβ8 integrin, whereas previous studies reported little or no expression of this integrin (see Figure 4) and (Monieri et al. (2023) Int J Biol Sci 19: 156-661). Figure 37 – Peptide 5a (coupled to a pH-sensitive dye, pHsDye) is efficiently internalized by PDAC and GBM cells. 5 Cells were seeded in a 96-well microtiter plate (40,000 cells per well) and incubated overnight. The following day, the culture medium was replaced with fresh medium containing either 10% fetal bovine serum (+ Serum) or serum-free medium (– Serum), along with varying concentrations of the indicated peptide–pHs-Dye conjugates. After 16-18 h of incubation, cells were washed with PBS, fixed, and the uptake of peptide–pHsDye conjugates was quantified10 using either a microplate reader or a scanner. A) Quantification of internalized peptide– pHsDye conjugates in BxPC-3 cells as a function of dye concentration using a Tecan Infinite 200 Pro plate reader (excitation / emission: 532 / 572 nm). Mean ± SEM from n=3–4 technical replicates are shown. B) Left panel: Representative image of microtiter wells seeded with LN- 229 cells and treated with the indicated concentrations of peptide–pHsDye conjugates. 15 Images were acquired using a Sapphire biomolecular imager scanner (FL 534 nm filter). The yellow signal indicates the presence of an internalized fluorescence dye. Center panel: Representative confocal images of cells incubated with 18 nM of 5a-, 2a-, or Cys–pHsDye and counterstained with DAPI (blue signal). Images were captured using an ImageXpress Micro Confocal Imaging System with a 40× objective. Right panel: Quantification of 20 internalized peptide–pHsDye conjugates in LN-229 cells as a function of pHs-Dye concentration. Mean±SEM from n=3–4 technical replicates. Figure 38 – Sodium azide inhibits the internalization of 5a-pHsDye in LN-229 cells. A) Representative image of microtiter wells seeded with LN-229 cells and incubated with 100 nM 5a-pHsDye, Cys-pHsDye, or vehicle (None) in the absence (–) or presence (+) of sodium 25 azide (0.2%) after 2 h of incubation at 37°C, 5% CO2.Images were acquired using a Sapphire biomolecular imager scanner. B) Quantification of internalized fluorescence intensity in LN- 229 cells. Mean± SEM of n=3 replicate wells per condition. ****, p<0.001; **, p<0.01, by unpaired two tail t-test. Figure 39 – Peptide 5a and CG_A20FMDV2 are internalized to a similar extent by BxPC-3 30 cells but not by LN-229 cells. BxPC-3 and LN-229 cells were seeded in 96-well microtiter plates (30,000 cells / well). After 16 h, cells were incubated with increasing concentrations of 5a-pHsDye, CG_A20FMDV2- pHsDye, or Cys-pHsDye (negative control) for an additional 18–20 h. Following incubation, 20 P128268PCT cells were washed, and internalized fluorescence was quantified using a Sapphire biomolecular scanner. Data represent mean ± SEM from n = 5–8 replicate wells per condition. Results shown are representative of two independent experiments, each performed with four technical replicates. For BxPC-3 cells, the maximum fluorescence intensities of internalized 5 5a and CG_A20FMDV2 were normalized to 100% to enable direct comparison. For LN-229 cells, the maximum intensity of internalized 5a was normalized to 100% and used to assess the relative internalization of CG_A20FMDV2-pHsDye. Figure 40 – Peptide 5a internalization is dependent on αvβ6 / αvβ8 integrin recognition. A) Flow cytometry analysis of integrin expression in wild-type) and ITGB6 / B8 double-knockout 10 (ITGB6 / B8-KO) T3M-4 cells using the monoclonal antibodies (mAb) against αvβ6, αvβ8, αvβ5, α5β1, β1, and αv. T3M-4 cells show surface expression of αvβ6 and αvβ8, which is abrogated in the KO cells. Expression of other integrins (αvβ5, α5β1, β1, αv) is comparable in both cell lines. B) Peptide 5a binding assay. T3M-4 and ITGB6 / B8-KO cells were incubated with increasing concentrations of peptide-IRDye conjugates (5a-, Cys-, and 2a-IRDye) in 96-well 15 plates at 37^°C, 5% CO₂ for 18–20^h. After washing, bound fluorescence was detected using a Sapphire biomolecular imager (Azure Biosystems). A representative fluorescence image of the microtiter wells is shown (left), along with quantitative analysis of binding (right). Data represent mean ± SE from 3-4 technical replicates. C) Peptide 5a internalization assay. T3M- 4 and ITGB6 / B8-KO cells were incubated with increasing concentrations of 5a- or Cys- 20 pHsDye). After washing, internalized fluorescence was quantified using a Sapphire biomolecular imager. Internalization was strongly reduced in KO cells compared to WT. A representative fluorescence image of the microtiter wells seeded with the indicated cell lines incubated with various amounts of 5a-pHsDye at 37°C, 5% CO2for 18-20 h shown (left), along with quantitative analysis of binding (right). Cumulative data from 2 independent experiments 25 are shown. Mean ± SE from 4-8 technical replicates. Figure 41 – Binding of peptibody 4Δ-mFc1 to human T3M-4 pancreatic ductal adenocarcinoma cells. A) Competition of 4∆-mFc1 binding to T3M-4 cells with i) peptide A20FMDV (a known ligand of αvβ6), ii) acetyl-HGRGDLGRLKK-amide peptide, derived from LAP-TGFβ3 (a ligand of 30 αvβ6), or iii) peptide 5a (a bispecific ligand of αvβ6 and αvβ8). One nanomolar 4∆-mFc1 was mixed with the indicated peptides (100 nM) and added to T3M-4 cells. After 1 h of incubation on ice, the cells were washed, and bound 4∆-mFc1 was detected by FACS using a 488- labeled anti-mouse Fc polyclonal antibody. B) Binding of 4∆-mFc1 labelled with IRDye800 (4∆-mFc1 / IRDye), to wild-type T3M-4 and ITGB6 / B8-KO T3M-4 cells. Cells were incubated 21 P128268PCT with the indicated amounts of 4∆-mFc1 / IRDye or with negative controls (a murine Fc or cysteine labeled with the same dye, called mFc / IRDye and Cys / IRDye, respectively). After 1 h of incubation at 37°C / 5% CO₂, cells were washed and fixed with formaldehyde; cell-bound fluorescence was then quantified using an Azure Biosystems scanner (see Methods). 5 Representative image of microtiter wells seeded with the indicated cells and treated with the indicated amounts of conjugates (left panels); conjugate binding curves (mean ± SE of technical quadruplicates) (right panels). Figure 42 – Internalization of 4Δ-mFc1 / pHsDye in αvβ6- and / or αvβ8-expressing cell lines and in αvβ6 / αvβ8-deficient cells. 10 Various concentrations of 4Δ-mFc1 / pHsDye or control mFc / pHsDye were incubated with T3M- 4 and ITGB6 / B8-KO cells (A), BxPC-3 cells (B), and LN-229 cells (C) seeded in 96-well microtiter plates. Following 18–20 h of incubation at 37^°C and 5% CO₂, cells were washed, and the cell-associated fluorescence was acquired using a Sapphire scanner. Representative fluorescence images (yellow signal) and corresponding quantification plots are shown. 4Δ- 15 mFc1 / pHsDye displayed robust internalization in integrin-positive cell lines (T3M-4, BxPC-3, LN-229), but not in the integrin-deficient ITGB6 / B8-KO cells, where fluorescence levels were comparable to those of the negative control (mFc / pHsDye). Data represent mean ± SEM of 2 technical duplicates. Figure 43 – 4Δ-mFc1 enables efficient internalization of antibody conjugates. 20 A) Schematic representation of the experimental setup: a goat anti-mouse polyclonal IgG labeled with a pH-sensitive dye (pAb / pHsDye) was pre-complexed with either 4Δ-mFc1, control mFc or diluent alone, and added to BxPC-3 cells. After 20 h incubation, cells were washed, and the plate was scanned with a Shappire imager scanner. After 20 h of incubation, the cells were washed and scanned using a Sapphire imager. Representative images of the 25 microtiter plate wells are shown. Yellow fluorescence indicates an intracellular signal. B) Quantification of internalized fluorescence. Data represent mean±SE of technical duplicates. **, P < 0.001 by two-tailed t-test. Figure 44 - 4Δ-mFc1 binds to αvβ6 / αvβ8 double-positive BxPC-3 pancreatic cancer cells and is internalized into the lysosomal compartment 30 4Δ-mFc1, control mFc, or diluent were mixed with an anti-mouse IgG polyclonal antibody conjugated to a pH-sensitive dye (anti-mouse IgGs-pHsDye) at the indicated concentrations. After 10 minutes of pre-incubation, the mixtures were added to BxPC-3 cells and incubated overnight at 37^°C in 5% CO₂. The following day, cells were washed and stained with 22 P128268PCT LysoTracker™ Deep Red (25^nM, 15^min) to label lysosomes (red), and with Hoechst 33258 to visualize nuclear DNA (blue). Fluorescent signals were acquired using a wide-field confocal microscopy with a 40× objective. 4Δ-mFc1 (green), but not control mFc, showed strong colocalization with the lysosomal marker (yellow, merge), indicating its internalization into the 5 lysosomal compartment. Figure 45 – Peptibody 4Δ-mFc1 synergizes with anti-PD-L1 mAb to reduce TS / A tumor size and improve survival of tumor-bearing mice. A) Experimental scheme. TS / A mammary adenocarcinoma cells were subcutaneously injected into 23 mice. On day 6 post-implantation, mice were randomized into four 10 experimental groups (n=5-6 per group). Mice were injected intraperitoneally with diluent (Vehicle) or with 50 µg of 4∆-mFc1 (3 treatments / weak, administered every other day, for a total of nine treatments) or with 100 µg of an anti-PD-L1 mAb (on days 6 and 20) or a combination of 4∆-mFc1 and anti-PD-L1 mAb (with anti-PD-L1 mAb administered 10 min before 4∆-mFc1). B) Growth curves of tumors (mean±SE, n=5-6). *, P<0.05; **, P< 0.01; ***, 15 P< 0.001; ****, P< 0.0001). Statistical differences between the groups were analyzed with GraphPad Prism software using a two-way ANOVA followed by Tukey's multiple comparisons test (from day 6 to day 20, when all the mice were alive). C) Waterfall plots showing the percentage change in tumor volume for individual mice over the indicated time. D) Kaplan- Meier survival curves. *, P< 0.05; **, P< 0.01, by long rank Mantel-Cox test. Mice were 20 euthanized when tumors reached 800-1000 mm3or when severe tumor ulceration or other signs of distress were observed. E) Body weight of mice bearing TS / A tumors after treatment with the indicated compounds (mean±SE, n=5-6 mice / group). Figure 46 – Expression, purification, and functional characterization of the peptibody 4Δ-hFc. A) Schematic workflow for the production and purification of 4Δ-hFc as monitored by PAGE 25 analysis. The peptibody was transiently expressed in mammalian XtenCHO cells (left gel) and purified from the culture medium by protein-A affinity chromatography (central gel). Selected fractions were pooled, dialyzed, sterile-filtered, and analyzed under reducing and non- reducing conditions (right gel). B) Binding of 4Δ-hFc and hFc (negative control) to immobilized αvβ6, αvβ8, and αIIbβ3 integrins, as measured by solid-phase binding assays. Left panels: 30 schematic representation of assay design. Right panels: dose response curves. A representative experiment is shown. Data points represent mean±SE of technical duplicates (n=2). Apparent dissociation constants (Kd) are indicated and were obtained from 2 independent experiments. 23 P128268PCT Figure 47 – Further illustrative peptibodies with a silenced-human fragment crystallizable domain (hsFc). Schematic diagrams showing arrangements of peptibodies with an Fc domain with peptide linked at the N-terminus, C-terminus, or both. Sequences of the signal peptide, peptide and 5 linkers are also shown. Figure 48 – Further illustrative peptide-PEG conjugates Arrangement of peptide-PEG conjugates, including sequence data of the peptide, and structural arrangement of the PEG. DETAILED DESCRIPTION OF THE INVENTION 10 Peptide In one aspect, the invention provides a product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to four amino acid substitutions, additions or deletions. In one aspect, the invention provides a product comprising a peptide, wherein the peptide 15 comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions. Preferably, the peptide binds to an integrin, such as αvβ6 or αvβ8 integrin, preferably the peptide binds to αvβ6 integrin and αvβ8 integrin. The present inventors have identified that targeting αvβ6 and / or αvβ8 integrins via a peptide described herein may mediate anti-tumour effects, and also reshape the tumour 20 microenvironment. Without wishing to be bound by theory, peptides of the present invention may accumulate in αvβ6- or αvβ8-positive tumours, and inhibit TGFβ activation by cancer cells. Integrin αvβ6 is weakly expressed or absent in adult tissue, and only up-regulated during wound healing, tissue remodelling and carcinogenesis. In addition, integrin αvβ8 is overexpressed by various carcinoma cells and by tumor-infiltrating regulatory T cells (Treg). 25 Suitably, the peptide may comprise or consist of an amino acid sequence derived from an arginine-glycine-aspartate (RGD) containing protein capable of binding integrins. Suitably, the peptide may contain a consensus motif for integrin binding. Integrins may be in multiple conformations, including a bent, inactive conformation, and extended higher affinity conformations where ligand binding is possible. Such extended 30 conformations may be termed active states. Without wishing to be bound by theory, the 24 P128268PCT peptide of the invention may bind to integrins when in their active conformation. Suitably the peptide may contain a consensus motif for integrin binding wherein the integrin is in an active conformation. The peptide of the present invention may exert anti-tumor activity through its interaction with 5 integrin αvβ6 or αvβ8. Without wishing to be bound by theory, integrins αvβ6 and αvβ8 can activate TGFβ which is a potent immunosuppressive cytokine via interaction with the RGD sequence of latency-associated peptide (LAP)-TGFβ, releasing active TGFβ. Peptide binding to the integrin(s) may block this activity, resulting in reduced immunosuppression. The peptide, or variant thereof may provide anti-tumor activity to tumour cells expressing integrin αvβ6 10 and / or αvβ8. In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions. SEQ ID NO: 1 (4a peptide): 15 CFETLRGDLRILSILRHQNLLKELQD The variant may have, for example, up to two amino acid substitutions, additions or deletions. The variant may have, for example, one amino acid substitution, addition or deletion. In some embodiments, the variant of SEQ ID NO: 1 has at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 1. Preferably, the variant retains the 20 ability to bind to integrin αvβ6 and integrin αvβ8. Preferably, the peptide comprises RGDL motif, more preferably wherein RGDL motif is in the position in the peptide where the RGDL motif is in SEQ ID NO: 1, which is amino acid positions 6-9 of the peptide (where the amino acids are numbered with respect to the positions in SEQ ID NO: 1). 25 In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, or a variant thereof having up to three amino acid substitutions, additions or deletions. SEQ ID NO: 2 (5a peptide): CFETLRGDLRILSILRX1QNLX2KELQD 30 wherein X1is propargylglycine and X2is azidolysine. 25 P128268PCT The variant may have, for example, up to two amino acid substitutions, additions or deletions. The variant may have, for example, one amino acid substitution, addition or deletion. In some embodiments, the variant of SEQ ID NO: 2 has at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 2. Preferably, the variant retains the ability to bind to 5 integrin αvβ6 and integrin αvβ8. In some embodiments, in the variant of SEQ ID NO: 2, X1and X2are both cysteine. In some embodiments, the peptide (e.g. comprising SEQ ID NO: 2) has a modified C terminus. For example, the peptide (e.g. comprising SEQ ID NO: 2) may have an amidated C-terminus. Suitably, the peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 20, 10 or a variant thereof having up to three amino acid substitutions, additions or deletions. SEQ ID NO: 20 (5a amidated peptide): CFETLRGDLRILSILRX1QNLX2KELQD-CONH2wherein X1is propargylglycine and X2is azidolysine, and wherein CONH2represents C- terminal amidation. 15 In some embodiments, the peptide has an alternative N-terminal residue. In some embodiments, the peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 52 or a variant thereof having up to three amino acid substitutions, additions or deletions. SEQ ID NO: 52 (alternate 5a amidated peptide): AFETLRGDLRILSILRX1QNLX2KELQD-CONH220 wherein X1is propargylglycine and X2is azidolysine, and wherein CONH2represents C- terminal amidation. In some embodiments, the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 3, or a variant thereof having up to three amino acid substitutions, additions or deletions. 25 SEQ ID NO: 3 (4Δ peptide): FETLRGDLRILSILRHQNLLKEL The variant may have, for example, up to two amino acid substitutions, additions or deletions. The variant may have, for example, one amino acid substitution, addition or deletion. 26 P128268PCT In some embodiments, the variant of SEQ ID NO: 3 has at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 3. Preferably, the variant retains the ability to bind to integrin αvβ6 and integrin αvβ8. In some embodiments, the peptide comprises or consists of an amino acid sequence of SEQ 5 ID NO: 32 or a variant thereof having up to three amino acid substitutions, additions or deletions. SEQ ID NO: 32 (4 peptide): FETLRGDLRILSILRHQNLLKELQD The variant may have, for example, up to two amino acid substitutions, additions or deletions. 10 The variant may have, for example, one amino acid substitution, addition or deletion. In some embodiments, the variant of SEQ ID NO: 32 has at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 32. Preferably, the variant retains the ability to bind to integrin αvβ6 and integrin αvβ8. In some embodiments, the peptide comprises or consists of an amino acid sequence of SEQ 15 ID NO: 4. SEQ ID NO: 4 (7a peptide): CFETLRGDLRALSILRHQNLLKELQD In some embodiments, the peptide or variant thereof comprises one or more post-translational modification. Suitably, the peptide or variant thereof may comprise phosphorylation, 20 glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and / or amidation. In some embodiments, the peptide or variant thereof comprises modified or chemically synthesised amino acids. In some embodiments, the peptide or variant thereof may comprise intra- and / or inter-chain 25 cross-links and / or cyclisation, for example through disulphide crosslinks or a triazole bridge. In some embodiments, the peptide or variant thereof binds integrin αvβ6 and / or integrin αvβ8 with a higher affinity than wild-type chromogranin A (CgA). In some embodiments, the peptide or variant thereof binds integrin αvβ6 and / or integrin αvβ8 with a higher affinity than a wild- type CgA peptide, wherein the affinity is increased by at least 10%, at least 20%, at least 50%, 27 P128268PCT at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150- fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, 5 at least 5000-fold, or at least 10000-fold relative to the wild-type CgA. In some embodiments, the peptide or variant thereof binds integrin αvβ6 with a higher affinity than wild-type CgA, wherein the affinity is increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 10 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold relative to the wild-type CgA. In some embodiments, the peptide or variant thereof binds integrin αvβ8 with a higher affinity than wild-type CgA, wherein the affinity is increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 2- 15 fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50- fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, or at least 10000-fold relative to the wild-type CgA. 20 The skilled person is readily able to determine binding affinities using methods that are well known in the art, for example as described further herein. In some embodiments, the peptide or variant thereof binds integrin αvβ6 and / or integrin αvβ8 with a higher affinity than a peptide consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide or variant thereof binds integrin αvβ6 and / or integrin αvβ8 25 with a higher affinity than a peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the affinity is increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold relative to the peptide consisting 30 of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the peptide comprises or consists of SEQ ID NO: 4. Chromogranin A (CgA) is a neurosecretory protein precursor that is cleaved into various biologically active fragments that have roles in regulation of the cardiovascular system, metabolism, innate immunity, tissue repair and tumor physiology. Human CgA with E49L 35 substitution may have the sequence of SEQ ID NO: 19. 28 P128268PCT SEQ ID NO: 19 (human CgA1-439with E49L substitution): LPVNSPMNKGDTEVMKCIVEVISDTLSKPSPMPVSQECFETLRGDLRILSILRHQNLLKELQDLALQG AKERAHQQKKHSGFEDELSEVLENQSSQAELKEAVEEPSSKDVMEKREDSKEAEKSGEATDGARPQAL PEPMQESKAEGNNQAPGEEEEEEEEATNTHPPASLPSQKYPGPQAEGDSEGLSQGLVDREKGLSAEPG 5 WQAKREEEEEEEEEAEAGEEAVPEEEGPTVVLNPHPSLGYKEIRKGESRSEALAVDGAGKPGAEEAQD PEGKGEQEHSQQKEEEEEMAVVPQGLFRGGKSGELEQEEERLSKEWEDSKRWSKMDQLAKELTAEKRL EGQEEEEDNRDSSMKLSFRARAYGFRGPGPQLRRGWRPSSREDSLEAGLPLQVRGYPEEKKEEEGSAN RRPEDQELESLSAIEAELEKVAHQLQALRRG CgA and fragments thereof, such as vasostatin-1 (CgA amino acids 1-76), are capable of 10 selective binding of integrin αvβ6. A CgA-derived peptide comprising residues 39-63 is sufficient for this binding activity. This peptide comprises an arginine-glycine-aspartate (RGD) motif, which is an integrin-binding motif. Replacement of the glutamic acid present in the human CgA at position 46 with leucine generates a peptide with selective binding and strong affinity to both αvβ6 and αvβ8 integrins (FETLRGDLRILSILRHQNLLKELQD, peptide 4, SEQ 15 ID NO: 21) (Nardelli et al. (2019) Chem Commun 55: 14777-14780). Of note, no binding of CgA39-63 was observed to other integrins (such as α1β1, α6β4, α3β1, α9β1 α6β7, α5β1, αvβ3, αvβ5, and αvβ8) at low-nanomolar concentrations (Curnis et al. (2012) Cell. Mol. Life Sci.69: 2791–2803). Conjugates 20 Suitably, the peptide of the present invention is linked to one or more further agent to form a peptide conjugate. In some embodiments, the further agent is a polypeptide. In some embodiments, the further agent is a globular protein or a fragment thereof. In some embodiments, the further agent is a serum protein or a fragment thereof. In some embodiments, the further agent is human serum 25 albumin (HSA). In some embodiments, the further agent is an immunoglobulin, or a fragment thereof, preferably an Fc domain of an immunoglobulin. Human serum albumin The peptide may, for example, be linked to human serum albumin (HSA). Suitably, the HSA is a full-length HSA. 30 In some embodiments, the HSA comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a variant thereof. SEQ ID NO: 5 (Human Serum Albumin, Uniprot ID: P02768) 29 P128268PCT MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK TCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCA SLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK 5 ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL 10 In some embodiments, the HSA variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the HSA consists of the amino acid sequence of SEQ ID NO: 5. 15 In some embodiments, the HSA variant comprises or consists of the amino acid sequence of SEQ ID NO: 25, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. SEQ ID NO: 25: RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTL 20 FGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARL SQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVE NDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHEC YAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK 25 RMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEK ERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL In some embodiments, the HSA consists of the amino acid sequence of SEQ ID NO: 25. Fc domain The peptide may be linked to an Fc domain. Suitably, the Fc domain is the Fc domain of an 30 immunoglobulin G (IgG). Suitably, the Fc domain comprises a CH2 and CH3 domain. In some embodiments, the Fc domain comprises a hinge, a CH2 domain and a CH3 domain. Suitably, the Fc domain is a wild-type immunoglobulin Fc sequence or an engineered Fc sequence. Suitably, the Fc domain is the Fc domain of a human IgG. In some embodiments, the Fc domain is the Fc domain of a human IgG1. 35 In some embodiments, the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, or a variant thereof. SEQ ID NO: 6 (human IgG1 Fc): 30 P128268PCT EPKSQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 5 In some embodiments, the Fc domain variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the Fc domain consists of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the Fc domain is an Fc domain that has modulated effector functions, 10 for example complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP) (see, for example Abdeldaim et al. Fc-Engineered Therapeutic Antibodies: Recent Advances and Future Directions. LID - 10.3390 / pharmaceutics15102402 [doi] LID – 2402; Vaccaro et al. (2005) Nat Biotechnol 23: 1283-1288; Wang et al. (2017) Protein & Cell 9: 63-73). In some embodiments, 15 the Fc domain is an Fc domain that has one or more reduced effector function(s). In some embodiments, the Fc domain is an Fc domain that has one or more increased effector function(s). In some embodiments, the Fc domain is a wild type Fc domain or an engineered Fc domain that is unable to bind Fc-receptors. In some embodiments, the Fc domain comprises or 20 consists of the amino acid sequence of SEQ ID NO: 7, or a variant thereof. SEQ ID NO: 7 (4 mut human IgG1 Fc): EPKSQDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ 25 GNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the Fc domain variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the Fc domain consists of the amino acid sequence of SEQ ID NO: 7 30 In some embodiments, the Fc domain is a silenced Fc domain. In some embodiments, the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 30, or a variant thereof. SEQ ID NO: 30 (silenced hFc): 31 P128268PCT EPKSQDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 5 In some embodiments, the Fc domain variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some embodiments, the Fc domain consists of the amino acid sequence of SEQ ID NO: 30 PEG 10 The peptide may, for example, be linked to a polyethylene glycol (PEG). PEG is also known as poly(ethylene oxide) (PEO) or poly(oxyethylene) (POE). Suitably, the PEG may be of any suitable average molecular mass. For example, the PEG may be a PEG100, PEG200, PEG500, PEG1K, PEG2K, PEG5K, PEG10K, PEG20K, PEG50K or PEG100K. 15 In some embodiments, the PEG is a PEG5K or a PEG10K. In some embodiments, the PEG is a PEG10K. In some embodiments, the PEG is a low molecular weight PEG, for instance a PEG with few repeating units. In some embodiments, the PEG may be a PEG4 or a PEG8. In some embodiments, the PEG may be a thiol-PEG4 or a thiol-PEG8. 20 In some embodiments, the PEG is a linear PEG. In some embodiments, the PEG is a branched PEG. For example, multiple PEG polymers may be linked to a further chemical group, for example to form a linear backbone molecule forming brush architecture PEG (bPEG), or may be a Y-shaped PEG. Suitably, the PEG may comprise one or more chemically-reactive functional groups. In some 25 embodiments, the PEG comprises one chemically-reactive functional group, and may be termed monofunctional. In some embodiments, the PEG comprises two chemically-reactive functional groups, and may be termed bi-functional. In some embodiments, the PEG comprises three chemically-reactive functional groups, and may be termed tri-functional. In some embodiments, the PEG comprises more than three chemically-reactive functional 30 groups. In some embodiments, the chemically-reactive functional groups are at the end of the PEG polymer. In some embodiments, multiple functional groups are at the same end of the 32 P128268PCT PEG polymer. In some embodiments, the chemically-reactive functional groups are at the end of a branched PEG. In some embodiments, the peptide is linked to a PEG via a thioether bond to a sulfhydryl (- SH) group in the peptide (e.g. on a cysteine side chain). The PEG may have been maleimide- 5 activated. In some embodiments, the peptide of the invention is linked to a PEG, which is further linked to one or more further peptide (e.g. as described herein). In some embodiments, the one or more further peptide is a peptide of the invention. In some embodiments, the one or more further peptide is not a peptide of the invention. In some embodiments, the PEG which is 10 further linked to one or more further peptide is a bi-functional PEG, a tri-functional PEG, or a branched PEG. Suitably, the further agent to which the peptide is linked provides a beneficial property to the peptide of the invention. Suitably, the peptide linked to an agent as described herein has an increased half-life when compared to the peptide when lacking the further agent. In some 15 embodiments, the peptide linked to an agent as described herein has an increased serum half-life when compared to the peptide when lacking the further agent. In some embodiments, the peptide linked to an agent as described herein has a serum half-life increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 20 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, or at least 1000-fold relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to an agent as described herein has a serum half- life increased by at least 10-fold relative to the peptide when lacking the further agent. In some 25 embodiments, the peptide linked to an agent as described herein has a serum half-life increased by at least 35-fold relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to an agent as described herein has a serum half-life increased by at least 125-fold relative to the peptide when lacking the further agent. The skilled person is readily able to determine half-lives of agents, such as serum half-lives, 30 using methods that are well known in the art, for example as described further herein. Suitably, the further agent to which the peptide is linked does not adversely affect the integrin- binding properties of the peptide relative to the peptide when lacking the further agent. Suitably, the further agent does not adversely affect the affinity and / or selectivity of the peptide 33 P128268PCT for αvβ6 and αvβ8 integrins relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein may have an increased affinity and / or avidity for αvβ6 and αvβ8 integrins relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein 5 has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15- fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, or at least 10 1000-fold relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 3-fold relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 5-fold 15 relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 15-fold relative to the peptide when lacking the further agent. In some embodiments, the peptide linked to a further agent as described herein has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 25-fold relative to the peptide when lacking the 20 further agent. In some embodiments, the peptide linked to a further agent as described herein has an affinity for αvβ6 and αvβ8 integrins that is increased by at least 100-fold relative to the peptide when lacking the further agent. The skilled person is readily able to determine binding affinities using methods that are well known in the art, for example as described further herein. 25 Linker In some embodiments, the peptide is linked to the HSA, PEG or Fc domain by a linker. In some embodiments, the peptide is linked directly to the HSA, PEG or Fc domain (e.g. linked directly via a covalent bond). The peptide may be linked (e.g. conjugated) by any suitable means known in the art. The skilled person is readily aware of suitable methods in the art 30 through which this can be achieved. Preferably, the linking is operable linked (e.g. wherein the components that are linked are all able to perform their normal function substantially unhindered). 34 P128268PCT The presence of a linker may allow each of the linked components to act independently of one another whilst being spatially restricted and retaining connectivity. In some cases, linkers are formed from amino acids, however, other means of linking are known. Linkers are commonly used to link protein domains, for example when tagging a protein with GFP. Linkers are also 5 commonly used in peptide-protein and peptide-drug conjugates. In some embodiments, the linker is a chemical crosslinker. Suitable chemical crosslinkers are known in the art. Linkers may be cleavable or non-cleavable. Suitably, the linker is a non- cleavable linker. Non-cleavable linkers may be more stable in plasma and may be preferred to ensure serum stability of peptide conjugates. Examples of chemical linkers include amide 10 and thioether linkages (such as is formed through the use of a hetero-bifunctional cross-linker such as sulfo-SMCC), ester and amide linkages, carbamate linkers, ester and triazole linkages, hydrazone linkers, disulphide linkers, oxime linkers and triazoles. Suitably, non- cleavable linkers may include triazole linkages (such as is formed through the use of a sulfo- SMCC cross-linker) and oxime linkages. In some embodiments, the peptide is linked by a15 hetero-bifunctional chemical cross linker, for example 3-sulpho-N-succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC). Suitably, a hetero- bifunctional chemical cross linker is linked at one end to the further agent, for example by an amide bond, and at the other end to the peptide, for example by a succinimidyl thioether bond. In some embodiments, the linker is a peptide linker. The product may, for example, be a fusion20 protein. Suitable peptide linkers are known in the art. Peptide linkers may be cleavable or non- cleavable. Suitably, the peptide linker may be non-cleavable. Generally, flexible peptide linkers consist of small non-polar (for example glycine), or polar (for example serine) amino acids. Examples of commonly used flexible linkers include linkers comprising stretches of glycine and serine amino acids (GS linkers) and stretches of glycine amino acids. Peptide 25 linkers may be of any suitable length. Suitably, a peptide linker consists of an amino acid sequence of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30 or at least 50 amino acids. In some embodiments, a peptide linker consists of an amino acid sequence of fewer than 20 amino acids, fewer than 10 amino acids, or fewer than 5 amino acids. In some 30 embodiments, a peptide linker consists of glycine amino acids. In some embodiments, a peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 9 (Glycine linker): GGGG 35 P128268PCT In some embodiments, the peptide linker comprises a variant sequence of SEQ ID NO: 9, with up to 3 amino acid substitutions, additions, or deletions. Suitably, the linker may be chosen based on the further agent to be linked to. For example, further agents that are proteins, such as HSA or Fc domains, may be linked by a chemical 5 linker or peptide linker. Suitably, further agents that are not proteins, such as PEG, may preferably be linked by a chemical linker. In some embodiments, HSA is linked to a peptide of the invention by sulfo-SMCC (e.g. via a triazole bond). In some embodiments, a maleimide- activated PEG (for example, wherein an amide bond is formed linking the PEG to a crosslinker containing a maleimide, e.g. sulfo-SMCC) is linked to a peptide of the invention by a thioether 10 bond. In some embodiments, an Fc domain is linked to a peptide of the invention by a peptide linker consisting of the amino acid sequence of SEQ ID NO: 9. The linker may be linked via any suitable site on the peptide. For example, peptide linkers may be linked via a peptide bond at the N- or C- terminus of the peptide, or may be linked to an amino acid side chain of the peptide via, for example, an isopeptide bond. Suitably, chemical 15 linkers may be linked to any functional group present in the peptide. The identity of the functional group may depend on the specificity of the linker used. For example, a linker containing a maleimide (e.g. sulfo-SMCC) may be linked via a thioether bond to a sulfhydryl (-SH) group in the peptide (e.g. on a cysteine side chain). For example, a linker containing an amino-reactive group may be linked via an amide bond to a primary amine in the peptide. 20 In some embodiments, the peptide of the invention is linked to one or more further agent in various ratios. For example, the peptide of the invention may be linked to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 further agent(s). In some embodiments, the further agent (e.g. HSA) is linked to at least one, at least two, at 25 least three, at least four, at least five, or at least six peptides of the invention. In some embodiments, the further agent (e.g. HSA) is linked to about three peptides of the invention. In some embodiments, the peptide of the invention is linked to one or more further agent in a ratio of further agent to peptide of from about 10:1 to about 1:10. In some embodiments, the peptide of the invention is linked to one or more further agent in a ratio of further agent to 30 peptide of about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. 36 P128268PCT In some embodiments, a composition of the product of the invention is homogenous. Suitably, the composition of the product of the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homogeneity in terms of the ratio of further agent to peptide in individual peptide conjugates. 5 In some embodiments, a composition of the product of the invention is heterogeneous. Suitably, the composition of the product of the invention contains a mixture of ratios of further agent to peptide in individual peptide conjugates. Signal peptide Signal peptides, also known as leader peptides or signal sequences, are short peptides that 10 are able to influence the targeting of a protein within a cell, for example targeting a protein to a secretion pathway. In some embodiments, the product of the invention comprises a signal peptide. Suitably, the product comprises a signal peptide at its N-terminus. The signal peptide may, for example, be derived from a naturally occurring signal peptide from 15 a native protein. The signal peptide may, for example, be a synthetic signal peptide (e.g. Park et al. (2022) Applied Microbiology and Biotechnology 106: 3571–3582). The signal peptide may, for example, be derived from a murine or human native protein. In some embodiments, the signal peptide is derived from an IL2 signal sequence, or the signal peptide is derived from a human immunoglobulin heavy chain signal sequence. 20 In some embodiments, the signal peptide is an IL2 signal sequence. In some embodiments, the signal peptide is a human immunoglobulin heavy chain signal sequence. The peptide of the present invention may, for example, comprise a signal peptide sequence of SEQ ID NO: 8, 16, 17 or 29, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of SEQ ID NO: 8, 16, 17 or 29. 25 SEQ ID NO: 8 (signal peptide) MKHLWFFLLLVAAPRWVLS SEQ ID NO: 16 (IL2 signal peptide) MYRMQLLSCIALSLALVTNS SEQ ID NO: 17 (human IgG heavy chain signal peptide) 37 P128268PCT MEFGLSWVFLVALLRGVQC SEQ ID NO: 29 (HSA signal peptide) MKWVTFISLLFLFSSAYS Additional agents 5 In some embodiments, the peptide is linked to one or more further agents such as HSA, an Fc domain and / or a PEG. In some embodiments, a product according to the invention comprising one or more further agents as described above may also comprise one or more additional agents. In some embodiments, the peptide is linked to one or more of HSA, an Fc domain and / or a PEG, and the one or more additional agents are linked to the peptide directly 10 and / or via the HSA, Fc domain or the PEG. Suitably, the additional agent to which the peptide is linked provides a beneficial property to the peptide of the invention. In some embodiments, the additional agent provides an anti- tumour activity. In some embodiments, the additional agent is an anti-tumour and / or cytotoxic agent, for example a chemotherapeutic agent or radioactive agent. 15 In some embodiments, the product according to the invention does not comprise any further agents than (i) the peptide; (ii) one or more of HSA, an Fc domain and / or a PEG, and (iii) a linker. In some embodiments, the product according to the invention is not a photoacoustic agent. In some embodiments, the product according to the invention does not comprise a photoacoustic 20 agent. In some embodiments, the product according to the invention does not comprise a metal-based nanoparticle made of gold, silver or hybrid gold / silver or hybrid gold / iron, an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squarine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes or IRDye800. Exemplary products 25 In some embodiments, the peptide is linked to HSA by a chemical linker. Suitably, the peptide comprises the amino acid sequence of SEQ ID NO: 2 and is linked to HSA, wherein the HSA comprises the amino acid sequence of SEQ ID NO: 5, preferably wherein the linker is a chemical linker formed by conjugation of the HSA and peptide with sulfo-SMCC. In some embodiments, a product according to the invention comprises or consists of the 30 peptide linked to HSA by a chemical linker. In some embodiments, the product consists of the 38 P128268PCT peptide, a chemical linker and HSA. In some embodiments, the product comprises or consists of the peptide, a chemical linker wherein the chemical linker is sulfo-SMCC, and HSA. In some embodiments, the peptide is linked to a PEG that has been maleimide-activated. Suitably, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and is linked to a 5 PEG, wherein the PEG is a PEG10K, wherein the peptide is linked to the PEG via a thioether bond to the sulfhydryl (-SH) group of the N-terminal cysteine of the peptide. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 52, and is linked to a PEG, wherein the PEG is a PEG4 or PEG8. In some embodiments, a product according to the invention comprises or consists of the 10 peptide linked to PEG by a chemical linker. In some embodiments, the product consists of the peptide, a chemical linker and a PEG. In some embodiments, the product comprises or consists of the peptide, a chemical linker wherein the chemical linker is sulfo-SMCC, and a PEG. In some embodiments, the product comprises or consists of the peptide and a maleimide-activated PEG. 15 In some embodiments, the peptide is linked to an Fc domain by a peptide linker. Suitably, the peptide comprises the amino acid sequence of SEQ ID NO: 3 and is linked to the Fc domain wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 6, wherein the linker is a peptide linker, for example a glycine linker. In some embodiments, the product of the invention comprises or consists of the amino acid 20 sequence of SEQ ID NO: 54 or a variant thereof. SEQ ID NO: 54 (4Δ-hFc without signal peptide): FETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 25 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 54. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. 30 In some embodiments, the product of the invention further comprises a signal peptide. In some embodiments, the product of the invention further comprises a signal peptide of the amino acid sequence of SEQ ID NO: 17. 39 P128268PCT In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 22, or a variant thereof. SEQ ID NO: 22 (4Δ-hFc): MEFGLSWVFLVALLRGVQCFETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPELLGG 5 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at 10 least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention comprises a signal peptide of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the product of the invention comprises 15 or consists of the amino acid sequence of SEQ ID NO: 60. SEQ ID NO: 60 (4Δ-hFc SPii): MKHLWFFLLLVAAPRWVLSFETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY 20 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 60. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, 25 with up to three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to an Fc domain by a peptide linker. Suitably, the peptide comprises the amino acid sequence of SEQ ID NO: 3 and is linked to the Fc domain wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 7, wherein the linker is a peptide linker, for example a glycine linker. In some embodiments, the product of 30 the invention comprises or consists of the amino acid sequence of SEQ ID NO: 55 or a variant thereof. SEQ ID NO: 55 (4Δ-hFc_4 mut without signal peptide): FETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS 40 P128268PCT NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ 5 ID NO: 55. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention further comprises a signal peptide. In some embodiments, the product of the invention further comprises a signal peptide of the amino acid sequence of SEQ ID NO: 17. 10 In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 23, or a variant thereof. SEQ ID NO: 23 (4Δ-hFc_4_mut): MEFGLSWVFLVALLRGVQCFETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV 15 LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ 20 ID NO: 23. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention comprises a signal peptide of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 61. 25 SEQ ID NO: 61 (4Δ-hFc_4_mut SPii): MKHLWFFLLLVAAPRWVLSMEFGLSWVFLVALLRGVQCFETLRGDLRILSILRHQNLLKELGGGGEPK SQDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV 30 FSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 61. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. 41 P128268PCT In some embodiments, the peptide is linked to an Fc domain by a peptide linker wherein the Fc domain is a silenced Fc domain. In some embodiments, the peptide is linked to an Fc domain wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3 and is 5 linked to an Fc domain wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 30, wherein the linker is a peptide linker, for example a glycine linker. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 56 or a variant thereof. SEQ ID NO: 56 (4∆-hsFc N-ter peptibody without signal peptide): 10 FETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at 15 least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 56. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention further comprises a signal peptide. In some embodiments, the product of the invention further comprises a signal peptide of the amino 20 acid sequence of SEQ ID NO: 8. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 31, or a variant thereof. SEQ ID NO: 31 (4∆-hsFc N-ter peptibody): MKHLWFFLLLVAAPRWVLSFETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAEGA 25 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK In some embodiments, the variant comprises an amino acid sequence with at least 80%, at 30 least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 31. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the peptide is linked to HSA by a peptide linker. Suitably, the peptide comprises the amino acid sequence of SEQ ID NO: 3 and is linked to a HSA wherein the HSA 42 P128268PCT comprises the amino acid sequence of SEQ ID NO: 25, wherein the linker is a peptide linker, for example a glycine linker. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 57 or a variant thereof. SEQ ID NO: 57 (4Δ-HSA without signal peptide): 5 FETLRGDLRILSILRHQNLLKELGGGGRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP FEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA ADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAA 10 DFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFD EFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHA DICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS QAALGL 15 In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 57. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention further comprises a signal peptide. In some 20 embodiments, the product of the invention further comprises a signal peptide of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 24, or a variant thereof. SEQ ID NO: 24 (4Δ-HSA): 25 MKWVTFISLLFLFSSAYSFETLRGDLRILSILRHQNLLKELGGGGRGVFRRDAHKSEVAHRFKDLGEE NFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYG EMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQ RFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHC 30 IAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVE VSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEV DETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL 35 In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions. 43 P128268PCT In some embodiments, the peptide may be linked to the N- or C- terminus of an Fc domain or HSA. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3 or 32 and is linked to the N- or C- terminus of an Fc domain, preferably wherein the Fc domain is a silenced hFc domain. In some embodiments, the peptide is linked to an Fc domain 5 wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 30, and wherein the peptide comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 58 or a variant thereof. SEQ ID NO: 58 (hsFc-4 C-ter peptibody without signal peptide): 10 EPKSQDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSFETLRGDLRILSILRHQNLLKELQD In some embodiments, the variant comprises an amino acid sequence with at least 80%, at 15 least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 58. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 32, with up to three amino acid substitutions, additions or deletions. In some embodiments, the product of the invention further comprises a signal peptide. In some embodiments, the product of the invention further comprises a signal peptide of the amino 20 acid sequence of SEQ ID NO: 8. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 33, or a variant thereof. SEQ ID NO: 33 (hsFc-4 C-ter peptibody): MKHLWFFLLLVAAPRWVLSEPKSQDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVV 25 DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSFETLRGDLRIL SILRHQNLLKELQD In some embodiments, the variant comprises an amino acid sequence with at least 80%, at 30 least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 32, with up to three amino acid substitutions, additions or deletions. In some embodiments, one or more peptide may be linked to a single further agent. In some embodiments, one or more peptide may be linked to a single PEG, Fc or HSA. In some 44 P128268PCT embodiments, one or more peptide may be linked to a single Fc by a peptide linker. In some embodiments, two peptides are linked to a single Fc by peptide linkers. In some embodiments, the first peptide comprises or consists of an amino acid sequence of SEQ ID NO: 3 or 32, and the second peptide comprises or consists of an amino acid of SEQ ID NO: 3 or 32. In some 5 embodiments, the first peptide comprises or consists of an amino acid of SEQ ID NO: 3 and the second peptide comprises or consists of an amino acid of SEQ ID NO: 32, wherein the first and the second peptide are each linked to a single Fc by peptide linkers. In some embodiments, a first peptide comprising or consisting of an amino acid sequence of SEQ ID NO: 3 is linked to the N-terminus of an Fc domain by a peptide linker, and a second 10 peptide comprising or consisting of an amino acid sequence of SEQ ID NO: 32 is linked to the C-terminus of the Fc domain by a peptide linker. In some embodiments, the Fc domain is a silenced human Fc domain, optionally wherein the silenced human Fc domain has an amino acid sequence of SEQ ID NO: 30. In some embodiments, the product of the invention comprises a peptide of amino acid sequence of SEQ ID NO: 3, a silenced human Fc domain 15 of amino acid sequence of SEQ ID NO: 30, and a peptide of amino acid sequence of SEQ ID NO: 32. In some embodiments, the product of the invention comprises or consists of an amino acid sequence of SEQ ID NO: 59, or a variant thereof. SEQ ID NO: 59 (4∆-hsFc-4 N- and C-ter peptibody without signal peptide): FETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRT 20 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSFET LRGDLRILSILRHQNLLKELQD 25 In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 59. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, with up to three amino acid substitutions, additions or deletions, and retains the amino acid sequence of SEQ ID NO: 32, with up to three amino acid substitutions, additions or deletions. 30 In some embodiments, the product of the invention further comprises a signal peptide. In some embodiments, the product of the invention further comprises a signal peptide of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the product of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 34, or a variant thereof. 45 P128268PCT SEQ ID NO: 34 (4∆-hsFc-4 N- and C-ter peptibody): MKHLWFFLLLVAAPRWVLSFETLRGDLRILSILRHQNLLKELGGGGEPKSQDKTHTCPPCPAPEAEGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY 5 PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKGGGGSGGGGSFETLRGDLRILSILRHQNLLKELQD In some embodiments, the variant comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34. Suitably, the variant sequence retains the amino acid sequence of SEQ ID NO: 3, 10 with up to three amino acid substitutions, additions or deletions, and retains the amino acid sequence of SEQ ID NO: 32, with up to three amino acid substitutions, additions or deletions. Polynucleotide In one aspect, the invention provides a polynucleotide encoding the product of the invention (e.g. wherein the product does not comprise a peptide linked to a PEG). 15 In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, 20 additions or deletions linked to a protein as described herein. In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions linked to HSA or an Fc domain. In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ 25 ID NO: 3 and SEQ ID NO: 4, or variants thereof having up to three amino acid substitutions, additions or deletions, linked to HSA or an Fc domain. In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 3, or a variant thereof having up to three amino acid substitutions, additions or deletions, linked to an Fc domain. 30 In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 32, or a variant thereof having up to three amino acid substitutions, additions or deletions linked to a protein as described herein. In some embodiments, the polynucleotide encodes a peptide comprising an amino acid sequence of SEQ ID NO: 32, or 46 P128268PCT a variant thereof having up to three amino acid substitutions, additions or deletions linked to an Fc domain. The polynucleotide may, for example, comprise the nucleotide sequence of SEQ ID NO: 10, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence 5 identity thereto. The polynucleotide may, for example, be a codon-optimised polynucleotide (e.g. wherein the degeneracy of the genetic code is utilised to replace the codons with codons encoding the same amino acids that are more efficiently transcribed). In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 10 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 35, 39, 40, 41 or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 10 (4a nt): 15 x1x2x3ttcgagaccctgagaggcgacctgagaatcctgagcatcctgagacaccagaacctgctgaag gagctgcaggac wherein x1x2x3is selected from tgt or tgc, SEQ ID NO: 11 (4Δ nt): ttcgagaccctgagaggcgacctgagaatcctgagcatcctgagacaccagaacctgctgaaggagct 20 g SEQ ID NO: 39 (4Δ nt ii): tttgaaaccctgcgcggggacctgcgcatcctatcaatacttagacatcagaatctgctgaaagaact a SEQ ID NO: 40 (4Δ nt iii) 25 ttcgaaaccttgcggggagaccttcgtatccttagcattctgagacatcagaatctgctaaaggagct c SEQ ID NO: 12 (7a nt): 47 P128268PCT x1x2x3ttcgagaccctgagaggcgacctgagax4x5x6ctgagcatcctgagacaccagaacctgctga aggagctgcaggac wherein x1x2x3is tgt or tgc, and wherein x4x5x6is selected from gca, gcg, gct or gcc. SEQ ID NO: 35 (4 nt): 5 ttcgaaacgcttcggggcgatctgcgcattctgtccattctgcgccaccaaaacctcttgaaagaact tcaagacSEQ ID NO: 41 (4 nt ii): tttgagactctgcggggcgacctgcggattctcagtatactgagacaccagaatcttctgaaagagct ccaagac In some embodiments, the polynucleotide further encodes the HSA or the Fc domain. In some 10 embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13 or 14, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 13 (human Fc nt): GAGCCAAAGAGCCAGGACAAGACCCACACCTGCCCACCCTGCCCAGCCCCTGAACTGCTGGGCGGCCC 15 CAGCGTGTTCCTGTTTCCGCCCAAGCCAAAGGACACCCTGATGATCTCTAGAACACCAGAAGTGACTT GTGTGGTGGTGGATGTGTCCCACGAAGATCCTGAGGTGAAATTCAACTGGTACGTGGATGGTGTGGAA GTGCACAATGCCAAGACAAAGCCCCGGGAGGAGCAGTACAATTCTACTTACAGAGTGGTCTCAGTGCT GACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGAGTAATAAGGCCCTGC CCGCCCCCATCGAGAAAACCATCTCTAAGGCCAAGGGGCAGCCTAGAGAACCTCAGGTGTACACCCTG 20 CCCCCCAGCAGAGACGAGCTGACCAAGAATCAGGTGTCACTGACCTGTCTGGTTAAGGGCTTCTACCC TTCCGATATTGCCGTGGAGTGGGAAAGCAATGGCCAGCCCGAGAACAACTACAAGACTACCCCTCCCG TGCTGGACTCTGATGGAAGCTTTTTTCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAG GGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTC CCTGAGCCCAGGCAAGTGA 25 SEQ ID NO: 14 (human Fc 4 mut nt): GAGCCAAAGAGCCAGGACAAGACCCACACCTGCCCACCCTGCCCAGCCCCTGAAGCCGCCGGCGCCCC CAGCGTGTTCCTGTTTCCGCCCAAGCCAAAGGACACCCTGATGATCTCTAGAACACCAGAAGTGACTT GTGTGGTGGTGGATGTGTCCCACGAAGATCCTGAGGTGAAATTCAACTGGTACGTGGATGGTGTGGAA GTGCACAATGCCAAGACAAAGCCCCGGGAGGAGCAGTACAATTCTACTTACAGAGTGGTCTCAGTGCT 30 GACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGAGTAATAAGGCCCTGC CCGCCCCCATCGAGAAAACCATCTCTAAGGCCAAGGGGCAGCCTAGAGAACCTCAGGTGTACACCCTG CCCCCCAGCAGAGAGGAGATGACCAAGAATCAGGTGTCACTGACCTGTCTGGTTAAGGGCTTCTACCC TTCCGATATTGCCGTGGAGTGGGAAAGCAATGGCCAGCCCGAGAACAACTACAAGACTACCCCTCCCG TGCTGGACTCTGATGGAAGCTTTTTTCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAG 35 GGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCCCTGTC CCTGAGCCCAGGCAAGTGA In some embodiments, the polynucleotide further encodes a silenced Fc domain. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 42, 43 48 P128268PCT or 44, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. SEQ ID NO: 42 (hsFc i): GAACCAAAATCCCAGGATAAGACCCACACATGTCCTCCGTGCCCAGCCCCAGAGGCTGAGGGCGCCCC 5 AAGCGTGTTTCTCTTTCCTCCTAAACCCAAGGACACACTGATGATCTCCCGGACCCCCGAGGTAACTT GTGTTGTGGTCGATGTGTCACACGAGGATCCAGAGGTGAAGTTTAATTGGTATGTTGACGGCGTGGAG GTTCATAATGCCAAGACAAAACCACGCGAAGAGCAGTATAACAGCACTTATCGGGTGGTTTCCGTGTT GACCGTGCTACACCAGGACTGGCTCAACGGAAAGGAATATAAGTGTAAGGTGTCCAATAAAGCCCTGC CTAGCTCTATCGAGAAAACTATCTCCAAGGCAAAGGGTCAGCCCCGGGAGCCTCAAGTGTACACTTTG 10 CCGCCGAGCCGAGATGAATTGACCAAAAATCAGGTTAGCCTAACTTGCCTGGTGAAGGGATTCTATCC TTCCGATATCGCCGTGGAGTGGGAGAGCAACGGACAACCTGAGAATAATTATAAAACAACCCCTCCTG TCCTGGACTCTGACGGCTCTTTCTTCCTCTACAGCAAGCTTACGGTGGACAAGTCTAGATGGCAACAG GGCAACGTGTTCTCTTGCAGCGTCATGCACGAAGCCCTCCACAACCACTACACGCAGAAGAGCCTATC CCTCTCACCTGGAAAA 15 SEQ ID NO: 43 (hsFc ii): GAGCCTAAGAGTCAGGACAAGACCCACACGTGCCCACCCTGCCCTGCCCCGGAGGCCGAGGGCGCTCC ATCTGTGTTTCTGTTCCCCCCTAAGCCGAAAGACACACTTATGATTTCCCGCACTCCTGAGGTGACCT GCGTCGTAGTGGACGTTTCCCATGAGGATCCAGAAGTTAAGTTCAATTGGTACGTAGATGGCGTGGAG GTGCATAACGCAAAAACCAAGCCACGGGAGGAACAGTACAATAGCACGTATCGCGTGGTATCCGTCCT 20 TACCGTACTGCATCAGGATTGGCTGAACGGGAAGGAGTACAAATGTAAGGTGTCCAACAAAGCTCTTC CTTCTTCCATCGAGAAGACAATATCTAAAGCCAAGGGCCAGCCTCGCGAGCCACAGGTATATACACTG CCCCCCAGTAGGGATGAGCTGACCAAAAATCAGGTAAGCCTGACTTGCCTGGTAAAAGGCTTCTACCC CTCTGACATTGCAGTCGAGTGGGAATCTAATGGACAACCCGAGAATAACTACAAAACCACTCCGCCTG TCCTAGACAGCGACGGCAGCTTCTTCCTGTATTCTAAGCTAACAGTAGACAAGAGTCGATGGCAACAA 25 GGTAACGTGTTTTCATGTTCTGTGATGCACGAGGCCCTCCATAATCACTACACTCAAAAGAGCCTGAG TCTCTCCCCCGGTAAGGGAGGTGGGGGATCTGGAGGTGGAGGATCT SEQ ID NO: 44 (hsFc iii): GAACCTAAGTCTCAAGACAAGACTCATACTTGCCCCCCTTGTCCAGCACCCGAGGCCGAGGGTGCCCC CTCAGTTTTTCTGTTTCCTCCAAAACCCAAGGACACCCTGATGATATCTAGGACGCCAGAGGTTACTT 30 GTGTGGTGGTGGACGTCAGCCATGAGGATCCCGAAGTCAAGTTTAATTGGTACGTTGATGGTGTTGAG GTTCATAACGCTAAAACTAAACCTCGAGAAGAGCAATACAATAGCACATACCGAGTTGTCAGTGTGCT CACGGTATTGCATCAGGATTGGCTGAATGGCAAGGAATATAAGTGTAAGGTCTCCAATAAAGCTCTTC CCAGTTCTATCGAGAAGACCATCAGCAAAGCAAAGGGCCAGCCGCGGGAGCCTCAGGTCTACACTTTG CCACCATCCCGAGACGAGCTCACTAAAAATCAGGTGAGCCTGACTTGCCTGGTGAAGGGCTTTTATCC 35 CTCAGACATCGCCGTTGAATGGGAAAGCAATGGCCAGCCCGAAAACAACTATAAGACAACACCACCAG TTCTTGATAGTGACGGAAGTTTCTTTCTTTACAGTAAGTTGACAGTCGATAAGAGCAGATGGCAACAG GGGAACGTTTTCAGTTGCTCCGTCATGCACGAGGCCCTTCATAATCATTATACCCAAAAGTCCTTGAG TCTGTCCCCCGGCAAA In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 40 15, or a variant thereof having at least 80%, 90%, 95% or 99% sequence identity thereto. SEQ ID NO: 15 (signal peptide nt) ATGGAGTTTGGCCTGAGCTGGGTGTTTCTGGTGGCCCTGCTGAGAGGCGTGCAGTGT 49 P128268PCT In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45, 46, 47 or a variant thereof having at least 80%, 90%, 95% or 99% sequence identity thereto. SEQ ID NO: 45 (signal peptide nt ii): 5 ATGAAGCATCTTTGGTTCTTCCTCCTGCTCGTGGCAGCCCCTCGCTGGGTGTTGTCT SEQ ID NO: 46 (signal peptide nt iii): ATGAAGCATCTGTGGTTCTTTCTGCTCCTAGTCGCCGCCCCCCGGTGGGTTCTCAGC SEQ ID NO: 47 (signal peptide nt iv): ATGAAGCACCTGTGGTTCTTCTTGCTCCTGGTGGCCGCACCCAGATGGGTCCTGAGC 10 SEQ ID NO: 69 (signal peptide nt v): ATGAAGCACCTGTGGTTCTTCCTGCTGCTGGTGGCTGCCCCTAGGTGGGTGCTGAGC In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 39 and SEQ ID NO: 42 or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises 15 a nucleotide sequence of SEQ ID NO: 45, SEQ ID NO: 39 and SEQ ID NO: 42 or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide further comprises one or more nucleotide sequences encoding a linker, optionally wherein the nucleotide sequence encoding the linker comprises a nucleotide sequence of GGTGGGGGAGGC (SEQ ID NO: 48) or a variant thereof 20 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 43 and SEQ ID NO: 35 or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 46, SEQ ID NO: 43 and SEQ ID NO: 35 or variants 25 thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide further comprises one or more nucleotide sequences encoding a linker, optionally wherein the nucleotide sequence encoding the linker comprises a nucleotide sequence of GGAGGTGGGGGATCTGGAGGTGGAGGATCT (SEQ ID NO: 49) or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% 30 sequence identity thereto. 50 P128268PCT In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44 or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 47, SEQ ID NO: 40, SEQ ID 5 NO: 41 and SEQ ID NO: 44 or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide further comprises one or more nucleotide sequences encoding a linker, optionally wherein the nucleotide sequences encoding the linker comprise a nucleotide sequence of GGTGGAGGGGGG (SEQ ID NO: 50) and GGAGGAGGGGGATCCGGGGGCGGTGGGTCA 10 (SEQ ID NO: 51) or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the peptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding Fc. In some embodiments, the polynucleotide sequence comprises the nucleotide 15 sequence of SEQ ID NO: 10, 11 or 12, a nucleotide sequence encoding a peptide linker, and the nucleotide sequence of SEQ ID NO: 13 or 14. In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide linker. In some embodiments, the product comprises a peptide linked to a HSA by a peptide linker. In some embodiments, the product is encoded by a polynucleotide comprising or 20 consisting of the nucleotide sequence of SEQ ID NOs: 26, 27 or 28, or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the product is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NOs: 36, 37 or 38, or variants thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. 25 In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 26 or a variant thereof. SEQ ID NO: 26 (4Δ-hFc nt): ATGGAGTTCGGACTGAGCTGGGTGTTTCTGGTTGCCCTGCTGCGGGGCGTGCAGTGCTTCGAGACCCT 30 GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGCTGCTGGGCGGG CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG 35 CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT 51 P128268PCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGATGAACTGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC 5 AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAATGA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 10 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 26. In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide linker and comprises a signal peptide with an amino acid sequence of SEQ ID NO: 8, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 67 or a variant thereof. 15 SEQ ID NO: 67 (4Δ-hFc SPii nt): ATGAAGCACCTGTGGTTCTTCCTGCTGCTGGTGGCTGCCCCTAGGTGGGTGCTGAGCTTCGAGACCCT GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGCTGCTGGGCGGG CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC 20 CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGATGAACTGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC 25 CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAATGA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 30 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 67. In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide linker and comprises a signal peptide with an amino acid sequence of SEQ ID NO: 8, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 70 or a variant thereof. 35 SEQ ID NO: 70 (4Δ-hFc SPii nt ii): ATGAAGCATCTTTGGTTCTTCCTCCTGCTCGTGGCAGCCCCTCGCTGGGTGTTGTCTTTCGAGACCCT GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGCTGCTGGGCGGG CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC 40 CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG 52 P128268PCT CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGATGAACTGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC 5 CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAATGA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 70. 10 In some embodiments, the product comprises a peptide linked to an engineered Fc domain by a peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 27 or a variant thereof. SEQ ID NO: 27 (4Δ-hFc_4_mut nt): ATGGAGTTCGGACTGAGCTGGGTGTTTCTGGTTGCCCTGCTGCGGGGCGTGCAGTGCTTCGAGACCCT 15 GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGGCTGCCGGCGCA CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG 20 CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGAGGAAATGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC 25 AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 30 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27. In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide linker and comprises a signal peptide with an amino acid sequence of SEQ ID NO: 8, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 68 or a variant thereof. 35 SEQ ID NO: 68 (4Δ-hFc_4_mut SPii nt): ATGAAGCACCTGTGGTTCTTCCTGCTGCTGGTGGCTGCCCCTAGGTGGGTGCTGAGCTTCGAGACCCT GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGGCTGCCGGCGCA CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC 40 CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT 53 P128268PCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGAGGAAATGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC 5 AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 68. In some embodiments, the product comprises a peptide linked to an Fc domain by a peptide 10 linker and comprises a signal peptide with an amino acid sequence of SEQ ID NO: 8, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 71 or a variant thereof. SEQ ID NO: 71 (4Δ-hFc_4_mut SPii nt ii): ATGAAGCATCTTTGGTTCTTCCTCCTGCTCGTGGCAGCCCCTCGCTGGGTGTTGTCTTTCGAGACCCT 15 GAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACTGGGAGGCGGTG GGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGGCTGCCGGCGCA CCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACTCCCGAGGTGAC CTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGTGGACGGAGTGG AGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAGTGGTGAGTGTG 20 CTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGCAATAAGGCCCT GCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCAGGTGTATACCC TGCCCCCTTCCAGGGAGGAAATGACTAAGAACCAGGTATCACTGACCTGCCTGGTGAAGGGCTTCTAC CCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCCCC CGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTCTAGGTGGCAGC 25 AGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCACTGAGCCCCGGCAAA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 71. In some embodiments, the product comprises a peptide linked to an engineered silenced Fc 30 domain by a peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 36 or a variant thereof. SEQ ID NO: 36 (4∆-hsFc N-ter peptibody nt): ATGAAGCATCTTTGGTTCTTCCTCCTGCTCGTGGCAGCCCCTCGCTGGGTGTTGTCTTTTGAAACCCT GCGCGGGGACCTGCGCATCCTATCAATACTTAGACATCAGAATCTGCTGAAAGAACTAGGTGGGGGAG 35 GCGAACCAAAATCCCAGGATAAGACCCACACATGTCCTCCGTGCCCAGCCCCAGAGGCTGAGGGCGCC CCAAGCGTGTTTCTCTTTCCTCCTAAACCCAAGGACACACTGATGATCTCCCGGACCCCCGAGGTAAC TTGTGTTGTGGTCGATGTGTCACACGAGGATCCAGAGGTGAAGTTTAATTGGTATGTTGACGGCGTGG AGGTTCATAATGCCAAGACAAAACCACGCGAAGAGCAGTATAACAGCACTTATCGGGTGGTTTCCGTG TTGACCGTGCTACACCAGGACTGGCTCAACGGAAAGGAATATAAGTGTAAGGTGTCCAATAAAGCCCT 40 GCCTAGCTCTATCGAGAAAACTATCTCCAAGGCAAAGGGTCAGCCCCGGGAGCCTCAAGTGTACACTT TGCCGCCGAGCCGAGATGAATTGACCAAAAATCAGGTTAGCCTAACTTGCCTGGTGAAGGGATTCTAT 54 P128268PCT CCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGACAACCTGAGAATAATTATAAAACAACCCCTCC TGTCCTGGACTCTGACGGCTCTTTCTTCCTCTACAGCAAGCTTACGGTGGACAAGTCTAGATGGCAAC AGGGCAACGTGTTCTCTTGCAGCGTCATGCACGAAGCCCTCCACAACCACTACACGCAGAAGAGCCTA TCCCTCTCACCTGGAAAA 5 In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 36. In some embodiments, the product comprises a peptide linked to an engineered silenced Fc domain by a peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 37 or a variant thereof. 10 SEQ ID NO: 37 (hsFc-4 C-ter peptibody nt): ATGAAGCATCTGTGGTTCTTTCTGCTCCTAGTCGCCGCCCCCCGGTGGGTTCTCAGCGAGCCTAAGAG TCAGGACAAGACCCACACGTGCCCACCCTGCCCTGCCCCGGAGGCCGAGGGCGCTCCATCTGTGTTTC TGTTCCCCCCTAAGCCGAAAGACACACTTATGATTTCCCGCACTCCTGAGGTGACCTGCGTCGTAGTG GACGTTTCCCATGAGGATCCAGAAGTTAAGTTCAATTGGTACGTAGATGGCGTGGAGGTGCATAACGC 15 AAAAACCAAGCCACGGGAGGAACAGTACAATAGCACGTATCGCGTGGTATCCGTCCTTACCGTACTGC ATCAGGATTGGCTGAACGGGAAGGAGTACAAATGTAAGGTGTCCAACAAAGCTCTTCCTTCTTCCATC GAGAAGACAATATCTAAAGCCAAGGGCCAGCCTCGCGAGCCACAGGTATATACACTGCCCCCCAGTAG GGATGAGCTGACCAAAAATCAGGTAAGCCTGACTTGCCTGGTAAAAGGCTTCTACCCCTCTGACATTG CAGTCGAGTGGGAATCTAATGGACAACCCGAGAATAACTACAAAACCACTCCGCCTGTCCTAGACAGC 20 GACGGCAGCTTCTTCCTGTATTCTAAGCTAACAGTAGACAAGAGTCGATGGCAACAAGGTAACGTGTT TTCATGTTCTGTGATGCACGAGGCCCTCCATAATCACTACACTCAAAAGAGCCTGAGTCTCTCCCCCG GTAAGGGAGGTGGGGGATCTGGAGGTGGAGGATCTTTCGAAACGCTTCGGGGCGATCTGCGCATTCTG TCCATTCTGCGCCACCAAAACCTCTTGAAAGAACTTCAAGAC In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 25 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 37. In some embodiments, the product comprises a first peptide linked to an engineered silenced Fc domain by a first peptide linker, and a second peptide linked to the engineered silenced Fc domain by a second peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 38 or a variant thereof. 30 SEQ ID NO: 38 (4∆-hsFc-4 N- and C-ter peptibody): ATGAAGCACCTGTGGTTCTTCTTGCTCCTGGTGGCCGCACCCAGATGGGTCCTGAGCTTCGAAACCTT GCGGGGAGACCTTCGTATCCTTAGCATTCTGAGACATCAGAATCTGCTAAAGGAGCTCGGTGGAGGGG GGGAACCTAAGTCTCAAGACAAGACTCATACTTGCCCCCCTTGTCCAGCACCCGAGGCCGAGGGTGCC CCCTCAGTTTTTCTGTTTCCTCCAAAACCCAAGGACACCCTGATGATATCTAGGACGCCAGAGGTTAC 35 TTGTGTGGTGGTGGACGTCAGCCATGAGGATCCCGAAGTCAAGTTTAATTGGTACGTTGATGGTGTTG AGGTTCATAACGCTAAAACTAAACCTCGAGAAGAGCAATACAATAGCACATACCGAGTTGTCAGTGTG CTCACGGTATTGCATCAGGATTGGCTGAATGGCAAGGAATATAAGTGTAAGGTCTCCAATAAAGCTCT TCCCAGTTCTATCGAGAAGACCATCAGCAAAGCAAAGGGCCAGCCGCGGGAGCCTCAGGTCTACACTT TGCCACCATCCCGAGACGAGCTCACTAAAAATCAGGTGAGCCTGACTTGCCTGGTGAAGGGCTTTTAT 40 CCCTCAGACATCGCCGTTGAATGGGAAAGCAATGGCCAGCCCGAAAACAACTATAAGACAACACCACC AGTTCTTGATAGTGACGGAAGTTTCTTTCTTTACAGTAAGTTGACAGTCGATAAGAGCAGATGGCAAC 55 P128268PCT AGGGGAACGTTTTCAGTTGCTCCGTCATGCACGAGGCCCTTCATAATCATTATACCCAAAAGTCCTTG AGTCTGTCCCCCGGCAAAGGAGGAGGGGGATCCGGGGGCGGTGGGTCATTTGAGACTCTGCGGGGCGA CCTGCGGATTCTCAGTATACTGAGACACCAGAATCTTCTGAAAGAGCTCCAAGAC In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 5 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 38. In some embodiments, the polynucleotide does not encode a signal peptide. Exemplary polynucleotides encoding products of the invention that do not encode a signal peptide may comprise or consist of the nucleotide sequences of SEQ ID NO: 62, 63, 64, 65, 66 or a variant thereof. 10 SEQ ID NO: 62 (4Δ-hFc nt without SP): TTCGAGACCCTGAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACT GGGAGGCGGTGGGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGC TGCTGGGCGGGCCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACT CCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGT 15 GGACGGAGTGGAGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAG TGGTGAGTGTGCTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGC AATAAGGCCCTGCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCA GGTGTATACCCTGCCCCCTTCCAGGGATGAACTGACTAAGAACCAGGTATCACTGACCTGCCTGGTGA AGGGCTTCTACCCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAG 20 ACCACCCCCCCCGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTC TAGGTGGCAGCAGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGTCCCTGTCACTGAGCCCCGGCAAATGA SEQ ID NO: 63 (4Δ-hFc_4_mut nt without SP): TTCGAGACCCTGAGGGGGGATCTCCGGATCCTGAGCATCCTGCGGCACCAGAACCTGCTGAAAGAACT 25 GGGAGGCGGTGGGGAACCTAAGTCACAGGACAAAACCCACACCTGCCCACCTTGCCCAGCTCCCGAGG CTGCCGGCGCACCATCAGTGTTCCTCTTCCCACCTAAGCCAAAGGACACCCTGATGATCAGCCGCACT CCCGAGGTGACCTGTGTGGTGGTGGATGTGTCCCATGAAGACCCTGAGGTGAAGTTCAATTGGTACGT GGACGGAGTGGAGGTGCACAATGCCAAGACAAAGCCTAGAGAAGAACAGTACAATAGCACATACAGAG TGGTGAGTGTGCTGACAGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGTAAGGTGAGC 30 AATAAGGCCCTGCCCGCCCCCATTGAGAAGACCATCAGCAAAGCCAAAGGGCAGCCAAGAGAGCCCCA GGTGTATACCCTGCCCCCTTCCAGGGAGGAAATGACTAAGAACCAGGTATCACTGACCTGCCTGGTGA AGGGCTTCTACCCCTCTGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAG ACCACCCCCCCCGTGCTGGACAGCGATGGCTCTTTCTTTCTGTACTCTAAGCTGACTGTGGATAAGTC TAGGTGGCAGCAGGGCAATGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC 35 AGAAGTCCCTGTCACTGAGCCCCGGCAAA SEQ ID NO: 64 (4∆-hsFc N-ter peptibody nt without SP): TTTGAAACCCTGCGCGGGGACCTGCGCATCCTATCAATACTTAGACATCAGAATCTGCTGAAAGAACT AGGTGGGGGAGGCGAACCAAAATCCCAGGATAAGACCCACACATGTCCTCCGTGCCCAGCCCCAGAGG CTGAGGGCGCCCCAAGCGTGTTTCTCTTTCCTCCTAAACCCAAGGACACACTGATGATCTCCCGGACC 40 CCCGAGGTAACTTGTGTTGTGGTCGATGTGTCACACGAGGATCCAGAGGTGAAGTTTAATTGGTATGT TGACGGCGTGGAGGTTCATAATGCCAAGACAAAACCACGCGAAGAGCAGTATAACAGCACTTATCGGG TGGTTTCCGTGTTGACCGTGCTACACCAGGACTGGCTCAACGGAAAGGAATATAAGTGTAAGGTGTCC AATAAAGCCCTGCCTAGCTCTATCGAGAAAACTATCTCCAAGGCAAAGGGTCAGCCCCGGGAGCCTCA 56 P128268PCT AGTGTACACTTTGCCGCCGAGCCGAGATGAATTGACCAAAAATCAGGTTAGCCTAACTTGCCTGGTGA AGGGATTCTATCCTTCCGATATCGCCGTGGAGTGGGAGAGCAACGGACAACCTGAGAATAATTATAAA ACAACCCCTCCTGTCCTGGACTCTGACGGCTCTTTCTTCCTCTACAGCAAGCTTACGGTGGACAAGTC TAGATGGCAACAGGGCAACGTGTTCTCTTGCAGCGTCATGCACGAAGCCCTCCACAACCACTACACGC 5 AGAAGAGCCTATCCCTCTCACCTGGAAAA SEQ ID NO: 65 (hsFc-4 C-ter peptibody nt without SP): GAGCCTAAGAGTCAGGACAAGACCCACACGTGCCCACCCTGCCCTGCCCCGGAGGCCGAGGGCGCTCC ATCTGTGTTTCTGTTCCCCCCTAAGCCGAAAGACACACTTATGATTTCCCGCACTCCTGAGGTGACCT GCGTCGTAGTGGACGTTTCCCATGAGGATCCAGAAGTTAAGTTCAATTGGTACGTAGATGGCGTGGAG 10 GTGCATAACGCAAAAACCAAGCCACGGGAGGAACAGTACAATAGCACGTATCGCGTGGTATCCGTCCT TACCGTACTGCATCAGGATTGGCTGAACGGGAAGGAGTACAAATGTAAGGTGTCCAACAAAGCTCTTC CTTCTTCCATCGAGAAGACAATATCTAAAGCCAAGGGCCAGCCTCGCGAGCCACAGGTATATACACTG CCCCCCAGTAGGGATGAGCTGACCAAAAATCAGGTAAGCCTGACTTGCCTGGTAAAAGGCTTCTACCC CTCTGACATTGCAGTCGAGTGGGAATCTAATGGACAACCCGAGAATAACTACAAAACCACTCCGCCTG 15 TCCTAGACAGCGACGGCAGCTTCTTCCTGTATTCTAAGCTAACAGTAGACAAGAGTCGATGGCAACAA GGTAACGTGTTTTCATGTTCTGTGATGCACGAGGCCCTCCATAATCACTACACTCAAAAGAGCCTGAG TCTCTCCCCCGGTAAGGGAGGTGGGGGATCTGGAGGTGGAGGATCTTTCGAAACGCTTCGGGGCGATC TGCGCATTCTGTCCATTCTGCGCCACCAAAACCTCTTGAAAGAACTTCAAGAC SEQ ID NO: 66 (4∆-hsFc-4 N- and C-ter peptibody): 20 TTCGAAACCTTGCGGGGAGACCTTCGTATCCTTAGCATTCTGAGACATCAGAATCTGCTAAAGGAGCT CGGTGGAGGGGGGGAACCTAAGTCTCAAGACAAGACTCATACTTGCCCCCCTTGTCCAGCACCCGAGG CCGAGGGTGCCCCCTCAGTTTTTCTGTTTCCTCCAAAACCCAAGGACACCCTGATGATATCTAGGACG CCAGAGGTTACTTGTGTGGTGGTGGACGTCAGCCATGAGGATCCCGAAGTCAAGTTTAATTGGTACGT TGATGGTGTTGAGGTTCATAACGCTAAAACTAAACCTCGAGAAGAGCAATACAATAGCACATACCGAG 25 TTGTCAGTGTGCTCACGGTATTGCATCAGGATTGGCTGAATGGCAAGGAATATAAGTGTAAGGTCTCC AATAAAGCTCTTCCCAGTTCTATCGAGAAGACCATCAGCAAAGCAAAGGGCCAGCCGCGGGAGCCTCA GGTCTACACTTTGCCACCATCCCGAGACGAGCTCACTAAAAATCAGGTGAGCCTGACTTGCCTGGTGA AGGGCTTTTATCCCTCAGACATCGCCGTTGAATGGGAAAGCAATGGCCAGCCCGAAAACAACTATAAG ACAACACCACCAGTTCTTGATAGTGACGGAAGTTTCTTTCTTTACAGTAAGTTGACAGTCGATAAGAG 30 CAGATGGCAACAGGGGAACGTTTTCAGTTGCTCCGTCATGCACGAGGCCCTTCATAATCATTATACCC AAAAGTCCTTGAGTCTGTCCCCCGGCAAAGGAGGAGGGGGATCCGGGGGCGGTGGGTCATTTGAGACT CTGCGGGGCGACCTGCGGATTCTCAGTATACTGAGACACCAGAATCTTCTGAAAGAGCTCCAAGAC In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 62, 63, 64, 65 or 35 66. In some embodiments, the product comprises a peptide linked to a HSA by a peptide linker, and is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 28 or a variant thereof. SEQ ID NO: 28 (4Δ-HSA nt): 40 ATGAAATGGGTGACCTTTATCTCTCTGCTGTTCCTGTTCTCATCAGCCTACTCTTTCGAAACCCTGAG AGGCGACCTCAGAATTCTGAGCATCCTGAGACACCAGAACCTGCTGAAGGAGCTGGGAGGCGGAGGCC GCGGCGTGTTCAGAAGGGACGCCCACAAGAGCGAGGTGGCCCATAGATTCAAGGACCTGGGCGAGGAG AACTTCAAAGCCCTGGTGCTCATCGCCTTCGCCCAGTACCTGCAGCAGTGTCCCTTTGAGGACCACGT 57 P128268PCT GAAACTGGTGAACGAGGTGACAGAGTTCGCCAAGACATGTGTCGCAGACGAGAGTGCCGAGAACTGCG ATAAGAGCCTGCACACCCTGTTCGGCGACAAATTGTGTACCGTGGCCACCCTGAGGGAGACCTACGGC GAAATGGCCGATTGTTGCGCCAAGCAGGAGCCCGAGCGGAACGAATGCTTCCTGCAGCACAAAGATGA CAACCCCAATCTGCCCCGGCTGGTGCGCCCTGAGGTGGACGTGATGTGCACCGCCTTCCACGATAATG 5 AGGAGACCTTTCTGAAAAAATATCTCTACGAGATCGCCAGACGCCACCCCTACTTCTACGCCCCAGAA CTGCTGTTCTTCGCCAAGAGATATAAGGCAGCCTTCACCGAGTGCTGTCAGGCCGCTGACAAGGCCGC CTGCCTGCTGCCTAAGCTGGACGAGCTGAGAGACGAGGGAAAGGCTTCTTCCGCCAAACAGAGGCTGA AGTGCGCTTCTCTGCAGAAATTCGGCGAACGCGCCTTCAAGGCCTGGGCCGTGGCCAGGCTGAGCCAG CGGTTTCCTAAGGCCGAGTTCGCCGAAGTGTCAAAACTGGTGACCGACCTGACCAAAGTGCATACTGA 10 GTGCTGCCACGGCGACCTGCTGGAGTGCGCCGATGATAGGGCCGATCTGGCCAAGTACATCTGCGAGA ACCAGGACTCTATTAGCTCCAAGCTGAAAGAGTGTTGTGAGAAACCTCTGCTGGAGAAAAGCCATTGT ATCGCCGAGGTAGAGAACGACGAGATGCCCGCCGATCTGCCAAGCCTGGCCGCCGACTTCGTGGAATC CAAAGACGTGTGTAAGAACTATGCCGAGGCCAAGGACGTGTTCCTGGGAATGTTCCTGTATGAATACG CCAGGAGACACCCAGACTATTCCGTGGTGCTCCTGCTGAGGCTGGCCAAAACCTACGAAACAACACTG 15 GAAAAGTGTTGTGCCGCTGCCGATCCACACGAGTGCTATGCAAAAGTGTTTGACGAGTTCAAACCACT GGTGGAGGAGCCACAGAATCTGATCAAGCAGAATTGCGAGCTGTTCGAGCAGCTGGGCGAGTATAAAT TCCAGAACGCCCTGCTGGTGCGCTATACCAAAAAGGTGCCACAGGTGTCCACTCCAACCCTGGTCGAG GTGAGTCGGAATCTGGGCAAGGTGGGCTCTAAGTGCTGCAAACACCCCGAGGCTAAGAGAATGCCATG CGCCGAGGATTATCTGAGCGTGGTGCTGAATCAGCTGTGCGTGCTGCACGAGAAGACCCCCGTCAGCG 20 ACCGGGTGACCAAGTGCTGCACCGAATCCCTTGTGAACCGGAGACCATGTTTTTCCGCCCTGGAGGTA GACGAGACCTATGTGCCCAAGGAATTCAATGCTGAGACTTTCACCTTCCACGCCGACATCTGCACACT GTCTGAGAAAGAGCGGCAGATCAAGAAGCAGACGGCCCTGGTGGAGTTGGTGAAGCACAAACCTAAGG CCACAAAGGAGCAGCTGAAGGCCGTGATGGATGACTTTGCCGCCTTCGTCGAAAAATGCTGCAAGGCC GATGACAAAGAGACTTGCTTTGCCGAGGAGGGAAAGAAGCTGGTGGCCGCTTCTCAGGCCGCCCTGGG 25 CCTGTGA In some embodiments, the variant comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 28. 30 It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in 35 which the polypeptides are to be expressed. Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and 40 phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life-span of polynucleotides of interest. 58 P128268PCT Vector A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. The present invention further provides a vector comprising a polynucleotide of the invention. 5 Suitably, said vector may be used to introduce said polynucleotide into a host cell, for example, so that the host cell expresses and / or is capable of expressing a peptide according to the invention. The vector may be used to maintain said nucleic acid within the cell. Vectors comprising polynucleotides of the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation. 10 Transfection may refer to a general process of incorporating a nucleic acid into a cell and includes a process using a non-viral vector to deliver a polynucleotide to a cell. Transduction may refer to a process of incorporating a nucleic acid into a cell using a viral vector. Examples of vectors include, but are not limited to, plasmids, chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. 15 DNA). The vector of the invention may be a viral vector. Examples of viral vectors include, retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors. Viral vectors may be used to introduce a nucleic acid sequence to a host cell via transduction. The vector of the invention may be non-viral. Non-viral vectors may be used to introduce a 20 nucleic acid sequence to a host cell via DNA transfection. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, and cationic facial amphiphiles (CFAs). Pharmaceutical composition 25 The present invention also provides a pharmaceutical composition comprising a product, polynucleotide or vector of the present invention. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent or excipient. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The 30 pharmaceutical composition may comprise as (or in addition to) the carrier, excipient or 59 P128268PCT diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agent(s). The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides or compounds. Such a formulation may, for example, be in a form suitable for intravenous administration. 5 Subject and use The product, polynucleotide, vector or pharmaceutical composition may be for use as a medicament. The product, polynucleotide, vector or pharmaceutical composition may be for use in treating or preventing cancer in a subject in need thereof. In one aspect, the invention provides the product, polynucleotide or vector of the invention for 10 use in therapy. In one aspect, the invention provides the product, polynucleotide or vector of the invention for use in the treatment or prevention of cancer. The subject may be a human. The subject may be a non-human animal. Cancer 15 The subject may be afflicted with a cancer. The subject may be at risk of developing a cancer. The subject may be diagnosed with a cancer by any suitable means known to those of skill in the art. For example, subjects may be diagnosed by imaging, such as computerised tomography (CT) scan, magnetic resonance imaging (MRI), positron emission tomography (PET) scan, ultrasound or X-ray, or they may be diagnosed with a biopsy, a physical exam, or 20 via blood tests, such as blood smear, complete blood count, or analysis of circulating cancer biomarkers. The subject may have been previously determined to be at risk of developing a cancer. The increased risk may have been determined by genetic screening and / or by reviewing the subject’s family history. The subject may have been determined to express one or more 25 genetic markers indicative of an increased risk of developing a cancer. The skilled person will be aware of genetic risk factors (e.g. genetic markers) associated with increased risk of developing a cancer. The skilled person may use any suitable method or technique known in the art to determine whether the subject has an increased risk of developing a cancer. 60 P128268PCT The subject may have previously received treatment for the cancer. The subject may be in remission from the cancer. The subject may be resistant to chemotherapy. The cancer may, for example, be a primary or secondary cancer. Secondary cancer may be the result of metastasis of a primary cancer to a further location in the body. Metastatic disease 5 may present with a much lower survival rate than primary cancer, and be a significant cause of cancer-related morbidity and mortality. αvβ6 integrin can be upregulated in several solid tumour types, including tumours of the pancreas, head and neck, skin, lung, oesophagus, stomach, colon, breast, uterine cervix, and fallopian tube / ovary. αvβ8 can be upregulated in various carcinoma cells, and by tumor- 10 infiltrating regulatory T cells (Treg). The cancer may be a solid tumour that has upregulated and / or overexpressed levels of αvβ6 and / or αvβ8 integrin. Suitably, the αvβ6 and / or αvβ8 integrin may be upregulated and / or overexpressed compared to corresponding non-cancerous cell types or tissues. The αvβ6 and αvβ8 integrin expression level of the cancer may have been previously determined, and / or the 15 cancer may be a cancer type known in the art to express αvβ6 and αvβ8 integrin. Suitably, the αvβ6 and / or αvβ8 integrin may be upregulated and / or overexpressed at the RNA or protein level. Methods to determine whether a cancer expresses or overexpresses αvβ6 and / or αvβ8 integrin are known in the art, and include reverse transcription polymerase chain reaction (RT- PCR), Western blotting, flow cytometry, fluorescent microscopy, and immunohistochemistry. 20 Suitably, the cancer is a solid tumour, also known as an organ tumour. A solid tumour may refer to, for example, a carcinoma, a sarcoma, or a lymphoma. The cancer may be a solid tumour selected from the group consisting of: pancreatic cancer, breast cancer, prostate cancer, fibrosarcoma, oral or skin squamous cell carcinoma, head and neck cancer, ovarian cancer, lung cancer, kidney cancer, cervical cancer, colorectal cancer, 25 gastric cancer, liver cancer, melanoma and brain tumor (e.g. glioblastoma, astrocytoma or metastatic brain tumors). The cancer may be pancreatic ductal adenocarcinoma (PDAC). Secondary cancers (metastases) are the development of malignant growths at a secondary location away from the primary site of the cancer. They most commonly develop due to cancer 30 cells breaking away from the main tumour and entering the bloodstream or lymphatic system and establishing a tumour at a new location. 61 P128268PCT The liver is one of the most common sites for metastasis, accounting for nearly 25% of metastatic cases. The double blood supply to the liver of the portal vein and the hepatic artery is hypothesised to increase the deposition of circulating cancer cells there according to the “mechanical and hemodynamic hypothesis”. The “seed-and-soil” hypothesis considers that 5 some primary tumours selectively target the liver as a favourable metastatic location. The majority of liver metastases are carcinomas, particularly adenocarcinoma. In particular embodiments of the invention, the cancer may be metastatic disease. In some embodiments, the cancer may be metastatic pancreatic ductal adenocarcinoma, or metastatic melanoma. In some embodiments, the cancer may be liver metastases of pancreatic ductal 10 adenocarcinoma. In some embodiments, the cancer may be a metastatic brain tumour. The invention provides the use of the product, polynucleotide, vector and / or pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing a cancer in a subject in need thereof. The invention provides a method for treating or preventing a cancer, wherein the product, 15 polynucleotide, vector and / or pharmaceutical composition of the invention is administered to a subject in need thereof. The therapeutic applications provided by the invention may comprise administration of a therapeutically effective amount of the product, polynucleotide, vector and / or pharmaceutical composition of the invention. The term “therapeutically effective amount”, as used herein in 20 the context of cancer treatment or prevention, refers to an amount of product, polynucleotide, vector, and / or pharmaceutical composition of the invention which is required to achieve an appreciable prevention or cure of a cancer; a prevention or delay of a tumor growth; and / or a reduction in severity or amelioration of one or more symptoms of a cancer. In some embodiments, the product of the invention is capable of binding to tumor cells that 25 express the avβ6 integrin, the avβ8 integrin or both the avβ6 and avβ8 integrins. In some embodiments, the product binding may interfere with activation of latent TGFβ, and inhibit TGFβ activation by cancer cells. Fibrosis In one aspect, the invention provides the product, polynucleotide or vector of the invention for 30 use in the treatment or prevention of fibrosis. The subject may be afflicted with fibrosis. The subject may be at risk of developing fibrosis. In some embodiments, the fibrosis is selected from the group consisting of pulmonary fibrosis, 62 P128268PCT liver fibrosis, kidney fibrosis, and capsular fibrosis. In some embodiments, the fibrosis is associated with functions of ανβ6 integrin and / or ανβ8 integrin. The patient may be diagnosed with fibrosis using any suitable means known in the art. Such means may include, for example, a CT scan, an MRI scan, an ultrasound scan, a magnetic 5 resonance elastography (MRE) scan, an X-ray, and biopsy. Suitably, the diagnostic tests may depend on the organ affected. In some embodiments, diagnostic means may include known means in the art suitable to assess organ function. The patient may be determined to be at risk of developing fibrosis through any suitable means known in the art. In some embodiments, a risk of developing fibrosis may be determined from 10 assessment of lifestyle factors, for example excessive drinking or tobacco use (e.g. for liver fibrosis), or from the presence of pre-existing conditions, for example viral hepatitis or diabetes (e.g. for liver fibrosis). In some embodiments, a risk of developing fibrosis may be caused by injury or surgery, for example capsular fibrosis, also known fibrotic posterior capsular opacification (PCO), which is a common complication of cataract surgery. In some 15 embodiments, a risk of developing fibrosis may be determined from genetic factors, such as mutations in telomere maintenance-associated genes (DKC1, NAF1, PARN, RTEL1, TERC, TERT, TINF2, ZCCHC8) or in surfactant metabolism-associated genes (ABCA3, SFTPA1, SFTPA2, SFTPC) in the case of familial pulmonary fibrosis (FPF). The invention provides the product, polynucleotide, vector and / or pharmaceutical composition 20 of the invention for use in the treatment or prevention of fibrosis in a subject in need thereof. The invention provides the use of the product, polynucleotide, vector and / or pharmaceutical composition of the invention in the manufacture of a medicament for treating or preventing fibrosis in a subject in need thereof. The invention provides a method for treating or preventing fibrosis, wherein the product, 25 polynucleotide, vector and / or pharmaceutical composition of the invention is administered to a subject in need thereof. The therapeutic applications provided by the invention may comprise administration of a therapeutically effective amount of the product, polynucleotide, vector and / or pharmaceutical composition of the invention. The term “therapeutically effective amount”, as used herein in 30 the context of fibrosis treatment or prevention, refers to an amount of product, polynucleotide, vector and / or pharmaceutical composition of the invention which is required to achieve an appreciable prevention or cure of fibrosis and / or a reduction in severity or amelioration of one or more symptoms of fibrosis. 63 P128268PCT The invention provides a method for treating or preventing fibrosis, wherein a product of the invention is used to inhibit TGFβ activation by cells expressing avβ6 and / or avβ8 integrin. Administration The product, polynucleotide, vector and / or pharmaceutical composition of the invention may 5 be administered by a variety of routes that make the agent bioavailable. For example, the agent can be administered parenterally, intraperitoneally, intravenously, subcutaneously, transcutaneously, intramuscularly, and / or via local delivery for example by catheter or stent. Typically, a physician will determine the dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. In some 10 embodiments, the dosage is such that it is sufficient to have anti-tumour activity, such as to destroy tumour cells or reduce tumour growth. In other embodiments, the dosage is such that it is sufficient to have anti-fibrotic activity, such as to prevent TGFβ activation. Suitably, the product, polynucleotide, vector and / or pharmaceutical composition of the invention may be administered to a subject in one or more doses. In some embodiments, the 15 product, polynucleotide, vector and / or pharmaceutical composition may be administered in a single dose. In some embodiments, the product, polynucleotide, vector and / or pharmaceutical composition may be administered in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more doses. Suitably, the one or more doses are administered at defined intervals. In some embodiments, the product, 20 polynucleotide, vector and / or pharmaceutical composition may be administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every 12 hours, at least every 24 hours, at least every 48 hours, at least every 72 hours, at least biweekly, at least weekly, at least every 14 days, at least every 21 days, at least every month, at least every two months, at least every 25 three months, at least every four months, at least every five months, at least every six months, at least every nine months, at least every twelve months, at least every two years, or at least every five years. Combinations The product of the invention may be used in combination with one or more further therapeutic 30 agent. Suitably, the further therapeutic agent may be a therapeutic agent used in the treatment or prevention of cancer. In some embodiments, the further therapeutic agent may be selected 64 P128268PCT from immunotherapy, chemotherapy, targeted therapy, hormone therapy or radiotherapy. In some embodiments, the further therapeutic agent is an immunotherapy. Suitably, the immunotherapy may be selected from any immunotherapy used in the treatment or prevention of cancer. Such therapeutic agents are well-known in the art. Examples of 5 immunotherapeutic agents include immune-checkpoint inhibitors, cell-based therapies (e.g. CAR T-cell therapy, TCR-redirected T lymphocytes, tumour infiltrating lymphocytes (TILs), NK cells and NKT-cells), vaccines, immunostimulatory factors (e.g. cytokines), and monoclonal antibodies. In some embodiments, the immunotherapy is an immune-checkpoint inhibitor, for example anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody; a cell-based 10 therapy, for example tumour-directed CAR T-cell therapy; or an immunostimulatory factor, such as a cytokine, for example NGR-TNF, S-NGR-TNF or IL-2. In some embodiments, the product of the invention is used in combination with one or more immunotherapies. In some embodiments, the product of the invention is used in combination with an anti-PD-L1 agent. In some embodiments, the product of the invention is used in combination with an anti-PD-L1 15 antibody. In some embodiments, the product of the invention is used in combination with tumour necrosis factor alpha (TNFα) or a derivative thereof. In some embodiments, the product of the invention is used in combination with S-NGR-TNF. In some embodiments, the product of the invention is used in combination with an anti-PD-L1 agent and a TNFα or a derivative thereof. In some embodiments, the product of the invention is used in combination 20 with an anti-PD-L1 antibody and a TNFα or a derivative thereof. In some embodiments, the product of the invention is used in combination with an anti-PD-L1 antibody and S-NGR-TNF. In some embodiments, the anti-PD-L1 agent is a mAb that targets PD-L1. In some embodiments, the anti-PD-L1 is 10F.9G2. Suitably, the further therapeutic agent may be a therapeutic agent used in the treatment or 25 prevention of fibrosis. In one aspect, the invention provides a combination of the product with one or more further therapeutic agent. The combination may for example, be a composition or a kit of parts. In some embodiments, the invention provides a combination of the product with an anti-PD-L1 agent (e.g. antibody) and / or a TNFα or derivative thereof. In some embodiments, the invention 30 provides a combination of the product with an anti-PD-L1 antibody and S-NGR-TNF. In some embodiments, the invention provides a product comprising an amino acid of SEQ ID NO: 2 wherein the peptide is linked to HSA, in combination with an anti-PD-L1 antibody and / or S- NGR-TNF. 65 P128268PCT In one aspect, the invention provides the product of the invention for use in therapy, wherein the product is administered simultaneously, sequentially, or separately in combination with one or more further therapeutic agent. In one aspect, the invention provides the product of the invention for use in therapy, wherein 5 the product is administered simultaneously, sequentially, or separately in combination with an anti-PD-L1 agent (e.g. antibody) and / or a TNFα or derivative thereof. In one aspect, the invention provides the product of the invention for use in therapy, wherein the product is administered simultaneously, sequentially, or separately in combination with an anti-PD-L1 antibody and S-NGR-TNF. 10 The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein may refer to the combined administration of two or more agents simultaneously, sequentially or separately. The term “simultaneous” as used herein means that the agents are administered concurrently, i.e. at the same time. The term “sequential” as used herein means that the agents are 15 administered one after the other. The term “separate” as used herein means that the agents are administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Suitably, the product may be administered together with or separately to the one or more further therapeutic agent. In some embodiments, the product is administered simultaneously 20 with the one or more further therapeutic agent. In some embodiments, the product is administered together in a single composition with the one or more further therapeutic agent. In some embodiments, the product is administered at the same time as the one or more further therapeutic agent but as separate administrations. In some embodiments, the product is administered sequentially with the one or more further 25 therapeutic agent. In some embodiments, the product of the invention is administered at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes prior to the one or more further therapeutic agent. In some embodiments, the product is administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at 30 least 72 hours prior to the one or more further therapeutic agent. In some embodiments, the product is administered at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, or at least 14 days prior to the one or more further therapeutic agent. In some embodiments, the product is administered at 66 P128268PCT least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks prior to the one or more further therapeutic agent. In some embodiments, the product is administered at least one month, at 5 least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least 10 months, at least 11 months, or at least 12 months prior to the one or more further therapeutic agent. In some embodiments, the product of the invention is administered at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes 10 after the one or more further therapeutic agent. In some embodiments, the product is administered at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 72 hours after the one or more further therapeutic agent. In some embodiments, the product is administered at least one day, at least two days, at least three days, at least four days, at least 15 five days, at least six days, at least seven days, or at least 14 days after the one or more further therapeutic agent. In some embodiments, the product is administered at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks after the one or more further therapeutic agent. 20 In some embodiments, the product is administered at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least 10 months, at least 11 months, or at least 12 months after the one or more further therapeutic agent. Variants, derivatives, analogues, homologues and fragments 25 In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses the use of variants, derivatives, analogues, homologues and fragments thereof. In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been 30 modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring protein. 67 P128268PCT The term “derivative” as used herein, in relation to proteins or polypeptides of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its endogenous 5 functions. The term “analogue” as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics. Proteins used in the present invention may also have deletions, insertions or substitutions of 10 amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and 15 arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine. A substitution may involve replacement of an amino acid for a similar amino acid (a conservative substitution). A similar amino acid is one which has a side chain moiety with related properties as grouped together, for example as shown below: 20 (i) basic side chains: lysine (K), arginine (R), histidine (H); (ii) acidic side chains: aspartic acid (D) and glutamic acid (E); (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or (iv) non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline 25 (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C). Variant sequences may comprise amino acid substitutions, additions, deletions and / or insertions. Conservative substitutions, additions or deletions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same 30 line in the third column may be substituted for each other: 68 P128268PCT The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue), e.g. like-for-like substitution such as basic for basic, acidic for 5 acidic, polar for polar etc. Non-homologous substitution may also occur e.g. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids, such as ornithine. The term “variant” as used herein may mean an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term “homology” can be 10 equated with “identity”. A variant sequence may include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97%, or at least 99% identical to the subject sequence. Typically, the variants will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms 15 of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. A variant sequence may include a nucleotide sequence which may be at least 40%, 45%, 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97%, or at least 20 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity. Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire 25 length of the SEQ ID NO referred to. 69 P128268PCT Identity comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences. Percentage homology may be calculated over contiguous sequences, i.e. one sequence is 5 aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for 10 example, in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence may cause the following codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall 15 homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology. However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, 20 will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default 25 values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for 30 carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid – Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are 70 P128268PCT available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8). 5 Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix – the default matrix for the BLAST suite of programs. GCG 10 Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Once the software has produced an optimal alignment, it is possible to calculate percentage 15 homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- 20 length polypeptide or polynucleotide. Such variants may be prepared using standard recombinant DNA techniques such as site- directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient 25 restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. 30 This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are 71 P128268PCT written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not 5 exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed. Preferred features and embodiments of the invention will now be described by way of non- 10 limiting examples. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. 15 (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch.9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. 20 (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference. EXAMPLES 25 Example 1 – production of a conjugate consisting of peptide coupled to human serum albumin (HSA) with a high affinity and selectivity for αvβ6 and αvβ8 integrins. Preparation and characterization of 5a-HSA The peptide 5a (CFETLRGDLRILSILRX1QNLX2KELQD, SEQ ID NO: 2) with an amidated C- terminus was chemically coupled to human serum albumin (HSA) using the hetero-bifunctional30 sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) cross- linking agent (5a-HSA, lot #A). To assess the number of peptide moieties / HSA molecule in the final product, we analyzed the molecular weight of HSA, sulfo-SMCC-activated HSA 72 P128268PCT (*HSA), and 5a-HSA (lot #A) by SDS-PAGE under reducing and non-reducing conditions, mass spectrometry analysis, and gel-filtration chromatography. The results showed that this conjugate consists of a mixture of monomeric molecules with molecular weights ranging from ∼70 kDa to ∼90 kDa (Fig.1A-C). Considering that the molecular weight of HSA is ∼66.5 KDa 5 and that of the 5a peptide plus linker is ∼3.33 KDa, we estimate from these data that different HSA molecules bearing different numbers of peptides (ranging from 1 to 6, average ∼3) were present in the final product. Similar results were obtained by mass spectrometry analysis of a second preparation of 5a-HSA (lot #B), showing again a heterogeneous conjugate with an average of coupled peptides / HSA molecule of ∼4 (Fig.2). 10 5a-HSA binds recombinant human αvβ6 and αvβ8 The binding properties of 5a-HSA to purified human integrin αvβ6 and αvβ8 were investigated using direct- and competitive-binding assays. Direct integrin-binding assays, based on the use microtiter plates coated with purified human integrins αvβ6 or αvβ8, showed that 5a-HSA can bind both integrins with high affinity (Kd, 15 0.113 nM and 0.620 nM, respectively) (Fig 3A). Similar results were obtained using competitive binding assays based on the use of isoDGR-HRP conjugate as a probe for the RGD binding site of these integrins (Fig.3B). Other competitive binding assays, based on the use of the Latent TGFβ1 in the solid-phase and biotinylated αvβ6 or αvβ8 in the liquid-phase, showed that peptide 5a and 5a-HSA could compete the interaction of these compounds with 20 similar potencies (Fig.3C). These results, overall, indicate that coupling the peptide 5a to HSA preserves its capability to recognize αvβ6 and αvβ8. 5a-HSA recognizes αvβ6 and / or αvβ8-positive cancer cells The capability of 5a-HSA to recognize αvβ6+and / or αvβ8+cancer cells was then investigated. To this aim, we characterized, first, the expression of αvβ6 and αvβ8 by murine cells, including 25 TS / A mammary adenocarcinoma, WEHI-164 fibrosarcoma, TRAMP-C2 adenocarcinoma prostate, 5M7101 / GFP / CEA and K8484 / GFP / CEA pancreatic ductal adenocarcinoma, and human cells, including BxPC3 pancreatic ductal adenocarcinoma and 5637 bladder cancer, by FACS analysis with anti-αvβ6 and -αvβ8 antibodies. Based on the results (Fig. 4) we selected the 5637cell line (αvβ6pos, αvβ8pos), BxPC3 cells (αvβ6pos, αvβ8neg) and TRAMP-C2 30 cells (αvβ6neg, αvβ8pos) for 5a-HSA binding studies based on cell adhesion to microtiter plates coated with 5a-HSA or *HSA. The results of these assays showed that 5a-HSA, but not *HSA (a control conjugate without peptide 5a), could promote the adhesion of double- or single- positive cells (such as 5637, BxPC3, or TRAMP-C2 cells) (Fig.5A). Of note, the adhesion of 73 P128268PCT TRAM-C2 cells to 5a-HSA-coated plates was completely blocked by an excess of peptide 5a added to the cells, but not by peptide 2a (a control peptide containing RGE in place of RGD) (Fig.5B). These results, overall, suggest that 5a-HSA can interact with αvβ6 and / or αvβ8 also when expressed on the cell membrane. 5 Peptide 5a and 5a-HSA inhibit the production of active TGFβ by cancer cells To verify that 5a-HSA can efficiently inhibit TGFβ activation mediated by αvβ6 or αvβ8-positive cancer cells, we then investigated the effect of 5a-HSA, *HSA (negative control protein), 5a, and 2a (negative control peptide) on TGFβ production by WEHI-164, TS / A, TRAMP-C2, 5M7101 / GFP / CEA, and 5637 cancer cells. The amount of active TGFβ in the cell supernatant10was quantified after 48-72 h incubation using a bioassay based on HEK-Blue™ TGFβ cells.As expected, 5a and 5a-HSA, but not 2a and *HSA, could inhibit in a dose-dependent manner the production of TGFβ by these cells (Fig.6), presumably by inhibiting the integrin-mediated activation of its latent form. Peptide 5a inhibits Treg-mediated TGFβ activation15Considering that regulatory T cells, potentially present in the tumor microenvironment, areknown to express αvβ8 and that these cells can induce TGFβ activation via this integrin, we tested the hypothesis that peptide 5a could also bind to these cells and block their TGFβ- activating mechanisms. Binding experiments (by FACS) showed a significant binding of fluorescence-labeled peptide 5a (5a-IRDye680) to in vitro differentiated regulatory T cells20(iTregs) and little or no binding to PBMC or conventional human T cells (Tconvs) (Fig.7A-C).Furthermore, peptide 5a, but not 2a, blocked TGFβ activation in iTregs cultures (Fig.7D). No TGFβ activation or no effect of peptides was observed in conventional human T cells (Fig. 7D). Notably, although no binding of an anti-human αvβ8 mAb (clone EM13309) to iTregs was observed by FACS analysis, mRNA analysis of these cells revealed the production of the25mRNA coding for the β8 integrin (data not shown).These results indicate that iTreg cells may represent another important target of peptide 5a and its derivatives. 5a-HSA has a plasma half-life longer than that of peptide 5a The plasma half-life of fluorescence-labeled peptide 5a, injected i.p., is very short (~8 min). 30 To assess whether the conjugation of this peptide to HSA increases its plasma half-life, we administered 5a-HSA (50 µg, i.p.) to mice and analyzed its plasma levels at various time points by ELISA. The plasma half-life of 5a-HSA was 15-17 hours, as estimated from the curve 74 P128268PCT obtained. Notably, 2-2.5 days after injection, the circulating levels of 5a-HSA were still above the Kd values for both αvβ6 and αvβ8 integrins (Fig.8, upper panel). The plasma half-life of the intravenously injected 5a-HSA was 100 min (Fig.8, lower panel). 5a-HSA inhibits TGFβ-signaling in αvβ6- or αvβ8-positive tumors 5 To assess whether circulating 5a-HSA can reduce TGFβ activation in the tumor microenvironment, we analyzed the activation of intracellular TGFβ-signaling mediators (SMAD2 / 3) in different models of tumor-bearing mice after systemic administration of 5a-HSA. The first investigated model consists of murine K8484 pancreatic ductal adenocarcinoma (PDAC) cells genetically engineered to express the green fluorescent protein (GFP) and 10 carcinoembryonic antigen (CEA) (K8484 / GFP / CEA), intra-portally injected in immunocompetent mice to generate liver metastatic colonies. Preliminary FACS analysis of these cells with specific antibodies showed that these cells express αvβ6, but not αvβ8 (Fig. 4) and that they can bind 5a-IRDye680 (Fig. 9A). Immunohistochemical analysis of K8484 / GFP / CEA liver metastases with anti-αvβ6 antibodies confirmed the presence of this 15 integrin in tumor tissues (Fig.9B), while imaging studies with 5a-IRDye800, administered i.v. to tumor-bearing mice, showed efficient accumulation of this conjugate into metastatic colonies in vivo (Fig.9C). These findings indicate that K8484 / GFP / CEA metastatic colonies express αvβ6 in a functionally active form (in terms of peptide 5a binding), and that this model, therefore, is suitable for in vivo studies regarding the effect of 5a-HSA on TGFβ activation. 20 Thus, tumor-bearing mice were treated with or without 5a-HSA (50 µg / dose, 4 treatments in 4 consecutive days, i.p, n=5 mice / group) (see Fig.9D). The day after the last treatment, the mice were sacrificed, and their livers were explanted for TGFβ signaling (pSMAD2 / 3) analysis in metastases. Microscopic analysis of liver tissue sections showed that all mice of the control group had metastases (5 / 5) while only 2 / 5 mice of the 5a-HSA-treated group had liver25 metastases (Fig.9E and F). Immunohistochemical analysis of metastatic lesions with anti- pSMAD2 / 3 antibodies showed a significant reduction of p-SMAD2 / 3 in the metastases of mice treated with 5a-HSA, compared to control livers, suggesting that 5a-HSA reduced local TGFβ activation in this model (Fig.9G and H). Notably, 5a-HSA did not reduce nuclear p-SMAD2 / 3 expression in the adjacent non-neoplastic “healthy” liver, indicating that 5a-HSA could reduce 30 TGFβ activation only in the tumor microenvironment (Fig.9H). The effect of systemically administered 5a-HSA on TGFβ activation in the tumor microenvironment was then analyzed in another model based on TS / A mammary adenocarcinoma cells (αvβ6neg, αvβ8pos(low)) implanted subcutaneously in immunocompetent mice. The rationale, for including a model lacking αvβ6 and with low expression of αvβ8 by 75 P128268PCT cancer cells relies on the notion that αvβ8+Treg cells, likely present in the tumor microenvironment, can significantly contribute to TGFβ activation in the tumor microenvironment. Thus, 5a-HSA or its vehicle were administered to tumor-bearing mice (n=4 mice / group, 50 µg / dose, i.p., 4 treatments in 4 days, see Fig.10A and B); one day after the 5 last treatment, the tumors were excised, lysed, and processed for western blot analysis of phospho-SMAD3 with anti-phospho-SMAD3 or anti-total-SMAD3 antibodies (p-SMAD3 and t- SMAD3). Interestingly, a significant reduction of the p-SMAD3 / t-SMAD3 ratio was observed in the group of mice treated with 5a-HSA compared vehicle-treated mice (Fig. 10C-F), supporting the hypothesis that this conjugate can reduce TGFβ activation also in tumors 10 lacking αvβ6. 5a-HSA significantly increases the survival of mice bearing K8484 / GFP / CEA liver metastases (αvβ6-positive) The prolonged half-life of 5a-HSA and its capability to reduce TGFβ activation in the tumor microenvironment, a potentially important immunosuppressive mechanism, provided the 15 rationale for assessing its therapeutic activity. The anti-tumor efficacy of 5a-HSA was first investigated in the PDAC K8484 / GFP / CEA liver metastasis model, which expresses αvβ6, but not αvβ8 (see above). Mice with liver metastatic colonies (n=9) were injected with 5a-HSA, 50 µg / mouse, i.p., for 4 consecutive days per week for a total of 8 administrations or were left untreated (n=5) (see Fig.11A). Tumor growth was then monitored by MRI at day 21, after the 20 completion of the first cycle of treatment, and again at day 33 and 47, after the second cycle. The results showed that 5a-HSA eradicated liver metastases in 3 out 9 mice (33%) after the first cycle of treatment (Fig.11B). These mice were still tumor-free at day 33 and 47, while the remaining mice showed a significant delay in tumor growth, with an overall median survival time of 47 days (Fig.11B and C). In contrast, all control mice (n=5) died between days 32 and 25 38, with a median survival time of 34 days. Notably, the cumulative results of the experiments performed on the K8484 / GFP / CEA model, including the experiments reported in the above paragraph (3 / 5 mice with no metastases in the 5a-HSA groups) and in this paragraph (3 / 9 mice with no metastases), show that 6 / 14 mice 30 could reject tumors (~43%) in the 5a-HSA group, while no rejection was observed in the control group. Interestingly, cured mice re-challenged with a tumorigenic dose of K8484 / GFP / CEA cells (1×106cells / mouse, subcutaneously) showed effective delay / rejection of tumor development. In contrast, naïve control mice injected with the same cell suspension developed subcutaneous tumors, as expected (Fig.11D). 35 76 P128268PCT 5a-HSA inhibits the growth of subcutaneous lesions of TS / A mammary adenocarcinomas, WEHI-164 fibrosarcomas and TRAMP-C2 prostate adenocarcinomas in mice (αvβ8-positive) The anti-tumor activity of 5a-HSA was then evaluated in other syngeneic subcutaneous tumor 5 models based on cells that express various levels of αvβ8 and no αvβ6, including TS / A mammary adenocarcinoma (αvβ6neg, αvβ8pos(low)), WEHI-164 fibrosarcoma (αvβ6neg, αvβ8pos(low)), and TRAMP-C2 prostate carcinoma (αvβ6neg, αvβ8pos) (Fig. 4). Tumors were allowed to grow to 95–150 mm3and then injected with 5a-HSA (40-50 µg / mouse, i.p.) three times per week for a total of 5-6 administrations. This conjugate significantly delayed the tumor 10 growth in all tested models (Fig.12-13). Notably, in the case of TS / A tumors, 5a-HSA induced almost complete cancer regression in 2 / 6 mice, although the tumor recurred after treatment discontinuation (Fig.12-13). The antitumor activity of 5a-HSA was not associated with signs of toxicity, such as changes in animal body weight, animal behavior, and aspect of fur. These results suggest that 5a-HSA can reduce the growth of tumor cells implanted in mice in vivo, 15 even in animal models based on cancer cells that express low levels of αvβ8 and no αvβ6. The anti-tumor activity of 5a-HSA is inhibited by an anti-CD8 depleting antibody To assess whether the anti-tumor activity of 5a-HSA was related to the activation of an immune-response we then investigated the effect of 5a-HSA alone and in combination with an anti-CD8 depleting antibody in the WEHI-164 fibrosarcoma model. The results (Fig.14) 20 showed that the anti-tumor effects of 5a-HSA was significantly inhibited by the anti-CD8 antibody, but not by an irrelevant antibody used as negative control, lending support to the hypothesis that inhibition of TGFβ signalling in tumors can unleash an anti-tumor immune- response, possibly by reprogramming the tumor immune microenvironment. 5a-HSA exerts synergistic anti-tumor effects with an anti-PD-L1 monoclonal antibody 25 The anti-tumor activity of 5a-HSA in combination with a modulator of the immune response, such as the anti-PD-L1 mAb 10F.9G2, was then investigated. The expression of PD-L1 by tumor cell lines was characterized by FACS analysis. Since WEHI-164 fibrosarcoma cells express good levels of PD-L1 (Fig.15) and poorly respond to 5a-HSA (Fig.12 and 13), we decided to use these cells, implanted subcutaneously for the study. In this model, the 30 combined treatment (n=6 mice / group) induced significantly stronger anti-tumor effects than those obtained with single compounds (Fig. 16B-E). While 5a-HSA and anti-PD-L1 mAb, given alone, cured 0 / 6 and 1 / 6 mice, respectively, the combined treatment cured 4 / 6 mice, with no evidence of toxicity as judged from the loss of animal weight in control and treated 77 P128268PCT groups (Fig.16D-F). These results suggest that 5a-HSA and anti-PD-L1 mAb exert synergistic effects, possibly by reducing immunosuppressive mechanisms in the tumor microenvironment. Notably, the 4 mice cured with the combined treatment rejected a second challenge with WEHI-164 tumor cells at day 60 (Fig.17), indicating that the treatment with 5a- 5 HSA and anti-PDL1 mAb induced long-term anti-tumor immunity. The antitumor activity of 5a-HSA in combination with anti-PD-L1 mAb was also investigated in a mammary adenocarcinoma model based on TS / A cells (αvβ6neg,αvβ8pos(low), and PD-L1possee Fig.4 and Fig.15) implanted subcutaneously in immunocompetent mice. The results showed that the combined treatment induced significantly stronger anti-tumor effects than 10 those obtained with single compounds (Fig.18). The antitumor activity of 5a-HSA in combination with anti-PD-L1 mAb was then investigated in a PDAC model of liver metastases based on 5M7101 / GFP / CEA cells (αvβ6pos(low),αvβ8neg, see Fig.4). First, we checked the capability of peptide 5a to recognize 5M7101 / GFP / CEA liver metastases in vivo using the fluorescent 5a-IRDye800 conjugate, taking advantage from the 15 fact that these cells, genetically engineered to express GFP (green) and 5a-IRDye800 (red) can be analyzed by optical imaging ex vivo. A good overlap was observed between GFP and infrared signals in the livers of mice injected with PDAC cells (Fig. 19A), supporting the hypothesis that, indeed, the peptide 5a can home to liver metastases. Based on this finding, mice with liver metastases were randomized into 3 experimental groups (n=6 mice / group) and 20 treated with or without anti-PD-L1 mAb, alone or in combination with 5a-HSA (see Fig.19B for the experimental scheme). The effect of these treatments on tumor burden was assessed by MRI, 3 days after the last administration. While all untreated mice developed liver metastases, 1 / 6 and 3 / 6 mice had no liver metastases in the single and combined treatment groups, respectively (16.7% vs 50% of complete response) (Fig.19C and D), suggesting that 25 the combination therapy induces stronger anti-tumor effects than the individual therapy with anti-PD-L1 mAb. The mice were subsequently subjected to a second round of therapy and examined again by MRI, 10 days after the final administration. The proportion of mice with no signs of liver metastases increased to 50% in the anti-PD-L1 mAb group, while no further changes were observed in the group of mice that received the combined treatment (Fig.19C 30 and D). 5a-HSA exerts synergistic anti-tumor effects with S-NGR-TNF The anti-tumor activity of 5a-HSA in combination with S-NGR-TNF, a derivative of tumor necrosis factor alpha capable of homing to the tumor vasculature and promoting CD8+T cells infiltration in tumors, was then investigated in the WEHI-164 fibrosarcoma model. Again, the 78 P128268PCT combined treatment (n=12 mice / group) induced significantly stronger anti-tumor effects than those obtained with single compounds (Fig.19). While 5a-HSA and S-NGR-TNF individually were unable to cure mice in this model, the combined treatment cured 3 / 12 mice, with no evidence of toxicity as judged from comparable animal weight in control and treated groups 5 (not shown). These results suggest that 5a-HSA and S-NGR-TNF can exert synergistic effects. Finally, the triple combination of 5a-HSA, S-NGR-TNF, and anti-PD-L1 antibody was also tested in the same model. In this experiment, 0 / 6 mice were cured with S-NGR-TNF alone, 2 / 6 with 5a-HSA / S-NGR-TNF, and 5 / 6 mice with 5a-HSA / S-NGR-TNF / anti-PD-L1 antibody 10 (Fig.20), suggesting that the triple combination could induce stronger anti-tumor effects than the double combination. Conclusions The experimental evidence shows that 5a-HSA: a) has a high affinity and selectivity for αvβ6 and αvβ8 integrins, 15 b) can inhibit TGFβ activation by cultured cancer cells and human regulatory T lymphocytes (Tregs), c) can bind αvβ8-expressing immunosuppressive human Tregs in vitro, d) has plasma half-life more prolonged than that of peptide 5a, e) can reduce the TGFβ activation in vivo in the tumor microenvironment, 20 f) can exert significant anti-tumor effects in tumor-bearing mice, either when injected alone, or when injected in combination with immune checkpoint inhibitors or with S-NGR-TNF, a targeted inflammatory cytokine. Materials and Methods Human serum albumin (Albutein, cat. A4AFC02342, Grifols); sulfosuccinimidyl 4-(N- 25 maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce, cat. A39268); gel-filtration NAP 5 and PD10 columns (Cytivia, cat. 17-0853-01 and cat. 17-0851-01); dialysis tubing (Spectra / Por®, cut-off 25 KDa, VWR, cat.734-0504). Millex^GP syringe filters (Millipore, cat. SLGM33RS); phosphate buffered saline (Merk, cat. P3813-1PAK, called PBS-Sigma). Precast 4-20% gradient polyacrylamide gel (Mini Protean TGX precast, BIO-RAD, cat.456- 30 1093). Recombinant human integrins were from Bio-techne (αvβ1, cat.6579-AVB-050; αvβ3, 79 P128268PCT cat. 3050-AV-050; αvβ6, cat. 3817-AV-50; αvβ8, cat. 4135-AV-50 and α5β1, cat. 3230-A5- 050); biotinylated recombinant human αvβ6 and αvβ8 integrins were from ACROBiosystems (cat. IT6-H82E4 and cat, IT8-H82W5, respectively). Normal goat serum (NGS), HRP-labelled goat anti-rabbit polyclonal antibody (cat. A4914), and HRP-labelled streptavidin (cat. S5512) 5 were from Sigma. The following antibodies were used: mouse anti-human / mouse αvβ6 antibody, clone 10D5, IgG2a (Millipore, cat. MAB2077Z); rabbit anti-human / mouse β6 polyclonal antibody (Invitrogen, cat. PA5-35903); mouse anti-human / mouse αvβ8 antibody, clone ADWA-11 (a kind gift from Dr. Dean Sheppard); control isotype-matched murine IgG1, clone MOPC-31C 10 (Sigma, cat. M9035); rabbit anti-human αvβ8 monoclonal antibody, clone EM13309, IgG (Absolute Antibodies, cat. Ab00892); control isotype rabbit IgGs (Abcam, cat. ab37415); Alexa Fluor 488-labeled goat anti-mouse (Invitrogen, cat. A-11001) and goat anti-rabbit (Invitrogen, cat. A-11034); Alexa Fluor 647-labeled goat anti-mouse (Invitrogen, cat. A-32728); Human TGFβ (InvivoGen, cat. rcyc-htgfb1). Anti-mouse PD-L1 antibody (clone 10F.9G2, cat. 15 #BE0101) and anti-mouse CD8α mAb (clone 53-67, cat. #BP0004-1) were from Bio-Cell. S- NGR-mTNF, a recombinant TNF derivative, was prepared as described in Corti A, et al. (2020) Mol Pharm 17: 3813-3824. Cell lines Human BxPC-3 pancreatic ductal adenocarcinoma (cat. CRL-168), human 5637 bladder 20 carcinoma (cat. HTB-9), murine WEHI-164 fibrosarcoma cells (cat. CRL-1751) and murine TRAMP-C2 prostate cancer (cat. CRL-2731) cells were from ATCC (American Type Culture Collection). Murine TS / A mammary adenocarcinoma cells were from Sigma-Aldrich (cat. SCC177). Murine 5M7101 pancreatic cancer cells were isolated from spontaneous liver metastases of Ptf1a-Cre KrasG12D, p53+ / - KCP heterozygous mice, were a kind gift from Dr. 25 Hana Algül (Technische Universität München, Germany). Murine K8484 PDAC cells were established from KPC mice (PdxCre / LSL-KrasG12D-Trp53R172H) were a kind gift of Dr. Lorenzo Piemonti (San Raffaele Scientific Institute, Milan, Italy). 5M7101 and K8484 cells were engineered in-house to express green fluorescent protein (GFP) and human carcinogenic embryonic antigen (CEA), by lentiviral transduction (5M7101 / GFP / CEA and 30 K8484 / GFP / CEA cells) as described in Consonni M et al. (2021) Nat Commun 12(1):4844. Human T3M-4 pancreatic ductal adenocarcinoma cells were a kind gift of Dr. Monica Casucci (San Raffaele Scientific Institute). BxPC-3, 5637, TS / A, K8484 / GFP / CEA cells were cultured in RPMI-1640 medium with standard supplements. WEHI-164, TRAMP-C2, 5M7101, and 5M7101 / GFP / CEA cells were cultured in DMEM medium containing standard supplements 35 with an additional 1% non-essential amino acids added for 5M7101 and 5M7101 / GFP / CEA. 80 P128268PCT All cell lines were free of mycoplasma as routinely tested with the MycoAlert Control Set (Lonza). Preparation of induced regulatory T lymphocytes (iTregs) and conventional T lymphocytes 5 Regulatory T cells (iTregs), defined as CD4+CD25hiCD127lowcells, and CD4+conventional T cells (Tconvs), defined as CD25−CD127hiCD45RA+cells, were isolated from human peripheral blood mononuclear cells by fluorescence-activated cell sorting, essentially as previously described in Yoon et al. (2017) Blood 129(2):238-45, starting from buffy coats. For iTregs, sorted CD4+, CD25+T cells were stimulated with magnetic beads coated with an anti-CD3 / anti- 10 CD28 mAb using 3:1 beads:T cells ratio and cultured in X-vivo medium, containing 10% human serum, 1% penicillin, 1% streptomycin, glutamine, and 100 nM rapamycin. After 2 days, cells were supplemented with 500 U / ml IL-2. After 14 days, activation beads were magnetically removed, and cells were kept in expansion until day 21 in the presence of IL-2 but without rapamycin. On day 21, their phenotype was checked through cytometry evaluation.15For Tconv, sorted CD4+, CD25-T cells were stimulated with anti-CD3 / anti-CD28 magneticbeads in 3:1 beads:T cells ratio and cultured in RPMI 10% FBS, 1% penicillin, 1% streptomycin, glutamine, and homeostatic cytokines IL-7 and IL-15 at a concentration of 5 ng / ml. After 6 days, activation beads were magnetically removed and cells were kept in expansion until day 21 in the presence of IL-7 and IL-15. 20 Preparation and characterization of peptides Peptides were prepared by chemical synthesis as described in Monieri et al. (2023) Int J Biol Sci 19156-66 and Nardelli et al. (2019) Chem Commun 55: 14777-14780, dissolved in sterile water, and stored in aliquots at −20 °C until use. The concentration of peptide was determined by Ellman’s assay using 5,5-dithio-bis2-nitrobenzoic acid (DTNB, Ellman’s Reagent, Thermo 25 Fisher, cat.22582) and / or by BCA assay (Thermo Fisher, cat. A55864). The identity and purity of peptides were checked by mass spectrometry (MS) and reverse-phase HPLC analysis. Conjugation of 5a to IRDye fluorophore Peptide 5a (sequence: CFETLRGDLRILSILRX1QNLX2KELQD, SEQ ID NO: 2) with an amidated C-terminus, chemically stabilized by a triazole bridge between the propargylglycine 30 (X1) and azidolysine (X2) residues) was coupled, via its thiol group, to IRDye® 800CW maleimide or IRDye® 680RD maleimide (LI-COR, P / N: 929-80020 and 929-71050, respectively) as described in Monieri et al. (2023) Int J Biol Sci 19: 156-66. The resulting conjugates were called 5a-IRDye800 and 5a-IRDye680. 81 P128268PCT Preparation and characterization of 5a-HSA HSA (2.5 ml, 500 mg) was gel-filtered through a PD10 column pre-equilibrated in 10 mM phosphate buffer, 138 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4, containing 5 mM EDTA (PBS-E). The eluted product was sterilized by filtration, aliquoted, and stored at - 5 80°C until subsequent use. The product was then used to prepare the 5a-HSA conjugate or a control conjugate lacking peptide 5a, as follows: HSA (117.3 mg in 1.5 ml of PBS-E) was mixed with sulfo-SMCC (6.78 mg in 340 μl of water) and left to react for 1 h at room temperature (HSA: SMCC molar ratio, 1:9). The mixture was then purified by gel-filtration chromatography on a NAP-5 column (Cytiva) pre-equilibrated with PBS-E. The eluted product, 10 corresponding to activated-HSA, was then diluted to 4.5 ml with PBS-E and divided into two separate tubes (2.25 ml / each). To one tube, we added 4.80 µmols of peptide 5a in aliquots of 0.45 ml every 20 min (final peptide / HSA molar ratio of ∼6:1), while the other tube, we added 2-mercaptoethanol (negative control, lacking the peptide). The mixtures were left to react overnight at 4°C and quenched with 2-mercaptoethanol (1 mM, final concentration) for 0.5 h. 15 The resulting products (5 ml final volume in PBS-Sigma) were gel-filtered using 2 PD10 columns in parallel preequilibrated with PBS-Sigma. The products were pooled (7 ml final volume) and dialyzed (cut-off: 25 KDa) overnight against 50 mM sodium phosphate, pH 7.4, containing 150 mM sodium chloride. The products were then aliquoted and stored at –20°C. The protein concentration of each conjugate (called 5a-HSA and *HSA) was determined20spectrophotometrically (A280 nm, using an adsorption coefficient of 0.54). The identity andpurity of the final product were characterized by a) SDS-PAGE using 4-20% precast polyacrylamide gels; b) MALDI-TOF mass spectrometry analysis; and c) gel-filtration chromatography using a Superdex 75 HR column (Cytivia) connected to an HPLC instrument (AKTA purifier 10). 25 Integrin binding assays The capability of 5a-HSA to recognize αvβ6 and αvβ8 integrins was investigated using a) direct binding assay based on the use of integrin-coated plates in the capture step and an anti-HSA polyclonal antibody in the detection step (Assay 1), b) competitive assays based on the use of integrin-coated plates and isoDGR-HRP conjugate as a probe for the RGD-binding 30 site of integrins (Assay 2), or c) competitive assays based on the use of Latent TGFβ1-coated plates and biotinylated αvβ6 or αvβ8 followed by HRP-labelled streptavidin (Assay 3) (see Fig. 3 for a schematic representation of the assays). Direct integrin binding assay (Assay 1) 82 P128268PCT Ninety-six-well PVC microtiter plates (Carlo Erba, cat. FA5280100) were coated with or without human recombinant αvβ6 and αvβ8 integrins in Dulbecco’s phosphate-buffered saline containing calcium and magnesium (DPBS) (1 µg / ml, 50 µl / well, overnight at 4°C). After washing, the plates were blocked with 3% BSA in DPBS (150 µl / well) and incubated for 1 h at 5 room temperature. The plates were then washed with 25 mM Tris–HCl buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, and 1 mM manganese chloride and 0.05% Tween-20 (Buffer-1), and filled with various amounts of 5a-HSA (50 µl / well) in Buffer-1 containing 1% w / v BSA and 0.05% Tween-20 (Buffer-2). After 1.5 h of incubation, the plates were washed with Buffer-1 and filled with an anti-human HSA antibody 10 (Sigma, cat. A3293, 1:10000, 50 µl / well, in Binding-2 containing 1% normal goat serum (NGS) and 0.05% Tween-20 (Buffer-3). After 1 h of incubation, the plates were washed and filled with an HRP-labelled goat anti-rabbit polyclonal antibody (1:1000, 50 µl / well, in Buffer-3). After washing, bound peroxidase was detected by adding o-phenylenediamine, a chromogenic substrate, and reading the absorbance at 490 nm. Specific binding of 5a-HSA was determined 15 by subtracting the non-specific binding measured on wells coated without integrin. The affinity constant (Kd) was then calculated using the "one-site specific binding" equation of Prism software. Competitive integrin binding assays (Assay 2 and 3). 20 Competitive integrin binding assay 2 was performed as described in Nardelli et al. (2019) Chem Commun 55: 14777-14780. Briefly, various amounts of 5a-HSA were mixed with a fixed amount of isoDGR-HRP conjugate and added to integrin-coated plates. After 2 h of incubation, the plates were washed, and the bound peroxidase was detected, as described above. The inhibitory constant (Ki) was calculated as described in Nardelli et al. (2019) Chem Commun 25 55: 14777-14780. Competitive integrin binding assay 3 was performed as follows: ninety-six-well microtiter plates (Greiner Bio-One, cat. #675061) were coated with or without human recombinant Latent TGFβ1 (Acro Biosystems, cat. TG1-H524x) in 50 mM sodium carbonate buffer, pH 9.5, 30 containing 1 mM calcium chloride, 1 mM manganese chloride (2 µg / ml, 50 µl / well, overnight at 4 °C). After washing with 20 mM Tris-HCl pH 7.4, containing 150 mM sodium chloride, 1 mM manganese chloride,and 0.05% Tween-20 (Buffer-4), the plates were blocked with 2% BSA in Buffer-4 (150 µl / well) and incubated for 1.5 h at 37°C. The plates were then washed with Buffer-4 and filled with various amounts of peptide 5a or 5a-HSA and 10 ng / ml of 35 biotinylated recombinant human αvβ6 or αvβ8 integrins (50 µl / well) in Buffer-4 containing 0.5 % w / v BSA (Buffer-5). After 1 h of incubation, the plates were washed with Buffer-4 and then 83 P128268PCT filled with HRP-labelled streptavidin (1:2000 in Buffer-5, 50 µl / well, 1 h). After washing, the bound peroxidase was detected by adding the chromogenic substrate 3,3′,5,5′- tetramethylbenzidineo-phenylenediamine (Sigma, cat. T3405, prepared according to the manufacturer’s instruction) and, after 10 min, by reading the absorbance at 450 nm. 5 Cell adhesion assays Cell adhesion assays were carried out as described in Monieri et al. (2023) Int J Biol Sci 19: 156-66, except that the cells seeded into 96-well plates using their own cell culture medium containing 3% BSA. Flow cytometry analysis 10 Flow cytometry analysis of cell-surface αvβ6 and αvβ8 expression was carried out as described in Nardelli et al. (2019) Chem Commun 55: 14777-14780 and Monieri et al. (2023) Int J Biol Sci 19: 156-66. Binding assays of 5a-IRDye680 and Cys-IRDye680 to cells were carried out by incubating trypsin-EDTA detached cells with various amounts of conjugates (range 0-200 nM) in 25 mM HEPES buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM 15 magnesium chloride, 1 mM manganese chloride and 2% w / v BSA (1 h on ice). After washing, the cells were fixed with 2% paraformaldehyde in PBS; bound fluorescence was detected using a CytoFLEX S flow cytometer (Beckman Coulter) equipped with a 638 nm laser and 712 / 25 nm band-pass filter set. Flow cytometry data were analyzed using FlowJo software (BD BioScience). 20 In vivo studies in animal models All procedures involving laboratory mice were performed in accordance with protocols approved by the Animal Care and Use Committee at San Raffaele Hospital Animal Facilities (IACUC numbers 1082 and 1293) and by the Italian Minister of Health. The study was conducted at the San Raffaele Hospital (an authorized organization) according to institutional 25 guidelines and in compliance with national and international laws and guidelines. The antitumor properties of 5a-HSA were assessed using mouse models of pancreatic cancer liver metastasis, subcutaneous mouse tumor models of fibrosarcoma, mammary adenocarcinoma, and prostate cancer. Mouse models of pancreatic cancer liver metastasis. 30 5M7101 / GFP / CEA or K8484 / GFP / CEA pancreatic ductal adenocarcinoma cells were implanted into the liver of 8-10 weeks old C57BL / 6N female and male mice, respectively. For this purpose, 1.0 x105cells in 200 µl of phosphate-buffered saline were injected into the portal 84 P128268PCT vein. After 2-3 weeks, tumor growth was monitored using a 7-Tesla magnetic resonance imaging (MRI) system (BioSpec, Bruker BioSpin Gmbh) equipped with 450 / 675 mT / m gradients (slew rate: 3400–4500 T / m per second; rise time: 140 µs) coupled with a circular polarized mouse body volume coil (inner diameter of 40 mm) using gadoxetic acid as a 5 contrast agent (Gd-EOB-DTPA; Primovist, Bayer Schering Pharma, administered via the tail vein before imaging, 0.05 μmol / g of body weight). To quantify the tumor volume and the number of metastases, coronal fat-saturated T2 weighted MR images were acquired with axial sections across the entire abdomen (TurboRARE-T2: TR=3080 ms, TE=40 ms; voxel-size, 0.161×0.116 x 0.8 mm; averages=5) acquired immediately after Gd-EOB-DTPA injection and10 an axial fat-saturated T1-weighted scan (RARE-T1: TR=500 ms, TE=7.6 ms, voxel- size=0.161×0.116x0.8 mm, averages=5) acquired thereafter, during the hepatobiliary phase of contrast excretion (starting from 10 min after Gd-EOB-DTPA injection). Normal liver tissue appears hypo-intense in T2-weighted images and hyper-intense in T1-weighted images after administration of Gd-EOB-DTPA; accordingly, liver metastases were defined as focal lesions 15 characterized by slight hyper-intensity on T2-weighted images and concurrent hypo-intensity on contrast-enhanced T1-weighted images. The tumor volume and the number of metastases were quantified by a certified radiologist with >4 years of experience in preclinical abdominal MRI (blinded to any other information) using the open-source Medical Imaging Processing, Analysis & Visualization software (MIPAV, version 11.0.7, Biomedical Imaging Research 20 Services Section, ISL, CIT, National Institute of Health, USA). Regions-of-interest (ROIs) were drawn slice-by-slice on liver metastases, and then the volume-of-interest (VOIs) of each lesion was calculated by multiplying the ROIs areas (mm2) for the slice thickness (mm). Finally, the total metastatic volume was obtained by summing the volumes of all the single VOIs. Subcutaneous mouse tumor models 25 BALB / c or C57BL / 6N mice (Charles River Laboratories) were challenged with subcutaneous injection in the leftflank of 1.5×106cells WEHI-164fibrosarcoma (BALB / c female mice, weighing 18-20 g) or 3x105TS / A cells (BALB / c female mice, 18-20 g) or 2.5×106TRAMP-C2 prostate cancer cells (C57BL / 6N male mice, 7-8 weeks-old). Tumor growth was monitored by measuring tumor size with calipers. The tumor volume was estimated by calculating 30 r1×r2×r3×4 / 3π, where r1 and r2 are the longitudinal and lateral radii, and r3 is the thickness of the tumor protruding from the surface of normal skin. Animals were sacrificed before tumors reached a diameter of 1−1.5 cm. Tumor sizes are shown as mean ± SE. In vivo treatments 85 P128268PCT Mice were injected, i.p., with 5a-HSA alone (40-50 µg / mouse diluted in 0.9% sodium chloride solution containing 100 μg / mL of HSA) or in combination with anti-PD-L1 (10 mg / kg, i.p. in 0.9% sodium chloride) or with S-NGR-TNF (100 pg, 5 ng / kg, in 0.9% sodium chloride solution containing 100 μg / mL of HSA) as indicated in each corresponding Figure. 5 TGFβ bioassay Quantification of bioactive TGFβ in cell supernatants was carried out using TGFβ-Reporter HEK-Blue™ cells (InvivoGen). Detection of phospho-SMAD2 / 3 in tumors by western blot and immunohistochemical analyses 10 Western blot analysis of phospho-SMAD3 in tumor extracts The effect of 5a-HSA on TGFβ activation in tumors in in vivo animal models was assessed by western blot analysis of phospho-SMAD3, a critical intracellular mediator of TGFβ signaling, in tumor tissue extracts. Tumors were excised from euthanized mice after treatment with or without 5a-HSA and homogenized in 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.1% 15 (w / v) sodium dodecyl sulfate, 0.5% (w / v) sodium deoxycholate, 1% (v / v) NP-40 (1 ml / g tissue), containing a cocktail of protease and phosphatase inhibitors (Abcam, cat. #ab201119). Lysate protein concentration was measured using the BCATMprotein assay kit (ThermoFisher Scientific cat. #23227). Tumor lysates containing 100-350 µg of total protein were a) diluted 1:1 in 2x Laemmli sample buffer (Bio-Rad, cat. #1610737) supplemented with 10% v / v 2- 20 mercaptoethanol, b) boiled at 95°C on a heat block, c) separated by SDS-PAGE on a 10% polyacrylamide gel (Mini-PROTEAN® TGX™ Precast, Bio-Rad), and transferred to a polyvinylidene difluoride membrane (Transfer Packs, Bio-Rad cat. #170415) using a Trans- Blot Turbo Mini device (Bio-Rad). The membranes were then soaked for 1 h at room temperature with 20 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.1% Tween 20 (TBS-T) 25 containing 5% (w / v) bovine serum albumin (BSA) (Blocking Buffer). After washing in TBS-T, the membranes were incubated overnight at 4°C with the following primary antibodies in Blocking Buffer: a rabbit anti-phospho-SMAD3 (S423 and S425) mAb (EP823Y clone, Abcam, cat. #ab52903, 1:1000) or a rabbit anti-SMAD3 mAb (EP568Y clone, Abcam cat. #ab40854, 1:30000). Both antibodies were mixed with a rabbit polyclonal anti-actin antibody (Sigma cat. 30 #A2066, 1:5000-1:20000). Membranes were then washed five times with TBS-T (3 min each) and incubated with a horseradish peroxidase (HRP)-goat anti-rabbit polyclonal antibody conjugate (Sigma cat. #A4914, 1:40000, 1 h at room temperature). After additional washing, the membranes were incubated with a chemiluminescent HRP substrate (Amersham, cat. 86 P128268PCT #RPN2232). Antibody binding was detected using a ChemiDoc Imaging System (Bio-Rad); band densitometric analysis was performed using the ImageJ software. Immunohistochemical analysis of phospho-SMAD2 / 3 in tumor tissue sections The liver of mice with PDAC metastases was explanted from euthanized mice and fixed in 5 zinc-formalin solution for 24 h at room temperature. Subsequently, the livers were embedded in paraffin and processed according to standard immunohistochemical procedures at the San Raffaele Mouse Clinic facility. Phospho-SMAD2 / 3 staining in tumor sections was conducted as previously described in Jurukovski et al. (2005) Fibrosis Research: Methods and Protocols 161-75 with minor modifications. Tissue sections were rehydrated at room temperature, 10 followed by antigen retrieval using 10 mM sodium-citrate buffer, pH 6.0 (Sigma-Aldrich, cat. S1804). Endogenous peroxidase activity was then blocked by treating the sections with methanol containing 0.03% hydrogen peroxide for 10 min at room temperature. Subsequently, the sections were incubated 30 min at room temperature with a rabbit anti-phospho-SMAD2 / 3 polyclonal antibody (Santa Cruz Biotechnology, cat. SC-11769) (5 µg / ml) After washing, the 15 sections were incubated with a biotinylated goat anti-rabbit polyclonal antibody conjugate (Dako, cat. E0432) (1:500, 1 h at room temperature) followed by avidin-biotin-peroxidase complex, according to the manufacturer’s instructions (Vector Lab). Antibody binding was then detected using the 3,3′-diaminobenzidine (DAB, Dako) staining. The tissue slices were imaged using an Aperio Digital Pathology Slide Scanner (Leica Biosystems). The brown signal area, 20 corresponding to the reaction product precipitated into the cell nuclei, was quantified using the QuPath software as follows: small lesions were quantified by drawing a single Region of Interest (ROI) covering the entire liver metastasis; large lesions were quantified by drawing multiple ROIs randomly selected within each lesion ( >3 ROI per lesion). 25 Example 2 – production of a peptibody consisting of peptide fused to a 4-glycine spacer and a murine IgG1-Fc fragment with efficient cancer cell binding and TGFβ inhibition with an improved plasma half-life Expression and characterization of peptibody 4∆-mFc1 We produced, by recombinant DNA technology, a peptibody consisting of 30 FETLRGDLRILSILRHQNLLKEL (a CgA-derived peptide called 4∆, SEQ ID NO: 3) fused to the Fc domain of a murine IgG1 (see Figure 22). The resulting peptide-Fc fusion product (peptibody) was called 4∆-mFc1. This peptibody was transiently expressed in XtenCHO mammalian cells as a secreted protein and purified from the cell culture medium by protein-A affinity chromatography (yield: about 60 mg / l of culture). 35 87 P128268PCT SDS-PAGE analysis of the product, under reducing and non-reducing conditions, showed bands of ∼34 and ∼65 KDa, respectively, likely corresponding to the monomeric and dimeric forms of the protein (expected molecular weight: ∼30 KDa and ∼60 KDa, respectively). The slightly higher molecular weight observed was likely due to glycosylation. The purity of the 5 product was >90%, as determined by the densitometric analysis of the gel. Overall, these results indicate that the production of this peptibody is feasible. 4∆-mFc1 binds recombinant human αvβ6 and αvβ8 The capability of 4∆-mFc1 to recognize purified human integrins αvβ6 and αvβ8 was investigated using direct and competitive binding assays. 10 Direct integrin-binding assays based on the use microtiter plates coated with various human integrins showed that 4∆-mFc1 can bind purified αvβ6 and αvβ8 with high affinity (0.022 and 0.028 nM, respectively). In contrast, very low binding, or no binding at all, was observed to αvβ1, αvβ3, and α5β1 (Fig. 23A). Similar results were obtained using competitive binding assays based on the use of isoDGR-HRP conjugate as a probe for the RGD binding site of all 15 these integrins (Fig.23B). These results suggest that 4∆-mFc1 can bind αvβ6 and αvβ8 with high affinity and selectivity. 4∆-mFc1 binds to cultured αvβ6 / αvβ8-positive cancer cells The capability of 4∆-mFc1 to recognize αvβ6 / αvβ8-positive cancer cells was then investigated. To this aim, human T3M-4 PDAC cells (αvβ6posand αvβ8pos) were incubated with various 20 concentrations of 4∆-mFc1, at 4°C and 37°C; then, the binding of peptibody to cells was analyzed by FACS using a fluorescein-labeled anti-mouse IgG antibody. The results showed that 4∆-mFc1 could bind T3M-4 cells in a dose-dependent manner with an apparent affinity of 0.6 and 0.2 nM at 4°C and 37°C, respectively (Fig.24). Notably, the binding was inhibited by an excess of other known ligands of αvβ6 an αvβ8, such as peptide 5a, peptide A20FMDV2, 25 and a TGFβ3-derived peptide (ac-HGRGDLGRLKK-NH2) but not by a control peptide with RGE in place of RGD (not shown). These results suggest that the binding of 4∆-mFc1 to T3M-4 cells is mediated by αvβ6 and / or αvβ8, and that its RGD sequence is crucial for binding. 4∆-mFc1 inhibits the production of active TGFβ by cancer cells To assess whether 4∆-mFc1 can inhibit TGFβ activation mediated by αvβ6 or αvβ8-positive 30 cancer cells, we then investigated the effect of 4∆-mFc1 on TGFβ production by 5M701 / GFP / CEA PDAC cells (αvβ6pos,αvβ8neg) and 5637 bladder cancer cells 88 P128268PCT (αvβ6pos / αvβ8pos). In parallel, a control isotype murine IgG1 mAb and peptide 2a were used as negative controls, while peptide 5a was used as positive control. The amount of active TGFβ in the cell supernatant was quantified after 24-48 h incubation using a bioassay based on HEK-Blue™ TGFβ cells. As expected, 4∆-mFc1 and 5a, but not the IgG1 control isotype and 5 peptide 2a, could inhibit the activation of TGFβ by these cells in a dose-dependent manner (Fig.25). Remarkably, 4∆-mFc1 was about 8-14-fold more potent than the free peptide 5a, suggesting that peptide dimerization via Fc increased the binding avidity. 4∆-mFc1 binds to PDAC tumor tissue sections Next, we have investigated the capability of 4∆-mFc1 to bind PDAC tumor tissue sections by 10 immunohistochemistry. 4∆-mFc1, but not a control isotype IgG1 mAb (clone MOPC-C31), could efficiently bind PDAC tissues (Fig.26). 4∆-mFc1 shows a plasma half-life of about 5 h To assess the plasma half-life of 4∆-mFc1, we labeled 4∆-mFc1 with IRDye800 NHS ester. The resulting conjugate, called 4∆-mFc1-IRDye800, preserved the capability to bind αvβ6- 15 coated microtiter plates, showing an affinity similar to that of peptide 5a coupled to the same fluorophore (Fig.27). This finding suggests 4∆-mFc1 labelling did not impair its capability to recognize its receptors. Then, the 4∆-mFc1-IRDye800 conjugate was administered i.v. to healthy C57BL / 6 mice, and plasma samples were collected at various time points.4∆-mFc1- IRDye800 fluorescence analysis showed that this conjugate has a plasma half-life of 20 approximately 5 h. 4∆-mFc1 inhibits tumor growth in the WEHI-164 fibrosarcoma model The anti-tumor activity of 4∆-mFc1 was then evaluated in a murine model of fibrosarcoma based on WEHI-164 cells (αvβ6negand αvβ8pos(low)) implanted subcutaneously in syngeneic BALB / c mice. Tumors were allowed to grow to 140-250 mm3; then mice were injected with 4∆- 25 mFc1 (35 µg / mouse, i.p.) four times per week for a total of 8 administrations (Fig.28A). This product significantly delayed the tumor growth and extended the survival of some mice (Fig. 28B-D). The antitumor activity of 4∆-mFc1 was not associated with signs of toxicity, such as changes in animal body weight, animal behaviour, and aspect of fur (not shown). Conclusions 89 P128268PCT 4∆-mFc1) can bind efficiently to human PDAC tissue section and inhibit TGFβ activation by cancer cells more efficiently than the original peptide 5a. This example also shows that a fluorescent derivative of 4∆-mFc1 labeled with IRDye800 has a plasma half-life markedly higher than that of peptide 5a (~ 5 h versus 9 min, respectively). 5 Materials and methods Reagents, antibodies, and cell lines Chemicals, antibodies, cell lines, and other compounds used for characterizing the peptibody are those described in the Example 1. 10 Production of the peptibody 4∆-mFc1 The cDNA coding for 4∆-mFc1, with codons optimized for expression in mammalian cells, was synthetized by Proteogenix. This cDNA encoded a) a signal secretion peptide derived from a human heavy chain IgG1, b) a CgA-derived peptide (FETLRGDLRILSILRHQNLLKEL, called peptide 4∆, SEQ ID NO: 3), c) a 4-glycine spacer, and d) a murine IgG1-Fc fragment (see 15 Figure 22). The cDNA was subcloned into the pTXs1 expression plasmid (Proteogenix) using seamless cloning procedures. The resulting plasmid was used to transfect XtenCHO mammalian cells (Proteogenix) using the Mammalian Protein Expression kit of Proteogenix (cat. #PX-XTE-001). The transfected cells (in 80 ml of cell culture medium) were left to grow in a 250 ml Erlenmeyer baffled flask for 14 days. After cell centrifugation, the supernatant was 20 harvested, filtered through a 0.22 µm filter, diluted 1:1 in PBS, pH 7.4, and loaded onto a protein-A column (GE, cat.17040201) for affinity chromatography purification. After washing, the product was eluted from the column with 20 mM citric acid and immediately neutralized with 1 M Tris-HCl, pH 9.0. The product was then dialyzed against PBS (pH 7.5) and stored at -20°C. About 6 mg of 4∆-mFc1 was recovered from 80 ml of cell culture. The purity of the final 25 product was >90%, as determined by SDS-PAGE and densitometric analysis. 4∆-mFc1 protein sequence and corresponding cDNA sequence cloned into the pTXs1 plasmid used for protein expression in XtenCHO cells. 90 P128268PCT Upper sequence: 4∆-mFc1 protein (amino acids indicated with the single letter code) Lower sequence: 4∆-mFc1 cDNA (codons optimized for expression in mammalian cells) *: stop codon 5 Boxed sequence: human heavy-chain signal peptide for peptibody secretion into the culture medium. Arrow: expected cleavage site of the fusion protein. Bold: CgA-derived peptide 4∆. Gray box: 4-glycine spacer. 10 Underlined sequence: murine IgG1-Fc region, consisting of hinge plus CH2 and CH3 domains. Integrin binding assay The capability of peptibody 4∆-mFc1 to recognize different integrins (αvβ1, αvβ3, αvβ6, αvβ8, and α5β1) was investigated using a) a direct assay, based on the use of integrin-coated plates in the capture step and an HRP-labelled anti-Fc mAb in the detection step (assay 1), and b) a 15 competitive assay based on the use of integrin-coated plates and isoDGR-HRP conjugate (a probe for the RGD-binding site of integrins) (assay 2). See Fig. 23 for a schematic representation of each assay. Direct integrin binding assay (assay 1) 20 91 P128268PCT Ninety-six-well microtiter plates (Greiner Bio-One, cat. #675061) were coated with or without human recombinant integrins (αvβ1, αvβ3, αvβ6, αvβ8, and α5β1) in DPBS with calcium and magnesium (2 µg / ml, 50 µl / well, overnight at 4 °C). After washing, the plates were blocked with 3% BSA in DPBS containing calcium and magnesium (150 µl / well) and incubated for 2 h 5 at room temperature. The plates were then washed with 25 mM Tris–HCl buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, and 0.05% Tween 20 (Buffer-A) and filled with various amounts of peptibody (50 µl / well) in Buffer-A containing 1% w / v BSA and 1% v / v NGS (Buffer-B). After 1.5 h of incubation, the plates were washed with Buffer-A and then filled with an HRP-labelled goat anti-mouse Fc 10 polyclonal antibody (Chemicon, Millipore, cat. #AP127P, 1:2000, 50 µl / well, in Buffer-B, 1 h). After washing, the bound peroxidase was detected by adding the chromogenic substrate o- phenylenediamine and, after 20 min, by reading the absorbance at 490 nm. Competitive integrin binding assay (assay 2) 15 The competitive integrin binding assay 2 was performed as described in Nardelli et al. (2019) Chem Commun 55: 14777-14780. Briefly, various amounts of peptibody were mixed with a fixed amount of an isoDGR-HRP conjugate and added to integrin-coated plates. After 2 h of incubation, the plates were washed, and the bound peroxidase was detected as described 20 above. Binding of the peptibody to cells as detected by flow cytometry The binding of peptibody to human T3M-4 pancreatic ductal adenocarcinoma (PDAC) cells was carried out essentially as described in Monieri, et al. (2023) Int J Biol Sci 19156-66 and Nardelli et al. (2019) Chem Commun 55: 14777-14780 and detected using a goat anti-mouse 25 Alexa Fluor 488-labeled secondary antibody (5 µg / ml). Human sample collection Human samples were collected at the Clinical Department at Ospedale San Raffaele (Milan, Italy) under written informed consent in agreement with the Declaration of Helsinki. The research proposal has been reviewed and approved by the Ethical Committee. Surgical 30 specimens, obtained for routine diagnostic or monitoring purposes, were processed and stored by the institutional biobank Biological Resource Center (CRB-OSR) (Num ID CRB in BBMRI-ERIC: bbmri-eric:ID:IT_1383758011993577). 92 P128268PCT Binding of the peptibody to human PDAC sections Explanted human PDAC tissues were OCT embedded, sliced (5-7 µm), and left to air-dry for 1-24 h before use. Subsequently, dried sections were incubated with 25 mM Hepes buffer, pH 7.4, containing 1 mM magnesium chloride, 1 mM manganese chloride and 1% w / v BSA for 1 5 h at room temperature and then incubated with the same solution containing 5 µg / ml of 4∆- mFc1 or isotype control IgG1 (clone MOPC-31C) (1 h at room temperature). The sections were then washed twice with the same buffer (5 min each) and incubated with an HRP-labelled goat anti-mouse antibody (SignalStain^ Boost IHC Detection Reagent, Cell Signaling, cat. 8125) for 30 min. After washing with PBS, the binding of immunoglobulins was detected using 10 the DAB substrate. After washing with distilled water, the sections were fixed with formalin and counterstained with hematoxylin. Conjugation of peptibody to IRDye800 fluorophore 4∆-mFc1 was coupled, via its amine groups, to IRDye® 800CW NHS Ester (LI-COR, P / N: 929-70020) using a 3:1 ratio (dye:protein). 15 Example 3 – production of a PEG-coupled peptide conjugates with integrin αvβ6 and αvβ8 binding and improved affinity Characterization of PEGylated peptide 5a Peptide 5a was conjugated to linear methoxy PEG maleimide-activated molecules via its cysteine, using 5 KDa or 10 KDa PEG chains. The resulting conjugates were called 5a-PEG5K 20 and 5a-PEG10K. RP-HPLC analysis of these products showed a purity >95% (Fig.29A). Gel filtration chromatography showed that 5a-PEG5K and 5a-PEG10K elute as single peaks with a hydrodynamic size corresponding to that of ~36 and ~96 KDa globular proteins (Fig.29B), likely reflecting its non-globular, expanded structure. 5a-PEG5K and 5a-PEG10K bind human recombinant αvβ6 and αvβ8 integrins 25 The capability of these conjugates to recognize αvβ6 and αvβ8 was assessed by direct and competitive-binding assays based on purified human recombinant integrins. An 8 KDa PEG (PEG8K) was used as a negative control. Direct binding assays showed that both 5a-PEG5K and 5a-PEG10K could bind to αvβ6 and αvβ8, albeit with different potencies, 5a-PEG10K being surprisingly 3-4-fold more potent than 5a-PEG5K. As expected, little to no binding was 30 observed with PEG8K alone (Fig.30A and Table 1). Competitive binding assays, based on the use of an isoDGR-HRP conjugate as a probe for the RGD-binding site of the integrins, 93 P128268PCT confirmed the hypothesis that both conjugates can recognize αvβ6 and αvβ8, although with similar potencies in this assay (Ki values of about 0.3 nM for αvβ6 and 0.7 nM for αvβ8) (Fig. 30A and Table 1). Taken together, these findings indicate that both compounds are functional in terms of αvβ6- and αvβ8-integrin recognition and they can recognize their integrins with 5 higher affinity compared to peptide 5a. 5a-PEG5K and 5a-PEG10K bind αvβ6 / αvβ8-positive cancer cells The capability of 5a-PEG5K and 5a-PEG10K to recognize αvβ6 / αvβ8-positive cancer cells was then investigated. To this aim, human T3M-4 pancreatic ductal adenocarcinoma cells were incubated with various concentrations of PEGylate conjugates and analyzed by FACS 10 using anti-PEG antibodies. The results showed that 5a-PEG10K could bind these cells in a dose-dependent manner with an EC50of 0.298 nM (Fig 31). In contrast, 5a-PEG5K poorly bound these cells (Fig 31). Peptide 5a-pegylation extends its plasma half-life The plasma half-life of these conjugates was then investigated. These conjugated were15injected intravenously to RAG2 − / −γc− / −mice (8 months old). Plasma samples were collectedat various time points and analyzed by competitive PEG-ELISA. The results showed a half- life of 40 min for 5a-PEG5K and 110 min for 5a-PEG10K (Fig 32). 5a-PEG10K inhibits tumor growth in the WEHI-164 fibrosarcoma model Based on the results of pharmacokinetics studies, 5a-PEG10K was selected for further in vivo 20 studies in mice-bearing subcutaneous WEHI-164 fibrosarcomas (αvβ6negand αvβ8pos(low)). To this aim, tumors were allowed to grow to 140-250 mm3, and then mice were injected with 5a- PEG10K (20 or 100 µg / mouse, i.p.) four times per week for a total of 8 administrations (Fig. 33A). Both doses delayed the tumor grow in the first two weeks of the treatments, although the dose of 100 µg was slightly more potent than that of 20 µg (Fig.33B-D), suggesting that 25 also this conjugate is endowed of anti-tumor activity. Conclusions 5a-PEG5K and 5a-PEG10K respectively, can recognize the integrins αvβ6 and αvβ8 with improved affinity, compared to the free peptide. Experimental evidence shows: a) 5a-PEG5K and 5a-PEG10K shows a markedly increased plasma half-life compared to 30 peptide 5a, 94 P128268PCT b) 5a-PEG5K and 5a-PEG10K are endowed of anti-tumor activity, and c) 5a-PEG10K can surprisingly bind αvβ6 / αvβ8-double positive PDAC cells more efficiently than 5a-PEG5K. Materials and methods 5 Reagents, antibodies and cell lines Chemicals, antibodies, cell lines and other compounds used for characterizing the PEGylated peptide 5a are those described above. Preparation of PEGylated peptide 5a Peptide 5a was synthesized by InnoPep (San Diego, CA) and then conjugated to linear 10 methoxy PEG maleimide-activated molecules via its cysteine, using PEG molecules of 5 KDa or 10 KDa (JenKem Technology USA, cat: A3125 and A3119). The purity of the conjugates was determined by RP-HPLC analysis by InnoPep. The compounds, supplied as lyophilized powder, were redissolved in sterile water and stored in aliquots at −20 °C until use. The concentration of the conjugates was determined by BCA assay. 15 PEG‑ELISA The amount of 5a-PEG10K in plasma samples was quantified using the Polyethylene Glycol Backbone ELISA Kit (cat #PEG, Life Diagnostics, Inc). Table 1. Binding affinity of peptides 5a, 5a-PEG5K and 5a-PEG10K conjugates to human αvβ6 and αvβ8 integrins. 20 Peptide or KibEC50epeptide-PEG (by competitive integrin binding (by direct integrin binding conjugate code assay, nM) assay, nM) ncαvβ6nαvβ8nαvβ6nfαvβ85a 5 1.70 7 3.90 NA NA ± 0.26d± 1.31 5a-PEG5K 3 0.36 2 0.69 2 0.25 2 2.45 ± 0.14 ± 0.18 ± 0.05 ± 0.81 5a-PEG10K 3 0.30 3 0.78 2 0.06 2 0.99 95 P128268PCT ± 0.11 ± 0.33 ± 0.05 ± 0.23 a) 5a-PEG5K and 5a-PEG10K; peptide 5a chemically conjugate to linear PEG molecule of 5KDa or 10KDa, respectively. b) Ki, inhibitory constant. Mean ± SE. 5 c) n, number of independent experiments. d) Nardelli et al. (2019) Chem Commun 55: 14777-80. e) EC50, effective concentration 50. Mean ± SE. f) NA, not applicable. Example 4 – production of peptide derivatives with high affinity and selectivity for αvβ6 and 10 αvβ8 Development of CgA-derived peptides with improved affinity for αvβ6 and αvβ8 The human chromogranin A region 38-68 has the following sequence: C38FETLRGDERILSILRHQNLLKELQD63(SEQ ID NO: 18). Considering that the canonical αvβ6 binding site consists of the RGDLXXL / I motif, we have synthesized a mini library of CgA- 15 derived peptides consisting of the C38FETLRGDLRILSILRHQNLLKELQD63(peptide 4a, SEQ ID NO: 1) and its mutants in which the I48residue was replaced with various standard amino acids. Furthermore, other peptides with R47I48replaced with GR or QV, and R47I48L49replaced with ATI (i.e., with residues present in the integrin binding motif of other known αvβ6 / αvβ8 ligands, such latent-TGFβ1, A20FMDV peptide, and latent-TGFβ3, respectively) were also 20 prepared (see Table 2). The ability of these peptides to inhibit the interaction of isoDGR-HRP (a non-selective probe of the RGD-binding site of integrins) with αvβ8 was analyzed and compared to that of the CFETLRGDLRILSILRHQNLLKELQD peptide (peptide 4a, SEQ ID NO: 1) (αvβ8, Ki=10.24; αvβ6, Ki=3.26 nM). The results showed that the substitution of I48with A, V, E, S, D, L, M, or F increased ∼5-fold the affinity for αvβ8 (Table 3 and Figure 34). Similar 25 results were observed with peptides with G47R48or A47T48I49substitutions, whereas the peptide with the Q47V48substitutions was only 2-fold more potent. Notably, the peptides with I48substituted with A, V, or S were as potent as peptide 5a (Ki=3.9 nM) (Table 3 and Figure 34). The peptides with improved affinity for αvβ8 were then characterized for their αvβ6-binding 30 affinity. Although all these peptides inhibited the binding of isoDGR-HRP to αvβ6 to a similar 96 P128268PCT extent, the peptide with A48was more potent than the others (Ki=0.73 nM). Based on these findings, the peptide with A48replacement (called peptide 7a, SEQ ID NO: 4) was selected for further optimization studies. To achieve this, a second mini library of peptides was synthesized in which R47of peptide 7a (C38FETLRGDLR47ALSILRHQNLLKELQD63, SEQ ID NO: 4) was 5 substituted with any of the 20 standard amino acids (Table 4). These peptides were also synthetized with an amidated C-terminal residue. The binding properties of these peptides for αvβ6 and αvβ8 were then evaluated, as described above. The results showed that none of these peptides were significantly more potent than the parental peptide 7a (Table 4). 10 Thus, peptide 7a was selected for further studies. Integrin binding studies showed that the affinity of peptide 7a for αvβ6 and αvβ8 was about two-fold higher than those of peptide 5a (Table 5). Considering that peptide 7a, but not 5a, entirely consists of standard amino acids without the triazole bridge, this peptide represents a suitable tool for producing high-affinity peptide-protein fusion products by recombinant DNA technology. 15 Table 2. Molecular mass of synthetic peptides and purity, as determined by electrospray ionization (ESI) mass spectrometry (MS) and reversed-phase (RP)-HPLC. 97 P128268PCT a) Single letter code; -CONH2, C-terminal amidated; ac-, N-terminal acetylated; triazole-stapled residues (X1 and X2, propargylglycine and azidolysine, respectively). b) Molecular mass of peptides was calculated using average the of the occurring amino acid 5 residues. c) For peptide 5 and 5a, monoisotopic masses are reported. d) As determined by RP-HPLC. Table 3. Binding affinity of peptides 5, 5a, 4, 4a, and CgA-derived peptides in which the 10 RIL sequence was mutated for human αvβ6 and αvβ8 integrins. 98 P128268PCT a) Single letter code; ac-, N-terminal acetylated; -CONH2, C-terminal amidated; triazole-stapled residues (X1 and X2, propargylglycine and azidolysine, respectively). b) Ki, inhibitory constant. Mean ± SE. 5 c) n, number of independent experiments. d) Nardelli et al. (2019) Chem Commun 55: 14777-80. e) NA, not analyzed. f) Curnis et al. (2012) Cell Mol Life Sci.69: 2791-803. g) Monieri et al. (2023) Int J Biol Sci.19: 156-66. 10 Table 4. Binding affinity of chromogranin A-derived peptides for human αvβ6 and αvβ8 integrin. 99 P128268PCT a) Single letter code; -CONH2, C-terminal amidated. b) Ki, inhibitory constant. Mean ± SE. 5 c) n, number of independent experiments. Table 5. Binding affinity of chromogranin A-derived peptides for human αvβ6 and αvβ8 integrin. 100 P128268PCT a) Single letter code; ac-, N-terminal acetylated; -CONH2, C-terminal amidated; triazole-stapled residues (X1and X2, propargylglycine and azidolysine, respectively). b) Ki, inhibitory constant. Mean ± SE. 5 c) n, number of independent experiments. d) Nardelli et al. (2019) Chem Commun 55: 14777-80. e) Monieri et al. (2023) Int J Biol Sci.19: 156-66. Table 6. Binding affinity of human αvβ6 and αvβ8 integrins for peptides and conjugates. 4a CFETLRGDLRILSILRHQNLLKELQD 3.26 10.24 NA NA (SEQ ID NO: 1) ± 0.61 ± 4.08 101 P128268PCT a) Single letter code peptide sequence; -CONH2, C-terminal amidated; triazole-bridge between residues X1 and X2 (propargylglycine and azidolysine, respectively). b) Ki, inhibitory constant. Mean ± SE. 5 c) Kd, apparent dissociation constant. Mean± SE. d) NA, not analyzed. Table 7. Plasma half-life of peptide 5a, 5a-HSA, 4∆-mFc1 / IRDey800, and 5a-PEG10K conjugates. Code Administration Mouse Injected Plasma route strain dose half-life (nmoles) (min, (h)) 5aaIntravenous BALB / c 1.2 8 min (0.133 h) 5a-HSA Intravenous C57BL / 6 0.715 100 min (1.66 h) Intraperitoneal C57BL / 6 0.715 960 min (16 h) 5a-PEG10K Intravenous RAG2− / −γc− / −16 100 min (1.66 h) 4∆-mFc1a Intravenous C57BL / 6 0.715 270 min (4.5 h)a) Coupled to IRDye800 for spectrofluorimetric detection. 10 Example 5 – Peptide 5a is internalised by αvβ6 and / or αvβ8 positive cells Preparation of 5a-pHsDye conjugates To investigate the internalization properties of peptide 5a, the pH-sensitive dye pHsDye (Promega) was used (Figure 35A). pHsDye becomes fluorescent in acidic environments, such 15 as those present in endosomes and lysosomes. Peptide 5a was conjugated to this dye using maleimide-activated pHsDye (peptide:dye molar ratio, 1:3). In parallel, peptide 2a, which contains an RGE motif instead of RGD, was also coupled to pHsDye and served as a negative control. 102 P128268PCT Other conjugates were also prepared including a) an additional negative control in which the maleimido-groups of the maleimide-activated pHsDye was blocked with cysteine (Cys- pHsDye); b) a positive control consisting of the well-characterized αvβ6 integrin ligand A20FMDV2 with extra N-terminal CG residues (CGNAVPNLRGDLQVLAQKVART (SEQ ID 5 NO: 53 (called CG_A20FMDV2), Kiαvβ6 = ~2 nM, not shown). Reverse-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry analysis of the resulting conjugates 5a-, 2a-, Cys-, and CG-A20FMDV2-pHsDye showed that conjugation was successful (Fig. 35B and 35C). Both 5a- and CG_A20FMDV2-pHsDye retained their ability to bind αvβ6 integrin, as demonstrated by a competitive binding assay 10 using horseradish peroxidase (HRP)-labeled with peptide 5a as a probe (Fig.35D), whereas, as expected, 2a- and Cys-pHsDye showed no appreciable binding. Thus, the pHsDye moiety does not impair the αvβ6 binding properties of 5a and CG_A20FMDV2. Peptide 5a is efficiently internalized by BxPC-3 and LN-229 cells To assess the internalization efficiency of 5a-pHsDye in cancer cells expressing integrin αvβ6 15 and / or αvβ8, we evaluated the uptake of 5a-, 2a-, and Cys-pHsDye by human cell lines characterized by differential expression of αvβ6 and αvβ8, including a) BxPC-3 pancreatic ductal adenocarcinoma cells, which express both αvβ6 and αvβ8 (Figure 36A), and b) LN-229 glioblastoma cells, which express only αvβ8 (Fig. 37B). Since fetal bovine serum (FBS) contains natural ligands of αvβ6 / αvβ8 integrin (e.g. fibronectin and vitronectin) that may 20 competitively inhibit the binding of peptide 5a to these integrins, we performed internalization experiments in the absence and in the presence of 10% FBS. 5a-pHsDye was internalized in both conditions and in a dose-dependent manner by BxPC-3 cells. The uptake of 5a-pHsDye was significantly greater than that of 2a- and Cys-pHsDye (Fig.37A). Quantitative analysis of 5a-pHsDye data showed an EC50of 0.14 nM in the absence 25 of serum, and 0.53 nM in its presence, indicating that serum exerts only a modest inhibitory effect on the internalization of this conjugate (Table 8). 5a-pHsDye was also efficiently internalized by LN-229 cells. However, in this case, the uptake was markedly enhanced in the presence of serum (Fig. 37B), suggesting that serum-derived factors may facilitate internalization by these cells. Indeed, in this case, the EC50of 5a-pHsDye was 5 nM and 25 30 nM in the absence and in the presence of FCS, respectively (Table 8). In contrast, internalization of the control conjugates 2a- and Cys-pHsDye remained minimal and largely unaffected by serum (Fig. 37A and B). Confocal microscopy of LN-229 cells incubated with 18 nM 5a-pHsDye showed a punctate fluorescence pattern, consistent with 103 P128268PCT endosomal or lysosomal localization (Fig. 37B, central panel). No intracellular fluorescence was observed in cells treated with 2a- or Cys-pHsDye. Together, these results demonstrate that 5a-pHsDye is selectively and efficiently internalized by both αvβ6 / αvβ8-positive and αvβ8- positive cancer cells. Moreover, internalization is largely serum-independent in BxPC-3 cells, 5 while it appears to be enhanced by serum in LN-229 cells, highlighting potential cell line- specific differences in the uptake mechanisms. Based on these findings, all the subsequent internalization studies were performed using serum-containing cell media. Sodium azide significantly inhibits the internalization of 5a-pHsDye To assess whether the internalization of 5a-pHsDye is an active, receptor-mediated process, 10 the uptake of this conjugate was analyzed in the absence and in the presence of sodium azide, a metabolic inhibitor that disrupts ATP production, thereby inhibiting energy-dependent endocytic pathways. LN-229 cells were treated with 100 nM 5a- or Cys-pHsDye with or without 0.2% mM sodium azide, and internalization was assessed by quantitative fluorescence analysis after 2 h of incubation. As expected, 5a-pHsDye, but not Cys-pHsDye, exhibited 15 strong and intracellular fluorescence, consistent with efficient receptor-mediated internalization (Fig. 38). Treatment with sodium azide led to a marked reduction in the internalization of 5a-pHsDye (Fig.38). Quantitative analysis revealed an approximately 40% decrease in 5a-pHsDye signal in the presence of sodium azide (Fig. 38), suggesting that internalization is an energy-dependent process, likely occurring through receptor-mediated 20 endocytosis. Peptide 5a and CG_A20FMDV2 are internalized by BxPC-3 cells to a similar extent The internalization properties of 5a-pHsDye were compared with those of CG_A20FMDV2- pHsDye. These products are internalized to a similar extent by BxPC-3 cells, which express both αvβ6 and αvβ8 integrins (Fig.39 and Table 8). In contrast, a significant difference was25 observed with the LN-229 cells, which express only αvβ8 integrins: in these cells, while 5a- pHsDye was efficiently internalized, the uptake of CG_A20FMDV2-pHsDye was considerably lower (EC50: 5 nM vs 180 nM, respectively). These findings are in line with the concept that peptide 5a is a bi-specific ligand of both αvβ6 and αvβ8 integrins, whereas CG_A20FMDV2 is selective for αvβ6. 30 αvβ6 / αvβ8 integrin recognition is necessary for peptide 5a internalization by T3M-4 cells To verify whether the 5a-pHsDye mechanism of internalization in cells was indeed mediated by αvβ6 and αvβ8 integrins, T3M-4 cells (a PDCA cell line double-positive for αvβ6 / αvβ8) were 104 P128268PCT genetically engineered to suppress the expression of both integrins using CRISPR-Cas9 technology (ITGB6 / B8-KO cells). Flow cytometry analysis confirmed that αvβ6 and αvβ8 expression was drastically reduced in ITGB6 / B8-KO cells compared to wild-type controls (Fig. 40A). Consistent with these results, 5a-IRDye binding was almost undetectable in the 5 knockout cells (Fig.40B). In contrast, wild-type cells displayed strong binding with high affinity (Kd ≈ 1 nM). Notably, both wild-type and ITGB6 / B8-KO cells expressed comparable levels of other integrins, including α5β1, αvβ5, β1, and the αv subunit (Fig.40A), supporting that peptide 5a is selective for αvβ6 and αvβ8 integrins. These results validate ITGB6 / B8-KO cells as a suitable model for investigating the internalization mechanism of peptide 5a. Comparative 10 internalization assays showed that 5a-pHsDye was efficiently internalized by wild-type cells but little or not by the double-knockout cells (Fig. 40C). All together, these findings demonstrate that peptide 5a internalization depends on the recognition of αvβ6 and αvβ8 integrins on the cell surface. Conjugate Cell lineaBxPC-3 LN-229 T3M-4 + Serum – Serum + Serum – Serum + Serum N EC50N EC50N EC50N EC50N EC50b (nM)c(nM) (nM) (nM) (nM) Cys-pHsDye 8 >> 100 2 >> 100 1 >>100 1 >>100 3 >>100 CG-A20FMDV2 / pHsDye 2 0.32 ± NAe2 160±65 NA 1 2.44 0.05 4∆-mFc1 / pHsDye 4 2.89 ±NA 1 60 NA 2 8.20 ± 1.32 2.04 mFc / pHsDye 1 >>100 NA 1 >>100 NA 1 >>100 15 a) Cells were incubated with conjugates for 18-20 h in complete DMEM FluoroBrite culture medium supplemented with 10% fetal bovine serum (+ Serum) or with 0.5% of bovine serum albumin (- Serum). b) N, number of independent experiments. c) EC50, effective concentration 50, determined using various concentrations of conjugates (range 0-100 nM) and >3 technical replicates per condition. The EC50 was determined by subtracting the non-specific binding of 20 Cys / pHsDye or mFc / pHsDye from the total binding of 5a-pHsDye or 4∆-mFc1 / pHsDye, respectively. Data were analyzed using Prism software. d) >>, maximum concentration tested, which failed to induce a significant internalization. e) NA, not analyzed. 105 P128268PCT Materials and methods Conjugation of Peptide 5a to a pH-Sensitive Dye Peptide 5a, containing an N-terminal cysteine, was coupled to a pH-sensitive dye using maleimide-activated pHAb (Promega, cat. G98314). Other peptides, including peptide 2a 5 (negative control) and a derivative of A20FMDV2 with CG residues at its N-terminus (CGNAVPNLRGDLQVLAQKVART (SEQ ID NO: 53), referred to as CG_A20FMDV2, positive control), were labelled in parallel. Cysteine was also coupled to the dye to generate an additional negative control. Peptides were incubated with the dye in PBS (pH 7.2) for 16 h at 4°C (3:1 peptide:dye molar ratio). The efficiency of conjugation was assessed by reverse- 10 phase HPLC (C18 Luna column, Phenomenex) and mass spectrometry analysis. The resulting conjugates (5a-, 2a-, CG_A20FMDV2-, and Cys-pHsDye) were used in internalization assays without further purification. Cell internalization assay The internalization of peptide- or protein / pHsDye conjugates by BxPC-3, LN-229, and T3M-4 15 cells was analyzed as follows. Cells were grown in 96-well microtiter plates (Greiner, cat: 655090, 3-4×104cells / well, 100 µl / well, seeded 24 h before the experiment) in their own cell culture medium. On the day of the assay, the cell culture medium was replaced with DMEM FluoroBrite (Gibco, cat. A1896701) supplemented with 1% glutamine, 1% penicillin / streptomycin and 10% FBS (Internalization buffer) and peptide- or protein-pHsDye 20 conjugates (0.10–200 nM). After 18-20 h of incubation at 37 °C, 5% CO2, the medium was removed, the cells were washed with Dulbecco’s Phosphate Buffer Saline (Euroclone, cat. ECB4004L) and then fixed with DPBS containing 2% paraformaldehyde and 3% sucrose for 15 min at room temperature. The internalization of the conjugates was then quantified by scanning the wells filled with DBPS (100 µl / well) with a Sapphire Biomolecular Imager system 25 (Azure Biosystems, model: Sapphire RGBNIR) and using the following settings: excitation filter: 520 nm; emission filter: 565 nm (BP24 nm); Intensity: 7 or 8, Speed: Highest. Resolution: 50 µm; Focus: 2.5 mm. In some experiments, internalized fluorescence was measured using a Tecan Infinite 200 Pro plate reader (bottom reading; excitation / emission: 532 / 572 nm, excitation bandwidth: 9 nm; emission bandwidth: 20 nm, Gain: 255 optimal (100%); number 30 of flashes: 75; integration time: 20 µs; Lag Time: 0 µs; settle time: 0 ms) as an alternative to the Azure scanner. Example 6 - 4Δ-mFc1 is internalised by αvβ6 and / or αvβ8 positive cells and synergises with anti-PD-L1 mAb 106 P128268PCT 4Δ-mFc1 binds to cultured αvβ6 / αvβ8-positive cancer cells It was previously shown that 4Δ-mFc1 recognizes αvβ6 / αvβ8-positive cancer cells, such as human T3M-4 PDAC cells (αvβ6posand αvβ8pos) with an EC50of about 200-600 pM (as determined by FACS analysis (Fig. 4). Notably, the binding of 4Δ-mFc1 to these cells was 5 inhibited by an excess of known ligands of αvβ6 and αvβ8, such as peptide 5a, peptide A20FMDV2, or a TGFβ3-derived peptide (ac-HGRGDLGRLKK-NH2), but not by a control peptide with RGE in place of RGD (Fig. 41A). These results suggest that 4Δ-mFc1 can recognize human αvβ6 / αvβ8-positive cancer cells and that its RGD sequence is crucial for binding. To verify that the binding to cells was indeed mediated by αvβ6 and αvβ8 integrins, we performed10 additional peptibody-cell binding assays using wild-type T3M-4 cells and αvβ6 / αvβ8 double- knockout cells (ITGB6 / B8-KO) (αvβ6negand αvβ8neg, Fig.41B). The results showed that 4Δ- mFc1, but not a murine Fc fragment (mFc) or Cys (negative controls), all labeled with the fluorescent dye IRDye800, could bind wild-type T3M-4 cells. In contrast, little or no binding was observed with ITGB6 / B8-KO cells. These findings confirm the hypothesis that peptibody- 15 cell binding occurs through the interaction of the peptibody moiety 4Δ with αvβ6 and αvβ8 integrins. Of note, no binding of peptibody occurred to ITGB6 / B8-KO, although these cells expressed α5β1, αvβ5, β1, and αv subunits (Fig. 40A). These findings are in line with the binding data obtained with the purified integrins, confirming that the peptibody is selective for αvβ6 and αvβ8. 20 4Δ-mFc1 is efficiently internalized by PDAC and GBM cells via αvβ6 / αvβ8 integrin recognition. The internalization properties 4Δ-mFc1 were next investigated. To this end, 4Δ-mFc1 was coupled to a pH-sensitive dye using N-hydroxysuccinimide ester chemistry at 1:10 protein-to- dye ratio. A murine Fc fragment was similarly labelled in parallel and served as a negative 25 control. The resulting conjugates, called 4Δ-mFc1 / pHsDye and mFc / pHsDye, were then characterized by UV / VIS spectrophotometric analyses to assess the labelling efficiency. The results confirmed that both conjugates carried approximately one dye molecule per protein molecule (DOL: ~1). The internalization ability of the conjugates was assessed in different cell lines expressing both αvβ6 and αvβ8 integrins (BxPC-3 and T3M-4), αvβ8 alone (LN-229), or 30 neither integrin (ITGB6 / B8-KO T3M-4). The results showed that 4Δ-mFc1 / pHsDye was efficiently internalized by BxPC-3, LN-229 and T3M-4 cells, but not by ITGB6 / B8-KO cells (Fig. 42 and Table 8). In contrast, mFc / pHsDye exhibited minimal uptake by all cell lines, consistent with its lack of targeting specificity. These findings confirm that 4Δ-mFc1 / pHsDye internalization is integrin-dependent and occurs efficiently in cells expressing either αvβ6 or 35 αvβ8, while its uptake is significantly reduced in integrin-deficient cells. Notably, the fact that 107 P128268PCT 4Δ-mFc1 / pHsDye exhibited a higher EC₅₀ compared to the peptide 5a-pHsDye is likely due to isolated and specific partial degradation of the peptibody that presumably occurred during its preparation, as supported by mass spectrometry analysis (data not shown). 4Δ-mFc1 allows cell internalization of a large cargo 5 It was next investigated whether pH-sensitive dye-labelled secondary antibodies could be exploited to study the internalization capacity of 4Δ-mFc1 without the need for direct labelling. This approach avoids potential inactivation of the targeting moiety, consequent to dye conjugation to the two accessible amino groups present on 4Δ-mFc1 (one at the N-terminus and one on the lysine residue). To test this, a goat anti-mouse IgG labeled with a pH-sensitive 10 dye (DOL = 3.6) was prepared and incubated (30 nM) with either 4Δ-mFc1 (10 nM), control mFc (10 nM), or diluent alone. These mixtures were then added to BxPC-3 cells and left to incubate for 24 h. The internalized fluorescence was then measured. The results demonstrated that only the complex containing 4Δ-mFc1 enabled robust internalization of the labelled secondary antibody, as indicated by the strong intracellular fluorescence signal (Fig. 15 43). In contrast, neither the complex with mFc nor the secondary antibody alone exhibited significant internalization. These findings clearly demonstrate that 4Δ-mFc1 can efficiently mediate the internalization of large protein assemblies, such as antibodies, into target cells. This highlights the potential properties of the peptibody as a versatile delivery system for macromolecular therapeutics. 20 4Δ-mFc1 exerts synergistic anti-tumor effects with an anti-PD-L1 monoclonal antibody The anti-tumor activity of 4Δ-mFc1 in combination with anti-PD-L1 mAb 10F.9G2, a modulator of the immune response, was then investigated. Since TS / A mammary adenocarcinomas cells express PD-L1 antigen (Fig.15), we decided to use these cells implanted subcutaneously for the study. In this model, the combined treatment (see Fig.45A for the schedule of treatments) 25 induced anti-tumor effects and increased survival more efficiently than treatment with single compounds (Fig.45B-D). No evidence of toxicity, as judged from the loss of animal weight, was obtained in all groups (Fig. 45E). These results suggest that 4Δ-mFc1 and anti-PD-L1 mAb exert synergistic effects, possibly by reducing immunosuppressive mechanisms in the tumor microenvironment. 30 Materials and methods Conjugation of peptibody 4Δ-mFc1 to a pH-Sensitive Dye 108 P128268PCT Peptibody 4Δ-mFc1 was coupled to NHS-ester pHAb (Promega, cat. G98414), an activated form of the dye that reacts with primary amines. A recombinant mouse Fc fragment (mFc) was included as a negative control and labeled under the same conditions. Conjugation was performed in PBS (pH 7.2) for 16 h at 4°C (1:10 protein:dye molar ratio), followed by 5 purification using a NAP-5 column (Cytiva) to remove the unreacted dye. The resulting conjugates were called 4Δ-mFc1 / pHsDye and mFc / pHsDye. Protein concentration and degree of labelling (DOL) were determined by UV-Vis spectroscopy, measuring the absorbance at 280 nm and 532 nm, according to the manufacturer’s instructions. Cell internalization assay 10 The internalization of peptide- or protein / pHsDye conjugates by BxPC-3, LN-229, and T3M-4 cells was analyzed as follows. Cells were grown in 96-well microtiter plates (Greiner, cat: 655090, 3-4×104cells / well, 100 µl / well, seeded 24 h before the experiment) in their own cellculture medium. On the day of the assay, the cell culture medium was replaced with DMEM FluoroBrite (Gibco, cat. A1896701) supplemented with 1% glutamine, 1% 15 penicillin / streptomycin and 10% FBS (Internalization buffer) and peptide- or protein-pHsDye conjugates (0.10–200 nM). After 18-20 h of incubation at 37 °C, 5% CO2, the medium was removed, the cells were washed with Dulbecco’s Phosphate Buffer Saline (Euroclone, cat. ECB4004L) and then fixed with DPBS containing 2% paraformaldehyde and 3% sucrose for 15 min at room temperature. The internalization of the conjugates was then quantified by 20 scanning the wells filled with DBPS (100 µl / well) with a Sapphire Biomolecular Imager system (Azure Biosystems, model: Sapphire RGBNIR) and using the following settings: excitation filter: 520 nm; emission filter: 565 nm (BP24 nm); Intensity: 7 or 8, Speed: Highest. Resolution: 50 µm; Focus: 2.5 mm. In some experiments, internalized fluorescence was measured using a Tecan Infinite 200 Pro plate reader (bottom reading; excitation / emission: 532 / 572 nm, 25 excitation bandwidth: 9 nm; emission bandwidth: 20 nm, Gain: 255 optimal (100%); number of flashes: 75; integration time: 20 µs; Lag Time: 0 µs; settle time: 0 ms) as an alternative to the Azure scanner. Example 7 - Expression and characterization of peptibody 4Δ-hFc The feasibility of producing a peptibody consisting of peptide 4Δ fused to a human IgG1 Fc 30 domain (referred to as 4Δ-hFc) was evaluated using the same expression strategy previously adopted for 4Δ-mFc1. In this case, 4Δ-hFc was transiently expressed for 10 days, rather than 14, and was efficiently secreted into the culture medium (see Figure 46A). The fusion protein was purified from the conditioned medium using protein-A affinity chromatography, yielding approximately 60 mg per liter of culture. SDS-PAGE analysis under 109 P128268PCT reducing and non-reducing conditions revealed bands at ~34 kDa and ~65 kDa, respectively, consistent with the expected monomeric and dimeric forms of the glycosylated fusion protein (expected molecular weights: ~30 kDa and ~60 kDa). Densitometric analysis indicated a purity >90%. Binding assays performed on immobilized αvβ6, αvβ8, and αIIbβ3 integrins 5 demonstrated that 4Δ-hFc can bind both αvβ6 and αvβ8 integrins, but not to αIIbβ3 (Figure 46B, upper panel). As expected, a human Fc domain, i.e., a negative control lacking the 4∆ peptide, failed to bind these integrins (Figure 46B, lower panel), confirming that 4Δ moiety is crucial for αvβ6 and αvβ8 binding. The apparent Kd of 4Δ-hFc for αvβ6 and αvβ8 were approximately 40 pM and 80 pM, respectively. 10 Together, these results demonstrate that the expression of a functional peptibody incorporating a human Fc domain is feasible and that the resulting fusion protein retains selective integrin-binding properties. Example 8 – hFc peptibody and PEG-peptide conjugates 15 Further illustrative peptibodies (Figure 47) and PEG-peptide conjugates (Figure 48) according to the invention are presented. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described products and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the 20 invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention, as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. 25 110

Claims

P128268PCT CLAIMS 1. A product comprising a peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein: 5 (i) the peptide is linked to: (a) human serum albumin (HSA), (b) a polyethylene glycol (PEG), or (c) an Fc domain; and / or 10 (ii) the peptide comprises the amino acid sequence of SEQ ID NO: 4; and wherein the peptide is less than or equal to 50 amino acids in length.

2. A product according to claim 1, wherein the peptide is linked to HSA, a PEG or an Fc domain, and wherein the peptide comprises or consists of an amino acid sequence selected from the group consisting of: 15 (i) SEQ ID NO: 2, (ii) SEQ ID NO: 3, and (iii) SEQ ID NO: 4, or a variant thereof having up to three amino acid substitutions, additions or deletions.

3. A product according to claim 1 or 2, wherein the peptide is linked to the HSA, PEG or 20 Fc domain by a chemical crosslinker or a peptide linker.

4. A product according to any preceding claim, wherein the HSA comprises or consists of the amino acid sequence of SEQ ID NO: 5, or a variant thereof having at least 80% sequence identity thereto.

5. A product according to any one of claims 1-3, wherein the PEG is selected from the 25 group consisting of PEG100, PEG200, PEG500, PEG1K, PEG2K, PEG5K, PEG10K, PEG20K, PEG50K and PEG100K, preferably wherein the PEG is PEG5K or PEG10K, more preferably wherein the PEG is PEG10K. 111P128268PCT 6. A product according to any one of claims 1-3, wherein the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7 or 30 or a variant thereof having at least 80% sequence identity thereto.

7. A product according to any one of claims 1-4, wherein the peptide comprises or 5 consists of the amino acid sequence of SEQ ID NO: 2, and wherein the peptide is linked to HSA.

8. A product according to any one of claims 1-3 or 5, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, and wherein the peptide is linked to a PEG. 10 9. A product according to any one of claims 1-3 or 6, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO: 3, and wherein the peptide is linked to an Fc domain.

10. A product according to claim 9, wherein the Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, 30 or a variant thereof having at least 80% sequence 15 identity thereto.

11. A product according to claim 10, wherein the product comprises or consists of the amino acid sequence of SEQ ID NO: 54, 56 or a variant thereof having at least 80% sequence identity thereto.

12. A product according to claim 11, wherein the product further comprises a signal 20 peptide.

13. A product according to claim 12, wherein the product comprises or consists of the amino acid sequence of SEQ ID NO: 60, 31 or a variant thereof having at least 80% sequence identity thereto.

14. A product according to any one of claims 1-2 or 6, wherein the peptide comprises or 25 consists of the amino acid sequence of SEQ ID NO:

4.

15. A polynucleotide encoding a product according to any one of claims 1-4, 6, 7 or 9-14, wherein the peptide is (i) linked to HSA or an Fc domain, and / or the peptide comprises the amino acid sequence of SEQ ID NO:

4.

16. A polynucleotide according to claim 15, wherein the polynucleotide encodes a peptide 30 linked to an Fc domain, and wherein the polynucleotide comprises the nucleotide sequence 112P128268PCT of any one of SEQ ID NOs: 11, 39, 40, 35, 41, 10, 12 or a variant thereof having at least 80% sequence identity thereto.

17. A polynucleotide according to claim 16, wherein the product comprises i) the nucleotide sequence of any one of SEQ ID NOs: 11, 39, 40 or a variant thereof 5 having at least 80% sequence identity thereto, and ii) the nucleotide sequence of any one of SEQ ID NOs: 13, 14, 42, 43, 44 or a variant thereof having at least 80% sequence identity thereto.

18. A polynucleotide according to claim 16 or 17, wherein the polynucleotide comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 62, 64, 63, 65, 66 or a variant 10 thereof having at least 80% sequence identity thereto.

19. A polynucleotide according to any one of claims 16 to 18, wherein the polynucleotide further encodes a signal peptide, wherein the polynucleotide comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 26, 36, 27, 37, 38 or a variant thereof having at least 80% sequence identity thereto. 15 20. A product according to any preceding claim for use in therapy.

21. A product according to any one of claims 1-19 for use in the treatment or prevention of cancer.

22. A product for use according to claim 21, wherein the cancer is ανβ6 integrin positive and / or ανβ8 integrin positive. 20 23. A product according to any one of claims 1-19 for use in the treatment or prevention of fibrosis.

24. A product according to any one of claims 1-19 for use in therapy, wherein the product is administered simultaneously, sequentially, or separately in combination with one or more further therapeutic agent, optionally wherein the one or more further therapeutic agent is an 25 anti-PD-L1 antibody and / or a TNFα or derivative thereof. 113

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