Combination of a βIG-h3 antagonist and a PDFG-AA antagonist for the treatment of cancer

Abstract

The invention relates to the combination of a βig-h3 antagonist and a PDGF-AA antagonist, for use in the prevention or treatment of a patient affected with cancer disease. In particular, the combination restore CD8+ T cell activation and thus anti-tumoral cytotoxic response. The cancer may be pancreatic ductal adenocarcinoma. PDGF-AA antagonist may be an anti-PDGF-AA neutralizing antibody or aptamer. Said βig-h3 antagonist may be an anti-βig-h3 neutralizing antibody or aptamer. The cancer is more particularly a cancer associated with a stroma expressing / secreting PDGF-AA and βig-h3. The invention also relates to methods of treatment of cancer disease with said combination.

Description

[0001] COMBINATION OF A 0IG-H3 ANTAGONIST AND A PDFG-AA ANTAGONIST FOR THE TREATMENT OF CANCER

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to compositions and methods for preventing or treating cancer disease, such as pancreatic ductal adenocarcinoma (PDAC).

[0004] BACKGROUND OF THE INVENTION:

[0005] Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related death in the industrialized world and is predicted to become the second leading cause of cancer-related death by 2030 (Hidalgo 2010). PDAC develops through the preceding formation of acinar-to-duct metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which are primarily driven by oncogenic Kras activation (Ying 2016). In addition, PDAC is associated with an abundant stromal reaction that usually surrounds islands of cancer cells and accounts for 50-80% of the tumor volume (Erkan 2008; Neesse 2011).

[0006] Accumulating evidence indicates the presence of close and complex paracrine interactions mediating bidirectional crosstalk between tumor cells and the cellular and noncellular stroma that facilitates cancer progression (Gore 2014). While the stroma might provide a barrier limiting the dissemination and metastasis of pancreatic cancer cells, it also stimulates aggressive behaviors in pancreatic cancer cells and helps these cells escape host immune surveillance (Kai 2019; Pickup 2014). Moreover, the high density of the PDAC- associated stroma further impairs drug delivery and immune cell infiltration, e.g., by providing a physical barrier and establishing mechanical tissue stiffness (Ali 2013). Indeed, mechanical tissue stiffness is associated with poor survival in PDAC patients (Laklai 2016; Perez 2021 ; Rice 2017). It is now a well-established fact that activated pancreatic stellate cells (PSCs) are primarily responsible for the development of the stroma (Feig 2012). PSCs represent approximately 4% of all pancreatic cells in the steady state. Upon inflammation, PSCs are activated and converted into cancer-associated fibroblasts (CAFs), which are the main source of ECM proteins and growth factors (Apte 1998).

[0007] Enhancement of intrinsic PDGFR signaling induced by mutational activation of Pdgfra, but not Pdgfrb, systemically induced fibroblastic hyperplasia and increased ECM deposition similar to that observed in collagen diseases (Olson 2009). PDGFRA point mutations (gain of function) were reported to be associated with gastrointestinal tumors (Chompre 2004; Corless 2005). The secretion of PDGFs by platelets and their effects on fibroblasts play a role in wound healing (Andrae 2008). Exogenous PDGF-BB accelerates ulcer healing in diabetic patients (Papanas 2008). High expression of PDGFA has been reported to predict a poor prognosis in esophageal squamous cell carcinoma (Han 2021). PDGF-AA binds primarily to PDGFRa, while PDGF-AB and PDGF-BB bind to PDGFRa as well as other receptor subtypes, such as PDGFRp (Liang 2020).

[0008] The purpose of the present invention is therefore to address a medical need by providing a new therapeutical target for treating cancer by restoring beneficial anti-tumor immunity, especially in PDAC.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention relates to a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for use in the prevention or treatment of a patient affected with cancer disease.

[0011] The invention also relates to a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF- AA antagonist, for the simultaneous or sequential use in the prevention or treatment of a patient affected with cancer disease.

[0012] The present invention also relates a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for use in a method to activate the anti-tumoral CD8+T cell response of a patient affected with a cancer.

[0013] The present invention also relates to the use of a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for the manufacture of a medicament for the prevention or treatment of a patient affected with cancer disease.

[0014] The present invention also relates to the use of a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for the manufacture of a medicament for activating the anti-tumoral CD8+T cell response of a patient affected with a cancer.

[0015] The present invention also relates to a method for preventing or treating a patient affected with cancer disease, comprising administering to said patient efficient amounts of both (i) a pig-h3 antagonist, and (ii) a PDGF-AA antagonist.

[0016] The present invention also relates to a method for activating the anti-tumoral CD8+T cell response of a patient affected with cancer disease, comprising administering to said patient efficient amounts of both (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist.

[0017] In the methods of the invention, the |3ig-h3 antagonist and the PDGF-AA antagonist may be administered simultaneously or sequentially to said patient.

[0018] Cancers concerned by these objects are especially PDGF-AA and |3ig-h3 expressing cancers. In a particular embodiment, the cancer is a cancer associated with a stroma expressing / secreting PDGF-AA and |3ig-h3. In a particular embodiment of these different objects, the cancer disease is pancreatic ductal adenocarcinoma (PDAC), more particularly a PDAC associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0019] Thus, the invention particularly relates to:

[0020] - a combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for use in the prevention or treatment of a patient affected with cancer disease, such as PDAC;

[0021] - a combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for the simultaneous or sequential use in the prevention or treatment of a patient affected with cancer disease, such as PDAC;

[0022] - a combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for use in a method to activate the anti-tumoral CD8+T cell response of a patient affected with a cancer, such as PDAC; wherein said PDGF-AA antagonist directly binds to PDGF-A, preferably said PDGF-AA antagonist is an anti-PDGF-AA neutralizing antibody or aptamer; and / or said |3ig-h3 antagonist directly binds to |3ig-h3, preferably said |3ig-h3 antagonist is an anti-|3ig-h3 neutralizing antibody or aptamer.

[0023] The invention also relates to a pharmaceutical composition comprising a |3ig-h3 antagonist, a PDGF-AA antagonist and a pharmaceutically acceptable carrier. Preferably, said PDGF-AA antagonist directly binds to PDGF-A, and may typically be an anti-PDGF- AA neutralizing antibody or aptamer. Preferably, said |3ig-h3 antagonist directly binds to |3ig- h3, and may typically be an anti-pig-h3 neutralizing antibody or aptamer. This composition is particularly dedicated to be used in the prevention or the treatment of a patient affected with cancer disease, such as PDAC.

[0024] The invention also relates to:

[0025] - a method for preventing or treating a patient affected with cancer disease, such as PDAC, comprising administering to said patient efficient amounts of a |3ig-h3 antagonist and a PDGF-AA antagonist;

[0026] - a method for activating the anti-tumoral CD8+T cell response of a patient affected with cancer disease, such as PDAC, comprising administering to said patient efficient amounts of a pig-h3 antagonist and a PDGF-AA antagonist; wherein the |3ig-h3 antagonist and the PDGF-AA antagonist may be administered simultaneously or sequentially to said patient; and / or wherein said PDGF-AA antagonist directly binds to PDGF-A, preferably said PDGF-AA antagonist is an anti-PDGF-AA neutralizing antibody or aptamer; and / or said |3ig-h3 antagonist directly binds to |3ig-h3, preferably said |3ig-h3 antagonist is an anti-|3ig-h3 neutralizing antibody or aptamer.

[0027] DETAILED DESCRIPTION OF THE INVENTION:

[0028] Here, we show for the first time that targeting PDGF signaling through a ligand trap approach is able to inhibit tumor progression by the reprogramming the activation status of the CAFs. Despite the previously described heterogeneity of CAF populations (Costa et al., 2018) (Elyada et al., 2019; Ohlund et al., 2017), we showed here that PDGFRa and CD61 were able to define two activation states reflecting the stiffness of the TME and that physical constraint was able to remodel the immune response outcome and the consequent tumor progression. As tumors evolve, they proliferate, produce PDGF ligands and instruct CAFs via a paracrine effect. PDGFRa+CAFs become siCAFs capable of inhibiting T-cell responses in situ. By using a PDGF-AA ligand trap approach, neoplastic tissue homeostasis can be restored. Neutralization of the PDGF-AA leads to PDGRFa+CAF maintenance associated with soft conditions and an efficient T-cell response. Our study provides support for the translational potential of using a PDGF ligand trap strategy. Furthermore, we have previously reported that big-h3 was highly expressed in stiff conditions and that the use of an anti-TGFBI / big-h3 antibody was able to reduce TME stiffness (Bachy et al., 2022; Goehrig et al., 2019). We are proposing targeting both PDGF-AA and |3ig-h3 (also written big-h3 herein) as a new therapeutic combination. We show here that the concomitant neutralization of both PDGF-AA and TGFBI leads to a synergistic effect increasing anti- tumoral cytotoxic response and decreasing the number of tumor cells.

[0029] Therapeutic methods and uses:

[0030] The present invention provides methods and compositions (such as pharmaceutical compositions) combining (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for preventing or treating cancer, and especially a pancreatic ductal adenocarcinoma. The present invention also provides methods and compositions for inhibiting or preventing pancreatic ductal adenocarcinoma.

[0031] These methods and compositions lead to decreasing the number of tumor cells. These methods and compositions lead to a reduction of the tumor growth.

[0032] In particular, the |3ig-h3 antagonists and the PDGF-AA antagonists individually restore CD8+ T cell activation and thus anti-tumoral cytotoxic response. Without willing to be bound to theory, it is deemed the combination synergistically restore CD8+ T cell activation and thus anti-tumoral cytotoxic response. Altogether, these effects result in the decrease of tumor cell number and in the reduction of tumor growth.

[0033] Thus, the present invention relates to a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist, for use, especially for the simultaneous or sequential use, in the treatment of a patient affected with cancer disease, in particular a solid tumor, e.g. a pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC).

[0034] The present invention also relates to a method for preventing or treating a patient affected with cancer disease, comprising administering to said patient effective amounts of (i) a pig-h3 antagonist, and (ii) a PDGF-AA antagonist.

[0035] In the context of the invention, the term "treatment or prevention" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in reducing the number of malignant cells. Most preferably, such treatment leads to the complete depletion of the malignant cells.

[0036] As used herein, the term “subject” or “patient” refers to any mammal, such as human or non-human mammal (such as a rodent (mouse, rat), a feline, a canine, or a primate. Preferably, the patient is affected or likely to be affected with cancer. Preferably, the patient is a human. Preferably, the patient is affected or likely to be affected with PDAC.

[0037] As used herein, the terms "cancer" and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the use of the invention, diseases, namely tumors with a stroma that expresses / secretes PDGF-AA and |3ig-h3 are most likely to respond to the combination.

[0038] In a particular embodiment, the cancer is a cancer associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0039] As used herein, the term “stroma “ or “microenvironment” has its general meaning in the art and refers to extracellular matrix and specialized connective tissue cells, including fibroblasts and mesenchymal stromal cells. Tumors have stroma and require stroma for nutritional support and the removal of waste products.

[0040] The inventors demonstrate that PDGF-AA-PDGFRa and pig-h3-aVp3 integrin interactions play a key role in the early establishment of tissue stiffness independent of CAF origin and subtype. Tumor associated tissue rigidity resulted in the emergence of stiffness- induced CAFs capable of inhibiting T-cell responses in situ. By using the present combination, neoplastic tissue homeostasis and CD8+ T cell activation can be restored. The cancer that may treated by methods and compositions of the invention include, but are not limited to cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease.

[0041] In particular, the cancer may be associated with a solid tumor. Examples of cancers that are associated with solid tumor formation include breast cancer, uterine / cervical cancer, oesophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, non-small cell lung cancer stomach cancer, tumors of mesenchymal origin (i.e; fibrosarcoma and rhabdomyoscarcoma), tumors of the central and peripheral nervous system (i.e; including astrocytoma, neuroblastoma, glioma, glioblatoma), thyroid cancer.

[0042] In particular, the invention relates to treating one of these cancers, which is associated with a stroma expressing or secreting PDGF-AA and |3ig-h3.

[0043] In a particular embodiment, the cancer is associated with a stiffness score of at least 0.37, and more particularly of at least 0.40. In a particular embodiment, the cancer is a cancer associated with a stiffness score comprising between 0,37 and 0,50.

[0044] As used herein, the term " stiffness score” has its general meaning in the art and refers to the stiff of the tumor, mainly determined by the extracellular matrix and specialized connective tissue cells, including fibroblasts and mesenchymal stromal cells. Matrix stiffness is critical for the progression of various types of cancers. In solid cancers such as mammary and pancreatic cancers, tumors often contain abnormally stiff tissues, mainly caused by stiff extracellular matrices due to accumulation, contraction, and crosslinking. The stiffness score can be measured based on the expression of genes as disclosed in Brielle S, et al., Proc Natl Acad Sci U S A. 2021 , incorporated by reference herein. In particular embodiment, the cancer is selected from the group consisting of nonsmall cell lung carcinoma, lung squamous cell carcinoma, lung adeno carcinoma, breast carcinoma, pancreatic cancer, ovarian serous cystadenocarcinoma, uterine carcinosarcoma, gastrointestinal stroma tumors such as stomach adenocarcinoma and cholangiocarcinoma, head and neack squamous carcinoma, oesophageal carcinoma, kidney renal clear cell carcinoma, sarcoma, mesothelioma and colorectal adenocarcinoma such as rectum adenocarcinoma and colon adenocarcinoma. In particular, the invention relates to treating one of these cancers, which is associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0045] More preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), preferably associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0046] Thus, in some embodiment, the invention refers to a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist for the use, especially for the simultaneous or sequential use, in the prevention or the treatment of a patient affected with a cancer disease by restoring CD8+ T cell activation.

[0047] In some embodiment, the invention refers to a combination of (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist for the use, especially for the simultaneous or sequential use, in the prevention or the treatment of a patient affected with PDAC by restoring CD8+ T cell activation.

[0048] Thus, in some embodiment, the invention refers to a method for preventing or treating a patient affected with cancer disease, comprising administering to said patient efficient amounts of both (i) a |3ig-h3 antagonist, and (ii) a PDGF-AA antagonist and obtaining restoring CD8+ T cell activation. In the method, the |3ig-h3 antagonist and the PDGF-AA antagonist may be administered simultaneously or sequentially to said patient.

[0049] In some embodiment, the invention refers to a method for preventing or treating a patient affected with PDAC, comprising administering to said patient efficient amounts of both (i) a pig-h3 antagonist, and (ii) a PDGF-AA antagonist and obtaining restoring CD8+ T cell activation. In the method, the |3ig-h3 antagonist and the PDGF-AA antagonist may be administered simultaneously or sequentially to said patient.

[0050] The terms "anti-tumoral CD8+T cell response" means the natural ability of the CD8+T cell to lyse cancer cells (Robbins PF, Kawakami Y. Human tumor antigens recognized by T cells. Curr Opin Immunol. 1996 Oct;8(5):628-36; Raskov, H., Orhan, A., Christensen, J.P. et al. Cytotoxic CD8+ T cells in cancer and cancer immunotherapy. Br J Cancer 124, 359-367 (2021)).

[0051] The terms " activate anti-tumoral CD8+T cell response" means the enhancement, the restoration the natural ability of the CD8+T cell to lyse cancer cells.

[0052] The present invention further contemplates a method of preventing or treating a cancer by restoring CD8+ T cell activation in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a PDGF-AA antagonist and of a |3ig-h3 antagonist.

[0053] In one aspect, the present invention provides a method of inhibiting tumor growth by restoring CD8+ T cell activation in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a PDGF-AA antagonist and of a |3ig-h3 antagonist.

[0054] An "pig-h3 antagonist" refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of |3ig- h3 including, for example, reduction or blocking the interaction between |3ig-h3 and aV 3 integrin and / or reduction or blocking the interaction between pig-h3 and collagen. pig-h3 antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include, antagonist variants of the protein, siRNA molecules directed to a protein, antisense molecules directed to a protein, aptamers, and ribozymes against a protein. For instance, the pig-h3 antagonist may be a molecule that binds to pig-h3 and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of pig-h3 (such as blocking the anti-tumoral immune response). More particularly, the pig-h3 antagonist according to the invention is an anti-pig-h3 antibody.

[0055] By "biological activity" of a pig-h3 is meant inhibiting CD8+ T cell activation (blocking the anti-tumoral immune response) and inducing stiffening of tumor microenvironment (TME or tumoral stroma). pig-h3 signals through binding to CD61 which is the 3 integrin subunit (hereinafter CD61 / 3 ) covalently associated with av integrin subunit in av 3 integrins. CD61 is present on T cell surface. “Neutralization” induced by pig-h3 antagonist may result from the inhibition of the interaction between pig-h3 and CD61 or 3, and ultimately to av 3 integrin. Tests for determining the capacity of a compound to be |3ig-h3 antagonist are well known to the person skilled in the art. In a preferred embodiment, the antagonist specifically binds to |3ig-h3 in a sufficient manner to inhibit the biological activity of |3ig-h3. Binding to pig-h3 and inhibition of the biological activity of |3ig-h3 may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as |3ig-h3 antagonist to bind to |3ig-h3. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd / Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist that "specifically binds to pig-h3" is intended to refer to an inhibitor that binds to human pig-h3 polypeptide with a KD of 1 M or less, 100nM or less, 10nM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of pig-h3. The functional assays may be envisaged such evaluating the ability to inhibit a) induction of stiffening of TME and / or b) inhibition of CD8+ T cell activation (see example / method related with Functional T cell Suppression Assay in W02020 / 079164, incorporated herein by reference).

[0056] The skilled in the art can easily determine whether a pig-h3 antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of pig-h3. To check whether the pig-h3 antagonist binds to pig-h3 and / or is able to inhibit stiffening of TME and / or blocking the inhibiting CD8+ T cell activation in the same way than the initially characterized blocking pig-h3 antibody and / or binding assay and / or a collagen I thick fiber assay and / or or a inhibiting CD8+ T cell activation assay may be performed with each antagonist. For instance inhibiting CD8+ T cell activation can be assessed by detecting cells expressing activation markers with antibody anti-CD69 and anti-CD44 (CD8+ T cells) as described in Patry 2015 (or see the Functional T cell Suppression Assay in example method in W02020 / 079164) and collagen I thick fiber assay can be measured by atomic force microscopy or polarized light after Sirius Red staining (see example section in in W02020 / 079164).

[0057] Accordingly, the pig-h3 antagonist may be a molecule that binds to pig-h3 selected from the group consisting of antibodies, aptamers, and polypeptides.

[0058] The skilled in the art can easily determine whether a pig-h3 antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of pig-h3: (i) binding to pig-h3 and / or (ii) inducing stiffening of TME and / or (iii) inhibiting CD8+ T cell activation. Accordingly, in a specific embodiment the |3ig-h3 antagonist directly binding to |3ig-h3 and inhibits the inhibition of CD8+ T cell activation (or restore CD8+ T cell activation) and stiffening of TME.

[0059] As used herein, the expression “tumor microenvironment (TME) " or " tumoral stroma” (both expressions will be used interchangeably) has its general meaning in the art and refers to the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signalling molecules and the extracellular matrix (ECM) (Joyce, JA. et al. 2015, Science Magazine pp. 74-80; Spill, F. et al. Current Opinion in Biotechnology 40: 41-48)). The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells (Korneev, KV et al, January 2017, Cytokine. 89: 127-135).

[0060] The pancreatic tumor stroma consists of a variety of cellular and noncellular components. A broad range of extracellular matrix (ECM) proteins, such as collagens, fibrous and nonfibrous glycoproteins, and proteoglycans contribute to the structural formation of the noncellular stromal compartment. In addition, the ECM also contains nonstructural components, such as growth factors and matricellular proteins (e.g Gamradt P., SPARC / osteonectin, osteopontin, periostin, and pig-h3 / TGFpip) (Neesse 2011 , Gore 2014, Gamradt 2021). The cellular compartment of the stroma includes immune cells, such as lymphocytes, macrophages, mast cells, and myeloid-derived suppressor cells (MDSCs), fibroblasts, along with vascular and neural elements (endothelial cells and neurons, respectively) (Pearce 2018, Torphy 2020, Oya 2020).

[0061] In an embodiment, the pig-h3 antagonist is an antibody (the term including antibody fragment or portion) that can block the interaction of pig-h3 with aVp3 integrin.

[0062] In preferred embodiment, the pig-h3 antagonist may consist in an antibody directed against the pig-h3, in such a way that said antibody impairs or inhibit the binding of a pig- h3 to CD61 / p3 or aVp3 integrin ("neutralizing antibody").

[0063] Then, for this invention, neutralizing antibodies of pig-h3 are selected as above described for their capacity to (i) bind to pig-h3 and / or (ii) impair or inhibit the binding of a pig-h3 to CD61 / p3 or aVp3 integrin. The neutralizing antibodies selected are those which (iii) inhibit tumor cell growth and / or (iv) reduce stiffening of TME and / or (v) block the inhibiting CD8+ T cell activation, and / or (vi) restore CD8+ T cell activation. An "PDGF-AA antagonist" refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of PDGF-AA including, for example, reduction or blocking the interaction between PDGF-AA and PDGFRa. PDGF-A antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Antagonists also include, antagonist variants of the protein, siRNA molecules directed to a protein, antisense molecules directed to a protein, aptamers, and ribozymes against a protein. For instance, the PDGF-AA antagonist may be a molecule that binds to PDGF-AA and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of PDGF-AA (such as inducing tumor cell growth).

[0064] By "biological activity" of a PDGF-AA is meant inducing tumor cell growth and inhibiting CD8+T cell activation (blocking the anti-tumoral response).

[0065] Tests for determining the capacity of a compound to be PDGF-AA antagonist are well known to the person skilled in the art. In a preferred embodiment, the antagonist specifically binds to PDGF-AA in a sufficient manner to inhibit the biological activity of PDGF-AA. Binding to PDGF-AA and inhibition of the biological activity of PDGF-AA may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as PDGF-AA antagonist to bind to PDGF- AA. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd / Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist that "specifically binds to PDGF-AA" is intended to refer to an inhibitor that binds to human PDGF-AA polypeptide with a KD of 1 pM or less, 100nM or less, 10nM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of PDGF-AA. The functional assays may be envisaged such evaluating the ability to inhibit a) induction of tumor cell growth and / or b) inhibition of CD8+T cell activation (see examples with neutralizing anti-PDGF-AA antibody and Figures 5 and 6).

[0066] The skilled in the art can easily determine whether a PDGF-AA antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of PDGF-AA. To check whether the PDGF-AA antagonist bind to PDGF-AA and / or is able to inhibit tumor cell growth and / or blocking the inhibiting CD8+T cell activation in the same way than the initially characterized blocking PDGF-AA antibody and / or binding assay and / or a cell proliferation assay and / or or a inhibiting CD8+ T cell activation assay may be performed with each antagonist. For instance inhibiting CD8+T cell activation can be assessed by detecting cells expressing activation markers with antibody anti-CD69 and anti-CD44 (CD8+T cells) as described in the Examples section and cell proliferation assay can be measured by CFSE-proliferation assay.

[0067] The skilled in the art can easily determine whether a PDGF-AA antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of PDGF-AA: (i) binding to PDGF-AA and / or (ii) inducing tumor cell growth and / or (iii) inhibiting CD8+ T cell activation.

[0068] Accordingly, the PDGF-AA antagonist may be a molecule that binds to PDGF-AA selected from the group consisting of antibodies, aptamers, and polypeptides.

[0069] In a particular embodiment, the PDGF-AA antagonist is a neutralizing antagonist.

[0070] Accordingly, in a specific embodiment the PDGF-AA antagonist directly binds to PDGF-AA and inhibits the inhibition of CD8+ T cell activation (or restore CD8+ T cell activation).

[0071] The present invention also relates to a PDGF-AA antagonist for use in a method to activate the anti-tumoral CD8+T cell response of a patient affected with a cancer.

[0072] In a particular embodiment, the PDGF-AA antagonist according to the invention is an antibody.

[0073] In a particular embodiment, the PDGF-AA antagonist is an antibody (the term including antibody fragment or portion) that can block the interaction of PDGF-AA with PDGFRa.

[0074] In a particular embodiment, the PDGF-AA antagonist is an antibody binding PDGF- AA.

[0075] In particular embodiment, the PDGF-AA antagonist is an antibody binding specifically PDGF-AA. In particular embodiment, the PDGF-AA antagonist is an antibody binding PDGF-AA and / or PDGF-AB.

[0076] In preferred embodiment, the PDGF-AA antagonist may consist in an antibody directed against the PDGF-AA, in such a way that said antibody impairs the binding of a PDGF-AA to PDGFRa ("neutralizing antibody").

[0077] Then, for this invention, neutralizing antibody of PDGF-AA are selected as above described for their capacity to (i) bind to PDGF-AA and / or (ii) inhibiting tumor cell growth and / or (iii) blocking the inhibiting CD8+T cell activation. As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

[0078] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as PDGF-AA, while having relatively little detectable reactivity with non- PDGF-AA proteins or structures, or such as |3ig-h3 while having relatively little detectable reactivity with non-pig-h3 proteins or structures. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10:1 , about 20:1 , about 50:1 , about 100:1 , 10.000:1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is Cath-D). Many different competitive binding assay format(s) which can be used include, but are not limited to, competitive assay systems using techniques such western blots, radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1 , John Wiley & sons, Inc., New York). For example, the BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.

[0079] In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody.

[0080] In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody. In a particular embodiment, the antibody of the invention is an antibody fragment selected from the group consisting of Fab, F(ab’)2, Fab’, dsFv, diabodies and scFv.

[0081] Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice- monthly or monthly) with antigenic forms of the concerned antigen, |3ig-h3 or PDGFD-AA. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

[0082] Briefly, the recombinant |3ig-h3 or the recombinant PDGF-AA may be provided by expression with recombinant cell lines. Recombinant form of |3ig-h3 or of PDGF-AA may be provided using any previously described method. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analysed for the presence of antibodies of the desired specificity, i.e. , that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

[0083] Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

[0084] It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and / or Fc / pFc' regions to produce a functional antibody.

[0085] This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761 , 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761 , and WO 90 / 07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

[0086] In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include lgG1 , lgG2, lgG3, lgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and / or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., Mol. Biol. 294:151 , 1999, the contents of which are incorporated herein by reference.

[0087] Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591 ,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

[0088] In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0089] Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so- called single chain antibodies.

[0090] As used herein, the term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond. As used herein, the term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin. As used herein, the term "Fab1" refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2. As used herein, the term "single chain Fv" ("scFv") polypeptide is a covalently linked VH:VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. As used herein, the term "dsFv" is a VH:VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0091] The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGI, lgG2, lgG3 and lgG4.

[0092] In another embodiment, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

[0093] Example of anti-PDGF-AA polyclonal antibody includes a neutralizing PDGF-AA polyclonal antibody supplied by Merck under reference 06-127. Example of anti-PDGF-AA monoclonal antibody includes an anti-PDGF-A antibody as disclosed in JP Pat application JPH07118298A (expressly incorporated herein by reference), and AF-221-NA a neutralizing PDGF-AA antibody. The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of PDAC as disclosed herein.

[0094] Example of neutralizing anti-pig-h3 monoclonal antibody is described, for example, in Bae JS et al., Acta Physiol 2014, 212, 306-315 and in W02020 / 079164, both incorporated herein by reference. The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs, the VH and VL domains, the paratope) and generate humanized antibodies for treatment of PDAC as disclosed herein.

[0095] Examples of humanized neutralizing anti-|3ig-h3 antibodies are disclosed in W02023 / 006919, incorporated herein by reference. Examples in the present description are using humanized antibodies described in W02023 / 006919.

[0096] In a particular embodiment, the |3ig-h3 antagonist consists in the neutralizing anti- ig- h3 antibody (murine 18B3 antibody) comprising a heavy chain having a sequence set forth as SEQ ID NO: 13 and a light chain having a sequence set forth as SEQ ID NO: 14 as described in W02020 / 079164. In a particular embodiment, the murine or chimeric 18B3 neutralizing antibody is an antibody comprising (a) a heavy chain comprising a variable domain having a sequence set forth as SEQ ID NO: 16, and (b) a light chain comprising a variable domain having a sequence set forth as SEQ ID NO: 17. The H-CDRs and L-CDRs thereof are as disclosed in W02020 / 079164, and are further presented in bold in the following Table 1 in the sequences SEQ ID NO: 16 and 17. The 18B3 may be a murine version, or a chimeric version, comprising the disclosed 6 CDRs or VH and VL.

[0097] In a particular embodiment, the |3ig-h3 antagonist consists in a neutralizing antibody that competes for binding to |3ig-h3 with the neutralizing anti-pig-h3 antibody (18B3 antibody) or one of the humanized antibodies described in W02023 / 006919.

[0098] As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope may typically be a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about 10'1° M or less, or about 10'11M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1 ,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a nonspecific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0099] Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard |3ig-h3 binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to |3ig-h3 demonstrates that the test antibody can compete with that antibody for binding to |3ig-h3; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on |3ig-h3 as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein (such as the 18B3 antibody). As used herein, an antibody “competes” for binding when the competing antibody inhibits |3ig-h3 binding of an antibody or antigen binding fragment of the invention by more than 50,51 ,52,53,54,55,56,57,58,59,60,61 ,62,63,64,65,66,67,68,69,70,71 ,72,73,74,75,76,77,7 8,79,80,81 ,82,83,84,85,86,87,88,89,90,91 ,92,93,94,95,96,97,98 or 99% in the presence of an equimolar concentration of competing antibody.

[0100] In other embodiments, the antibodies or antigen binding fragments of the invention bind to one or more epitopes of |3ig-h3. In some embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes.

[0101] The antibodies of the invention may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such western blots, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1 , John Wiley & sons, Inc., New York).

[0102] > Humanized anti-pig-h3 antibodies as described in W02023 / 006919

[0103] In some embodiments, the |3ig-h3 antagonist is a humanized neutralizing monoclonal antibody. Examples of such antibodies derived from 18B3 are described hereinafter.

[0104] The sequences of interest in the present application are indicated in the following

[0105] Table 1 :

[0106]

[0107] Legend : in VL and VH, the CDRs according to Kabat are in bold, the other amino acids are in the FRs, specific amino acids differing from the murine are sub-lined.

[0108] These VH and VL domains, including the mutated one, can be combined in order to design antibody binding domains H-330 / L-41 , H-330 / L-228, H-330 V1.2 / L-41 , H-330 V1.2 / L-228, H-311 / L-41 , and H-311 / L-228. Thus, the neutralizing anti-|3ig-h3 antibody and the binding fragment thereof comprise one of these combinations of VH and VL domains, thus encompassing any one of the following combinations H-330 / L-41 , H-330 / L-228, H-330 V1 .2 / L-41 , H-330 V1 .2 / L-228, H-311 / L-41 , and H-311 / L-228. CDRs and domain sequences are presented in Table 1 above.

[0109] H-330 V1.2 is a mutated version of H-330 with the mutation of the last amino acid of H-CDR-3 as the sole difference with respect to H-330. Cysteine 102 become serine. This mutated version is called H330 V1.2 (whereas original H330 is V1) or H330C102S, and has a VH-1 sequence as set forth in SEQ ID NO: 27. This mutation occurs in the H-CDR-3, and the mutated CDR3 noted H’-CDR3 has sequence as set forth in SEQ ID NO: 26.

[0110] In an embodiment, the neutralizing anti-|3ig-h3 antibody and the binding fragment thereof comprise a VH domain comprising the three CDRs of H-330 and a VL domain comprising the three CDRs of L-41 or L-228.

[0111] In an embodiment, the neutralizing anti-|3ig-h3 antibody and the binding fragment thereof comprise a VH domain comprising the three CDRs of H-330 V1 .2 and a VL domain comprising the three CDRs of L-41 or L-228.

[0112] In an embodiment, the neutralizing anti-|3ig-h3 antibody and the binding fragment thereof comprise a VH domain comprising the three CDRs of H-311 and a VL domain comprising the three CDRs of L-41 or L-228.

[0113] In an embodiment, said humanized anti-pig-h3 antibody comprises: a heavy chain comprising a variable domain as disclosed above, such as H-330, H-330 V1.2, or H-311 , and a constant domain CH, such as a CH having a sequence set forth as SEQ ID NO: 14; a light chain comprising said variable domain, such as L-41 or L-228, and a constant domain CL, such as a CL having a sequence set forth as SEQ ID NO: 15.

[0114] The same CH and CL domains may be used in a chimeric version of 18B3 monoclonal antibody.

[0115] > Aptamer

[0116] In another embodiment, the |3ig-h3 antagonist is an aptamer directed against |3ig-h3, and / or the PDGF-AA antagonist is an aptamer directed against PDGF-AA. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0117] Then, for this invention, neutralizing aptamers of |3ig-h3 are selected as above described for their capacity to (i) bind to |3ig-h3 and / or (ii) impairs or inhibit the binding of a pig-h3 to CD61 / p3 or aVp3 integrin. The neutralizing aptamers selected are those which (iii) inhibit tumor cell growth and / or (iv) reduce stiffening of TME and / or (v) block the inhibiting CD8+ T cell activation, and / or (vi) restore CD8+ T cell activation.

[0118] For this invention, neutralizing aptamers of PDGF-AA are selected as above described for their capacity to (i) bind to PDGF-AA and / or (ii) inhibit tumor cell growth and / or (iii) blocking the inhibiting CD8+ T cell activation.

[0119] > Inhibitor of gene expression

[0120] In still another embodiment, the pig-h3 antagonist is an inhibitor of pig-h3 gene expression and / or the PDGF-AA antagonist is an inhibitor of PDGF-A gene expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an "inhibitor of gene expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of the targeted gene. In a preferred embodiment of the invention, said inhibitor of |3ig-h3 or PDGF-A gene expression is a siRNA, an antisense oligonucleotide, a nuclease or a ribozyme.

[0121] Inhibitors of gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of pig-h3 or PDGF-A mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of |3ig-h3 or PDGF-A and PDGF- AA, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding pig-h3 or PDGF-A can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131 ; 6,365,354; 6,410,323; 6,107,091 ; 6,046,321 ; and 5,981 ,732).

[0122] Small inhibitory RNAs (siRNAs) can also function as inhibitors of pig-h3 or PDGF-A gene expression for use in the present invention. pig-h3 gene expression can be reduced by using small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that pig-h3 or PDGF-A gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see U.S. Pat. Nos. 6,573,099 and 6,506,559; and WO 01 / 36646, WO 99 / 32619, WO 01 / 68836).

[0123] Examples of said siRNAs against pig-h3 include, but are not limited to, those described in Chaoyu Ma (2008) Genes & Development 22:308-321.

[0124] Examples of said siRNAs against PDGF-A include, but are not limited to, those described in Lee J, et al. Autocrine DUSP28 signaling mediates pancreatic cancer malignancy via regulation of PDGF-A; Sci Rep. 2017 Oct 6;7(1):12760.

[0125] Inhibitors of gene expression according to the present invention may be based nuclease therapy (like Talen or Crispr).

[0126] The term “nuclease” or “endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALEN or CRISPR recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.

[0127] The guide RNA (gRNA) sequences direct the nuclease (i.e. Cas9 protein) to induce a site-specific double strand break (DSB) in the genomic DNA in the target sequence.

[0128] Restriction endonucleases (also called restriction enzymes) as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. In contrast, some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical. Moreover, also preferably the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and / or reduced catalytic activity when compared to the wildtype restriction endonuclease.

[0129] According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonuclease subunits. Preferably, in accordance with the present invention the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. The preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides. Preferably, the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity. The type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of: Pvull, EcoRV, BamHI, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJI, Bse6341 , BsoBI, BspD6l, BstYI, Cfr101 , Ecl18kl, EcoO109l, EcoRI, EcoRII, EcoRV, EcoR124l, EcoR124ll, HinP11 , Hindi, Hindlll, Hpy99l, Hpy188l, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAI, Pabl, Pad, PspGI, Sau3AI, Sdal, Sfil, SgrAI, Thai, VvuYORF266P, Ddel, Eco57l, Haelll, Hhall, Hindll, and Ndel.

[0130] Ribozymes can also function as inhibitors of |3ig-h3 or PDGF-A gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of |3ig-h3 or PDGF-A mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUO. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0131] Antisense oligonucleotides, siRNAs and ribozymes useful as inhibitors of |3ig-h3 or PDGF-A gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0132] Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA or ribozyme nucleic acid to the cells and preferably cells expressing |3ig-h3 or PDGF-AA. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0133] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

[0134] Preferred viruses for certain applications are the adeno- viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUCI9, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

[0135] As used herein, the term “active ingredients of the invention” is intended to refer to the pig-h3 antagonist compound and the PDGF-AA antagonist compound as defined above.

[0136] The active ingredients of the invention may be administered in the form of a pharmaceutical composition, as defined below, or may be administered separately as disclosed herein, in particular either simultaneously or sequentially.

[0137] By a "therapeutically effective amount" of a PDGF-AA antagonist as above described is meant a sufficient amount of the antagonist to prevent or treat a pancreatic ductal adenocarcinoma. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day. Preferably, the compositions contain 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of each active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of each active ingredient, preferably from 1 mg to about 100 mg of each active ingredient. An effective amount of each drug or active ingredient is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0138] Since association of PDGF-AA and |3ig-h3 antagonists has a synergistic effect on pancreatic cancer cells, the PDGF-AA antagonist and / or the |3ig-h3 antagonist can advantageously be used at low doses, or lower doses than in a treatment regimen wherein it is administered alone.

[0139] In the frame of the present invention, by “low dose” is meant a dose that is inferior to the recommended dose that would be given to the patient when the PDGF-AA antagonist or the pig-h3 antagonist is administered in the absence of the other compound. Said low dose is preferably inferior by at least 10%, 15%, 20%, 25%, 50% or 75% to the recommended dose when combined to the therapeutic dose of the single compound.

[0140] In a preferred embodiment, the active ingredients of the invention are preferably administered by the intravenous route.

[0141] According to the invention, the active ingredients of the invention may be administered as a combined preparation for simultaneous, separate or sequential use in the treatment of a cancer disease or a solid tumor.

[0142] The above method and use comprise the step of measuring the level of PDGF-AA protein in a blood sample obtained from said patients and compared to a reference control value.

[0143] Typically, a high level of PDGF-AA is intended by comparison to a control reference value. Said reference control values may be determined in regard to the level of PDGF-AA present in blood samples taken from one or more healthy subject or to the PDGF-AA distribution in a control population. A high level of PDGF-AA is predictive of a high risk of having or developing a pancreatic ductal adenocarcinoma and means that PDGF-AA antagonist must be used.

[0144] Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of PDGF-AA in blood samples previously collected from the patient under testing.

[0145] A “control reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the PDGF-AA levels (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the PDGF-AA level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders to pancreatic ductal adenocarcinoma treatment. In one embodiment of the present invention, the threshold value may also be derived from PDGF-AA level (or ratio, or score) determined in a blood sample derived from one or more subjects who are not affected with pancreatic ductal adenocarcinoma. Furthermore, retrospective measurement of the PDGF-AA levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

[0146] Typically, a body fluid sample is obtained from the subject and the level of PDGF-AA is measured in this sample. Indeed, statistical analyses revealed that decreasing PDGF-AA levels would be particularly beneficial in those patients displaying high levels of PDGF-AA.

[0147] Pharmaceutical compositions according to the invention

[0148] The PDGF-AA antagonist and the |3ig-h3 antagonist as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. There can be a single composition containing both ingredients or active principles; or two separate composition, one containing the PDGF-AA antagonist and the other containing the |3ig-h3 antagonist, intended to be administered to the same patient as described above. In particular, the present invention also provides a pharmaceutical composition comprising: i. a pig-h3 antagonist (as defined here above), ii. a PDGF-AA antagonist (as defined here above); and iii. a pharmaceutically acceptable carrier.

[0149] The present invention also relates to a pharmaceutical composition for use in the prevention or treatment of cancer comprising a PDGF-AA antagonist according to the invention, a |3ig-h3 antagonist according to the invention, and a pharmaceutically acceptable carrier. In particular, the invention relates to treating one of these cancers, which is associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0150] The present invention also relates to a pharmaceutical composition for use in the prevention or treatment of pancreatic ductal adenocarcinoma comprising a PDGF-AA antagonist according to the invention, a |3ig-h3 antagonist according to the invention, and a pharmaceutically acceptable carrier. In particular, the pancreatic ductal adenocarcinoma (PDAC), is associated with a stroma expressing / secreting PDGF-AA and |3ig-h3.

[0151] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0152] The choice of the formulation ultimately depends on the intended way of administration, such as e.g. an intravenous, intraperitoneal, subcutaneous or oral way of administration, or a local administration via tumor injection.

[0153] The pharmaceutical composition according to the invention may be a solution or suspension, e.g. an injectable solution or suspension. It may for example be packaged in dosage unit form.

[0154] In a preferred embodiment, the |3ig-h3 antagonist and the PDGF-AA antagonist of the invention, and the pharmaceutical composition of the invention, are administered by the intravenous route.

[0155] The pharmaceutical compositions of the present invention may be suited to oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration. They can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0156] The appropriate unit forms of administration include forms for oral administration, such as tablets, gelatine capsules, powders, granules and solutions or suspensions to be taken orally, forms for sublingual and buccal administration, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.

[0157] In the pharmaceutical compositions of the present invention, each one active principle is generally formulated as dosage units containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more preferably from 2 to 200 mg of said active principle per dosage unit for daily administrations.

[0158] When preparing a solid composition in the form of tablets, a wetting agent such as sodium laurylsulfate can be added to the active principles optionally micronized, which is then mixed with a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose, with various polymers or other appropriate substances or else they can be treated so as to have a prolonged or delayed activity and so as to release a predetermined amount of active principle continuously.

[0159] A preparation in the form of gelatin capsules is obtained by mixing the active principles with a diluent such as a glycol or a glycerol ester and pouring the mixture obtained into soft or hard gelatine capsules.

[0160] A preparation in the form of a syrup or elixir can contain the active principles together with a sweetener, which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.

[0161] The water-dispersible powders or granules can contain the active principles mixed with dispersants or wetting agents, or suspending agents such as polyvinyl-pyrrolidone, and also with sweeteners or taste correctors.

[0162] Rectal administration is effected using suppositories prepared with binders which melt at the rectal temperature, for example cacao butter or polyethylene glycols.

[0163] Parenteral, intranasal or intraocular administration is effected using aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersants and / or wetting agents, for example propylene glycol, butylene glycol, or polyethylene glycol.

[0164] Thus a cosolvent, for example an alcohol such as ethanol or a glycol such as polyethylene glycol or propylene glycol, and a hydrophilic surfactant such as Tween. RTM. 80, can be used to prepare an aqueous solution injectable by intravenous route. The active principle can be solubilized by a triglyceride or a glycerol ester to prepare an oily solution injectable by intramuscular route.

[0165] Transdermal administration is effected using multilaminated patches or reservoirs into which the active principle is in the form of an alcoholic solution.

[0166] Administration by inhalation is effected using an aerosol containing for example sorbitan trioleate or oleic acid together with trichlorofluoromethane, dichlorotetrafluoroethane or any other biologically compatible propellant gas.

[0167] The active principle can also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

[0168] Among the prolonged-release forms which are useful in the case of chronic treatments, implants can be used. These can be prepared in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium.

[0169] The active principle can also be presented in the form of a complex with a cyclodextrin, for example .alpha.-, .beta.- or .gamma. -cyclodextrin, 2-hydroxypropyl-. beta. -cyclodextrin or methyl-. beta. -cyclodextrin.

[0170] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0171] Figure 1 : Identification of PDGF signaling signature associated with tissue stiffness.

[0172] (A) Violin plots displaying the gene expression scores for the PDGF signaling pathway calculated from the WikiPathways (WP) or the Reactome databases. (B) Violin plots displaying the gene expression of PDGRFA, PDGFRB, PDGFA, PDGFB and ITB3 by CAFs and ducts obtained from six-week-old KC and 4KC mice determined by scRNAseq (C-G). ***p <0.001 ;****p <0.0001 ; ns not significant.

[0173] Figure 2: Increased tissue stiffness favors the accumulation of siCAFs.

[0174] (A) Representative FACS dot plots showing the surface expression of PDGFRa and CD61 on PDGFRa+CAFs and siCAFs (PDGFRa-) in pancreata from six-week-old KC (top) and 4KC (bottom) mice. Cells were gated on viable CD45-CD31- Lectin PNA-EpCAM- CAFs. (B) Frequencies of PDGFRa+CAFs and siCAFs among CD45-CD31-Lectin PNA- EpCAM' CAFs in pancreata from six-week-old KC (squares) and 4KC mice (triangles). (C) Quantification of the PDGFRa staining in stromal area in six-week-old KC and 4KC mice. The mean values ± SEMs are displayed. *p <0.05; **p <0.01 ; ****p <0.0001.

[0175] Figure 3: Neoplastic cells instruct the emergence of siCAFs at early tumor stages.

[0176] (A-B) Frequencies of (A) PDGFRa+CAFs and (B) siCAFs among CD45'CD3TLectin PNA'EpCAM' CAFs determined by FACS analysis of pancreata from KC (squares) and 4KC mice (triangles) harvested at 1 , 1.5, 3, 4, and 6 months of age. (C) Mean fluorescence intensity (MFI) of intracellular GFAP in PDGFRcF CAFs and siCAFs determined by FACS analysis. (D) Representative FACS dot plots showing the surface expression of PDGFRa and CD61 on PDGFRa+CAFs and siCAFs generated by coculturing wild-type (WT) PSCs with acinar cells (Ac) from KC or 4KC mice in the absence or presence of the soluble activin A inhibitor ActRIIBFc. (E) Frequencies of PDGFRa+CAFs and siCAFs generated in the absence or presence of ActRIIBFc. (F) MFI of intracellular PDGFRa in PDGFRa+CAFs and siCAFs determined by FACS analysis. (G) Tissue levels of PDGF-AA (pg / g) determined by ELISA analysis of pancreata from six-week-old KC and 4KC mice. (A, B) Results from five mice per group and timepoint are shown. (C, F) Representative data from two individual experiments with five mice are shown. (G) Representative data from two individual experiments with three mice and technical triplicates are shown. The mean values ± SEMs are displayed. *p <0.05; **p <0.01 ; ***p <0,001.

[0177] Figure 4: siCAFs prevent efficient of T cell activation.

[0178] (A, B) FACS analysis of the frequencies of CD45+and CD8a+cells in the pancreata of 6 weeks old KC and 4KC mice. (C, D) Frequencies of CD62L|OWcells among CD45+ / CD8a+ / CD11c- T cells in the spleen and pancreata of KC and 4KC mice. (E) CFSE dilution in CD8+T cells cocultured with BMDCs and CD3 / CD28 activation beads in PDGFRa+CAF / tumor cell- (blue line) or siCAF / tumor cell-conditioned medium (green line). (F-G) Proliferating (F) and Granzyme B (G)-producing CD8+T cells at the indicated division numbers after coculture with BMDCs and CD3 / CD28 activation beads in PDGFRa+CAF / tumor cell- (blue line) or siCAF / tumor cell-conditioned medium (green line). (A-D) Cumulative data from at least two individual experiments with 3-4 mice per group are shown. (E-G) Representative data from two individual experiments with technical replicates are shown. PDGFRa+CAFs and siCAFs were isolated from three six-week-old 4KC mice. *p < 0.05; **p < 0.01. ***p <0,001. Unpaired t test.

[0179] Figure 5: PDGF neutralization reduces tumor growth and promotes PDGFRa surface expression. (A) Graphical scheme representing the treatment schedule for 4KC mice: at 3, 4, and 5 weeks of age, 200 pg of neutralizing anti-PDGF antibody 06-127 (Merck) diluted in 100 pl of PBS was administered i.p. (the anti-PDGF antibody is designated “aPDGF”); age- matched control mice received PBS alone. One week after the last injection, the mice were sacrificed, and harvested pancreata were subjected to FACS and IHC analyses. (B) Weight (mg) of pancreata excised from six-week-old 4KC mice. Lines connect age-matched littermates treated with the anti-PDGF antibody (white) or PBS alone (black). (C) Representative FACS dot plots showing the surface expression of PDGFRa and CD61 on CAFs in pancreata from six-week-old 4KC mice treated with the anti-PDGF antibody (right) or PBS (left). Cells were gated on viable CD45'CD31 'Lectin PNA'EpCAM' CAFs. (D) Frequencies of PDGFRcF CAFs and siCAFs among CD45'CD31 'Lectin PNA'EpCAM' CAFs in pancreata from six-week-old 4KC mice treated with the anti-PDGF antibody or PBS. (E- H) FACS analysis of the percentages of CD44+among CD8+T cells (E), CD31+cells among CD45- cells (F), and Lectin PNA+(G) and EpCAM+(H) cells among CD45'CD3T cells isolated from the pancreas of six-week-old 4KC mice treated with the anti-PDGF antibody or PBS.

[0180] Figure 6: PDGF and big-h3 neutralization reduces tumor growth

[0181] (A) Graphical scheme representing the treatment schedule for implanted C57BI6 mice with 4KC-GFP cell line: at day 10, 17, and 22, 200 pg of neutralizing anti-PDGF antibody 06-127 (Merck) or of anti-pig-h3 antibody 18B3 or lgG1 control diluted in 100 pl of PBS was administered i.p. (the anti-PDGF antibody is designated “aPDGF”). One week after the last injection, the mice were sacrificed, and harvested pancreata were subjected to FACS in order to count the number of tumor cells (GFP+). (B) Quantification of tumor cells in s.c tumor implants with control, 18B3 murine antibody, 06-127 polyclonal antibody, and Combo = both 18B3 and 06-127 antibodies.

[0182] Methods

[0183] Mouse models

[0184] All animal protocols were reviewed and approved in accordance with the guidelines provided by the Cancer Research Center Lyon Animal Care and Use Committee (CECCAPP_CLB_2019_002). The generation of Acvrl bflox / floxmutant mice has been previously described (Ripoche et al., 2013). Acvrl bfl0X / fl0X;LSL-KrasG12D / +;Ptf1a-Cre mice (termed 4KC mice) were generated by crossing Acvrl bflox / floxmice with previously established LSL-KrasG12D / +;Ptf1a-Cre mice (termed KC mice) (Hingorani et al., 2003).

[0185] Atomic force microscopy (AFM)

[0186] The mechanical properties of pancreatic tissue areas were determined by AFM as described previously (Milani et al., 2014). Briefly, in AFM, the tip of a cantilever is pushed against sample tissue, and its deflection is monitored. Based on the stiffness constant of the cantilever, the deflection indicates the resisting force of the sample (Milani et al., 2014). The applied protocol allows the measurement of tissue stiffness very locally in a minimally invasive manner by deforming the sample down to a depth of 100 nm.

[0187] Cell isolation from mouse tissues

[0188] Excised mouse pancreata were washed in phosphate-buffered saline (PBS) and minced into small fragments, followed by incubation in a collagenase solution (1 mg / ml collagenase P obtained from Sigma-Merck in HBSS) at 37°C for 20 minutes. A single- pancreatic cell suspension was obtained by sequentially filtering the digested tissue through a 100 pm cell strainer followed by a 70 pm cell strainer. Spleens were homogenized by filtration through a 100 pm cell strainer to obtain single-cell suspensions. Red blood cells were lysed using NH4CI lysis buffer.

[0189] Human CAF generation

[0190] Small pancreatic tissue blocks were obtained from patients with resectable PDAC during pancreatic surgery. The experimental procedure relating to the use of patient-derived pancreatic tumor pieces was performed after approval by the South Mediterranean Personal Protection Committee under reference 2011-A01439-32. Primary CAFs were isolated as previously described (Leca et al., 2016). Briefly, tumors were cut into small pieces (1 mm3) using a razor blade. The tissue pieces were dissociated using the Tumor Dissociation Kit (Miltenyi Biotec; 130-095-929) according to the manufacturer’s recommendations. The cells were then resuspended, passed through a cell strainer (100 pM), and plated. Primary CAFs were used between passages 4 and 8. Primary CAF features were verified by flow cytometry with positive a-SMA and FAP staining. Immortalized CAFs were generated from primary CAFs of limited passage via retrovirus- mediated expression of human telomerase reverse transcriptase (hTERT).

[0191] Human primary CAFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) / F-12 supplemented with 10% fetal bovine serum (Biosera FB-1001 / 500), 2 mmol / l l-glutamine (Invitrogen; 25030-024), 1% antibiotic-antimycotic (Invitrogen; 15240- 062), and 0.5% sodium pyruvate (Invitrogen; 11360-039). Human immortalized CAFs were cultured in DMEM / F-12 supplemented with 10% fetal bovine serum and 1 % antibiotic- antimycotic. The pancreatic cancer cell line PANC-1 was obtained from ATCC and cultured in DMEM GlutaMAX (Gibco 10566016) supplemented with 10% fetal bovine serum and 1 % antibiotic-antimycotic. For coculture experimental conditions, primary CAF medium was used. Cells were authenticated through an STR profile report (LGC Standard) and confirmed to be mycoplasma free (Lonza, LT07-318). PANC-1 cells were plated 24 h before coculture with CAFs in triplicate for each experimental condition and treated with 0.5 pg / ml ActRllbFc inhibitor. The following day, human primary or immortalized CAFs were plated in monoculture or coculture according to the experimental conditions at a 2:1 ratio with PANC-1 cells and were treated with 0.5 pg / ml ActRllbFc inhibitor. Half of the cell culture medium was refreshed every 48 h with the addition of 0.5 pg / ml ActRllbFc inhibitor until day 6 of culture. Cells were detached using Stem Pro Accutase cell dissociation reagent (Gibco A1110501) and washed once with PBS. Samples were resuspended in FACS buffer (0.5% BSA and 2 mM EDTA in PBS) and surface stained with the following fluorochrome-coupled antibodies: anti-CD61 (BioLegend 370010), anti-CD140a / PDGFRa (BD Biosciences 742666), anti-FAP (R&D Biotechne FAB3715R), and anti-EpCAM (BioLegend 324204). Intracellular staining using anti-a-SMA (R&D Biotechne IC1420P) and anti-FSP-1 (BioLegend 370010) antibodies was performed following fixation and permeabilization with BD Cytofix / Cytoperm (554714). Samples were analyzed on a BD LSRFortessa X20 cell analyzer.

[0192] Flow cytometric analysis

[0193] Single-cell suspensions were stained with fluorochrome-labeled antibodies for 20 minutes at 4°C. Fixable viability dye (BioLegend) or DAPI staining was performed to exclude dead cells from the analysis. The stained cells were fixed directly by incubation in FACS buffer supplemented with 4% paraformaldehyde (PFA) for 20 minutes at 4°C, or intracellular staining was performed using the Fixation / Permeabilization Kit (BD Biosciences). No fixation / permeabilization was performed if the cells were subjected to FACS sorting. Stained cells were acquired on a BD Fortessa flow cytometer (BD Biosciences) for FACS analysis. For CAF and ductal cell isolation, cells were acquired on a BD FACSAria (BD Biosciences), and the sorted cells were collected in DM EM containing 10% (functional assays) or 20% fetal calf serum (FCS) (RNA isolation). FACS data were analyzed using FlowJo software (TreeStar).

[0194] Single-cell RNA sequencing (scRNAseq)

[0195] FACS-purified CAFs and ductal tumor cells from a pool of five KC or 4KC mice were partitioned into nanoliter-scale gel bead-in-emulsions (GEMs) with the Chromium Single Cell Controller (10x Genomics) at the in-house Single Cell Platform (CLB / CRCL). After cell encapsulation and barcoding, library preparation followed the standard 10x Genomics 3’scRNAseq protocol comprising reverse transcription, amplification, and indexing. Sequencing was performed using a NovaSeq Illumina device (Illumina). Illumina bcl files were basecalled, demultiplexed and aligned to the mouse mm10 genome using CellRanger software (10x Genomics).

[0196] PSC isolation and CAF differentiation PSCs were isolated from wild-type (WT) C57BL / 6 mice as previously described (Apte et al., 1998; Ohlund et al., 2017). Briefly, a single-pancreatic cell suspension was resuspended in 9 ml of GBSS containing 0.3% BSA and 43.75% Histodenz (Sigma-Merck), placed into a 15 ml conical tube and overlaid with 6 ml of GBSS containing 0.3% BSA. After gradient centrifugation, the cells within the gray band just above the interface between the GBSS and Histodenz layers were harvested and used for CAF differentiation.

[0197] Acinar cells (Acs) were isolated from KC and 4KC pancreata after digestion in a collagenase / soybean trypsin inhibitor solution (1 mg / ml collagenase P and 25 pg / ml soybean trypsin inhibitor, both obtained from Sigma-Merck, in HBSS).

[0198] PSCs and acinar cells were labeled using a CellTrace-CFSE or CellTrace-Violet proliferation kit (Invitrogen), respectively, and cocultured in DMEM (Gibco) containing 10% FCS, penicillin / streptomycin, and 0.2 mg / ml soybean trypsin inhibitor (Sigma-Merck) at a ratio of 1 :2 in 24-well plates equipped with discs made of rat tail collagen (Sigma-Merck). For certain conditions, the activin A inhibitor ActRIIBFc (gift from Olli Ritvos, Helsinki, Finland) was added at a final concentration of 0.5 pg / ml. After six days, the cells and collagen plates were recovered, and FACS analysis was performed to evaluate the differentiation of WT PSCs into CAFs.

[0199] CAF-conditioned medium

[0200] For the generation of conditioned medium, CAF subpopulations isolated by FACS were cocultured with cells from the KIC tumor cell line (Goehrig et al., 2019) at a ratio of 1 :1 in DMEM containing 10% FCS. After a 48 h incubation at 37°C and 5% CO2, the supernatants were collected and stored at -20°C until further use.

[0201] Bone marrow-derived dendritic cell (BMDC) generation

[0202] To generate bone marrow-derived monocytes, bone marrow cell suspensions were isolated by flushing the femurs and tibias of 8- to 12-week-old C57BL / 6 mice (Charles River) with DMEM containing 10% FCS as previously described (Inaba et al., 1992). Cell aggregates were dislodged by passing the suspension through a 70 pm cell strainer. Lysis of red blood cells was performed with ammonium-chloride-potassium (AKC) lysis buffer. The obtained cells were incubated for 6 days at 37°C and 5% CO2, and every other day, fresh DMEM containing 10% FCS and GM-CSF was added.

[0203] T-cell proliferation assay

[0204] Spleen and lymph nodes from 8 to 12-weeks old C57BL / 6 mice were harvested, mechanically dissociated and cell suspension was incubated with anti-CD8a magnetic beads following manufacturer’s protocol (Miltenyi Biotec). MACS-purified CD8+T cells were labeled with a CellTrace-CFSE proliferation kit (Life Technologies) according to the manufacturer’s protocol. The CSFE-labeled CD8+T cells were cultured for 2 days in the presence of BMDCs (T cells: BMDCs=16:1). Mouse T-Cell-Activator CD3 / CD28 Dynabeads (Gibco) were added to the coculture (T cells:Beads=1 :1). The proliferation of the CD8+ T cells was evaluated at the end of the culture period by analyzing CSFE dilution using flow cytometry.

[0205] Enzyme-linked immunosorbent assay (ELISA)

[0206] To determine the amount of PDGF-AA in tissue samples, freshly excised mouse pancreata were weighed and finely minced. Then, 500 pl of 1x PBS was added to the tissue fragments of each pancreas and thoroughly mixed. After centrifugation for 5 minutes at 300 g, the supernatant was collected, and a mouse PDGF AA ELISA kit obtained from Abeam was used according to the manufacturer’s protocol.

[0207] Statistical analysis

[0208] GraphPad Prism was used for graphical representation of the data and for statistical analysis. P values were calculated using Student’s test. For multiple comparisons, one-way analysis of variance with Tukey’s post-hoc test was used. Significance was indicated as follows: *p<0.05, **p<0.01 , ***p<0.001 , and ****p<0.0001.

[0209] Results

[0210] Sustained PDGF signaling is increased in stiff tissue conditions

[0211] Recently, the complex heterogeneity of CAFs has been revealed by other studies, indicating the existence of CAF subpopulations equipped with pro- and antitumorigenic qualities (Costa et al., 2018; Elyada et al., 2019; Huang et al., 2019; Ohlund et al., 2017). Although CAFs have been determined to be the main ECM producers, to our knowledge, there are no data available linking stromal tissue stiffness to the phenotypic and functional properties of CAFs after their initial instruction / activation by neoplastic cells. Thus, we took advantage of the KC and 4KC mouse models representing opposing ends of the tissue stiffness scale and performed scRNAseq analysis of pancreatic CAFs and neoplastic enriched cell fractions.

[0212] We performed pathway analysis revealed that the PDGF signaling pathway was one of the most upregulated pathways under the stiff conditions (Fig. 1 A, B ; WikiPathways and REACTOME). Next, we sought to determine the expression of PDGFRa, PDGFRb, PDGFA and PDGFB in merged RNAseq data. We identified the expression of the two major receptors PDGFRa and PDGFRb exclusively in the CAF compartment and of the ligands PDGFA and PDGFB exclusively in the duct compartment (Fig. 1C-F). In addition, we analyzed the expression of the integrin (ITG) b3 / CD61 , which has been shown to be key in CAF-mediated tumor cell invasion via the assembly of the ECM protein fibronectin (Attieh et al., 2017) and thereby might promote tissue stiffness. Moreover, CD61 is known to interact with the ECM protein big-h3 / TGFbi (Tumbarello et al., 2012), which has been described as a key ECM protein in the pancreatic TME hampering conventional (Goehrig et al., 2019; Patry et al., 2015) and unconventional T-cell responses (Lecker et al., 2021). We observed that CD61 was also expressed in the CAF compartment (Fig. 1G). Altogether, these results indicate that PDGF-PDGFR interactions play a key role in the early establishment of tissue stiffness independent of CAF origin and subtype.

[0213] Increased tissue stiffness is associated with the loss of PDGFRa surface expression on CAFs

[0214] Based on these observations, we sought to further examine the TME of 4KC and KC mice by determining the phenotype of CAFs by multicolor flow cytometry. Therefore, we applied the same gating strategy as that used in FACS sorting and focused on the CAF populations. FACS analysis of the two markers PDGFRa and CD61 identified a CAF population that was positive for both markers (PDGFRa+CD61+) and present in both KC mice and 4KC mice at frequencies of 75.8% and 52.9%, respectively (Fig. 2A, B). In addition, we detected a PDGFRa'CD61+CAF population that was significantly increased in 4KC pancreata (18.5%) compared to KC pancreata (6.1%) (Fig. 2 A, B). Given the association of these two CAF populations with the opposing tissue stiffness explored in KC and 4KC mice, we termed them stiffness-induced CAFs (siCAFs) and PDGFRa+CAFs. The fact that we did not detect the siCAF population by scRNAseq analysis highlighted that the disruption of ALK4 signaling in 4KC mice might be a result of continuous signaling through the PDGF-PDGFR system at the protein level.

[0215] Spatial analysis by IHC displayed an almost exclusive site of PDGFRa+stromal cells at the outer edge of pancreatic lesions that was more pronounced in 4KC than in KC pancreata. We detected a diminished staining for PDGFRa in 4KC (Fig. 2C) conditions confirming the results obtained by FACS. Linking the spatial distribution of PDGFRa and CD61 expression with the phenotype determined by FACS analysis, we concluded that PDGFRa+CAFs were located at the edge of lesions whereas siCAFs were mainly found within 4KC lesion centers. Taken together, our data demonstrate that stiffness-promoting ductal cells lead to specific instruction of CAFs, characterized by a unique phenotypic signature based on two markers: PDGFRa and CD61.

[0216] Loss of PDGFRa surface expression on siCAFs is a tumor cell-driven early event accompanied by PDGF ligand accumulation

[0217] To better understand the instruction of CAFs by ductal cells and the contribution of CAFs to altered tissue mechanics, we determined the kinetics of PDGFRa+and siCAFs emergence in KC and 4KC mice by FACS performed at 1 , 1 .5, 2, 3, 4, and 6 months of age. In KC mice, the frequencies of PDGFRa+CAFs and siCAFs stably represented -66.9% and 5.6% of all CAFs, respectively (Fig. 3A, B). In contrast, although the frequencies in 4KC mice were comparable to those in KC mice at one month of age, a significant decrease and increase in PDGFRa+CAFs and siCAFs, respectively, could be observed as early as 1.5 months of age. Moreover, these changes in the CAF populations of 4KC mice stabilized until the end of the experiment at six months of age, with PDGFRa+CAFs representing 36.9% and siCAFs representing 27.1% of all CAFs (Fig. 3A, B). Notably, the previously mentioned PDGFRa-CD61- CAF population remained unchanged in both KC mice and 4KC mice over the course of the experiment. These results indicated that early CAF instruction was maintained and that PDGFRa+CAFs and siCAFs most likely represented two different CAF activation states associated with the opposing tissue stiffness phenotypes of KC and 4KC mice. In line with these findings, we observed that siCAFs had significantly lower expression of the qPSC marker glia fibrillary acidic protein (GFAP) than PDGFRa+CAFs (Fig. 3C), further highlighting the different activation statuses of these two cell populations.

[0218] We next aimed to verify that the instruction of PDGFRa+CAFs into siCAFs in 4KC mice is driven through altered signals mediated by neoplastic ductal cells lacking ALK4 signaling rather than by increased release and / or availability of the ALK4 ligand activin A, which is still produced in ALK4-KO tumor cells. Therefore, we differentiated PSCs isolated from WT mice into CAFs in the presence of neoplastic cells from KC or 4KC pancreata and added ActRIIBFc, a soluble inhibitor of activin A. Previously, injection of ActRIIBFc into KC mice has been shown to result in a 4KC-like phenotype (Zhao et al., 2020). Similar to our previous ex vivo data, even in the absence of ActRIIBFc, PSCs differentiated into more siCAFs when cocultured with 4KC acinar cells rather than KC acinar cells, which produced significantly more PDGFRa+CAFs (Fig. 3D, left panel, and 3E). However, when ActRIIBFc was added to the coculture containing KC acinar cells, the frequency of siCAFs was similar to that observed in the coculture containing 4KC acinar cells. The addition of ActRIIBFc to the coculture containing 4KC acinar cells did not affect the expression of CD61 or PDGFRa on CAFs (Fig. 3D, right panel and 3E).

[0219] We next aimed to investigate the reason for the downregulation of PDGFRa surface expression in 4KC CAFs as an indicator of CAF activation. PDGFRs are internalized after binding to their ligands. Ultimately, most internalized PDGFRs are subjected to lysosomal or proteasomal degradation (Rogers and Fantauzzo, 2020). FACS analysis revealed that siCAFs had lower intracellular PDGFRa expression than PDGFRa+CAFs (Fig. 3F). We speculated that the increased availability of PDGF ligands in 4KC mice results in receptor internalization, which in turn promotes cell proliferation and ECM deposition (Eitner et al., 2008; Kuo et al., 2019; Ostendorf et al., 2012). Although PDGF ligand expression was not different between KC and 4KC mice on a per cell basis we hypothesized that in 4KC mice, higher overall PDGF ligand levels must be found due to the increased presence of ductal cells. Indeed, ELISA analysis revealed significantly increased levels of the ligand PDGF-AA per gram of 4KC mouse pancreas (Fig. 3G). siCAFs hamper antitumor immune responses

[0220] Next, we were therefore interested in investigating whether the siCAF signature associated with high tissue stiffness is also linked to immune suppression. We did not observe altered frequencies of infiltrating hematopoietic CD45+cells, including CD8+T cells (Fig. 4A, B), CD4+T cells, CD4+ / Foxp3+regulatory T cells (Tregs), NK-p46 natural killer cells, TCRgd T cells or neutrophils, between KC and 4KC pancreata. Further analysis of CD8+T cells revealed a significant reduction in T-cell activation in pancreata from 4KC mice, as indicated by the lower frequencies of CD62L|OW(Fig. 40). In contrast, CD8+T-cell activation was significantly increased in the spleen of 4KC mice (Fig.4D). Taken together, these data indicate that although peripheral T-cell activation appears to be efficient in 4KC mice and CD8+T cells are recruited into the pancreas at similar frequencies in KC and 4KC mice, T-cell responses are hampered within the pancreatic TME.

[0221] To determine whether the two CAF populations affect CD8+T-cell activation within the pancreas of 4KC mice, we isolated PDGFRa+CAFs and siCAFs from 4KC mice by FACS and cocultured them with a primary tumor cell line (Goehrig et al., 2019) to generate CAF- conditioned medium. Next, WT CD8+T cells were cocultured in the presence of BMDCs and CD3 / CD28 activation beads using the obtained PDGFRa+CAF- or siCAF-conditioned medium. Reduced proliferation rates were detected with the siCAF-conditioned medium, as indicated by less CFSE dilution compared to that achieved with the PDGFRa+CAF- conditioned medium (Fig. 4E, F). Moreover, the production of Granzyme B (GrzB) was reduced in proliferating CD8+T cells when they were cultured in the siCAF-conditioned medium (Fig. 4G).

[0222] PDGF neutralization reduces tumor growth and favors CD8+T-cell response

[0223] Our results show that the instruction of siCAFs in 4KC mice occurs early between 1- 1.5 months after birth and it develops in a PDGF / PDGFR signaling-dependent manner. Moreover, siCAFs prevent an efficient CD8+T-cell immune response, which is key for tumor elimination (Ene-Obong et al., 2013; Galon et al., 2006; Raskov et al., 2021). Thus, we hypothesized that the prevention of siCAF development in 4KC mice could improve the course of the disease. Therefore, we intraperitoneally (i.p.) injected 4KC mice at 3, 4, and 5 weeks of age with a neutralizing polyclonal anti-PDGF antibody 06-127 (Merck), which recognizes PDGF-AA, PDGF-AB, and PDGF-BB dimers. Control age-matched littermates were injected with 1x PBS. At 6 weeks of age, we analyzed pancreata collected from anti- PDGF- and PBS-injected mice (Fig. 5A). First, we observed a decrease in pancreas weight in 4KC mice injected with anti-PDGF, indicating diminished tumor growth (Fig. 7B). We next performed FACS analysis and evaluated the frequencies of siCAFs and PDGFRa+CAFs among total CAFs. We observed decreased siCAFs in anti-PDGF-injected 4KC mice compared to PBS-injected 4KC mice (Fig. 5C, D). In addition, we observed an increased percentage of activated CD8+T cells (CD44+CD8+T cells) pancreas in the anti-PDGF- treated conditions (Fig. 5E). Furthermore, we observed increased CD31+endothelial cells (Fig. 5F), decreased lesions (Fig. 5G) and increased EPCAM expression (Fig. 5H) in lesions, suggesting an early tumor phenotype. CD8+T-cell activation, indicated by the upregulation of CD44 and CD69 as well as the downregulation of CD62L, was similar in the spleen of anti-PDGF-injected or PBS-injected 4KC mice. Taken together, our data show that the PDGF neutralization leads to the reprogramming of the tumor environment.

[0224] PDGF and big-h3 neutralization leads to further tumor growth reduction

[0225] We assessed next the benefit of combining anti-PDGF (antibody 06-127 (Merck)) and big-h3 (antibody 18B3) neutralization on tumor growth. For this purpose we injected sc primary tumor cell line isolated from 4KC mice tagged with GFP and let the tumor establish for 10 days. Starting from day 10 we injected every 7 days (day 10, 17 and 22) either the isotype control lgG1 or anti-PDGF, or 18B3 or the combination of both and then the mice were sacrificed 7 days after the last injections (day 31). We recover the tumors and quantified the number of tumor cells by counting the number GFP+ cells by FACS analysis. As shown in Figure 6, the use of 18B3 or anti-PDGF diminished the number of tumor cells. More importantly, the number of tumor cells was further diminished by the use of both antibodies in combination. This results suggest that targeting both big-h3 and PDGF-AA has an increased impact on tumor reduction compared to use of the 18B3 or anti-PDGF standalone.

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Claims

CLAIMS1 . A combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for use in the prevention or treatment of a patient affected with cancer disease.

2. A combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for the simultaneous or sequential use in the prevention or treatment of a patient affected with cancer disease.

3. A combination of a |3ig-h3 antagonist and a PDGF-AA antagonist, for use in a method to activate the anti-tumoral CD8+T cell response of a patient affected with a cancer.

4. The combination for use according to any one of claims 1 to 3, wherein said PDGF-AA antagonist directly binds to PDGF-AA.

5. The combination for use according to claim 4, wherein said PDGF-AA antagonist is an anti-PDGF-AA neutralizing antibody or aptamer.

6. The combination for use according to any one of claims 1 to 5, wherein said pig-h3 antagonist directly binds to |3ig-h3.

7. The combination for use according to claim 6, wherein said |3ig-h3 antagonist is an anti-|3ig-h3 neutralizing antibody or aptamer.

8. The combination for use according to any one of claims 1 to 7, wherein the cancer is a cancer associated with a stroma expressing or secreting PDGF-AA and |3ig-h3.

9. The combination for use according to any one of claims 1 to 8, wherein the cancer is pancreatic ductal adenocarcinoma.

10. A pharmaceutical composition comprising a |3ig-h3 antagonist, a PDGF-AA antagonist and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claims 10, wherein said PDGF-AA antagonist directly binds to PDGF-AA, preferably said PDGF-AA antagonist is an anti-PDGF-AA neutralizing antibody or aptamer.

12. The pharmaceutical composition according to claim 10 or 11 , wherein said pig-h3 antagonist directly binds to pig-h3, preferably said pig-h3 antagonist is an anti-pig- h3 neutralizing antibody or aptamer.

13. The pharmaceutical composition according to any one of claims 10 to 12, for use in the prevention or the treatment of a patient affected with cancer disease, preferably a cancer associated with a stroma expressing or secreting PDGF-AA and pig-h3.

14. The pharmaceutical composition for use according to claim 13, wherein said solid cancer is pancreatic ductal adenocarcinoma.

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