WO2025068180A1 - Methods of treatment of cancer by targetting cancer - associated fibroblasts - Google Patents

Abstract

The present invention relates to antagonist of PDGF-AA protein, for use in the treatment of cancer. Here in the present invention, they 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 (Cancer -Associated Fibroblasts). The inventors found that PDGF-AA bind directly on CD8⁺ T cells by reducing their activation and cytotoxic properties. Furthermore, the use of neutralizing PDGF-AA antibodies in PDAC mouse model, reduced tumor growth by enhancing CD8⁺ T cell anti-tumoral response. Thus, neutralizing PDGF-AA which acts as a novel immunological check-point target in PDAC therefore allows to restore beneficial anti-tumor immunity in cancer, and especially in PDAC.

Description

METHODS OF TREATMENT OF CANCER BY TARGETTING CANCER -

ASSOCIATED FIBROBLASTS

FIELD OF THE INVENTION:

The present invention relates to a method for preventing or treating cancer disease by targeting cancer-associated fibroblasts (CAFs), such as pancreatic ductal adenocarcinoma (PDAC).

BACKGROUND OF THE INVENTION:

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 (1). 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 (2). 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 (3, 4).

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 (4-6). The cellular compartment of the stroma includes immune cells, such as lymphocytes, macrophages, mast cells, and myeloid-derived suppressor cells (MDSCs), along with vascular and neural elements (endothelial cells and neurons, respectively) (7-9).

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 (5). 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 (10, 11). Mechanical tissue stiffness is associated with poor survival in PDAC patients (12-14). It is now a well-established fact that activated pancreatic stellate cells (PSCs) are primarily responsible for the development of the stroma (15). 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 (16). Several mouse studies have shown that CAF depletion abolishes immune suppression (17). Surprisingly, contrary to the initial preclinical results (18), several publications have shown that the stromal response mediated by hedgehog signaling inhibits tumor progression and that its ablation would be harmful in PDAC. However, it has been shown that high stromal activity, as represented by a-smooth muscle actin (a-SMA) expression, is associated with a poor prognosis in patients with pancreatic cancer (3). All these results show that tumor-stroma interactions are complex. Indeed, several populations of CAFs with different functions related to antitumor immune responses have been described in both breast cancer (19) and pancreatic cancer (20, 21), indicating that the modulation of stromal activity rather than overall depletion of the stroma would be a therapeutic approach of choice.

Members of the TGF-B superfamily, including TGF-0, activins, inhibins, bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs) and nodal, have growth-stimulatory or growth-inhibitory effects in different types of tumors (22). Inactivating mutations in ALKA, the receptor of activin A, have been identified in pancreatic cell lines derived from patients (23, 24). These mutations are associated with increased tumor aggressiveness and a poor survival prognosis (23). Recently, our group discovered that activin A secreted by neoplastic cells acts as a protective senescence-associated secretory phenotype (SASP) protein that limits tumor progression even during the early stage of ADM by preventing massive ECM deposition (25). Here, inventors aimed to investigate the role of early tissue mechanical alterations in driving CAF differentiation and the consequent impact on the immune response.

Enhancement of intrinsic PDGFR (Platelet-derived growth factor Receptor) signaling induced by mutational activation of Pdgfira, but not Pdgfirb, systemically induced fibroblastic hyperplasia and increased ECM deposition similar to that observed in collagen diseases (Olson and Soriano, 2009). PDGFRA point mutations (gain of function) were reported to be associated with gastrointestinal tumors (Chompret et al., 2004; Corless et al., 2005). The secretion of PDGFs by platelets and their effects on fibroblasts play a role in wound healing (Andrae et al., 2008). Exogenous PDGF-BB accelerates ulcer healing in diabetic patients (Papanas and Maltezos, 2008). High expression of PDGFA has been reported to predict a poor prognosis in esophageal squamous cell carcinoma (Han et al., 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 et al., 2020). 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.

SUMMARY OF THE INVENTION:

A first object of the invention relates to a PDGF-AA (Platelet-derived growth factor- AA) antagonist for use in the prevention or treatment of a patient affected with cancer disease by restoring CD8+ T cell activation.

In a particular embodiment the cancer disease is pancreatic ductal adenocarcinoma (PDAC).

DETAILED DESCRIPTION OF THE INVENTION:

Here the inventors investigated the correlation between PDGF-AA and biological findings from PDAC patients. Here in the present invention, they 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. PDGFRoC 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 PDGRFot, 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.

These results show that PDGF-AA contributes to the anti-tumoral CD8⁺T cell response blockade. Neutralizing PDGF-AA which acts as a novel immunological check-point target in PDAC therefore allows to restore beneficial anti-tumor immunity in pancreatic cancer. Therapeutic methods and uses:

The present invention provides methods and compositions (such as pharmaceutical compositions) for preventing or treating cancer, an especially a pancreatic ductal adenocarcinoma, by restoring CD8+ T cell activation. The present invention also provides methods and compositions for inhibiting or preventing pancreatic ductal adenocarcinoma.

Accordingly, in a first aspect, the present invention relates to a PDGF-AA antagonist for use in the prevention or the treatment of a patient affected with a cancer by restoring CD8+ T cell activation.

In other words, 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.

As used herein, the term “PDGF” also known as “Platelet-derived growth factor” (PDGF) is one among numerous growth factors that regulate cell growth and division. In humans PDGF is encoded by the TGFBI gene In particular, PDGF plays a significant role in blood vessel formation, the growth of blood vessels from already-existing blood vessel tissue, mitogenesis, i.e. proliferation, of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells and mesenchymal stem cells as well as chemotaxis, the directed migration, of mesenchymal cells.

PDGF (Heldin CH (1992). " EMBO J. 11 (12): 4251-4) is a potent mitogen for cells of mesenchymal origin, including fibroblasts, smooth muscle cells and glial cells. In both mouse and human, the PDGF signalling network consists of five ligands, PDGF-AA through - DD (including -AB), and two receptors, PDGFRalpha and PDGFRbeta. All PDGFs function as secreted, disulphide-linked homodimers, but only PDGFA and B can form functional heterodimers. Though PDGF is synthesized, stored (in the alpha granules of platelets), and released by platelets upon activation, it is also produced by other cells including smooth muscle cells, activated macrophages, and endothelial cells (Kumar, Vinay (2010). Robbins and Coltran Pathologic Basis of Disease. China: Elsevier, pp. 88-89).

There are five different isoforms of PDGF that activate cellular response through two different receptors. Known ligands include: PDGF-AA (Platelet-derived growth factor subunit A is a protein that in humans is encoded by the PDGFA gene (Gene ID 5154)), -BB (Platelet- derived growth factor subunit B is a protein that in humans is encoded by the PDGFB gene), - CC (Platelet-derived growth factor subunit C is a protein that in humans is encoded by the PDGFC gene), and -DD (Platelet-derived growth factor subunit D is a protein that in humans is encoded by the PDGFD gene), and -AB (a PDGFA and PDGFB heterodimer). The gene product of PDGFA gene can exist either as a homodimer (PDGF-AA) or as heterodimer (PDGF-AB). The ligands interact with the two tyrosine kinase receptor monomers, PDGFRa (PDGFRA) and -RP (PDGFRB).( Fredriksson, Linda; Li, Hong; Eriksson, Ulf (August 2004). "The PDGF family: four gene products form five dimeric isoforms". Cytokine & Growth Factor Reviews. 15 (4): 197-204.). Platelet-derived growth factor is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB), two C subunits (PDGF-CC), or two D subunits (PDGF-DD).

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.

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 an human. Preferably, the patient is an human affected or likely to be affected with PDAC.

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 are most likely to respond to the PDGF-AA antagonist.

In particular embodiment, the cancer is a cancer associated with a stroma expressing/secreting PDGF-AA.

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.

The inventors demonstrate that PDGF-AA-PDGFRa 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 a PDGF-AA antagonist, 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.

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.

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.

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 the example (stiffness CAF signature comprising 29 genes, see Table 1) or in Brielle S., et al. Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling. Proc Natl Acad Sci U S A. 2021, incorporated by reference herein.

In particular embodiment, the cancer is selected from the group consisting of non- small 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.

More preferably the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

Thus, in some embodiment, the invention refers to a PDGF-AA antagonist for use in the prevention or the treatment of a patient affected with PDAC by restoring CD8+ T cell activation.

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)).

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.

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-AA 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).

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

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 IpM or less, lOOnM or less, lOnM 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 example with blocking PDGF-AA antibody and Figures 2 and 3).

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 (figure 2) and cell proliferation assay can be measured by CFSE-proliferation assay.

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.

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).

Accordingly, the PDGF-AA antagonist is an inhibitor of PDGF-A gene expression or an inhibitor of PDGF-AA activity.

Accordingly, the PDGF-AA antagonist may be a molecule that binds to PDGF-AA selected from the group consisting of antibodies, aptamers, and polypeptides. In particular embodiment, the PDGF-AA antagonist is a neutralizing antagonist.

• Antibody

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

In 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.

In particular embodiment, the PDGF-AA antagonist is an antibody binding PDGF-AA.

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.

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 or inhibit the binding of a PDGF-AA to PDGFRa ("neutralizing antibody").

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.

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. 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.

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.

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.

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 PDGF-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.

Briefly, the recombinant PDGF-AA may be provided by expression with recombinant cell lines. Recombinant form of PDGF-AA and recombinant form of PDFG-Amay 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 analyzed 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 nondenaturing 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. 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.

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.

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.

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 IgGl, IgG2, IgG3, IgG4, 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., I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

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.

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.

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.

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.

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 IgGl, IgG2, IgG3 and IgG4.

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.

Example of anti- PDGF-AA antibody includes an anti-PDGF-AA monoclonal antibody as disclosed in JP Pat application JPH07118298A (expressly incorporated herein by reference), a neutralizing PDGF-AA polyclonal antibody supplied by R&D system under reference AF-22-NA and a neutralizing PDGF-AA polyclonal antibody supplied by Merck under reference 06-127. The skilled artisan can use routine technologies to use the antigenbinding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of PDAC as disclosed herein.

• Aptamer

In another embodiment, 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).

Then, 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.

• Inhibitor of PDGF-A gene expression

In still another embodiment, 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 PDGF-A gene expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of PDGF-A gene. In a preferred embodiment of the invention, said inhibitor of PDGF-A gene expression is a siRNA, an antisense oligonucleotide, a nuclease or a ribozyme.

Inhibitors of PDGF-A 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 PDGF-A mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PDGF-A, 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 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).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of PDGF-A gene expression for use in the present invention. PDGF-A 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 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 Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Examples of said siRNAs against PDGF-A include, but are not limited to, PDGF-A siRNA supplied by Santa Cruz Biotechnology under reference sc-39703 or sc-39704, PDGF- A siRNA supplied by Origene under reference sr303419 and tr310536, and those described in Lee J, Lee J, Yun JH, Choi C, Cho S, Kim SJ, Kim JH. Autocrine DUSP28 signaling mediates pancreatic cancer malignancy via regulation of PDGF-A. Sci Rep. 2017 Oct 6;7(1): 12760.

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

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.

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.

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.

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, BamHl, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfrlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pad, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, and Ndel. Examples of said CRISPR guide RNA against PDGF-A include, but are not limited to, CRISPR Kit supplied by Origene® under reference KN4114164, and CRISPR Kit supplied by Santa Cruz Biotechnology under reference sc-400711.

Ribozymes can also function as inhibitors of 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 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 GUC. 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.

Antisense oligonucleotides, siRNAs and ribozymes useful as inhibitors of 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.

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 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.

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).

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, pUC19, 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.

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.

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.

In particular embodiment, the patient have a high level of PDGF-AA in blood.

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 coincidental 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 the 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 the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug 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.

In particular embodiment, the cancer is PDAC.

Thus, the invention also relates to a method for treating or preventing PDAC in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a PDGF-AA antagonist.

In other words, the invention also relates to PDGF-AA antagonist for use in the prevention or treatment of a PDAC in a patient.

In particular embodiment, the patient have a high level of PDGF-AA in blood.

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.

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 (as disclosed in Shaw VE, et al. Serum cytokine biomarker panels for discriminating pancreatic cancer from benign pancreatic disease. Mol Cancer. 2014) and means that PDGF-AA antagonist must be used.

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.

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 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 cancer, such as 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.

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.

In some embodiment, the PDGF-AA antagonist for use according the invention can be administered in combination with any suitable agent, in particular with anti-cancer therapy.

As used herein, the term “anti-cancer therapy” has its general meaning in the art and refers to any compound, natural or synthetic, used for the treatment of cancer.

In a particular embodiment, the classical treatment refers to radiation therapy, antibody therapy, immune checkpoint inhibitor, antiandrogens, CAR Therapy, such as CAR T- , CAR M- or CAR NK-cell therapy, antibody-drug conjugates (ADC) or chemotherapy.

In some embodiment, the PDGF-AA antagonist is administered in combination with an antibody-drug conjugates. Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADC includes but are not limited to Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Polatuzumab vedotin, Enfortumab vedotin, Trastuzumab deruxtecan, Sacituzumab govitecan, Belantamab mafodotin, Moxetumomab pasudotox, Loncastuximab tesirine and Tisotumab vedotin-tftv.

In some embodiment, the PDGF-AA antagonist is administered in combination with a chemotherapeutic agent.

As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as antiestrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiment, the PGDF-AA antagonist is administered in combination with PROTAC or molecular glue degraders.

As used herein, the term “proteolyse targeting chimeric molecules”, also known as “PROTAC”, has its general meaning in the art and refers to a heterobifunctional molecule composed of two active domains and a linker, capable of removing specific unwanted proteins. PROTAC works by bringing together the E3 ligase with the target protein thus allowing its ubiquitination and degradation by the proteasome. As used herein, the term “molecular glue degraders” has its general meaning in the art and refers to monovalent compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting the proteasomal degradation of the former. Techniques for conjugating therapeutic agents such as PROTAC and molecular glue degraders, and in particular to antibodies, are well-known. (See, e.g., Conilh L, Sadilkova L, Viricel W, Dumontet C. Payload diversification: a key step in the development of antibody-drug conjugates. J Hematol Oncol. 2023 Jan 17;16(1):3.; Dragovich PS. Degrader-antibody conjugates. Chem Soc Rev. 2022 May 23;51(10):3886-3897.).

In some embodiment, the PGDF-AA antagonist is administered in combination with radiation therapy.

As used herein, the term “radiation therapy” has its general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

In some embodiment, the PGDF-AA antagonist is administered in combination with an immune checkpoint inhibitor.

As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T lymphocytes in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoints are the regulators of the immune system. They are crucial for selftolerance, which prevents the immune system from attacking cells indiscriminately. Immune checkpoints are targets for cancer immunotherapy due to their potential for use in multiple types of cancers. Typically, by using immune checkpoint inhibitors, the anti-tumoral response is reactivated by reactivation of cytotoxic T- lymphocytes. The anti-Cathepsin-D antibody antibody of the invention as described above can be combined with an immune checkpoint inhibitor to inhibit the recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells.

Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD 122, CD 137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7- H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a costimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD 152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3- dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3 -dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.

In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

In a particular embodiment, the immune checkpoint inhibitor is an antibody.

Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, the immune checkpoint inhibitor is an anti -PD-1 antibody such as Pembrolizumab (Keytruda), Nivolumab (Opdivo) and Cemiplimab (Libtayo).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as Atezolizumab (Tecentriq), Durvalumab (Imfinzi), Avelumab and BMS-936559 (BMS).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154. In some embodiments, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody such as Ipilimumab (Yervoy) and tremelimumab (Imjuno).

In some embodiments, the immune checkpoint inhibitor is an anti-LAG-3 antibody such as Relatlimab.

In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy -tryptophan, 5 -hydroxy -tryptophan, indole 3 -carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :

In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA- 170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

In some embodiments, the immune checkpoint inhibitor is an aptamer.

Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti -PD-1 aptamer. Particularly, the anti -PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

In some embodiments, the immune check point inhibitor is selected from the group consisting of PD-1 inhibitors such as Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), PD-L1 inhibitors such as Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), CTLA-4 inhibitors such as Ipilimumab (Yervoy) and tremelimumab (Imjuno) and LAG-3 inhibitors such as Relatlimab.

Pharmaceutical compositions of the invention:

The PDGF-AA antagonist as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

Accordingly, the present invention relates to a pharmaceutical composition comprising a PDGF-AA antagonist according to the invention and a pharmaceutically acceptable carrier. The present invention also relates to a pharmaceutical composition comprising a PDGF-AA antagonist according to the invention and a pharmaceutically acceptable carrier for use in the prevention or treatment of cancer, wherein said antagonist restores the CD8+ T cell activation.

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 and a pharmaceutically acceptable carrier, wherein said antagonist restores the CD8+ T cell activation.

The present invention also relates to a pharmaceutical composition comprising a PDGF-AA antagonist according to the invention and a pharmaceutically acceptable carrier for use for activating the anti-tumoral CD8+T cell response of a patient affected with a cancer.

"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.

In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An appropriate dosage of the pharmaceutical composition is readily determined according to any one of several well- established protocols. For example, animal studies (for example on mice or rats) are commonly used to determine the maximal tolerable dose of the bioactive agent per kilogram of weight. In general, at least one of the animal species tested is mammalian. The results from the animal studies can be extrapolated to determine doses for use in other species, such as humans for example. What constitutes an effective dose also depends on the nature and severity of the disease or condition, and on the general state of the patient's health.

In therapeutic treatments, the antagonist contained in the pharmaceutical composition can be administered in several dosages or as a single dose until a desired response has been achieved. The treatment is typically monitored and repeated dosages can be administered as necessary. Compounds of the invention may be administered according to dosage regimens established whenever inactivation of PDGF-AA is required.

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 the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug 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 10 mg/kg of body weight per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability, and length of action of that compound, the age, the body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local (such as tumor injection) or rectal administration, the active principle, alone or in combination with another active principle, 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.

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.

In the pharmaceutical compositions of the present invention, the 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.

When preparing a solid composition in the form of tablets, a wetting agent such as sodium laurylsulfate can be added to the active principle 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.

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

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

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

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

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.

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.

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

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

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

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. 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.

FIGURES:

Figure 1. Figure 1: ALK4 signaling disruption in tumor cells alters the tissue mechanics of the pancreatic TME.

(A) Quantification of the total collagen (left) and thick collagen fiber (right) content in the pancreata of KC and 4KC mice. Cumulative data from three individual experiments with 4-5 mice per group are shown. (B) Quantification of the elastic modulus (kPa) measured by AFM in tumor or stromal regions of KC and 4KC pancreata. Cumulative data from three independent mice per group are shown. One hundred force curves per zone of interest were measured. (B) The mean values ± SEMs are displayed. **p <0.01; ****p <0.0001.

Figure 2: Identification of increased interactions between components in stiff condition.

(A) Frequencies of CAFs (Lectin PNA-EpCAM-) and ductal cells (Lectin PNA- EpCAM+) among viable CD45-CD31- cells detected by FACS analysis. The mean values ± SEMs are displayed. *p <0.05; **p <0.01. (B) Number of interactions (Ducts-CAFs and CAFs-Ducts) in 4K and 4KC conditions. *p <0.05; **p <0.01 Two-sided test of proportions.

Figure 3. Identification of PDGF signaling signature associated with tissue stiffness.

(A-E) Violin plots displaying the gene expression of PDGRFA, PDGFRB, PDGF A, 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.

Figure 4. Increased tissue stiffness favors the accumulation of siCAFs.

Frequencies of PDGFRa+ CAFs and siCAFs among CD45-CD31 -Lectin PNA- EpCAMCAFs in pancreata from six-week-old KC (squares) and 4KC mice (triangles). Cumulative data from three individual experiments with three to four mice per group are shown. The mean values ± SEMs are displayed. *p <0.05; **p <0.01; ****p<0.0001.

Figure 5: Neoplastic cells instruct the emergence of siCAFs at early tumor stages.

(A-B) Frequencies of (A) PDGFRa+ CAFs and (B) siCAFs among CD45-CD31- Lectin PNAEpCAM-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 PDGFRa+ CAFs and siCAFs determined by FACS analysis. (D) Frequencies of PDGFRa+ CAFs and siCAFs generated in the absence or presence of ActRIIBFc. (E-F) siCAFs were generated by coculturing iPSCs with 4KC cell line in the absence or presence of aPDGF Ab for 7 days. (E) Number of iPSC and total CAFs and (F) number of siCAFs determined by FACS analysis. (G) siCAFs were generated by coculturing iPSCs with 4KC cell line or in the presence of rPDGF-AA for 7 days. MFI of extracellular PDGFRa determined by FACS analysis. (H) MFI of intracellular PDGFRa in PDGFRa+ CAFs and siCAFs determined by FACS analysis (I) 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, E) Representative data from two individual experiments with five mice are shown. (F) Representative data from two individual experiments with three mice and technical triplicates are shown. (E-H) Representative data from two individual experiments and technical triplicates are shown. The mean values ± SEMs are displayed. *p <0.05; **p <0.01; ***p <0.001, ****p <0.0001.

Figure 6: siCAFs prevent efficient of T cell activation.

(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 CD62Llow cells among CD45+/CD8a+/CDl lc- 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). (G) Cytokine and chemokines profiles detected in PDGRFa and siCAF in conditioned media used in E-G. (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.

Figure 7: PDGF neutralization reduces stromal activation 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 diluted in 100 pl of PBS was administered i.p.; 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-EpCAMCAFs. (D) Frequencies of PDGFRa+ CAFs and siCAFs among CD45-CD31 -Lectin PNAEpCAM-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-CD31- cells isolated from the pancreas of six-week-old 4KC mice treated with the anti-PDGF antibody or PBS. *p < 0.05; **p < 0.01.

Figure 8: Tumor cells instruct siCAFs in human PDAC.

(A) FACS analysis of the frequencies of PDGRFa+ CAFs and siCAFs among viable cells after CAF isolation. (B-F) FACS analysis of the MFIs of PDGRFa (B), CD61 (C), CD29

(D), FAP (E), and FSP1 (F) on PDGRFa+ CAFs and siCAFs. (G) Representative FACS dot plots showing the surface expression of PDGFRa and CD61 on EpCAM-FSPl+ CAFs after coculturing with or without PANC-1 tumor cells in the absence or presence of the soluble activin A inhibitor ActRIIBFc. Frequencies of siCAFs generated in the absence or presence of ActRIIBFc.

Table 1: siCAF signature. EXAMPLE J_:

Material & Methods

Mouse models

All animal protocols were reviewed and approved in accordance with the guidelines provided by the CRCL Animal Care Committee (CECCAPP CLB 2019 002). The generation of Acvrlb^flox/flox mutant mice has been previously described (26). Acvrlb^flox/flox;LSL126 Kras^G12D/+;Ptfla-Cre mice (termed 4KC mice) were generated by crossing Acvrlbflox/flox mice with previously established LSL-Kras^G12D/+;Ptfla-Cre mice (termed KC mice) (27).

Atomic force microscopy (AFM)

The mechanical properties of pancreatic tissue areas were determined by AFM as described previously (34). 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 (34). 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.

The stiffness patterns of different regions within a pancreatic lesion (stromal compartment and tumor cells) were determined at high resolution by applying quantitative nanomechanical mapping and force volume protocols (Bruker). In these protocols, the AFM probe oscillates at a low frequency while horizontally scanning the sample and generating a force curve each time the probe contacts the sample. The elastic modulus reflecting the stiffness of the sample is extracted from each curve by applying the Sneddon (Hertz) model, resulting in a 2D stiffness map in which each pixel represents one force curve.

Cell isolation from mouse tissues

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. scRNAseq: quality control and data analysis 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 (lOx Genomics) at the in-house Single Cell Platform (CLB/CRCL). After cell encapsulation and barcoding, library preparation followed the standard lOx 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 mm 10 genome using CellRanger software (lOx Genomics).

Count data (filtered barcode matrices) were obtained with CellRanger (lOxGenomics). All downstream analyses were performed using R/Bioconductor/CRAN packages, R version 4.2.2 (2022-11-10) [https://cran.r-project.org/; http://www.bioconductor.org/; https://cran.r- project.org/] on a Linux platform (x86_64-pc-linux-gnu [64-bit]). Filtered barcoded matrices were used to create Seurat objects (81) for each condition that were subsequently merged (package 'Seurat' v.4.1.1).

A total of 8878 cells (3638 4KC CAFs, 3664 KC CAFs, 901 4KC Ducts, and 675 KC Ducts) remained after filtering for quality parameters (number of features per cell between 1000 and 6000, fraction of mitochondrial genes < 10%). The Seurat SCTransform function was used to simultaneously normalize, identify variable features and scale the data. Following dimension reduction with principal component analysis (PCA), the first 30 dimensions were used to construct a shared nearest neighbor (SNN) graph using the FindNeighbors function. Clusters were identified with a resolution of 0.1 and projected in two-dimensional plots using UMAP [arXiv: 1802.03426v3], The markers of each cluster and DEGs in pairwise comparisons were identified using the Find AllMarkers and FindMarkers functions, respectively (main parameters: only.pos = F, min. pct = 0.25, and logfc. threshold = 0.25), with an adjusted p value threshold of 0.05. Fibroblasts (CAFs) were identified and re-clustered with a higher resolution (0.5). Known CAF markers(23, 24) were used to score each cell according to their myofibroblastic (myCAFs), inflammatory (iCAFs), and antigen-presenting (apCAFs) signatures, using the AUCell package (v.1.18.1). Pathway analyses were performed with the enrichment functions of the 'ClusterProfiler' package (v.4.7.1). Pathway scores were estimated with the Seurat AddModule Score function using 100 control features after downloading relevant pathways with the R packages enrichR v.3.1 (82, 83) and pathfindR v.1.6.3 (84).

'SingleCellSignalR' v.1.8.0 was used to study cell interaction networks on Seurat preprocessed data, using the major cell type labels (i.e. ducts, CAFs ), independently for the two study conditions (i.e. KC and 4KC). All receptorligand analyses were done in "paracrine" mode and visualized with the chord plot and heatmap functions of the same package. siCAF signature using the AUCell method to calculate a score for every single cell (46).

CAF differentiation assay: coculture of isolated PSCs and acinar cells

PSCs were isolated from wild-type (WT) C57BL/6 mice as previously described (17, 23). 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.

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).

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.

Mouse cells lines

The isolation and culture of cells were performed using a protocol adapted from previously published protocol (35). Tumor primary cell line 4KC was obtained from the pancreas of 2.5-months old 4KC mice using the same protocol. After several passages, the cells were infected with a lentivector expressing H2B GFP as previously described (36). Immortalized mouse pancreatic stellate cells (iPSCs) were obtained from Tuveson DA (23).

Human CAF generation

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 (37). 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).

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/1 1-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 ActRIIbFc 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 ActRIIbFc inhibitor. Half of the cell culture medium was refreshed every 48 h with the addition of 0.5 pg/ml ActRIIbFc inhibitor until day 6 of culture. Cells were detached using StemPro Accutase cell dissociation reagent (Gibco Al 110501) 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.

PDGFRa tyrosine phosphorylation and Western blot iPSCs were seeded onto 6 wells plate (4.0 xlO⁵ cells per wells). Treatments of various lengths (lOmin, 30min, 2h) were done with 4KC conditioned medium or mouse recombinant PDGF-AA protein (Biolegend, 776304) at 50ng/mL. For proteasome inhibition, cells were starved and treated with the proteasome inhibitor MG- 132 (Sigma Aldrich, M8699-1MG) at lOpM for 12h. Cell extracts were prepared from cultured cells lysed by scratching at 4°C in 80uL RIPA (ThermoFisher, 89900) supplemented with protease (Roche, 04693159001) and phosphatase inhibitors (Roche, 04906837001). Obtained lysates were sonicated and centrifugated at 13,000g for 15min at 4°C. Equal amount of proteins were separated by SDS- PAGE then transferred onto Immune-Blot PVDF membrane. Membranes were incubated in blocking buffer containing 5% milk or Bovine Serum Albumine (BSA) (Sigma Aldrich, A2153-100G) in Tris Buffered Saline/Tween 20 (TBST). The blots were then probed overnight at 4°C with the appropriate primary antibodies. The membranes were revealed with the appropriate secondary antibodies for Ih at RT. Detection was performed by enhanced chemiluminescence using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, 32106) according to the manufacturer’s protocol. Tubulin was used as a loading control. Antibodies and dilutions were as follows: anti-p-Ty754 Ab (Thermo Fisher, TF441008G), 1 :1000; anti-PDGFRa (Cell Signalling Technology, 3174S),l : 1000; anti-Tubulin (GenTex, GTX628802), 1 : 1000. Secondary HRP-conjugated anti-rabbit Ab (Jackson Immuno Research, 711-035-152) or anti-mouse Ab (Jackson Immuno Research, 715-035-150).

CAF and 4KC-conditioned medium

For the generation of conditioned medium, CAF subpopulations isolated by FACS were cocultured with cells from the KIC tumor cell line (35) 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. For the generation of conditioned medium, 4KC cells (1.5 xlO⁴ cells per wells) were seeded onto 6 wells plate in DMEM containing 10% FCS. After 7 days of incubation at 37°C and 5% CO2, the supernatants were collected and stored at -20°C until further use.

Bone marrow-derived dendritic cell (BMDC) generation

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 (36). 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.

T-cell proliferation assay

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: l). Mouse T-Cell-Activator CD3/CD28 Dynabeads (Gibco) were added to the coculture (T cells:Beads=l : l). The proliferation of the CD8+ T cells was evaluated at the end of the culture period by analyzing CSFE dilution using flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)

To determine the amount of PDGF-AA in tissue samples, freshly excised mouse pancreata were weighed and finely minced. Then, 500 pl of lx 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.

Flow cytometric analysis

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 (PF A) 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 DMEM containing 10% (functional assays) or 20% fetal calf serum (FCS) (RNA isolation). FACS data were analyzed using FlowJo software (TreeStar).

Statistical analysis

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

Results

ALK4 signaling disruption in neoplastic cells leads to early increased collagen deposition and tissue rigidity

Given that ablation of protective activin A signaling promotes the formation of ADM lesions (25), we sought to further evaluate the effect of ALK4 signaling disruption in neoplastic cells on the structural and mechanical qualities of the pancreatic tumor microenvironment (TME). Although oncogenic KRAS^G12D expression occurs during the prenatal state in both KC mice and 4KC mice (28), ADM lesions develop only at or shortly after the time of weaning. While no difference in pancreas weight was observed at three weeks of age, at six weeks of age, the pancreata of 4KC mice were already significantly enlarged than those of KC mice (data not shown). Histological analysis revealed significantly expanded areas of pancreatic lesions in 4KC mice compared to KC mice at six weeks of age, and while this expansion was mainly mediated by stroma formation, ADM formation was also accelerated in 4KC mice at this age (data not shown). In both KC pancreata and 4KC pancreata, collagen deposition was observed in the stromal compartment of lesions, as determined by histological analysis of Sirius Red staining (data not shown), and as expected, the total collagen amount was significantly higher in the 4KC pancreata (Fig. 1A), which was accompanied by palpable tissue induration. Interestingly, although more collagen was detected in 4KC pancreata than in KC pancreata, evaluation of Sirius Red staining under polarized light revealed no change in the thickness of the collagen fibers for either genotype (Fig. 1A).

To compare the rigidity within distinct tissue compartments between KC and 4KC pancreata, we performed AFM analysis in combination with IF microscopy. Stromal and ADM regions were identified by the expression of aSMA and CK19, respectively, and the elastic modulus was measured in three different regions per sample (Fig. IB). The data revealed increased tissue rigidity in the stroma of 4KC mice compared to that of age-matched KC mice (Fig. IB), which could be explained by increased deposition of collagen fibers that most likely form more interfiber connections, which create tissue stiffness (Fig. 1A). Of note, we also detected an increase in tissue stiffness in the neoplastic compartment of 4KC mice compared to that of KC mice (Fig. IB). Taken together, the results indicate that disruption of ALK4 signaling in neoplastic cells not only overcomes protective antitumorigenic mechanisms as previously shown (25) but also produces a strong paracrine effect at the early stage of ADM, resulting in excessive ECM secretion within the TME.

Sustained PDGF signaling is increased in stiff tissue conditions

Recently, the complex heterogeneity of CAFs has been revealed by other studies, indicating the existence of CAF subpopulations equipped with pro- and antitumorigenic qualities (19-21, 29). 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. FACS sorting was used to exclude hematopoietic and endothelial cells based on their expression of CD45 and CD31, respectively. Next, lectin PNA⁺ acinar cells were excluded (30) to enrich for CAFs (CD45'CD31 'Lectin PNA-EpCAM') or neoplastic ductal cells (CD45'CD31 'Lectin PNA'EpCAM⁺) (data not shown). Importantly, in 4KC mice, CAFs and ductal cells were present at significantly higher frequencies among pancreatic CD45-CD31- cells (Fig. 2A), but equal numbers of single cells from each sample (KC or 4KC) and each fraction (CAFs or Ducts) were captured and sequenced using a droplet-based approach.

In the CAFs-enriched fraction, 9 main clusters (CAFs 0-8) were identified in both KC mice and 4KC mice and the cluster proportions are indicated (data not shown). We identified cluster 7 as being pericytes stem cells (PeSC) that we recently identified in neoplasia (31). Previously described myCAFs, iCAFs and apCAFs (21) signature were identified as cluster 6, 3 and 8 in both conditions (data not shown). All of the previously identified populations of CAFs were significantly increased in proportions in the 4KC condition compared to KC (data not shown).

In order to identify any particular interactions between the CAF and duct cell population associated with increased tissue stiffness, we merged the 2 fractions and obtained 3 major clusters: CAFs, Ducts (Tumor) and in addition, a small fraction of PeSCs (data not shown). To determine how these 3 populations interact, we performed ligand-receptor based SingleCell SignalR algorithms (32) analysis among the CAFs, Ducts and PeSCs in the KC and 4KC conditions. The algorithm was used in “paracrine” mode with a large receptor: ligand database specific for mouse, as previously described (32). Our analysis revealed that there were increased number of interactions among the 3 clusters in the KC soft condition than in the 4KC stiff condition (data not shown) and the specific top 50 interactions are represented (data not shown). To test the hypothesis that early instruction from duct cells to CAFs contributes to the establishment of stiff phenotype, we analyzed the interactions between Ducts and CAFs in both soft (KC) and stiff conditions (4KC). As shown in Fig. 2B we determined an increased number of interactions either from Ducts towards CAFs or from CAFs towards Ducts in 4KC condition.

We analyzed in detailed the most upregulated interactions in both KC and 4KC conditions (data not shown). We identified platelet-derived growth factors (PDGF)-dependent interactions from Ducts to CAFs to be prevalent in 4KC while absent in KC. As PDGFs are considered fibrogenic growth factors, we next sought to determine the expression of PDGRFA, PDGRFB, PDGFA and PDGFB in merged scRNAseq data. While the two major receptors, PDGFRA and PDGFRB, were exclusively expressed in the CAF compartment, the ligands PDGFA et PDGFB were exclusively expressed in the duct compartment (Fig. 3A-D). 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 (33) and thereby might promote tissue stiffness. Moreover, CD61 is known to interact with the ECM protein big-h3/TGFbi (34), which has been described as a key ECM protein in the pancreatic TME hampering conventional (35, 36) and unconventional T-cell responses (37). We observed that CD61 was also expressed in the CAF compartment (Fig. 3E).

As shown in Fig. 3A, most CAFs expressed PDGFRA in both, KC and 4KC conditions (82% and 74% of CAFs, respectively). Therefore, we performed differential expression analysis in 4KC vs. KC, using only PDGFRA⁺ CAFs. This analysis resulted in 72 differentially expressed genes (DEGs), 29 of them upregulated and 43 downregulated in 4KC CAFs. DEGs were tested for enrichment in Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathway terms showing significant differences in ECM organization, assembly of collagen fibrils, and collagen formation (data not shown) in the 4KC stiff relative to KC soft condition. Furthermore, most of ECM pathways from several repositories were enriched in the DEGs of the PDGRFa⁺ vs PDGRFa' suggesting these pathways exhibit dependence on PDGRFA expression (data not shown). Based on the 29 4KC upregulated genes (Table 1) we generated a stiffness CAF signature. To specifically test for potential signaling activity, we interrogated the 29-gene CAF signature for kinase activity enrichment using two different kinase databases. In both cases, PDGFRA was among the top kinase activities significantly enriched in the CAF signature (data not shown). Furthermore, ligand-receptor interaction analysis was also performed separately for PDGFRA⁺ positive CAFs. More PDGF-related interactions from Ducts to CAFs were detected in the 4KC condition, relative to the KC condition (data not shown). We further looked for the distribution of siCAFs signature across the 9 CAF clusters. We applied the 29- gene siCAF signature using the AUCell method to calculate a score for every single cell (38). All clusters displayed at least one fraction of cells with relatively high score with the exception of cluster 7, previously identified as pericytes stem cells (31). 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.

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

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. 4). 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. 4). 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.

In addition to PDGFRa and CD61, we further evaluated the expression of the established CAF markers aSMA, FAP1, FSP1/S100-A4, and PDGFRa, as well as the integrin CD29/ITGbl (19, 39). Interestingly, aSMA and FSP1, which are both considered activated CAF markers (40, 41), were expressed on siCAFs and PDGFRa⁺ CAFs, respectively. While no differences in the expression of aSMA were found between KC and 4KC siCAFs, FSP1 expression was significantly increased on PDGFRa⁺ CAFs from 4KC mice (data not shown). Moreover, the expression of the integrins CD61 and CD29 was significantly higher on PDGFRa⁺ CAFs obtained from KC mice. In contrast, the mean fluorescence intensity (MFI) of CD61 was significantly higher on PDGFRa⁺ CAFs from 4KC mice (data not shown). Unsupervised t-distributed stochastic neighbor embedding (tSNE) analysis of siCAFs and PDGFRa⁺ CAFs revealed an increased siCAF population in the high-stiffness conditions (data not shown). Of note, we also detected a PDGFRa CD61‘ CAF population in both KC and 4KC pancreata, representing 16.8% and 26.7% of all CAFs, respectively (data not shown). However, since this population did not express any of the other evaluated CAF markers (data not shown), we excluded it from the current study. Nevertheless, given the relatively high frequency, this population should be subject to future analysis.

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 (data not shown). We detected a diminished staining for PDGFRa protein in 4KC although the PDGRFa mRNA detected by RNAscope was similar in both conditions (data not shown). 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. To compare the rigidity within distinct PDGRFa⁺ and PDGRFa' regions between KC and 4KC pancreata we performed AFM analysis in combination with IF microscopy. We detected increased elastic modulus in PDGRFa' stromal area in 4KC mice. In KC mice, PDGRFa⁺ area had significant diminished elastic modulus compared to PDGRFa' area (data not shown). These results demonstrate that siCAFs are localized in the stiff regions in mouse pancreata. 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.

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

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. 4). 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. 5A, B). Notably, the previously mentioned PDGFRa- CD61- CAF population remained unchanged in both KC mice and 4KC mice over the course of the experiment (data not shown). These results indicate 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. 5C), further highlighting the different activation statuses of these two cell populations.

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 (25). 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. 5D). 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. 5D).

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. First, we verified that PDGRFa was phosphorylated in PSCs after ligand binding in vitro. As neoplastic primary cells from 4KC mice as well as primary PSC from WT are difficult to generated in high numbers, we used alternatively immortalized PSC (iPSC) previously described (20) as well as 4KC-GFP primary cell line generated as previously described (31). iPSC displayed increased phosphorylation upon 4KC culture media or recombinant PDGRF-AA binding (data not shown). Next, we performed in vitro generation of siCAF for 7 days in presence or absence of a neutralizing polyclonal anti-PDGF antibody. The use of anti-PDGF neutralizing Ab was able to significantly reduce the phosphorylation of PDGRFa. Moreover, the number of siCAF diminished in the presence of anti-PDGF neutralizing Ab (Figure 5E-F). Adding recombinant PDGRF-AA induced PDGRFa surface expression downregulation on the surface of CAFs, although in a lesser extent than in the presence of 4KC-GFP cell line (Figure 5G). Ultimately, most internalized PDGFRs are subjected to lysosomal or proteasomal degradation (42). FACS analysis revealed that siCAFs had lower intracellular PDGFRa expression than PDGFRa⁺ CAFs (Fig. 5H) and the use of MG-132 proteasome inhibitor increased the accumulation of PDGRFa (data not shown). 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 (43-45). Although PDGF ligand expression was not different between KC and 4KC mice on a per cell basis (Fig.3D) we hypothesized that in 4KC mice, higher overall PDGF ligand levels must be found due to the increased presence of ductal cells (Fig. 2A). Indeed, ELISA analysis revealed significantly increased levels of the ligand PDGF-AA per gram of 4KC mouse pancreas (Fig. 51). siCAFs hamper antitumor immune responses

Next, we investigated whether the siCAF signature associated with high tissue stiffness was also linked to immune suppression. We did not observe altered frequencies of infiltrating hematopoietic CD45⁺ cells, including CD8⁺ T cells (Fig. 6A, B), CD4⁺ T cells, CD4⁺/Foxp3⁺ regulatory T cells (Tregs), NK-p46 natural killer cells, TCRgd T cells or neutrophils (data not shown), 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^low (Fig. 6C). In contrast, CD8⁺ T-cell activation was significantly increased in the spleen of 4KC mice (Fig. 6D). 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.

To determine whether the 2 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 (35) 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. 6E, 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. 5F). Similar results were obtained with KC PDGFRa⁺ CAF- and siCAF-conditioned medium (data not shown). T cells cocultured in KC or 4KC PDGFRa⁺ CAF-conditioned medium showed proliferation and GrzB production similar to those cultured in control conditioned medium obtained from tumor cells alone (data not shown). In order to determine which soluble factors could be responsible for T cell mediated inhibition, we performed customed LegendPlex array for cytokines and chemokines that have been previously reported to be produced by CAFs (Fig. 6H). We detected a significant increase of VEGF, CCL2 and CXCL12 amount siCAFs condition media compared to PDGRFa⁺ CAFs condition media. Low quantities of IL-6, TGF- bl and IL-10 were detected in both conditions. VEGF was previously shown to have direct immunosuppressive effect on T cell proliferation and activation (46) suggesting that siCAF T cell activation inhibition might be at least in part mediated by VEGF.

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

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 (47-49). 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, which recognizes PDGF-AA, PDGF-AB, and PDGF-BB dimers. Control age-matched littermates were injected with lx PBS. At 6 weeks of age, we analyzed pancreata collected from anti-PDGF- and PBS-injected mice (Fig. 7A). 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. 7C, 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. 7E). Furthermore, we observed increased CD31⁺ endothelial cells (Fig. 7F), decreased lesions (Fig. 7G) and increased EPCAM expression (Fig. 7H) 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 (data not shown). In addition, IHC analyses revealed the prevention of PDGFRa downregulation in pancreatic lesions by anti-PDGF injection (data not shown), as well as a reduced number of advanced lesions. Furthermore, Sirius Red staining showed decreased collagen fiber deposition in anti-PDGF condition (data not shown). Taken together, our data show that the PDGF neutralization leads to the reprogramming of the tumor environment.

Furthermore in order to illustrate that PDGF neutralization lead to collagen content modification, we performed in vitro coculture experiments. To do so we cocultured PSC with tumor cells (4KC cell line). At Day 0, 4KC (1.5 xl03 cells per wells) and PSCs (12.5 xl03 cells per wells) were seeded onto 6 wells plate alone or in co-culture (1.5x103 cells per wells and 12.5x103 cells per wells respectively) with IgGl rabbit control Ab or anti-PDGF neutralizing Ab. After 7 days of incubation, Sirius red staining was performed, the collagen area was quantified by Image! We observed decreased collagen content in the anti-PDGF neutralizing Ab condition compared to compared to IgGl control condition (Fig. 9) suggesting that inhibition of collagen deposition by siCAFs consistent with the in vivo data (reported in Fig. 7).

Identification of siCAFs in the human setting

To relate our findings to human PDAC patients, we performed FACS analysis of primary pancreatic CAFs obtained from 4 different donors. Similar to our mouse results, we detected an abundant siCAF population correlated with a reduced PDGFRa⁺ CAF population, indicating a differentiation-dependent connection between these two populations (Fig. 8A). Further analysis showed that the overall expression of the known CAF markers and integrins PDGFRa, CD61, CD29, FAP, and FSP-1 was significantly lower in siCAFs than in PDGFRoC CAFs (Fig. 8B-F). To determine whether it is possible to reprogram siCAFs in the human setting, we performed in vitro coculture of human CAFs with PANC-1 cells, a human PDAC cell line previously described to express ALK4 (24) and respond to Activin A (25), in the presence or absence of ActRIIBFc. FACS analysis showed that in the presence of PANC- 1 cells, CAFs lost expression of PDGFRa which was similar to the mouse results, and that the addition of ActRIIBFc did not have any effect on the amount of siCAFs. To get insight into the existence of siCAFs in human setting, we applied siCAFs signature to a scRNA dataset specific for pancreas (50). As shown in Fig. 8H, the annotated dataset includes a well-defined “Fibroblast” cluster. We extracted 10953 human fibroblasts (1037 and 9916 from non-tumor and tumor tissues, respectively), classified these fibroblasts according to human CAFs signatures (51) that distinguishes iCAFs, myCAFs and meCAF as well as normal fibroblasts and applied the 29-gene siCAF signature (38). As shown in Figure 81, the siCAF score was expressed in all 3 types of CAFs at significantly higher levels than normal fibroblasts. In order to determine if similar association between siCAFs and tissue stiffness exists in human tissues we took advantage of a previously published stiffness signature (data not shown) (52). This stiffness score was upregulated in tumor CAFs compared to normal fibroblasts (Figure 8J). Moreover, siCAF and stiffness scores were significantly correlated across all PDAC CAF cells (Figure 8K). Altogether, these data suggest for the first time that paracrine signaling between tumor cells and CAFs is able to induce siCAFs phenotype independent of the genetic background.

Discussion

In this study, 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 (19-21), 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.

PDGF-AA binds primarily to PDGFRa, while PDGF-AB and PDGF-BB bind to PDGFRa as well as other receptor subtypes, such as PDGFRp (60). We demonstrated here that continuous binding of PDGF-AA to PDGFRa induced receptor downregulation, leading to the emergence of siCAFs. The expression analysis of genes encoding CAF markers other than PDGFRa and PDGFRP (64, 65) showed that FSP1 expression was significantly increased on PDGFRa - CAFs from 4KC mice (stiff conditions). This finding was validated by the high expression of FSP1 observed in a human setting, suggesting that FSP-1 might be a universal bona fide marker of activated CAFs. The expression of integrins, such as CD29/ITGB1 (66, 67) or CD61/ITGB3 (68), on the surface of CAFs has been previously described. Here, we show that CD29 and CD61 expression was downregulated on siCAFs in both mouse and human settings, suggesting an active role in signaling.

Herein, the pro- and anti-inflammatory properties of CAFs are demonstrated to be highly dependent on intratumoral location and less dependent on origin. We demonstrated that PDGFRa⁺ CAFs had no potential to inhibit a T-cell response in vitro under the loose condition (KC condition). Under stiff conditions, this population had less inhibitory potential than siCAFs, highlighting the importance of the mechanical properties of the tissue in the education of CAFs. Several publications have demonstrated that these cells are important for sensing mechanical changes in tissues not only at homeostasis but also in pathological conditions, i.e., fibrosis. The important finding of this study is that despite the origin of the cells, the mechanical constraint together with the secretion of soluble factors breaks the heterogeneity of CAFs into a binary classification according to their “microenvironment sensing”, namely, PDGFa⁺ CAFs and siCAFs. This creates the option of targeting a pathway through a ligand trap approach that would reprogram siCAFs into PDGFRa⁺ CAFs rather than targeting a particular surface marker. This might also be beneficial since the elimination of CAFs by antibody-dependent cytotoxicity (ADCC) has been shown to be deleterious in PDAC (clinical trial failure).

A hallmark of the pathogenesis of solid cancers such as PDAC is escape from efficient antitumor immune responses. It has been demonstrated that the collagenous TME can restrain infiltrating CD8⁺ T cells from accessing tumor cells (70, 71). Additionally, the cytotoxic activity of tumor-infiltrating lymphocytes (TILs) can be reduced by insufficient T-cell priming (72). CD8⁺ T-cell exclusion from the tumor bed has been proven to be a key element in the antitumor response (48). We show here that early mechanical and paracrine education through PDGF ligands (PDGF-AA in particular) can skew an effective antitumor response in situ. Although we identified potent immune priming of CD4⁺ and CD8⁺ T-cell responses in the spleen, the number of activated CD8⁺ T cells in the stiff pancreas was diminished compared to that in the loose pancreas, indicating that local tissue mechanics are a key element in the outcome of the immune response. Further studies combining spatial detection of activated CD8⁺ T cells and tissue rigidity would provide indications of the outcome of immune checkpoint therapy. Interestingly, we also observed an increase in CD31 expression in PDGF-depleted conditions, suggesting that an increase in modified interactions at the level of blood vessels might involve other partners that prevent an efficient T-cell response (i.e., MDSCs or macrophages, data not shown). Further investigation linking the availability of PDGF-AA to angiogenesis should shed new light on this additional mechanism of action.

Based on our data, we established a model of the mechanism of action. As tumors evolve, they proliferate, produce PDGF ligands and instruct CAFs via a paracrine effect. PDGF Rot, 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 PDGRFot, 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. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A PDGF-AA ((Platelet-derived growth factor- AA) antagonist for use in the prevention or the treatment of a patient affected with a cancer, wherein said antagonist restores the CD8+ T cell activation.

2. 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.

3. The PDGF-AA antagonist for use according to claim 1 or 2, wherein the PDGF- AA antagonist directly binds to PDGF-AA.

4. The PDGF-AA antagonist for use according to any one claim 1 to 3, wherein the PDFG-AA antagonist is an inhibitor of PDGF-A gene expression or an inhibitor of PDGF- AA activity.

5. The PDGF-AA antagonist for use according to any one claim 1 to 4, wherein said antagonist is an anti-PDGF-AA neutralizing antibody or aptamer.

6. The PDGF-AA antagonist for use according to any one claim 1 to 5, wherein the cancer is a solid tumor.

7. The PDGF-AA antagonist for use according to claim 6, wherein the solid tumor is associated with a stiffness score of at least 0.37, and more particularly of at least 0.40.

8. The PDGF-AA antagonist for use according to claim 7, wherein the solid tumor is selected from the group consisting of non-small 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.

9. The PDGF-AA antagonist for use according to claim 8, wherein the solid tumor is pancreatic cancer.

10. The PDGF-AA antagonist for use according to claim 9, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

11. The PDGF-AA antagonist for use according to any one claim 1 to 10, wherein said PDGF-AA antagonist is administered in combination with anti-cancer therapy.

12. A pharmaceutical composition comprising a PDGF-AA antagonist and a pharmaceutically acceptable carrier.

13. The pharmaceutical comprising according to claim 11, wherein the PDGF-AA antagonist is an inhibitor of PDGF-A gene expression or an inhibitor of PDGF-AA activity.

14. The pharmaceutical comprising according to claim 11 or 12, wherein said antagonist is an anti-PDGF-AA neutralizing antibody or aptamer.