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WO2025068180A1 - Méthodes de traitement du cancer par ciblage des fibroblastes associés au cancer - Google Patents

Méthodes de traitement du cancer par ciblage des fibroblastes associés au cancer Download PDF

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Publication number
WO2025068180A1
WO2025068180A1 PCT/EP2024/076778 EP2024076778W WO2025068180A1 WO 2025068180 A1 WO2025068180 A1 WO 2025068180A1 EP 2024076778 W EP2024076778 W EP 2024076778W WO 2025068180 A1 WO2025068180 A1 WO 2025068180A1
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pdgf
antagonist
cancer
cells
cell
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Ana HENNINO
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Leon Berard
Universite Claude Bernard Lyon 1
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Leon Berard
Universite Claude Bernard Lyon 1
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • 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).
  • CAFs cancer-associated fibroblasts
  • PDAC pancreatic ductal adenocarcinoma
  • Pancreatic ductal adenocarcinoma 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).
  • ADM acinar-to-duct metaplasia
  • PanIN pancreatic intraepithelial neoplasia
  • 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.
  • ECM extracellular matrix
  • 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).
  • CAFs cancer-associated fibroblasts
  • a-SMA smooth muscle actin
  • 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).
  • BMPs bone morphogenic proteins
  • GDFs growth and differentiation factors
  • PDGFR Plate-derived growth factor Receptor
  • 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
  • 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.
  • 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.
  • PDGF-AA Platinum-derived growth factor- AA
  • the cancer disease is pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • the inventors investigated the correlation between PDGF-AA and biological findings from PDAC patients.
  • 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.
  • 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.
  • 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.
  • PDGFRoC CAFs become siCAFs capable of inhibiting T-cell responses in situ.
  • a PDGF-AA ligand trap approach 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mitogenesis i.e. proliferation
  • 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.
  • 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.
  • 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).
  • PDGF-AA Platinum-derived growth factor subunit A is a protein that in humans is encoded by the PDGFA gene (Gene ID 5154)
  • -BB Platinum-derived growth factor subunit B is a protein that in humans is encoded by the PDGFB gene
  • - CC Platinum-derived growth factor subunit C is a protein that in humans is encoded by the PDGFC gene
  • -DD Platinum-derived growth factor subunit D is a protein that in humans is encoded by the PDGFD gene
  • -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).
  • PDGFRA tyrosine kinase receptor monomers
  • PDGFRB tyrosine kinase receptor monomers
  • 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).
  • PDGF-AA two A subunits
  • PDGF-BB two B subunits
  • PDGF-AB two C subunits
  • PDGF-DD two D subunits
  • 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.
  • 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.
  • 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.
  • the patient is affected or likely to be affected with cancer.
  • the patient is an human.
  • the patient is an human affected or likely to be affected with PDAC.
  • 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.
  • the cancer is a cancer associated with a stroma expressing/secreting PDGF-AA.
  • 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.
  • 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; acid
  • the cancer may be associated with a solid tumor.
  • 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.
  • mesenchymal origin i.e; fibrosarcoma and rhabdomyoscarcoma
  • the central and peripheral nervous system i.e; including astrocytoma, neuroblastoma, glioma, glioblatoma
  • 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.
  • 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.
  • 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.
  • pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • 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.
  • 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)).
  • activate anti-tumoral CD8+T cell response means the enhancement, the restoration the natural ability of the CD8+T cell to lyse cancer cells.
  • 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.
  • 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).
  • biological activity of a PDGF-AA is meant inducing tumor cell growth and inhibiting CD8 + T cell activation (blocking the anti-tumoral response).
  • 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.
  • 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.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e.
  • Kd/Ka 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.
  • 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).
  • a PDGF-AA antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of PDGF-AA.
  • 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.
  • 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.
  • 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.
  • the PDGF-AA antagonist directly binds to PDGF-AA and inhibits the inhibition of CD8+ T cell activation (or restore CD8+ T cell activation).
  • the PDGF-AA antagonist is an inhibitor of PDGF-A gene expression or an inhibitor of PDGF-AA activity.
  • the PDGF-AA antagonist may be a molecule that binds to PDGF-AA selected from the group consisting of antibodies, aptamers, and polypeptides.
  • the PDGF-AA antagonist is a neutralizing antagonist.
  • the PDGF-AA antagonist according to the invention is an antibody.
  • the PDGF-AA antagonist is an antibody (the term including antibody fragment or portion) that can block the interaction of PDGF-AA with PDGFRa.
  • the PDGF-AA antagonist is an antibody binding PDGF-AA.
  • the PDGF-AA antagonist is an antibody binding specifically PDGF-AA.
  • the PDGF-AA antagonist is an antibody binding PDGF-AA and/or PDGF-AB.
  • 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").
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody.
  • 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.
  • 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.
  • 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.
  • the recombinant PDGF-AA may be provided by expression with recombinant cell lines.
  • Recombinant form of PDGF-AA and recombinant form of PDFG-A may be provided using any previously described method.
  • 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.
  • 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.
  • 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.
  • 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.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • compositions and methods that include humanized forms of antibodies.
  • 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.
  • 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.
  • 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.
  • 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.
  • KAMA human anti-mouse antibody
  • 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.
  • 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.
  • 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.
  • Fab 1 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.
  • scFv single chain Fv
  • 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.
  • a peptide linker such as divalent sc(Fv)2.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 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).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • 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.
  • 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).
  • 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.
  • gRNA guide RNA
  • 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.
  • 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.
  • 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.
  • the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonuclease subunits.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic 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).
  • 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
  • KRIEGLER A Laboratory Manual
  • MURRY Method of Recombinant retroviruses by the packaging cell line
  • Methods in Molecular Biology vol.7, Humana Press, Inc., Cliffton, N.J., 1991.
  • adeno-viruses and adeno-associated viruses 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • 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.
  • 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.
  • 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.
  • the patient have a high level of PDGF-AA in blood.
  • 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.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • 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.
  • the cancer is PDAC.
  • 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.
  • the invention also relates to PDGF-AA antagonist for use in the prevention or treatment of a PDAC in a patient.
  • 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.
  • 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.
  • ROC Receiver Operating Characteristic
  • 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.
  • 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.
  • 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.
  • 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.
  • the PDGF-AA antagonist for use according the invention can be administered in combination with any suitable agent, in particular with anti-cancer therapy.
  • anti-cancer therapy has its general meaning in the art and refers to any compound, natural or synthetic, used for the treatment of cancer.
  • 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.
  • CAR Therapy such as CAR T- , CAR M- or CAR NK-cell therapy, antibody-drug conjugates (ADC) or chemotherapy.
  • ADC antibody-drug conjugates
  • 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.
  • the PDGF-AA antagonist is administered in combination with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • 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 a
  • 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-pyrrolin
  • 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.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • 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.
  • the PGDF-AA antagonist is administered in combination with PROTAC or molecular glue degraders.
  • proteolyse targeting chimeric molecules also known as “PROTAC”
  • 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.
  • 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.
  • the PGDF-AA antagonist is administered in combination with radiation therapy.
  • 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.
  • 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.
  • the radiation therapy is external radiation therapy.
  • external radiation therapy examples 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
  • the PGDF-AA antagonist is administered in combination with an immune checkpoint inhibitor.
  • 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.
  • 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-H4 also called VTCN1
  • B7-H4 also called VTCN1
  • B and T Lymphocyte Attenuator (BTLA) and also called CD272 has HVEM (Herpesvirus Entry Mediator) as its ligand.
  • 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
  • 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.
  • an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade.
  • 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.
  • the immune checkpoint inhibitor is an antibody.
  • antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • the immune checkpoint inhibitor is an anti -PD-1 antibody such as Pembrolizumab (Keytruda), Nivolumab (Opdivo) and Cemiplimab (Libtayo).
  • the immune checkpoint inhibitor is an anti-PD-Ll antibody such as Atezolizumab (Tecentriq), Durvalumab (Imfinzi), Avelumab and BMS-936559 (BMS).
  • 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.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody such as Ipilimumab (Yervoy) and tremelimumab (Imjuno).
  • the immune checkpoint inhibitor is an anti-LAG-3 antibody such as Relatlimab.
  • the immune checkpoint inhibitor is a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • 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.
  • small organic molecules interfere with transduction pathway of PD-1 and Tim-3.
  • they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.
  • 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.
  • 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 thioh
  • 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.
  • 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 :
  • 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:
  • 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).
  • PD-L1 programmed death ligand-1
  • VISTA V-domain Ig suppressor of T cell activation
  • the immune checkpoint inhibitor is an aptamer.
  • the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
  • 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.
  • aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the aptamer is an anti -PD-1 aptamer.
  • the anti -PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
  • 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.
  • 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 Ip
  • compositions of the invention are provided.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 diluent such as a glycol or a glycerol ester
  • 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.
  • 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.
  • pharmacologically compatible dispersants and/or wetting agents for example propylene glycol, butylene glycol, or polyethylene glycol.
  • 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.
  • 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 are a diagrammatic representation of FIGURES.
  • 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-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 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.
  • 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.
  • 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.
  • FIG. 1 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.
  • FIG. 8 Tumor cells instruct siCAFs in human PDAC.
  • 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).
  • 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 were determined at high resolution by applying quantitative nanomechanical mapping and force volume protocols (Bruker).
  • 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.
  • 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.
  • PCA principal component analysis
  • the first 30 dimensions were used to construct a shared nearest neighbor (SNN) graph using the FindNeighbors function.
  • CMOS CMOS-derived neurotrophic factor
  • apCAFs antigen-presenting signatures
  • 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 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).
  • the activin A inhibitor ActRIIBFc gift from Olli Ritvos, Helsinki, Finland
  • 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).
  • 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 mm 3 ) 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.
  • 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.
  • 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.
  • 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.
  • PDGFRa tyrosine phosphorylation and Western blot iPSCs were seeded onto 6 wells plate (4.0 xlO 5 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.
  • MG- 132 Sigma Aldrich, M8699-1MG
  • 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).
  • BSA Bovine Serum Albumine
  • 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 PierceTM 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 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.
  • 4KC cells 1.5 xlO 4 cells per wells
  • 4KC cells 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.
  • BMDC bone marrow-derived dendritic cell
  • 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.
  • APC ammonium-chloride-potassium
  • PDGF-AA human PDGF AA ELISA kit obtained from Abeam was used according to the manufacturer’s protocol.
  • RNA isolation 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).
  • ALK4 signaling disruption in neoplastic cells leads to early increased collagen deposition and tissue rigidity
  • 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.
  • 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.
  • 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).
  • 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.
  • CAFs 0-8 9 main clusters (CAFs 0-8) were identified in both KC mice and 4KC mice and the cluster proportions are indicated (data not shown).
  • cluster 7 was 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).
  • CD61 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.
  • 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).
  • siCAFs stiffness-induced CAFs
  • PDGFRa + CAFs stiffness-induced CAFs
  • Loss of PDGFRa surface expression on siCAFs is a tumor cell-driven early event accompanied by PDGF ligand accumulation
  • 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.
  • GFAP qPSC marker glia fibrillary acidic protein
  • PDGFRs are internalized after binding to their ligands.
  • PDGRFa was phosphorylated in PSCs after ligand binding in vitro.
  • iPSC alternatively immortalized PSC
  • 4KC-GFP 4KC-GFP primary cell line generated as previously described
  • CD8 + T 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.
  • 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).
  • VEGF vascular endothelial growth factor
  • 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
  • pancreata collected from anti-PDGF- and PBS-injected mice (Fig. 7A).
  • Fig. 7B we observed a decrease in pancreas weight in 4KC mice injected with anti-PDGF, indicating diminished tumor growth.
  • Fig. 7C we observed decreased siCAFs in anti -PDGF -injected 4KC mice compared to PBS-injected 4KC mice.
  • Fig. 7E we observed an increased percentage of activated CD8 + T cells pancreas in the anti -PDGF -treated conditions pancreas in the anti -PDGF -treated conditions.
  • siCAFs signature to a scRNA dataset specific for pancreas (50). As shown in Fig.
  • the annotated dataset includes a well-defined “Fibroblast” cluster.
  • the siCAF score was expressed in all 3 types of CAFs at significantly higher levels than normal fibroblasts.
  • PDGF-AA binds primarily to PDGFRa
  • PDGF-AB and PDGF-BB bind to PDGFRa as well as other receptor subtypes, such as PDGFRp (60).
  • PDGFRp PDGFRp
  • 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
  • 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.
  • CD29/ITGB1 66, 67
  • CD61/ITGB3 68
  • TME tumor-infiltrating lymphocytes
  • TILs tumor-infiltrating lymphocytes
  • CD8 + T-cell exclusion from the tumor bed has been proven to be a key element in the antitumor response (48).
  • PDGF-AA early mechanical and paracrine education through PDGF ligands (PDGF-AA in particular) can skew an effective antitumor response in situ.
  • TGFBI transforming growth factor beta induced
  • pancreatic stellate cell a star on the rise in pancreatic diseases. J Clin Invest. 2007;117(l):50-9.
  • beta-catenin-activated autocrine PDGF/Src signaling is a therapeutic target in pancreatic cancer. Theranostics. 2019;9(2):324-36.
  • PDGF-C is a proinflammatory cytokine that mediates renal interstitial fibrosis. J Am Soc Nephrol. 2008;19(2):281-9.
  • VEGF directly suppresses activation of T cells from ovarian cancer patients and healthy individuals via VEGF receptor Type 2. Int J Cancer. 2012;130(4):857-64.
  • pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatum oral compartment of pancreatic ductal adenocarcinoma. Gastroenterology. 2013; 145(5): 1121-32.
  • Hosein AN Huang H, Wang Z, Parmar K, Du W, Huang J, et al. Cellular heterogeneity during mouse pancreatic ductal adenocarcinoma progression at single-cell resolution. JCI Insight. 2019; 5.

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Abstract

La présente invention concerne un antagoniste de la protéine PDGF-AA, destiné à être utilisé dans le traitement du cancer. Dans la présente invention, il a été démontré pour la première fois que la signalisation de PDGF par l'intermédiaire d'une approche de piège à ligand peut inhiber la progression tumorale par la reprogrammation de l'état d'activation des CAF (fibroblastes associés au cancer). Les inventeurs ont découvert que PDGF-AA se lie directement aux lymphocytes T CD8+ par réduction de leur activation et leurs propriétés cytotoxiques. En outre, l'utilisation d'anticorps PDGF-AA neutralisants dans un modèle de souris PDAC permet de réduire la croissance tumorale par amélioration de la réponse antitumorale des lymphocytes T CD8+. Ainsi, la neutralisation de PDGF-AA qui agit en tant que nouvelle cible de point de contrôle immunologique dans PDAC permet donc de restaurer l'immunité antitumorale bénéfique dans le cancer, et en particulier dans PDAC.
PCT/EP2024/076778 2023-09-25 2024-09-24 Méthodes de traitement du cancer par ciblage des fibroblastes associés au cancer Pending WO2025068180A1 (fr)

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