P21-engineered monocytes and radiotherapy in solid tumors

Abstract

The present invention concerns monocytes that over-express the cyclin-dependent kinase inhibitor p21 protein and therapeutic compositions comprising thereof, and in particular their use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

Description

[0001] P21 -ENGINEERED MONOCYTES AND RADIOTHERAPY IN SOLID TUMORS

[0002] Field of the invention

[0003] The present invention concerns monocytes that over-express the cyclin-dependent kinase inhibitor p21 protein and therapeutic compositions comprising them, and in particular their use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

[0004] Technical background

[0005] Radiation therapy is a cornerstone of cancer treatment. Even though more than half of cancer patients receive radiotherapy during their treatment, many limitations affect the use and the efficacy of radiotherapy for the treatment of solid tumors such as pancreatic ductal adenocarcinoma (PDAC). Several intrinsic or acquired resistance mechanisms involving a protective DNA damage response (Huang, R. X. & Zhou,) tumor aneuploidy (Schwartz, J. L. et al.) the anti-apoptotic proteins such as BCL-2 and BCL-XL (Li, J. Y. et al.), the alteration of glucose metabolism pathway (Shimura, T. et al.), hypoxia (Doi, N. et al.) or the overexpression of negative immune modulators such as the ecto-5'-nucleotidase CD73 (Nguyen, A. M. et al.), the ligand of the programmed death 1 (PD-1) receptor PD-L1 (Jeong, H. et al.) or the “don’t eat me” signal CD47 (Candas-Green, D. et al.) have been associated with the absence or a partial tumor regression after radiotherapy.

[0006] Thus, the optimization of radiation therapy protocols and / or the identification of novel combinatorial strategies to sensitize cancer cells to death and / or to enhance the recognition of irradiated cancer cells by the immune effectors are urgently needed to improve cancer patient’s outcomes after radiotherapy. The present invention provides p21 -engineered monocytes, or of composition comprising them which can be used to address this need.

[0007] The inventors had demonstrated that the cyclin-dependent kinase inhibitor CDKN1A (p21) was shown to be a negative transcriptional repressor of the phagocytosis inhibitory receptor SIRPa is known to impair tumor phagocytosis through its interaction with CD47 (Allouch, A. et al., also in WO2021 / 013764). It was also observed that monocyte-derived macrophages overexpressing p21 after lentiviral transduction engulf CD47+ cancer cells and undergo proinflammatory activation. Interestingly, it was demonstrated that the adoptive transfer of p21 -engineered monocytes (Mos) into mouse models of human T-cell acute leukemia leads to the differentiation of p21-engineered Mos into phagocytosis-proficient tumor-associated macrophages (TAMs), which, after tumor cell engulfment undergo proinflammatory activation and support leukemia regression, thus revealing the potential of adoptive myeloid cell therapies using p21 -engineered Mos to act as an immunotherapy against liquid cancers.

[0008] However, no effect of p21 -engineered monocytes, or of composition comprising them, on solid tumors, in particular on sensitization of solid tumors to radiation therapy, let alone on cancers resistant to radiation therapy, had been demonstrated at the time.

[0009] Detailed description of the invention

[0010] The present invention stems from the demonstration, as supported in the example part, by the inventors, that adoptive transfer of p21 -engineered Mos significantly reduces the growth of established PDAC tumors and elongates the survival of infused mice. Importantly, their work also reveals that this novel adoptive myeloid cell therapy sensitizes PDAC tumors to radiotherapy. As a result, combinatorial therapy, combining the p21 -engineered monocytes and radiotherapy, strongly impairs the growth of irradiated PDAC tumors and leads to the survival of all treated mice. Mice cured by the combinatorial therapy rejected the rechallenge with PDAC tumors, demonstrating long-lasting antitumor immunity. These results thus evidence that combination treatment of adoptive transferred p21 -engineered Mos followed by radiotherapy represents a strategy that may be very effective to treat solid cancers.

[0011] Therefore, p21 -engineered monocytes or pharmaceutical compositions comprising them, may be useful in the treatment of solid cancers susceptible to be treated by radiation therapy, such as for instance cancers resistant to radiation therapy, where p21 -engineered monocytes may improve an otherwise sub-effective therapeutic response, or cancers sensitive to radiation therapy, where p21 -engineered monocytes may further enhance the therapeutic response in cancers which are responsive to radiation therapy.

[0012] The present invention therefore concerns cell compositions and their incorporation into pharmaceutical compositions for use in the treatment of solid cancers, more particularly, for use in the treatment of solid cancers by immunotherapy. In another embodiment, it is possible to have the circulating monocytes transfected in situ, resulting in p21 -engineered monocytes according to the invention. In other terms, the invention also pertains to a polynucleotide encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, for use in the treatment of solid cancers, more particularly, for use in the treatment of solid cancers by immunotherapy, preferably in combination with radiotherapy. The cells of the mononuclear phagocytic system comprise peripheral blood monocytes, their bone marrow or blood precursors and tissue macrophages. Monocytes are formed in the bone marrow, which they leave after maturation, passing from the peripheral blood to the tissues. Human monocytes circulating in the blood have a half-life of approximately 3 days. When it reaches the tissues, the monocyte is called a macrophage. The total number of tissue macrophages greatly exceeds the number of circulating monocytes, by a factor of approximately 400. Macrophages are found everywhere in the body, but are especially numerous in the liver (Kupffer cells), in the lymph nodes, in the lungs, in the peritoneum and in the skin (Langerhans' cells). The passage of monocytes from the general circulation to the tissues is irreversible.

[0013] Monocytes and macrophages are known to have numerous and important functions, including induction of immune responses in acute phases, regulation of haematopoiesis, activation of the immune system, coagulation, destruction of organisms and of tumour cells and tissue repair and cicatrisation.

[0014] Monocytes-macrophages can also be used in adoptive immunotherapy for the treatment of some types of cancer in man. Typically, these cells can be purified from the circulating blood of patients, cultured ex vivo and activated with interferon y to induce their differentiation and increase their tumoricidal power, then reinjected into the patients. It is also possible, using suitable vectors, to transfer genes ex vivo into monocyte-derived macrophage cells, thereby enabling them to be endowed with superior properties in terms of cytotoxicity and of stimulation of the immune system.

[0015] In a first aspect, the present invention therefore relates to a monocyte which overexpresses the cyclin-dependent kinase inhibitor p21 protein, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. Such monocyte once engrafted in the tissues will differentiate into macrophages. In some embodiments, it is possible to administer a recombinant nucleic acid in vivo in order to have the monocytes or their precursors transfected in situ. To this end, the monocyte may for instance be modified by the introduction, i.e. the transfection or transduction into the monocyte, of exogenous polynucleotide such as mRNA, DNA or cDNA encoding the protein p21 to be overexpressed. In particular embodiments, the monocyte which overexpresses p21 is transfected with a vector (plasmid or virus or mRNA) that targets monocytes, macrophages and / or precursor cells thereof in vivo resulting in overexpression of p21 in said cells. Thus, the present invention also relates to a vector (mRNA, DNA or cDNA) coding for cyclin- dependent kinase inhibitor (p21) protein that targets monocytes, macrophages and / or precursor cells for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. Preferably the mammal is a human.

[0016] The invention also pertains to a polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, for use in the treatment of solid cancers, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

[0017] In particular, the present invention concerns the use of a monocyte which overexpresses the cyclin-dependant kinase inhibitor p21 protein, for preparing a medicament intended to be used in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. The present invention also concerns the use of a vector (mRNA, DNA or cDNA) coding for cyclin- dependent kinase inhibitor (p21) protein that targets monocytes, macrophages and / or precursor cells for preparing a medicament intended to be used in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. In some embodiments, the mammal is administered the monocyte which overexpresses the p21 protein or the vector coding for p21 protein that targets monocytes, macrophages and / or precursor cells, prior to, during (concurrent with) or after radiation therapy.

[0018] The invention also pertains to the use of a polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, for preparing a medicament intended to be used in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

[0019] The vector may be a viral vector comprising a nucleic acid encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells. The vector may be a non-viral vector or plasmid comprising a nucleic acid encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells. The choice of the vector and of the regulatory sequences should be done by the skilled person keeping in mind that the final expression of p21 by the macrophages of the invention is preferably enhanced by at least two or three times as compared with mock-transfected control macrophages. Suitable regulatory sequences include a monocyte-specific promoter or enhancer or miRNA, macrophage-specific promoter or enhancer or miRNA, or myeloid-specific promoter or enhancer or miRNA. The vector may be an mRNA encoding p21 protein, which can be delivered to target monocytes, macrophages and / or precursor cells (i.e., a monocyte-specific promoter, macrophagespecific promoter, or myeloid-specific promoter). For example the mRNA may be delivered by nanoparticles, liposomes, or an LNP linked to a targeting moiety that targets monocytes, macrophages and / or precursor cells. Examples include lipid-based nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, lipid nanoparticles (LNPs) and lipoplexes (LPX), polymer-based polyplexes, lipid shell coated lipopolyplexes (LPPs), cationic lipid-assisted nanoparticles (CLANs), inorganic nanoparticles, cationic nanoemulsions. Suitable targeting moieties are known in the art and include antibody fragments, receptor-binding ligand fragments, peptides, or glycans.

[0020] In a further aspect, the present invention also relates to a pharmaceutical composition comprising said monocyte, and a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. The present invention also relates to a pharmaceutical composition comprising a vector coding for the cyclin-dependent kinase inhibitor p21 protein, that preferably targets monocytes, macrophages and / or precursor cells, and optionally a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. In some embodiments, the mammal is administered the monocyte which overexpresses the p21 protein or the vector coding for p21 protein that targets monocytes, macrophages and / or precursor cells, prior to, during (concurrent with) or after radiation therapy.

[0021] The present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, and a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. The present invention also concerns the use of a monocyte which overexpresses the cyclin-dependant kinase inhibitor p21 protein, or of a pharmaceutical composition comprising said monocyte and a pharmaceutically acceptable excipient, in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

[0022] The present invention also concerns the use of a polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, or of a pharmaceutical composition comprising said polynucleotide and a pharmaceutically acceptable excipient, in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

[0023] The present invention also concerns a method for treating a mammal suffering from a solid cancer preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, wherein the mammal is administered a monocyte which overexpresses the cyclin-dependant kinase inhibitor p21 protein, or a pharmaceutical composition comprising said monocyte and a pharmaceutically acceptable excipient. The present invention also concerns a method for treating a mammal suffering from a solid cancer preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, wherein the mammal is administered a vector coding for the cyclin- dependent kinase inhibitor p21 protein, that preferably targets monocytes, macrophages and / or precursor cells, or a pharmaceutical composition comprising said vector coding for the cyclin-dependent kinase inhibitor p21 protein, that preferably targets monocytes, macrophages and / or precursor cells, and a pharmaceutically acceptable excipient. In some embodiments, the mammal is administered the monocyte which overexpresses the p21 protein or the vector coding for p21 protein that targets monocytes, macrophages and / or precursor cells, prior to, during (concurrent with) or after radiation therapy.

[0024] The present invention also concerns a method for treating a mammal suffering from a solid cancer preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, wherein the mammal is administered a polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, or a pharmaceutical composition comprising said polynucleotide and a pharmaceutically acceptable excipient. Preferably, the monocyte is modified to overexpress the cyclin-dependent kinase inhibitor p21 protein, preferably it is modified in that it comprises exogenous polynucleotide such as mRNA, DNA or cDNA encoding the cyclin-dependent kinase inhibitor p21 protein.

[0025] In a preferred embodiment the monocyte is genetically modified, more preferably the monocyte is genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein.

[0026] Thus in a preferred embodiment, the present invention relates to a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. In some embodiments, the monocyte is administered, or the monocyte is genetically modified in situ by administering the vector, prior to, during (concurrent with) or after radiation therapy.

[0027] In another preferred embodiment, the present invention also relates to a pharmaceutical composition comprising a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein, and a pharmaceutically acceptable excipient, or comprising a vector coding for the cyclin-dependent kinase inhibitor p21 protein, that preferably targets monocytes, macrophages and / or precursor cells, and a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. This pharmaceutical composition is hereafter also referred to as the “composition of the invention”, the “pharmaceutical composition of the invention”, or the “cell composition of the invention”.

[0028] In a preferred embodiment of the invention, the solid cancer is a cancer overexpressing at least CD47.

[0029] In the context of the invention, the terms “solid cancer resistant to radiation therapy” or “solid tumor resistant to radiation therapy” refer to solid tumors which are known as or can be identified as being resistant to radiation therapy, that is to say as tumors which are known as or can be identified as having a therapeutic response below average to radiation therapy.

[0030] In the context of the invention, a therapeutic response is considered as below average when it is significantly lower than the mean therapeutic effect obtained in cancers or tumors of reference, when treated with the same treatment. The person skilled in the art may thus identify on this basis whether a specific solid cancer or tumor is resistant to radiation therapy.

[0031] Some cancers are known in the art as being resistant to radiation therapy.

[0032] For instance, a first category are cancers or tumors known as being resistant to radiation therapy are cancers or tumors which have demonstrated resistance to radiation therapy based on clinical criteria. More specifically solid tumors or solid cancers are generally considered in the art as “resistant to radiation therapy” if recurrences, also called local relapses, are observed within six months following the first course of radiation therapy (Hutchinson, MK.N.D. et al.). In other terms, solid tumors which have been treated with a first course of radiation therapy and have relapsed within six months following the end of said first course are generally considered in the art as meeting the criteria for “solid tumor resistant to radiation therapy”.

[0033] A second category are cancers or tumors known as being resistant to radiation therapy, based on biological criteria. More specifically solid tumors or solid cancers are also generally considered in the art as “resistant to radiation therapy” if they can be associated with biological markers of intrinsic or acquired resistance mechanisms involving the DNA damage response, tumor aneuploidy, the anti-apoptotic proteins such as BCL-2 and BCL- XL, the alteration of glucose metabolism pathway, hypoxia or the overexpression of negative immune modulators such as the ecto-5'-nucleotidase CD73, PD-L1 or CD47.

[0034] A solid tumor or solid cancer can be considered as associated with biological markers of intrinsic or acquired resistance mechanisms involving a protective DNA damage response for instance when the Mre11-Rad50-Nbs1 complex (encoded respectively by the genes Gene ID: 4361 , 10111 , 821254) or the protein TRIP13 (encoded by the gene: Gene ID: 9319), or the protein Ku80 (encoded by the gene: Gene ID: 7520), is overexpressed in the tumor or in the biological tissues surrounding it, compared with healthy tissues of similar embryonic origin. Such cancer or tumor may be designated as overexpressing the Mre11- Rad50-Nbs1 complex, TRIP13, or Ku80.

[0035] A solid tumor or solid cancer can be considered as associated with biological markers of tumor aneuploidy for instance when several cells in the tumors contain an abnormal number of chromosomes, as measured by the karyotype. Such cancer or tumor may be designated as aneuploid. A solid tumor or solid cancer can be considered as associated with resistance mechanisms that imply BCL-2 and / or BCL-XL when BCL-2 (encoded by the gene: Gene ID: 596), and / or BCL-XL (encoded by the gene: Gene ID: 598), is overexpressed in the tumor or in the biological tissues surrounding it. Such cancer or tumor may be designated as overexpressing BCL-2 and / or BCL-XL.

[0036] A solid tumor or solid cancer can be considered as associated with biological markers of the alteration of glucose metabolism pathway, such as AKT-mediated enhanced aerobic glycolysis, when the protein AKT (encoded by the gene: Gene ID: 5207), is overexpressed in the tumor or in the biological tissues surrounding it. Such cancer or tumor may be designated as overexpressing AKT.

[0037] A solid tumor or solid cancer can be considered as associated with biological markers of hypoxia, such as the HIF subunit genes, when the protein HIF-1a and / or HIF-2a (encoded respectively by the genes: Gene ID: 3091 and 2034), is overexpressed in the tumor or in the biological tissues surrounding it. Such cancer or tumor may be designated as overexpressing HIF-1a and / or HIF-2a.

[0038] A solid tumor or solid cancer can be considered as associated with overexpression of negative immune modulators, for instance when CD73, PD-L1 or CD47 (encoded respectively by the genes: Gene ID: 4907, 100196322 and 961), is overexpressed in the tumor or in the biological tissues surrounding it. Such cancer or tumor may be designated as overexpressing CD73, PD-L1 or CD47.

[0039] In a preferred embodiment, the solid cancer resistant to radiation therapy is a cancer overexpressing a biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, PD-L1 , CD47, and any combination thereof.

[0040] In a yet preferred embodiment, the solid cancer resistant to radiation therapy is a cancer overexpressing CD47, and optionally one or more biological selected from the Mre11- Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, and PD-L1.

[0041] Some types of cancers are generally known in the art as more prone to developing or presenting with resistance to radiation therapy, for instance pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), head and neck cancer, preferably head and neck squamous cell carcinoma, breast cancer, in particular triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer , and glioblastoma.

[0042] In a preferred embodiment of the invention, the solid cancer, in particular the solid cancer resistant to radiation therapy, is thus selected from the list consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), head and neck cancer, preferably head and neck squamous cell carcinoma, breast cancer, in particular triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer, and glioblastoma.

[0043] Such cancers may present with one or more of the biological markers defined above, that is to say the biological markers of intrinsic or acquired resistance mechanisms involving the DNA damage response, tumor aneuploidy, the anti-apoptotic proteins such as BCL-2 and BCL-XL, the alteration of glucose metabolism pathway, hypoxia or the overexpression of negative immune modulators such as the ecto-5'-nucleotidase CD73, PD-L1 or CD47.

[0044] In a preferred embodiment, the solid cancer, in particular the solid cancer resistant to radiation therapy, is selected from the list consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), head and neck cancer, preferably head and neck squamous cell carcinoma, breast cancer, in particular triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer, and glioblastoma; and is a cancer overexpressing a biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, PD-L1 , CD47, and any combination thereof; preferably CD47 and optionally one or more biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, and PD-L1.

[0045] In a preferred embodiment, the solid cancer, in particular the solid cancer resistant to radiation therapy, is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC), and is a cancer overexpressing a biological marker selected from the Mre11- Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, PD- L1 , CD47, and any combination thereof; preferably CD47 and optionally one or more biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, and PD-L1.

[0046] In a preferred embodiment, the solid cancer, in particular the solid cancer resistant to radiation therapy, is selected from the list consisting of pancreatic cancer, in particular pancreatic ductal adenocarcinoma (PDAC), head and neck cancer, preferably head and neck squamous cell carcinoma, breast cancer, in particular triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer, and glioblastoma and is a cancer overexpressing CD47, and optionally one or more biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, and PD-L1.

[0047] In a preferred embodiment, the solid cancer, in particular the solid cancer resistant to radiation therapy, is pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC), and is a cancer overexpressing CD47, and optionally one or more biological marker selected from the Mre11-Rad50-Nbs1 complex, TRIP13, Ku80, BCL-2, BCL-XL, AKT, HIF-1a, HIF-2a, CD73, and PD-L1.

[0048] In the context of the invention, the terms “solid cancer sensitive to radiation therapy” or “solid tumor sensitive to radiation therapy” refer to solid tumors which can be identified as being sensitive to radiation therapy, that is to say as having a therapeutic response above average to radiation therapy.

[0049] In the context of the invention, a therapeutic response is considered as above average when it is significantly higher than the mean therapeutic effect obtained in tumors or cancers of reference, when treated with the same treatment. In the context of the invention, such tumors or cancers of reference may be cancers of the same type, arising in the same type of biological tissue, and / or of the same stage. The person skilled in the art may thus identify on this basis whether a specific solid cancer or tumor is sensitive to radiation therapy.

[0050] Some cancers are known in the art as being sensitive to radiation therapy, such as for instance seminomas, medulloblastoma, neuroblastoma, Wilm’s tumor, early cervical carcinoma, vaginal carcinoma, for which radiation therapy results in a positive response in most patients. Thus preferably the solid cancer is chosen from the list consisting of seminomas, medulloblastoma, neuroblastoma, Wilm’s tumor, early cervical carcinoma, and vaginal carcinoma.

[0051] Preferably, the polynucleotide encoding protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, the monocyte or the pharmaceutical composition comprising a monocyte as defined herein are for improving the prognosis or enhancing the chance of regress of the solid cancer, preferably when administered previous to radiation therapy. Preferably, the polynucleotide encoding protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, the monocyte or the pharmaceutical composition comprising a monocyte as defined herein are for treating the solid cancer or for ameliorating tumor regress of the solid cancer, preferably when administered previous to radiation therapy.

[0052] In the context of the invention, the term "prognosis” should be construed as generally understood in the art, that is to say as the likelihood projection or prediction of the disease outcome (including overall survival “OS” and progression-free survival “PFS”). In the context of the invention, the terms "improving the prognosis” should be construed as generally understood in the art, that is to say as improving the projected or predicted disease outcome (including overall survival “OS” and progression-free survival “PFS”).

[0053] The terms “enhancing the chance of regress of the solid cancer” or “ameliorating tumor regress” should be construed as generally understood in the art, that is to say as indicating an improvement in the chances of cancer regression, wherein the improvement may concern the degree or rate of cancer regression, for instance as measured by imagery or histopathological evaluation, or the speed of cancer regression, for instance measured as median time to full regression. The enhancement or amelioration is to be understood as a comparison with a similar medical case wherein the treatment as claimed herein would not be administered to the mammal.

[0054] Preferably, the polynucleotide encoding protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, the monocyte or the pharmaceutical composition comprising a monocyte as defined herein are for preventing, limiting, or delaying the risk of metastasis and / or cancer recurrence, and / or as antitumoral vaccination, and / or for stimulating immunological memory.

[0055] As indicated above, the invention relies on a monocyte which overexpresses the cyclin- dependant kinase inhibitor p21 protein, or a pharmaceutical composition comprising said monocyte and a pharmaceutically acceptable excipient.

[0056] As used herein, the term “p21 protein” designates interchangeably the cyclin-dependent kinase inhibitor 1 which is also known as “p21CiP1”, “p21Waf1”, “Waf1”, “CDKN1A”, “CAP20”, “CIP1”, “MDA-6”, “SDI1” and “CDK-interacting protein 1”. This protein binds to and inhibits the activity of the cyclin-CDK1 , CDK2 and CDK4 / 6 complexes and thus functions as a regulator of cell cycle progression at G1 and S phase. The binding of p21 to CDK complexes occurs through p21’s N-terminal domain, which is homologous to the other CIP / CDK inhibitors p27 and p57. As a major target of p53 activity, it is usually associated with linking DNA damage to cell cycle arrest. This protein is encoded by the CDKN1A gene of SEQ ID NO:1 (NM_078467) located on the chromosome 6 (6p21.2) in humans. In mice, the protein is encoded by the CDKN1A gene of SEQ ID NO:3 (NM_007669). The human p21 protein has the amino acid sequence of SEQ ID NO:2 (NP_000380), whereas the mouse p21 protein has the amino acid sequence of SEQ ID NO:4 (NP_031695).

[0057] The term “p21 protein” herein also encompass functional variants and / or fragments of the above-mentioned p21 proteins.

[0058] “Functional variants” are for example the wild-type p21 proteins of animal species other than human or mouse (e.g., from horse, dog, cats, or cattle animals). These proteins are now well-characterized and their sequence can be easily retrieved from conventional data bases. “Functional variants” are also mutated version of the natural p21 proteins, whose amino acid sequence share a percentage of identity of at least 75%, preferably of at least 80%, more preferably of at least 90% with the wild-type protein of the corresponding species (for a human therapy, with SEQ ID NO:2, for a mouse therapy, with SEQ ID NO:4, etc).

[0059] In the context of the invention, the identity percentage between said two homologous sequences is identified by a global alignment of the sequences in their entirety, this alignment being performed by means of an algorithm that is well known by the skilled person, such as the one disclosed in Needleman and Wunsch (1970). Accordingly, sequence comparisons between two amino acid sequences or two nucleotide sequences can be performed for example by using any software known by the skilled person, such as the “needle” software using the “Gap open” parameter of 10, the “Gap extend” parameter of 0.5 and the “Blosum 62” matrix.

[0060] “Functional fragments” of the p21 protein are any fragment of the wild-type p21 protein or of functional variants thereof, that retains the function of p21 protein to enhance the programmed cell removal (PrCR) of tumor cells by macrophages.

[0061] By “overexpressing the cyclin-dependant kinase inhibitor p21 protein”, it is herein meant that the overall expression level of the p21 protein is higher in the monocyte, and / or the macrophages contained in the composition of the invention than in conventional non-treated monocytes and / or macrophages. This overexpression can be detected by any conventional means enabling the measurement of protein levels, such as western blot. To be used in the composition of the invention, the monocytes and / or macrophages are genetically modified so that the final expression of p21 is preferably at least two or three times higher than in untreated control macrophages. The stable integration of p21 gene in the monocyte genome ensures the durability of the expression the cyclin-dependant kinase inhibitor p21 protein in the differentiated macrophages engrafted in the tissues. Besides considering the long longevity of macrophages in the tissues, the durability of p21 protein expression is further ensured.

[0062] Preferably, the monocyte is modified to overexpress the cyclin-dependent kinase inhibitor p21 protein, yet preferably the monocyte is genetically modified to overexpress the cyclin- dependent kinase inhibitor p21 protein.

[0063] In the context of the invention, the terms “is modified to overexpress the cyclin-dependent kinase inhibitor p21 protein" refer to any modification made to the monocyte using biotechnological tools, and leading to transient or stable overexpression of the cyclin- dependent kinase inhibitor p21 protein. The engineering of the monocytes can for instance be performed by the use of the non-viral transfer of in vitro transcribed cyclin-dependent kinase inhibitor p21 mRNA in the monocytes.

[0064] Thus, in a preferred embodiment the monocyte of the invention comprises exogenous polynucleotide such as mRNA, DNA or cDNA encoding the cyclin-dependent kinase inhibitor p21 protein, as defined above. In the context of the invention, the term exogenous should be construed as referring to a polynucleotide, preferably mRNA, DNA or cDNA, which is from a different source than the cell, that is to say the monocyte, it is introduced into. In the context of the invention, the term exogenous does not imply a difference in the organism of origin of the polynucleotide from that of the monocyte. Preferably, the exogenous polynucleotide is synthetic polynucleotide, that is to say it is a polynucleotide from a synthetic source, for example produced by genetic engineering. In this context, the sequence of the mRNA encoding the cyclin-dependent kinase inhibitor p21 protein may be optimized for the purpose of improving its stability and translatability, by methods and means known in the art such as for example the modification of rare codons (“also known as codon-optimization”) with synonymous frequently occurring codons, for increasing expression levels, or the modification of the 5’ mRNA cap with 5'-phosphorothiolate dinucleotide cap analogues, for inhibiting RNA decapping and improving resistance to enzymatic degradation, or the incorporation of base modified nucleosides to amplify the translation of mRNA in the cell (such as reviewed by Bornewasser L et al.). To this end, the monocyte may for instance be modified by the introduction, i.e. the transfection or transduction into the monocyte, of exogenous polynucleotide such as mRNA, DNA or cDNA encoding the protein p21 to be overexpressed. For example, the monocyte may be modified using mRNA-based cell engineering, wherein mRNA encoding for the cyclin-dependent kinase inhibitor p21 protein, such as in vitro transcribed cyclin-dependent kinase inhibitor p21 mRNA, is directly transferred into the monocyte using known intracellular mRNA delivery methods such as electroporation, lipid nanoparticles (LNP), cell penetrating peptides, zwitterionic amino lipids (ZALs, such as disclosed by Miller J.B. et al.), polyplexes and polymeric micelles.

[0065] LNP formulations are typically composed of (1) an ionizable or cationic lipid or polymeric material, bearing tertiary or quaternary amines to encapsulate the mRNA; (2) a zwitterionic lipid that resembles the lipids in the cell membrane (such as for instance 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine [DOPE]); (3) cholesterol to stabilize the lipid bilayer of the LNP; and (4) a polyethylene glycol (PEG)-lipid. Use of LNP to introduce in vitro transcribed mRNA with such technique was described for instance by Billingsley MM, et al..

[0066] Polyplexes are particles, usually spherical, made up from polymer molecules engaging in electrostatic interactions with negatively charged nucleic acids. They can be made of poly(ethyleneimine) (PEI), poly(amino acids) (such as P(Lys) or P(Asp)), polyesters (such as poly(lactic-co-glycolic acid), poly( / ?-amino ester)s or poly(amine-co-ester)), natural polymers (chitosan, Protamine) or poly(amidoamine) (PAMAM).

[0067] In the context of the invention, the terms “is genetically modified to overexpress the cyclin- dependent kinase inhibitor p21 protein" refer more specifically to modifications made to the monocyte as defined above, and based on recombinant DNA technology.

[0068] In a preferred embodiment, the monocytes comprised in the pharmaceutical composition of the invention contain a replication defective recombinant virus encoding the cyclin- dependent kinase inhibitor p21 or a non-viral recombinant nucleic acid containing the gene encoding p21 placed under the control of regulatory elements permitting its expression. This recombinant virus or nucleic acid makes it possible to overexpress the cyclin-dependant kinase inhibitor p21 protein. Said recombinant nucleic acid is preferably a DNA plasmid. We can also use the non-viral Sleeping Beauty stable transposition of p21 gene from supercoiled minimal DNA vectors called minicircles. The use of a vector (plasmid or virus or in vitro translated mRNA) makes it possible to improve the administration of the nucleic acid encoding p21 in the target cells, and also to increase the stability of said nucleic acid into said cells, thereby enabling a long-lasting effect to be obtained.

[0069] In a preferred embodiment, said vector is chosen in the group consisting of: adenovirus, adeno-associated virus (AAV), herpesvirus, lentivirus, vaccinia virus, cytomegalovirus (CMV) and the like, that have been shown to effectively transfect macrophages (Singh G. et al, F1000 research 2015).

[0070] In a preferred embodiment, the vector is a virus, optionally adenovirus, adeno-associated virus (AAV), herpesvirus, lentivirus, vaccinia virus, cytomegalovirus (CMV); plasmid; or mRNA, optionally delivered via nanoparticle, liposome or LNP. In a preferred embodiment, the vector is a viral or non-viral vector comprising a nucleic acid operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells thereof in vivo, optionally a monocyte-specific promoter or enhancer or miRNA, macrophagespecific promoter or enhancer or miRNA, or myeloid-specific promoter or enhancer or miRNA. Yet preferably, the mRNA is delivered via nanoparticle, liposome or LNP linked to a targeting moiety that targets monocytes, macrophages and / or precursor cells thereof in vivo.

[0071] Advantageously, said virus is a defective virus. The term “defective virus” denotes a virus incapable of replicating in the target cell. Generally, the genome of the defective viruses used in the context of the present invention hence lacks at least the sequences needed for the replication of the said virus in the infected cell. These regions may be either removed (wholly or partially), or rendered non-functional, or replaced by other sequences, in particular by the recombinant nucleic acid. Preferably, the defective virus nevertheless retains the sequences of its genome which are needed for encapsulation of the viral particle.

[0072] In particular, the AAV vector display several advantages such as i) a long-lasting expression of synthesized genes, ii) a low risk for pathogenic reactions (because they are artificially manufactured and not toxic), iii) they trigger low immunogenic response and iv) they do not integrate the human genome. In order to increase the efficacy of gene expression, and prevent the unintended spread of the virus, genetic modifications of AAV can be performed. These genetic modifications include the deletion of the E1 region, deletion of the E1 region along with deletion of either the E2 or E4 region, or deletion of the entire adenovirus genome except the cis-acting inverted terminal repeats and a packaging signal. Such vectors are advantageously encompassed by the present invention. We can also use the non-viral Sleeping Beauty stable transposition of p21 gene from supercoiled minimal DNA vectors called minicircles. The genetic engineering of the monocytes could also be performed by the use of the non-viral transfer of in vitro translated p21 mRNA in the monocytes. The plasmids in these two methods are delivered by transfection of monocytes through electroporation.

[0073] Another advantageous vector for the preparation of the cell compositions according to the invention is an adenoviral vector. Indeed, Haddada H. et al. Biochem. Biophys. Res. Commun (1993) showed that adenoviruses are capable of very effectively infecting cells of the monocyte-macrophage line, of being maintained stably therein and of expressing a therapeutic gene. Different serotypes of adenovirus exist, the structure and properties of which vary somewhat but which are not pathogenic for man, and in particular for nonimmunosuppressed subjects. Moreover, these viruses do not integrate in the genome of the cells they infect, and can incorporate large fragments of exogenous DNA. Among the different serotypes, it is preferable in the context of the present invention to use adenoviruses type 2 or 5 (Ad 2 or Ad 5). In the case of Ad 5 adenoviruses, the sequences needed for replication are the E1A and E1 B regions. These sequences are preferable deleted from the recombinant nucleic acid used in the present invention.

[0074] Another advantageous vector for the preparation of the cell compositions according to the invention is a lentivirus. Lentiviruses like HIV have the capacity to infect non-dividing and dividing cells and to integrate into the host cell genome. Due to these characteristics, HIVbased lentiviral vectors have been proposed as good delivery system candidates for gene therapy, but the attempt to use them in clinical trials has raised concerns about their safety including the risk of genetic recombination leading to the generation of replication- competent retrovirus in humans. Further modifications in the packaging and genetic components of viral genes have been carried out to develop safer HIV-based lentiviral vector systems. Today, a number of safe HIV-based lentiviral vectors have been designed for efficiently transducing target genes into differentiated monocyte-derived macrophages (Leyva F. et al, BMC biotechnology (2011). Any of these vectors can be used in the context of the present invention.

[0075] Preferred lentiviral vectors are those that have been modified so as to be safely administered into mammals. These vectors are for example the HIV I SIV vectors known to be useful in human or mammal gene therapy, as disclosed in Neschadim A. et al. Biol Blood Marrow Transplant. 2007 Dec;13(12):1407-16. The most interesting vectors to use are the HIV and SIV based lentiviral Self Inactivating vectors (Neschadim A. et al. Biol Blood Marrow Transplant. 2007 Dec;13(12):1407-16.), the adenoviral vectors (Haddada H. et al. Biochem. Biophys. Res. Commun (1993)) and the sleeping Beauty transposon non- viral vectors (Aronovich et al. Human. Molecular. Genetics (2011)).

[0076] The vector used in the example i.e., the HIV-1 based Self inactivated (SIN) lentiviral vector encoding the p21 protein, can be used. This vector can encode the p21 protein alone or fused with another protein such as AIP (aryl hydrocarbon receptor interacting protein) or fused to small protein tags such Flag tag or hemagglutinin (HA) tag.

[0077] In a particularly preferred embodiment, the composition of the invention also contains Viral Like Particles (VLPs) containing SlVmac-VPX to induce the degradation of factors that impair the lentiviral infection (Berger G., Gene Therapy (2009)). The SlVmac-VPX degrades SAMHD1 , which was identified as an HIV-1 restriction factor that hydrolyzes dNTPs required for retroviral replication (Lahouassa et al., Nat Immunol (2012)).

[0078] In an even more particularly preferred embodiment, the monocytes contained in the cell composition of the invention have been transduced by a SIN lentiviral vector containing the nucleic acid sequence SEQ ID NO:5 encoding for p21 protein:

[0079] As stated above, the p21 encoding gene is placed under the control of regulatory elements permitting its expression. These regulatory elements generally consist of transcription promoter sequences. These can be sequences which are naturally responsible for the expression of p21 , when these sequences are capable of functioning in monocytes- macrophages. They can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic genes). In particular, they can be promoter sequences of eukaryotic or viral genes. For example, they can be promoter sequences originating from the genome of the monocyte which it is desired to infect. Similarly, they can be promoter sequences originating from the genome of a virus. In this connection, the promoters E2F1 (E2 promoter binding factor 1) or the promoters of EFS (elongation factor 1a short), SFFV (silencing-prone spleen focus forming virus), CMV (cytomegalovirus), RSV (Rous sarcoma virus), and the like, genes may be mentioned for example. In addition, these expression sequences may be modified by the addition of activator sequences, regulatory sequences, and the like.

[0080] The choice of the vector and of the regulatory sequences should be done by the skilled person keeping in mind that the final expression of p21 by the macrophages of the invention should be enhanced by at least two or three times as compared with mock-transfected control macrophages.

[0081] Methods to construct expression vectors containing coding sequences and appropriate transcriptional / translational control signals are well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. The nucleic acids may be isolated and obtained in substantial purity, then introduced into suitable host cells using a variety of techniques available in the art.

[0082] All the techniques of construction of vectors derived from adenoviruses, lentiviruses, or from AAV, and incorporation of heterologous nucleic acid sequences in same, have been described in the literature and can be used in the context of the present invention. The methods traditionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenolchloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in a saline medium, transformation in Escherichia coli, and the like, are well known to a person skilled in the art and are amply described in the literature [Maniatis T. et al., “Molecular Cloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, 1987],

[0083] Once the genome of the viruses has been genetically modified, the viruses are multiplied and recovered and purified according to standard techniques of molecular biology.

[0084] The pharmaceutical composition of the invention contains monocytes that overexpress the p21 protein.

[0085] As used herein, the term “monocyte-derived macrophages” designates mononuclear cells that have been cultivated and differentiated into macrophages from peripheral blood monocytes (PBMCs), or from their bone marrow or blood precursors, under the conditions detailed in the examples (see also Andressen R. et al., Cancer Res. 1990; Bartholeyns J et al., Anticancer Res. (1991)). As precursors, it is also possible to use pluripotent stem cells, myeloid stem cells (CFU-GEMM), myelomonocytic stem cells (CFU-GM), CFU-M, monoblasts or promonocytes. The intravenous injections of monocytes induced their engraftment and their differentiation into macrophages in the bone marrow, in the spleen and in the liver.

[0086] Since PBMCs are found in the blood, a PBMCs sample can be obtained by a completely harmless and non-invasive blood collection from the subject.

[0087] Withdrawal and isolation of PBMCs I monocyte-macrophage cells or their precursors may be performed by any technique known to a person skilled in the art. These different techniques can involve physical separation steps (centrifugation, cell sorting (FACS), and the like), and selection with immunological compounds (specific antibodies for cell markers and the like) or biochemical compounds (membrane receptor ligands), and the like. Cultivating the isolated cells may be performed in different media known to a person skilled in the art (for example RPMI, IMDM), supplemented, inter alia, with serum and amino acids. The culture of the cells is carried out under sterile conditions, preferably at 37° C, as illustrated in the examples. It may be performed in culture plates, or preferably in teflon bags.

[0088] In a preferred embodiment, the cells contained in the composition of the invention have been obtained by cultivating PBMCs under suitable conditions permitting their differentiation. Human blood monocyte differentiation into macrophages can for example be induced in vitro using three different methods, namely by culturing PBMCs in 1) human serum (HS), 2) fetal bovine serum (FBS) with granulocyte-macrophage colony-stimulating factor (GM-CSF) or 3) FBS with macrophage colony-stimulating factor (M-CSF).

[0089] In a particular embodiment, the transduction of the purified monocytes from PBMC cells with the recombinant nucleic acids is performed ex vivo, then the transduced cells are differentiated I cultured into macrophages ex vivo. In this embodiment, the cell composition of the invention contains differentiated macrophages overexpressing the p21 protein.

[0090] In another particular embodiment, the recombinant nucleic acids of the invention have been transduced in the cells once they have been differentiated I cultured into macrophages ex vivo. In this embodiment, the cell composition of the invention also contains differentiated macrophages overexpressing the p21 protein, but the differentiation of the cells into macrophages has been performed prior to the transduction with the recombinant nucleic acids of the invention.

[0091] In another particular embodiment, the recombinant nucleic acids of the invention have been transduced in the cells at their monocytic stage, ex vivo. Then the cells are administered to the subject where their differentiation into macrophages occurs. In this embodiment, the cell composition of the invention contains undifferentiated monocytes overexpressing the p21 protein.

[0092] In another particular embodiment, it is possible to administer the recombinant nucleic acid in vivo in order to have the circulating monocytes transfected in situ.

[0093] As disclosed herein, the terms “in vitro" and “ex vivo" are equivalent and refer to studies or experiments that are performed using biological components (e.g. cells or population of cells) that have been isolated from their usual host organisms (e.g. animals or humans). In contrast, the terms “in vivo” or “in situ” refer to studies that are conducted on whole living organisms (e.g., humans), after administration of the composition of the invention in a living subject.

[0094] In another embodiment, the cell composition of the invention contains purified monocytes from PBMCs that have been transformed ex vivo, but have not been differentiated ex vivo into macrophages. Their differentiation will occur in vivo, in the host. In this case, the compositions of the invention possess more than 80% of monocytes or, more preferably more than 90% of monocytes, still more preferably more than 99% of monocytes. This means that the cell composition of the invention contains very few other cells, if any.

[0095] The monocytes purified from PBMCs contained within the cell composition of the invention are monocyte cells that have been recovered from the peripheral blood of an individual by conventional means. These monocytes purified from PBMCs are positive for the markers: CD14, CD11b and CD16 but negative for the markers CD56 (which is a marker of NK cells), CD3 (marker of T cells) and CD20 (marker of B cells). Preferably, the cell composition of the invention contains more than 90%, preferably more than 95% and ideally more than 99% of such cells. The presence of these markers can be assessed by any conventional means, e.g., by cytometry (FACS).

[0096] Macrophages differentiated in the tissues after the intravenous injections of the cell composition of the invention are monocyte-derived cells that are positive for the markers: CD14, CD11b, CD71 , CD163 and CD206, but negative for the markers CD56 (which is a marker of NK cells), CD3 (marker of T cells) and CD20 (marker of B cells). Preferably, the cell composition of the invention contains more than 90%, preferably more than 95% and ideally more than 99% of such cells. The presence of these markers can be assessed by any conventional means, e.g., by cytometry (FACS). The transformation of the cells to be included in the composition of the invention with the recombinant nucleic acid of the invention is to be performed in a sterile medium, under conditions adjusted by a person skilled in the art. Notably, the multiplicity of infection has to be adjusted in accordance with the vector used. One example with a SIV virus is given in the examples below.

[0097] When a lentiviral vector is used, the cells to be included in the composition of the invention are for example contacted with 50 to 250 pfu per cell of purified virus, and more preferably 50 to 100 pfu / cell. Depending on the transformation conditions, the percentage of cells modified by insertion of the recombinant nucleic acid can vary from 30 to 95%.

[0098] The modified cells thereby obtained may then be packaged for the purpose of immediate use, and / or stored for the purpose of subsequent use. For an immediate readministration, the cells are generally suspended in a phosphate buffer or in physiological saline at a concentration varying from 30x106to 109cells per dose. For their storage, the cells may be frozen, preferably in the presence of preservatives such as glycerol, DMSO, and the like.

[0099] The cells transformed ex vivo according to the invention, especially by using the recombinant viral vector disclosed above, are a tool of choice for the preparation of a pharmaceutical composition, in particular of a composition intended to strengthen a patient's immune and haematopoietic system.

[0100] The cells of the composition of the invention can originate from the patient himself (the composition therefore contains autologous cells) or from a donor (the composition therefore contains allogeneic cells). For allogeneic cells, HLA compatibility and matching between the donor and the patient receiving the cells is required.

[0101] The pharmaceutical composition contains, as active principle, the transformed cells or the polynucleotide or the vector, optionally for in vivo cell transformation,, as described above. It moreover contains a pharmaceutically acceptable excipient.

[0102] The term “pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Compositions for the treatment of cancer can usually be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means. A typical route of administration is intravenous or intratumoral, although other routes can be equally effective.

[0103] For intravenous administration, the composition of the invention will be under liquid form. It will thus contain, apart from the cells, a pharmaceutically-acceptable diluent that does not affect the biological activity of the cells of the invention. Example of such diluents are physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0104] In a preferred embodiment, the composition of the invention is under a liquid form.

[0105] The polynucleotide, the vector, the monocyte or the pharmaceutical compositions for use according to the invention can be administered alone or combined with another pharmaceutical composition or another active principle. In particular, the pharmaceutical compositions of the invention can contain the polynucleotide, the vector, the monocyte or the pharmaceutical compositions for use according to the invention as well as another active principle, combined in the same container.

[0106] This active principle can be for example a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol™), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate.

[0107] As used herein, the term “combined” does not imply that the cells of the invention and the other active principle are necessarily administered simultaneously. It also extends to any use or presentation involving their administration at different time intervals, or in separate containers.

[0108] In another embodiment, the polynucleotide the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with a chemotherapeutic agent for example as defined above. In another embodiment, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with (or contains) an effective dose of an agent that increases patient haematocrit, for example erythropoietin stimulating agents (ESA). Such agents are known and used in the art, including, for example, Aranesp® (darbepoetin alfa), Epogen®NF / Procrit®NF (epoetin alfa), Omontys® (peginesatide), Procrit®, etc.

[0109] Other combination therapies include administration with cell-specific antibodies, for example antibodies selective for tumor cell markers, radiation therapy, surgery, and / or hormone deprivation.

[0110] Therefore, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with (or contains) an effective dose of said cell-specific antibodies.

[0111] A number of antibodies are currently in clinical use for the treatment of cancer, and others are in varying stages of clinical development. For example, there are a number of antigens and corresponding monoclonal antibodies for the treatment of B cell malignancies. One target antigen is CD20. Rituximab is a chimeric unconjugated monoclonal antibody directed at the CD20 antigen. CD20 has an important functional role in B cell activation, proliferation, and differentiation. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for treatment of chronic lymphocytic leukemia. CD22 is targeted by a number of antibodies, and has recently demonstrated efficacy combined with toxin in chemotherapy-resistant hairy cell leukemia. Two new monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes. Alemtuzumab (Campath) is used in the treatment of chronic lymphocytic leukemia; Gemtuzumab (Mylotarg) finds use in the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin) finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) finds use in the treatment of colon cancer.

[0112] Angiogenesis inhibitors can also be combined with (or contained in) the compositions of the invention.

[0113] In another embodiment, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with (or contains) an effective dose of an immune checkpoint modulator, in particular of an immune checkpoint inhibitor (ICI). “Immune checkpoint inhibitors” (ICI) include anti-PD1 antibodies (such as Nivolumab or Pembrolizumab or Pidilizumab), anti-PD-L1 antibodies (such as Atezolizumab or Durvalumab), anti-CTLA-4 antibodies (such as Ipilimumab or Tremelimumab) and anti-PD- L2 antibodies.

[0114] Preferably, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with radiation therapy. In an embodiment, the polynucleotide, the vector, the monocyte or pharmaceutical composition is administered prior to radiation therapy. In another embodiment, the polynucleotide, the vector, the monocyte or pharmaceutical composition is administered during (concurrent with) radiation therapy. In yet another embodiment, the polynucleotide, the vector, the monocyte or pharmaceutical composition is administered after radiation therapy, preferably after low- dose radiotherapy.

[0115] The terms “radiation therapy” should be construed as generally understood in the art, that is to say as referring to treatment using ionizing radiation, generally provided as part of cancer therapy to either kill or control the growth of malignant cells. The term “high dose radiation therapy”, also referred to as “hypofractionated radiotherapy”, should be construed as generally understood in the art that is to say as referring to a radiation therapy approach where the total radiation dose is delivered in large individual doses per session (typically >2 Gy per fraction), The term “low-dose radiotherapy” should be construed as generally understood in the art that is to say as referring to a radiation therapy approach where the total radiation dose is delivered in small individual doses per session (typically <2 Gy per fraction). Protocols for low-dose radiotherapy have been described for instance by Herrera FG, et al. Preferably, the low-dose radiotherapy comprises radiation dose of 0.5-2 Gy per session, yet preferably the low-dose radiotherapy comprises radiation dose of about 1Gy per session, In a yet preferred embodiment, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use according to the invention is combined with radiomimetic agents, preferably neocarzinostatin or bleomycin, or with radiosensitazing agents, preferably chosen from gold nanoparticles or gadolinium. In the context of the invention the terms “radiomimetic agents” should be construed as generally understood in the art, that is to say as referring to compounds producing similar symptoms in living organisms as ionising radiation does. Preferably the radiomimetic agent is neocarzinostatin.

[0116] In the context of the invention the terms “radiosensitazing agents”, should be construed as generally understood in the art, that is to say as referring to compounds enhancing sensitivity to radiation therapy in a mammal. Preferably the radiomimetic agent is chosen in the list consisting of gold nanoparticles, gadolinium nanoparticles and PARP inhibitors, wherein the PARP inhibitor is preferably chosen in the list consisting of nirapribn olaparib, talazoparibn rucaparib, talazoparib, veliparib, pamiparib, CEP 9722 and E7016.

[0117] The invention further pertains to products containing (a) the polynucleotide encoding protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, the monocyte modified to overexpress the cyclin-dependent kinase inhibitor p21 protein, or the pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, as defined herein, and (b) an agent that increases the haematocrit; a chemotherapeutic agent; a cell-specific antibody; an immune checkpoint inhibitor (ICI); a radiomimetic agent, preferably neocarzinostatin or bleomycin; or a radiosensitazing agent, preferably gold nanoparticles or gadolinium nanoparticles; as a combined preparation for simultaneous, separate or sequential use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, preferably wherein the solid cancer resistant to radiation therapy is a cancer overexpressing at least CD47.

[0118] Preferably, in embodiments wherein the polynucleotide, the vector, the monocyte or the pharmaceutical composition comprising a monocyte or a polynucleotide or vector is combined with other therapeutic agents or procedures, such as for instance the agents and therapeutic procedures described herein, the anti-tumoral effect of the polynucleotide, the vector, the monocyte or the pharmaceutical composition, preferably of p21 expressed in the monocyte or the pharmaceutical composition, is thereby enhanced.

[0119] The invention also pertains to a combination of radiation therapy and of a polynucleotide encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, a vector comprising thereof as defined herein, a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin- dependent kinase inhibitor p21 protein, or a pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy. In the context of the invention, the terms “for use in the treatment of a mammal suffering from a solid cancer” and “for use in the treatment of a mammal suffering from a solid cancer chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy” should be construed as equivalent to the phrases “for use in the treatment of a solid cancer” and “for use in the treatment of a solid cancer chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy”

[0120] The invention also pertains to the use of a polynucleotide encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells; a vector comprising thereof; a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein; or a pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, for preparing a medicament for the treatment of a solid cancer, in combination with radiation therapy.

[0121] With respect to embodiments wherein the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use in the invention is combined with radiotherapy, it may be administered prior to, during (concurrent with) or after radiotherapy. In an embodiment of the combination, the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use in the invention is administered prior to radiation therapy. In another embodiment of the combination, the polynucleotide, the vector, the monocyte or pharmaceutical composition for use in the invention is administered during (concurrent with) radiation therapy. In yet another embodiment of the combination, the polynucleotide, the vector, the monocyte or pharmaceutical composition for use in the invention is administered after radiation therapy, preferably after low-dose radiotherapy.

[0122] With respect to embodiments wherein the polynucleotide, the vector, the monocyte or the pharmaceutical composition for use in the invention is combined with other therapeutic agents, such as with an agent that increases the patient haematocrit, with a chemotherapeutic agent, with a cell-specific antibody, with an immune checkpoint inhibitor (ICI), with radiomimetic agents, preferably neocarzinostatin, or with radiosensitazing agents, preferably gold nanoparticles, gadolinium nanoparticles and PARP inhibitors, wherein the PARP inhibitor is preferably chosen in the list consisting of nirapribn olaparib, talazoparibn rucaparib, talazoparib, veliparib, pamiparib, CEP 9722 and E7016.

[0123] "Concomitant administration" of said active principle with the pharmaceutical composition of the present invention means administration with the recombinant cells at such a time that both the active principle and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the active principle with respect to the administration of a compound of the invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0124] The in vivo delivery of the polynucleotide, the vector, or the cell composition of the invention, containing the obtained p21 -expressing monocytes, in a subject in need thereof, is herein proposed as a simple and effective way of treating cancer, specifically solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, in particular when combined with radiation therapy.

[0125] Usually, the said subject is a human, but nonhuman mammals may also be treated, e.g. companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like.

[0126] A “subject in need thereof’, as herein meant, is therefore a mammal, preferably a human being, that is suffering from cancer. Said cancer can be a liquid (e.g. leukemia) or a solid cancer, preferably is a solid cancer such as, without limitation, a lymphoma, a carcinoma, a melanoma, a glioblastoma, a sarcoma, a myeloma, colon rectal tumours, etc. as primary or metastatic cancers, in another preferred embodiment said “subject in need thereof” is a human or another mammal suffering from a solid cancer preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, as defined herein.

[0127] The present invention encompasses treating methods in which the cell composition of the invention is administered to said subject in need thereof by injection, preferably by intravenous injection. A systemic injection may be also carried out by perfusion. These injections are harmless for the treated subject. The intravenous administration of the cell composition of the invention is able to increase the in vivo phagocytosis of the tumor cells present in the blood of the subject, thereby reducing the amount of tumor cells in said subject.

[0128] A particular treatment method according to the invention comprises:

[0129] 1 . Withdrawal and isolation of monocytes or their precursors or pluripotent stem cells from blood or bone marrow or umbilical cord, from the subject in need thereof or from an healthy donor,

[0130] 2. Culturing of these cells as disclosed above, so as to obtain I isolate a monocyte population, 3. Transformation of these cells with the recombinant nucleic acid as defined above,

[0131] 4. Optionally, packaging and / or storage of the cells thereby obtained, and then

[0132] 5. Their administration to the patient.

[0133] The treatment of the present invention affords many advantages relative to other treatments in adoptive immunotherapy with LAK, TIL or NK (natural killers), such as, for example, the absence of toxic mediators released by the cells, the fact that the ratio of effector to target cells for the cytotoxicity is lower than in other treatments, and the fact that it does not necessitate the simultaneous injection of cytokine such as IL-2, the side effects of which are deleterious. Furthermore, it makes it possible to infect only a defined and controlled cell population, it enables the multiplicity of infection (number of viral particles per cell) to be chosen, it enables the tissues to be reached irreversibly from the blood circulation, and it makes it possible to turn to good account the central role of the macrophages in the body, both by their antitumour or anti-infectious activity and in their activity of stimulation or regulation of the immune system, as explained above. Besides the proinflammatory reprogramming of phagocytic macrophages enhances the innate and adaptive anti-cancer immune response, which in turn establishes durable anti-tumour growth microenvironment. In addition, and taking account of the considerable longevity of macrophages, a handicapping repetition of treatments for the patient could be avoided.

[0134] The present invention hence affords new possibilities of more effective treatment which are less demanding for the patient, less expensive and more reproducible.

[0135] As used herein, the terms “treat”, “treating”, “treatment”, and the like refer to reducing or ameliorating the symptoms of a disorder (e.g., leukemia), and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0136] Effective doses of the therapeutic entity of the present invention, e.g. for the treatment of cancer, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages can be titrated to optimize safety and efficacy.

[0137] For prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of disease in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In these prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0138] Conversely, in therapeutic applications, a relatively high dosage (50x106monocytes per injection dose per patient) at relatively short intervals (typically each week) is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease.

[0139] Figure legends

[0140] Figure 1. p21 Mo cellular therapy and radiotherapy combination induces long-lasting PDAC KPC tumor regression and elongation of mice survival.

[0141] Figure 1 a Schematic representation showing the adoptive transfer of control (Co. Mos ACT) and p21 -genetically engineered (p21 Mos ACT) murine Mos into KPC tumor-bearing immunocompetent syngeneic C57BL / 6J mice (when tumor volumes reached 100 mm3) and 8 Gy irradiation (RT) received by KPC tumors 6 days after ACT.

[0142] Figure 1 b,c Individual (b) or average (c) tumor growth curves of established KPC tumors treated as indicated. Figure 1 b: the data are individual tumor growth from n=5 mice / group. Figure 1 c: the data are presented as the mean+SEM from n=5 mice / group.

[0143] Figure 1 d Kaplan-Meier survival curves of mice bearing established KPC tumors, treated either with Co. Mos ACT or p21 Mos ACT and receiving or not 8 Gy (RT).

[0144] Figure 1 e-g Individual (e) and average (f) tumor growth curves of naive or cured mice (treated with p21 Mos ACT and 8 Gy (RT)) that were rechallenged with KPC cells on day 65 after p21 Mos ACT.

[0145] Figure 2. SIRPa overexpression represses antitumor activity of p21 Mo+RT combination.

[0146] Figure 2 a-d Individual tumor growth curves of established KPC tumors treated as indicated.

[0147] Figure 2 e Kaplan-Meier survival curves of mice bearing established KPC tumors, infused with Co. Mo, p21Mo or p21 / SIRPa Mo and receiving or not 8 Gy (RT). Data are from n=5 mice / group. *p<0.05, ***p<0.001 and ****p<0.0001 are determined with log-rank Mantel- Cox test (e).

[0148] Figure 3. The antitumor activity of p21 Mo+RT combination is immune-dependent.

[0149] Figure 3 a-h Individual tumor growth curves of established KPC tumors treated as indicated. Abcissia: days after Mo injection.

[0150] Figure 3 i Kaplan-Meier survival curves of C57BL / 6N or Rag2- / -IL2rg- / - C57BL / 6N bearing established KPC tumors, infused with Co. Mo or p21Mo Mo and receiving or not 8 Gy (RT). Data are n=5 mice / group. *p<0.05, **p<0.01 , ***p<0.001 and ****p<0.0001 are determined with log-rank Mantel-Cox test (i).

[0151] Examples

[0152] The examples given below are not limiting but are an illustration of the therapeutic effect underlying the intended medical use of the p21 -engineered monocyte therapy disclosed herein in solid cancers.

[0153] Example 1

[0154] 1. Material and methods

[0155] Primary cells and cell lines.

[0156] Murine Mos were obtained from femur bone marrow cells. Briefly, the 5x108 bone marrow cells were obtained after flushing femur bones of 20 naive non tumor-bearing C57BL / 6J mice (6-8-week old). Then, bone marrow Mos were purified by negative selection using EasySep Mouse Monocyte Isolation Kit (STEMCELL#19861), according to manufacturer’s recommendations. Murine PDAC KPC cells (KrasG12D p53R172H / +) were kindly provided by Dr. Florent Ginhoux (Gustave Roussy, Villejuif) and cultured in DMEM medium supplemented with 10% HI FBS.

[0157] Plasmids, lentiviral vectors and transduction.

[0158] Self-inactivated lentiviral vectors and murine p21 cDNA sequence were used for genetic engineering of murine Mos. Mos (1x106) were treated for 1 h at 37°C with 1 ml viral-like particles containing the Vpx protein (VLPs-Vpx+) containing 10 pg / ml polybrene (Sigma, #H9268) before the addition of 200 ng CAp24 of each control or / and encoding-gene lentiviral vectors with 10 pg / ml polybrene for overnight transduction in 1 ml Opti-mem medium (containing 2% HI FBS and 100 ll / rnl penicillin, 100 pg / ml streptomycin). VLPs- Vpx+ were produced as previously described by Allouch, A. et al. and viral stocks were quantified for viral CAp24 content by ELISA (Perkin Elmer, # NEK050A).

[0159] In vivo PDAC model and adoptive cellular transfer.

[0160] Mouse studies were performed in accordance with protocols approved by the French Ethical Committee (Comite d'Ethique en Experimentation Animale N°026 (CEEA26)) of the Ministere de I’education nationale, de I’enseignement superieur et de la recherche (Project N°2020-064-27337) and following recommendations for proper use and care during animal experimentation. Six- to eight-week-old female C57BL / 6J mice (from Janvier Laboratories) were maintained in specific pathogen-free (SPF) grade room (at 20°C-22°C ambient temperature, humidity (45-60%) and 12h (7:00 a. m. -7:00 p.m.) light / dark cycle) and randomized to homogenous mouse body weight groups (20 g to 23 g) before experiments. KPC tumor cells (0.5x105cells / mouse) were then subcutaneously injected into the right flank of C57BL / 6J mice. When tumor volumes reached 100 mm3, mice were randomized into treatment groups. Control or p21 -engineered Mos were then adoptively transferred in C57BL / 6J mice by intravenous injections into the tail using local application of lidocaine as painkiller (1x106Mos / mouse). Six days after Mo transfer, tumors of two treatment groups that were infused with control or p21 -engineered Mos were locally irradiated with single dose 8 Gy ionizing radiation (IR) using Small Animal Radiation Research Platform (SARRP) (Xstrahl Inc., Swanee, GA, USA). Tumor growth was monitored and tumor volumes calculated as 0.5 x length x width as described by Schwartz, J. L. et al.. In tumor cell rechallenge experiments, KPC (0.5x105) cells were inoculated subcutaneously into the left flank of cured mice. Naive C57BL / 6J mice were inoculated with the same number of cells as control group. Tumor growth was monitored as described above.

[0161] Statistics.

[0162] Statistical analysis was performed with GraphPad Prism 8.0 (GraphPad). For all figures, statistical significances are indicated as *p <0.05, ***p<0.001 , and ****p<0.0001.

[0163] 2- Results

[0164] Adoptive transfer of p21 -engineered Mos reduces the growth of established PDAC solid tumors and significantly elongates the survival of treated mice Considering that CD47 blockade with monoclonal antibody or depletion of SIRPa (Bian, Z. et al., Nishiga, Y. et al.) did not affect growth of radioresistant tumors such pancreatic KPC tumors, we explored the antitumor effects of adoptively transferred, p21 -engineered Mos into syngeneic immunocompetent C57BL / 6 mice bearing Pdx1-Cre, lox-stop-lox-KrasG12D / +, lox-stop-lox- tp53R172H / + mouse-derived PDAC (KPC) tumors. When tumor volume reached 100 mm3, tumor-bearing mice were infused with control or p21 -engineered Mos and analyzed for tumor growth and survival (Fig. 1a). Mice that were infused with p21 -engineered Mos exhibited significantly slower KPC tumor growth (Fig. 1b,c) and an increased survival (Fig. 1d), as compared to mice adoptively transferred with control Mos (Fig. 1b-d). These results indicate that the adoptive transfer of p21 -engineered monocytes drives the regression of pancreatic KPC tumors.

[0165] The combination of p21 -engineered monocyte therapy and radiotherapy leads to the complete regression of radioresistant KPC tumors

[0166] Given that KPC tumors are radioresistant and SIRPa was identified as a master regulator of tumor resistance to radiotherapy (Nishiga, Y. et al.), we next asked whether a combinatorial therapeutic strategy involving adoptive p21 -engineered Mo therapy followed by radiotherapy (p21 Mos + RT) should be an effective treatment for established radioresistant KPC tumors. As shown in Fig. 1b-d, a durable regression of KPC tumors is found when p21 -engineered Mo therapy and radiotherapy are combined. In most cases, complete clearances of KPC tumors are detected (Fig. 1 b,c) and associated with survival of treated mice (Fig. 1d). Interestingly, a significant delay in the growth of KPC tumors is detected when comparing tumor-bearing mice that were infused with p21 -engineered Mos alone and tumor-bearing mice that were treated with p21 Mos + RT (Fig. 1 b,c). Altogether, these results reveal that the adoptive transfer of p21- engineered Mos sensitize KPC tumors to radiotherapy, but also highlight the ability of radiotherapy to enhance the antitumor activity of p21 -engineered Mos.

[0167] The combination of p21 -engineered monocyte therapy and radiotherapy confer a memory response against tumor rechallenge

[0168] To determine whether long-lasting tumor immunity is associated with tumor clearances shown in Fig. 1 b,c, we rechallenged tumor-eradicated mice and naive control mice with KPC cells on the opposite flank of the primary tumor bed. Subcutaneous injected KPC cells failed to grow in tumor-eradicated mice (Fig. 1e,f), as compared to those injected in naive mice that did not prevent tumor recurrence and associated lethality (Fig. 1g). These results demonstrate that the combination treatment of adoptive transferred p21 -engineered Mos and radiotherapy supports the development of a memory response against tumor rechallenge.

[0169] Similar experimental conditions as those of example 1 were reproduced, using p21 -engineered Mo co-transduced with a SIRPa cDNA sequence resistant to p21 repression (p21 / SIRPaMo).

[0170] 1. Material and methods

[0171] Primary cells and cell line.

[0172] Bone marrow cells were obtained after flushing femur bones of six- to eight-week-old female C57BL / 6J or C57BL / 6N mice and were purified by negative selection using EasySep Mouse Monocyte Isolation Kit (STEMCELL#19861), according to manufacturer’s recommendations. Murine pancreatic ductal adenocarcinoma (PDAC) KPC cells (KrasG12D p53R172H / +) were kindly provided by Dr. Florent Ginhoux (Gustave Roussy, Villejuif) and cultured in DMEM medium supplemented with 10% HI FBS.

[0173] Plasmids, lentiviral vectors and transduction.

[0174] Murine monocytes were engineered with self-inactivated lentiviral vectors, murine p21 and SIRPa cDNA sequences, as previously described (Allouch et al., Nat Commun (2022)).

[0175] In vivo PDAC model and adoptive cellular transfer.

[0176] Studies using were performed in accordance with protocols approved by the French Ethical Committee (Comite d'Ethique en Experimentation Animale N°026 (CEEA26)) of the Ministere de I’education nationale, de I’enseignement superieur et de la recherche (Project N°2020-064- 27337) and following recommendations for proper use and care during animal experimentation. C57BL / 6J mice, control and Rag2' / '-IL2rg_ / ' C57BL / 6N (from Janvier Laboratories) were randomized to homogenous mouse body weight groups and subcutaneously injected with KPC tumor cells (0.5x105cells / mouse). When tumor volumes reached 100 mm3, mice were randomized and intravenously infused with control or p21-engineered Mo (106Mo / mouse). Six days after infusion, subcutaneous tumors were stereotactically irradiated with single dose 8 Gy ionizing radiation (IR) using Small Animal Radiation Research Platform (SARRP) (Xstrahl Inc., Swanee, GA, USA). Then, tumor volumes were calculated as 0.5 x length x width and overall mice survival was determined. Statistics.

[0177] Statistical analysis was performed with GraphPad Prism 8.0 (GraphPad). For all figures, statistical significances are indicated as *p <0.05, **p<0.01 , ***p<0.001 , and ****p<0.0001.

[0178] 2. Results

[0179] SIRPa repression dictates in vivo antitumor efficacy of combining p21 -engineered monocytes with stereotactic radiotherapy

[0180] To evaluate the impact of p21-dependent SIRPa repression on tumor regression elicited by the combination of the adoptive p21 -engineered monocyte (p21Mo) therapy with stereotactic radiotherapy (p21 Mo+RT), p21 -engineered Mo were co-transduced with a SIRPa cDNA sequence resistant to p21 repression (p21 / SIRPaMo) and their antitumoral activities were compared to p21Mo and Control Mo (Co. Mo). Considering that the identity of SIRPa gene sequences between humans and mice is over 70% (Feng et al., Eur J Immunol (2023)), p21- engineered mouse Mo were co-transduced with human SIRPa cDNA sequence that was previously published (Allouch et al., Nat Commun (2022)). Engineered Mo were then adoptively transferred in mice harboring 100 mm3subcutaneous KPC tumors. Six days post-infusions, KPC tumors were stereotactically irradiated (or not) with 8 Gy, and the tumor growth and the survival of treated mice were analyzed. It was observed that p21 Mo+RT combination significantly impaired the growth of KPC tumors (Fig. 2c) and drastically elongated the survival of treated mice (Fig. 2e), as compared to control or irradiated mice (Fig. 2a, b and e). Interestingly, mice infused with p21+SIRPaMo and whose tumors were irradiated with 8 Gy (p21 / SIRPaMo+ RT) did not show tumor regression (Fig. 2d) and prolonged mouse survival (Fig. 2e). These results highlight the central role of SIRPa repression by p21 in the antitumor efficacy of p21Mo+RT combination.

[0181] The antitumor efficacy of combining p21 -engineered monocytes with stereotactic radiotherapy depends on the immune system

[0182] The immune system’s role on the antitumoral effect observed following the adoptive transfer of p21 Mo and its combination with RT was assessed. Control C57BL / 6N mice and immunodeficient Rag2' / “IL2rg_ / ' C57BL / 6N mice were subcutaneously engrafted with KPC cancer cells. When KPC tumors reached a volume of 100 mm3, mice were infused with Co. Mo or p21 Mo, and were stereotactically irradiated with 8 Gy 6 day post-infusion. As reported in Figure 2, adoptive transfer of p21 Mo and its combination with stereotactic RT significantly repressed KPC tumor growth (Fig. 3d) and elongated survival of C57BL / 6N mice (Fig. 3i), as compared to mice infused with Co. Mo (Fig. 3a, i) or with p21Mo (Fig. 3b, i) or to mice infused with Co. Mo and stereotactically irradiated (Fig. 3c, i). Such biological effects were not observed in Rag2' / '-IL2rg_ / ' C57BL / 6N immunodeficient mice (Fig. 3e-i), thus revealing the central role of the immune system in the antitumor activity observed following p21Mo+RT combination.

[0183] Table 1: sequences referred to herein Bibliographic references

[0184] Allouch, A. et al., Nat Commun 13, 6739 (2022)

[0185] Andressen R. et al., Cancer Res. (1990) 50:7450

[0186] Arandjelovic S and Ravichandran KS, Nat Immunol (2015) Sep;16(9):907-17

[0187] Aronovich EL et al., Human. Molecular. Genetics (2011) Vol. 20, Review Issue 1 R14-R20

[0188] Bartholeyns J et al., Anticancer Res. 11 (1991) 1201-1204

[0189] Berger G., Gene Therapy (2009) 16, pages159-163

[0190] Bian, Z. et al. Nat Commun 12, 3229 (2021)

[0191] Billingsley MM, et al. Small. 2024 Mar;20(11): :e2304378

[0192] Bornewasser L et al.. Chem Sci. 2022 Mar 3;13(17):4753-4761

[0193] Candas-Green, D. et al. Nat Commun 11 , 4591 (2020)

[0194] Doi, N. et al. Exp Cell Res 333, 249-260 (2015)

[0195] Haddada H. et al. Biochem. Biophys. Res. Commun (1993) Sep 30; 195(3): 1174-83.

[0196] Herrera FG, et al. . Cancer Discov. 2022 Jan;12(1):108-133. doi: 10.1158 / 2159-8290.CD- 21-0003. Epub 2021 Sep 3.

[0197] Huang, R. X. & Zhou, P. K. Signal Transduct Target Ther5, 60 (2020))

[0198] Hutchinson, MK.N.D. et al.. Oncogene 39, 3638-3649 (2020)

[0199] Jeong, H. et al. Blood Res 51 , 157-163 (2016)

[0200] Leyva F. et al, BMC biotechnology (2011) ; 11 : 13

[0201] Li, J. Y. et al.. J Exp Clin Cancer Res 31 , 102 (2012)

[0202] Miller J. B. et al. Angew. Chem. Int. Ed. Engl. 2017; 56: 1059-1063

[0203] Neschadim A. et al. Biol Blood Marrow Transplant. 2007 Dec;13(12):1407-16.

[0204] Nishiga, Y. et al. Nat Cancer 3, 1351-1366 (2022)

[0205] Nguyen, A. M. et al. Mol Cell Proteomics 19, 375-389 (2020)

[0206] Shimura, T. et al. Radiother Oncol 112, 302-307 (2014) Singh G. et al, F1000 research 2015, 4:495

[0207] Schwartz, J. L. et al. BrJ Cancer79, 744-747 (1999)

Claims

CLAIMS1. Combination of radiation therapy and of a polynucleotide encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells; a vector comprising thereof; a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein; or a pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, for use in the treatment of a solid cancer.

2. The combination for use according to claim 1 , wherein the solid cancer is chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.

3. The combination for use according to claim 1 or 2, wherein the polynucleotide encoding p21 protein, operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, a vector comprising thereof, a monocyte genetically modified in that said monocyte contains a vector coding for the cyclin-dependent kinase inhibitor p21 protein, or a pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, is administered prior to, concurrent with, or after radiation therapy.

4. The combination for use according to any one of claims 1 to 3, wherein the solid cancer is a cancer overexpressing at least CD47.

5. The combination for use according to any one of claims 2 to 4, wherein the solid cancer resistant to radiation therapy is a cancer overexpressing at least CD47.

6. The combination for use according to any one of claims 1 to 5, wherein the vector is a virus, optionally adenovirus, adeno-associated virus (AAV), herpesvirus, lentivirus, vaccinia virus, cytomegalovirus (CMV), plasmid; or mRNA, optionally delivered via nanoparticle, liposome or LNP.

7. The combination for use according to any one of claims 1 to 6, wherein the vector is a viral or non-viral vector comprising a nucleic acid operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells thereof in vivo, optionally a monocyte-specific promoter or enhancer or miRNA, macrophage-specific promoter or enhancer or miRNA, or myeloid-specific promoter or enhancer or miRNA.

8. The combination for use according to claim 6, wherein the mRNA is delivered via nanoparticle, liposome or LNP linked to a targeting moiety that targets monocytes, macrophages and / or precursor cells thereof in vivo.

9. The combination for use according to any one of claims 1 to 8, wherein said solid cancer is selected from the list consisting in pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC); head and neck cancer, preferably head and neck squamous cell carcinoma; breast cancer, preferably triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer and glioblastoma.

10. A polynucleotide encoding p21 protein, such as mRNA, DNA or cDNA encoding the protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, a monocyte modified to overexpress the cyclin-dependent kinase inhibitor p21 protein, or a pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, for use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy.11 . The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for use according to claim 10, comprising a vector coding for cyclin-dependent kinase inhibitor (p21) protein, and a pharmaceutically acceptable carrier; wherein the vector targets monocytes, macrophages and / or precursor cells thereof in vivo resulting in overexpression of p21 in said cells12. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for use according to claim 10 or 11 wherein the vector is a virus, optionally adenovirus, adeno-associated virus (AAV), herpesvirus, lentivirus, vaccinia virus, cytomegalovirus (CMV); plasmid; or mRNA, optionally delivered via nanoparticle, liposome or LNP.

13. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for use according to any of claims 10 to 12 wherein the vector is a viral or non-viral vector comprising a nucleic acid operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells thereof in vivo, optionally a monocyte-specific promoter or enhancer or miRNA, macrophage-specific promoter or enhancer or miRNA, or myeloid-specific promoter or enhancer or miRNA14. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for use according to claim 12 wherein the mRNA is delivered via nanoparticle, liposome or LNP linked to a targeting moiety that targets monocytes, macrophages and / or precursor cells thereof in vivo.

15. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 14, wherein the solid cancer is a cancer overexpressing at least CD47.

16. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition, for its use according to any one of claims 10 to 15, wherein the solid cancer resistant to radiation therapy is a cancer overexpressing at least CD47.

17. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 16, wherein it is for improving the prognosis or enhancing the chance of regress of the solid cancer, preferably when administered prior to radiation therapy, or wherein it is for preventing, limiting, or delaying the risk of metastasis and / or cancer recurrence, and / or as for inducing antitumoral vaccination, and / or for stimulating immunological memory.

18. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 17, wherein said solid cancer is selected from the list consisting in pancreatic cancer, preferably pancreatic ductal adenocarcinoma (PDAC); head and neck cancer, preferably head and neck squamous cell carcinoma; breast cancer, preferably triple-negative breast cancer (TNBC), prostate cancer, kidney cancer, lung cancer, preferably non-small cell lung cancer and glioblastoma.

19. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 18, wherein it is combined with an agent that increases the patient haematocrit, with a chemotherapeutic agent, with a cellspecific antibody, with an immune checkpoint inhibitor (I Cl), with radiation therapy, preferably the monocyte or pharmaceutical composition comprising thereof is administered before radiation therapy, with radiomimetic agents, preferably neocarzinostatin, or with radiosensitazing agents, preferably chosen in the list consisting of gold nanoparticles, gadolinium nanoparticles and PARP inhibitors, wherein the PARP inhibitor is preferably chosen in the list consisting of nirapribn olaparib, talazoparibn rucaparib, talazoparib, veliparib, pamiparib, CEP 9722 and E7016.

20. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to claim 19, wherein the anti-tumoral effect of the polynucleotide, the vector, the monocyte or the pharmaceutical composition, preferably of p21 expressed in the monocyte or the pharmaceutical composition, is thereby enhanced.

21. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 20, wherein said monocyte contains a replication defective recombinant virus encoding the cyclin-dependent kinase inhibitor p21 under the control of regulatory elements permitting its expression.

22. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to claim 21 , wherein said virus is a replication defective lentivirus.

23. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to claim 22, wherein said replication defective lentivirus is the HIV- 1 based Self inactivated (SIN) lentiviral vector.

24. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 23, wherein said monocyte contains a Sleeping Beauty transposon system encoding the cyclin-dependent kinase inhibitor p21 under the control of regulatory elements permitting its expression.

25. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according any one of claims 10 to 24, wherein said p21 protein is SEQ ID NO:2, or a functional variant or fragment thereof, and / or wherein said virus or said transposon system contains the nucleic acid of SEQ ID NO:5, preferably under control of the SFFV promoter.

26. The polynucleotide, the vector, the monocyte, or the pharmaceutical composition for its use according to any one of claims 10 to 25, wherein it is formulated in an intravenous injectable form or in a perfusion form.

27. Products containing (a) the polynucleotide encoding protein p21 , preferably operably linked to a regulatory sequence that targets monocytes, macrophages and / or precursor cells, and / or vectors comprising thereof, the monocyte modified to overexpress the cyclin-dependent kinase inhibitor p21 protein, or the pharmaceutical composition comprising any one of them and a pharmaceutically acceptable excipient, as defined in any of the preceding claims, and (b) an agent that increases the haematocrit; a chemotherapeutic agent; a cell-specific antibody; an immune checkpoint inhibitor (ICI); a radiomimetic agent, preferably neocarzinostatin or bleomycin; or a radiosensitazing agent, preferably gold nanoparticles or gadolinium nanoparticles; as a combined preparation for simultaneous, separate or sequential use in the treatment of a mammal suffering from a solid cancer, preferably chosen from cancers resistant to radiation therapy and cancers sensitive to radiation therapy, preferably wherein the solid cancer resistant to radiation therapy is a cancer overexpressing at least CD47.

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