P21-expressing monocytes for cancer cell therapy

Abstract

There is a very high interest in identifying effective targets to attenuate the programmed cell removal (PrCR) of tumor cells by macrophages. The present inventors have determined that the cyclin-dependent kinase inhibitor p21 protein is a strong modulator of macrophage-mediated PrCR. Moreover, they have shown that adoptive transfer of monocytes overexpressing p21 induces macrophage PrCR and a shift from an anti-inflammatory to a pro-inflammatory phenotype in vivo, delays cancer progression and significantly improves the overall survival of mice engrafted with cancer cells. Accordingly, the present invention relates to therapeutic compositions comprising monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein, and to their use for treating a mammal suffering from cancer, in particular leukemia.

Description

TECHNICAL FIELD

[0001] It is of great interest to determine effective targets to attenuate the programmed cell removal (PrCR) of tumor cells by macrophages. The present inventors have determined that the cyclin-dependent kinase inhibitor p21 protein is a strong modulator of macrophage-mediated PrCR. Moreover, they have shown that the adoptive transfer of monocytes overexpressing p21 induces macrophage PrCR and the shift from an anti-inflammatory to a pro-inflammatory phenotype in vivo, delays cancer progression and significantly improves the overall survival of mice engrafted with cancer cells. The present invention thus relates to therapeutic compositions comprising monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein, and to their use for treating a mammal suffering from cancer, in particular leukemia. BACKGROUND

[0002] When properly activated, both innate and adaptive immune system effector cells have the capacity to successfully attack cancer cells. It has been proposed in the past to inhibit negative adaptive immune modulators such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), or to use adoptively transferred engineered patient's own immune cells such as chimeric antigen receptor (CAR) T cells to enhance the normal capacity of the patient's immune response. This treatment has led to significant progress in the treatment of solid cancers and hematological disorders (Pardoll DM., Nat Rev Cancer (2012) and Sharma SH et al., Cell (2015)).

[0003] However, to develop new cancer treatment strategies, a more comprehensive understanding of immune regulation is needed.

[0004] Programmed cell removal (PrCR) is a macrophage-mediated immune surveillance process by which target cells are recognized and phagocytosed (Jaiswal et al., Trends Immunol (2010)). Initially described as a key mechanism linking programmed cell death to the removal of dead cells (Arandjelovic and Ravichandran, Nat Immunol (2015)), PrCR is also involved in the clearance of live tumor cells (Majeti et al., Cell (2009)).

[0005] The efficacy of PrCR depends on the balance between the recognition of pro-phagocytic ("eat me") signals by macrophages and the inhibition of macrophages by target cancer cells through the activation of anti-phagocytic ("don't eat me") pathways (Chao et al., Nat Rev Cancer (2011)). The macrophage-secreted "eat me" signal calreticulin (CRT) was recently identified as the first signal favoring PrCR through the binding of sialoglycans to target cancer cells (Feng et al., Nat Comm (2018)). In contrast, the transmembrane protein CD47 was identified as the "don't eat me" signal, which inhibits PrCR through binding and activation of signal-regulatory protein alpha (SIRPa), a phagocytosis- inhibiting receptor expressed on macrophage membranes (Jaiswal et al., Cell (2009)). A study showed that blockade of the CD47-SIRPa axis with a monoclonal antibody blocking CD47 selectively induced phagocytosis of tumor cells and demonstrated efficacy in various preclinical models of lymphoma, bladder cancer, colon cancer, glioblastoma, breast cancer, acute lymphoblastic leukemia, and acute myeloid leukemia (Weiskopf K. Eur J Cancer (2017)). Recently, the combination of blockade of CD47 with Rituximab showed promising activity in patients with relapsed or refractory non-Hodgkin lymphoma (Advani R. et al., N Engl J Med (2018)), highlighting the fact that targeting macrophage immune checkpoints (MICs) to lift the PrCR restriction represents a major therapeutic opportunity.

[0006] Accordingly, it is of great interest to identify effective targets to alleviate the programmed cell removal (PrCR) of tumor cells by macrophages. The present invention addresses this need. SUMMARY

[0007] As disclosed in the following examples, the inventors found that depletion of the cell cycle protein-dependent kinase inhibitor p21 in primary human macrophages blocks the phagocytic capacity of said cells, thus reducing their immune surveillance Figure 1 of j and Figure 1 of k). They first identified the p21 protein as a strong modulator of macrophage-mediated PrCR. They showed that overexpression of p21 can act as an inhibitor of the macrophage immune checkpoint (MIC), thus tumor cells are eventually recognized, phagocytosed and destroyed. Accordingly, they showed that adoptive transfer of monocytes overexpressing p21 induces macrophage PrCR and shift from an anti-inflammatory to a pro-inflammatory phenotype in vivo, delaying cancer progression and significantly improving the overall survival of mice engrafted with cancer cells Figure 1 (r).

[0008] The present invention relates to a cell composition and its incorporation into a pharmaceutical composition which can be used for cancer treatment, more specifically for cancer immunotherapy. In particular, the present invention relates to the isolation, culture, activation and genetic modification of cells of the mononuclear phagocyte system and the use of said cells in cell therapy, for example in adoptive immunotherapy.

[0009] Cells of the mononuclear phagocyte system include peripheral blood monocytes, their bone marrow or blood precursors and tissue macrophages. Monocytes are formed in the bone marrow, leave the bone marrow after maturation and migrate to the tissues via the peripheral blood. The half-life of human monocytes circulating in the blood is about 3 days. When monocytes reach the tissues, they are called macrophages. The total number of tissue macrophages is much greater than the number of circulating monocytes, approximately 400 times. Macrophages are present throughout the body, but are especially numerous in the liver (Kupffer cells), lymph nodes, lungs, peritoneum and skin (Langerhans cells). The migration of monocytes from the systemic circulation to the tissues is irreversible.

[0010] Monocytes and macrophages are known to have many important functions, including inducing immune responses in the acute phase, regulating hematopoiesis, activating the immune system, coagulation, destroying organisms and tumor cells, and tissue repair and scarring.

[0011] Monocyte-macrophages can also be used in adoptive immunotherapy to treat some types of cancer in humans. Typically, these cells can be purified from the circulating blood of a patient, cultured ex vivo and activated with interferon gamma to induce their differentiation and increase their tumor killing capacity, and then injected into the patient. Genes can also be transferred ex vivo into monocyte-derived macrophages using suitable vectors, thereby endowing them with superior properties in terms of cytotoxicity and stimulation of the immune system.

[0012] Thus, in a first aspect, the present invention relates to a pharmaceutical composition comprising monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein. These monocytes differentiate into macrophages upon transplantation into tissues.

[0013] This pharmaceutical composition is hereinafter referred to as "the composition of the invention", "the pharmaceutical composition of the invention" or "the cell composition of the invention".

[0014] In particular, the present invention relates to the use of monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein for the manufacture of a medicament intended to enhance the immune and hematopoietic system of a patient for the treatment of cancer, in particular leukemia.

[0015] As used herein, the term "p21 protein" refers interchangeably to the cyclin-dependent kinase inhibitor 1, which is also known as "p21 Cip1 ", "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, thus acting as a regulator of cell cycle progression at the G1 and S phases. The binding of p21 to the CDK complex occurs through the N-terminal domain of p21, which is homologous to the other CIP / CDK inhibitors p27 and p57. As a major target of p53 activity, it is usually associated with the linkage of DNA damage and cell cycle arrest. The protein is encoded by the CDKN1A gene of SEQ ID NO: 1 (NM_078467) located on human chromosome 6 (6p21.2). 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), while the mouse p21 protein has the amino acid sequence of SEQ ID NO: 4 (NP_031695).

[0016] The term "p21 protein" herein also encompasses functional variants and / or fragments of the above p21 proteins.

[0017] A "functional variant" is for example a wild-type p21 protein of an animal species other than human or mouse, such as the animals horse, dog, cat or cow. These proteins are now well characterized and their sequences can be easily retrieved from conventional databases. A "functional variant" is also a mutated form of the natural p21 protein, whose amino acid sequence has at least 75%, preferably at least 80%, more preferably at least 90% of identity with the wild-type protein of the corresponding species (SEQ ID NO: 2 for human treatment, SEQ ID NO: 4 for mouse treatment, and so on).

[0018] In the context of the present application, the percentage of identity between the two homologous sequences is determined by global alignment of the sequences as a whole, by algorithms well known to the skilled person, such as the algorithm disclosed in Needleman and Wunsch (1970). Accordingly, the sequence alignment between two amino acid sequences or between two nucleotide sequences can be performed, for example, by using any software known to 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.

[0019] A "functional fragment" of the p21 protein is any fragment of the wild-type p21 protein or of a functional variant thereof, which fragment retains the function of the p21 protein to enhance the programmed cell removal (PrCR) of tumor cells by macrophages.

[0020] In a preferred embodiment, the monocytes comprised in the pharmaceutical composition of the application contain a replication-defective recombinant virus or a non-viral recombinant nucleic acid, wherein the replication-defective recombinant virus encodes the cyclin-dependent kinase inhibitor p21 and the non-viral recombinant nucleic acid contains a gene encoding p21 placed under the control of regulatory elements to allow its expression. This recombinant virus or nucleic acid makes it possible to overexpress the cyclin-dependent kinase inhibitor p21 protein. The recombinant nucleic acid is preferably a DNA plasmid. We can also use a non-viral Sleeping Beauty stable transposition of the p21 gene from a supercoiled minimal DNA vector, called minicircle. The genetic engineering of monocytes can also be performed by non-viral transfer of p21 mRNA translated in vitro in monocytes.

[0021] "Overexpression of the cyclin-dependent kinase inhibitor p21 protein" means herein that the total expression level of the p21 protein is higher in the macrophages contained in the composition of the application than in a conventional untreated macrophage. This overexpression can be detected by any conventional method able to measure the level of protein, such as Western blot. In order to be used in the composition of the application, the macrophages are genetically modified so that the final expression of p21 is at least two to three times higher than in untreated control macrophages. The stable integration of the p21 gene in the monocyte genome guarantees the sustained expression of the cyclin-dependent kinase inhibitor p21 protein in the differentiated macrophages grafted into the tissue. The durability of the p21 protein expression is further guaranteed in addition to considering the long life of macrophages in the tissue.

[0022] The use of a vector (plasmid or virus or mRNA translated in vitro) can improve the administration of the nucleic acid encoding p21 in the target cell and can also increase the stability of said nucleic acid into said cell, thus enabling a sustained effect.

[0023] In a preferred embodiment, the vector is chosen among adenovirus, adeno-associated virus (AAV), herpes virus, lentivirus, vaccinia virus, cytomegalovirus (CMV), etc., which have proven to efficiently transfect macrophages (Singh G. et al., F1000 research 2015).

[0024] Advantageously, the virus is a defective virus. The term "defective virus" means a virus that is not able to replicate in the target cell. Generally, the genome of the defective virus used in the context of the present application thus lacks at least the sequences required for the replication of the virus in the infected cell. These regions can be (totally or partially) removed or rendered non-functional, or replaced by other sequences, in particular by a recombinant nucleic acid. Preferably, the defective virus still retains the sequences of its genome that are required for the encapsidation of the viral particle.

[0025] In particular, AAV vectors show several advantages such as i) long-lasting expression of the synthetic gene, ii) low risk of pathogenic reaction (as they are artificially manufactured and non-toxic), iii) low immunogenic reaction they elicit and iv) they do not integrate into the human genome. AAVs can be genetically modified to improve the efficacy of the gene expression and to prevent accidental dissemination of the virus. These genetic modifications include deletion of the El region, deletion of the El region and deletion of the E2 or E4 region, or deletion of the entire adenoviral genome except for the cis-acting inverted terminal repeats and the packaging signal. The present application advantageously encompasses such vectors. We can also use non-viral Sleeping Beauty stable transposition of the p21 gene from a supercoiled minimal DNA vector, called minicircle. Gene engineering of monocytes can also be performed by non-viral transfer using in vitro translated p21 mRNA in monocytes. In both methods, plasmids are delivered by transfection of monocytes by electroporation.

[0026] Another advantageous vector for the preparation of the cellular composition according to the present application is an adenoviral vector. Indeed, Haddada H. et al., Biochem. Biophys. Res. Commun (1993) showed that adenoviruses are able to infect very efficiently cells of the monocyte-macrophage lineage, to be stably maintained therein and to express a therapeutic gene. There exist different serotypes of adenoviruses, which differ in their structure and properties, but which are not pathogenic for humans, in particular for non-immunosuppressed subjects. Moreover, these viruses do not integrate into the genome of the cells they infect and can incorporate large fragments of exogenous DNA. Among the different serotypes, use of adenovirus of type 2 or 5 (Ad 2 or Ad 5) is preferred in the context of the present application. In the case of Ad 5 adenovirus, the sequences required for replication are the El A and El B regions. These sequences are preferably deleted in the recombinant nucleic acid used in the present application.

[0027] Another advantageous vector for preparing the cellular composition according to the application is a lentivirus. Lentiviruses such as HIV have the ability to infect both non-dividing and dividing cells and to integrate into the host cell genome. Because of these properties, HIV-based lentiviral vectors have been proposed as good delivery system candidates for gene therapy, but attempts to use them in clinical trials have raised concerns about their safety, including concerns about the risk of gene recombination leading to the production of replication-competent retroviruses in humans. Further modifications of the packaging and genetic components of the virus have been made to develop safer HIV-based lentiviral vector systems. Today, many 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 application.

[0028] Preferred lentiviral vectors are those which have been modified so as to be safe for administration to a mammal. These vectors are for example HIV / SIV vectors known to be useful for human or mammalian 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 HIV and SIV-based lentiviral self-inactivating vectors (Neschadim A. et al., Biol Blood Marrow Transplant. 2007 Dec; 13(12): 1407-16.), adenoviral vectors (Haddada H. et al., Biochem. Biophys. Res. Commun (1993)) and Sleeping Beauty transposon non-viral vectors (Aronovich et al., Human. Molecular. Genetics (2011)).

[0029] A HIV-1 -based self-inactivating (SIN) lentiviral vector encoding the p21 protein can be used, as used in the examples. This vector can encode the p21 protein alone, or fused to another protein such as AIP (aryl hydrocarbon receptor interacting protein), or to a small protein tag such as a Flag tag or a hemagglutinin (HA) tag.

[0030] In a particularly preferred embodiment, the composition of the application also comprises a virus-like particle (VLP) containing SIVmac-VPX to induce the degradation of factors impairing lentiviral infection (Berger G., Gene Therapy (2009)). SIVmac-VPX degrades SAMHD1, identified as an HIV-1 restriction factor, which hydrolyzes dNTPs required for reverse-transcriptase viral replication (Lahouassa et al., Nat Immunol (2012)).

[0031] In an even more particularly preferred embodiment, the monocytes contained in the cellular composition of the application have been transduced with a SIN lentiviral vector containing the following nucleic acid sequence encoding the p21 protein SEQ ID NO: 5:

[0032]

[0033] As mentioned above, the gene encoding p21 is placed under the control of regulatory elements allowing its expression. These regulatory elements generally consist of transcriptional promoter sequences. These sequences can be the sequences naturally responsible for the expression of p21 when these sequences are able to function in monocyte-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 derived from the genome of the monocytes one wishes to infect. Similarly, they can be promoter sequences derived from the genome of a virus. In this respect, one can mention for example the following genes: promoter E2F1 (E2 promoter binding factor 1) or promoter EFS (elongation factor 1 alpha short), SFFV (spleen focus-forming virus easy), CMV (cytomegalovirus), RSV (Rous sarcoma virus), etc. Furthermore, these expression sequences can be modified by the addition of activation sequences, regulatory sequences, etc.

[0034] The skilled person will choose the vector and the regulatory sequences while keeping in mind that the final expression of p21 should be at least 2 or 3 times greater in the macrophages of the application compared to control macrophages mock-transfected.

[0035] The methods for constructing 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 can be isolated and obtained in substantial purity and then introduced into suitable host cells using the various techniques available in the art.

[0036] All techniques for constructing vectors derived from adenovirus, lentivirus or AAV and incorporating a heterologous nucleic acid sequence therein have been described in the literature and can be used in the context of the present application. Methods traditionally used in molecular biology, such as preparation of extracted plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradients, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol-chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in a saline medium, transformation in E. coli, etc. are well known to those skilled in the art and are fully 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].

[0037] After the genetic modification of the viral genome, the virus is propagated and recovered and purified according to standard techniques of molecular biology.

[0038] The pharmaceutical composition of the application contains mononuclear cells overexpressing p21 protein.

[0039] The term "mononuclear cell-derived macrophage" as used herein refers to a mononuclear cell cultured and differentiated into a macrophage from peripheral blood mononuclear cells (PBMC) or 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)). Pluripotent stem cells, myeloid stem cells (CFU-GEMM), bone marrow mononuclear stem cells (CFU-GM), CFU-M, monoblasts or promonocytes can also be used as precursors. Intravenous injection of mononuclear cells induces their engraftment into the bone marrow, spleen and liver and differentiation into macrophages.

[0040] Since PBMCs are present in the blood, a sample of PBMCs can be obtained by collecting blood from a subject entirely harmlessly and non-invasively.

[0041] PBMCs / monocyte-macrophages or their precursors can be extracted and isolated by any technique known to those skilled in the art. These different techniques can involve physical separation steps (centrifugation, cell sorting (FACS), etc.), and selection with immunological compounds (antibodies specific for cell markers, etc.) or biochemical compounds (membrane receptor ligands), etc. The isolated cells can be cultured in different culture media known to those skilled in the art (e.g. RPMI, IMDM), in particular culture media supplemented with serum and amino acids. The cells are cultured under sterile conditions, preferably at 37°C, as shown in the examples. The culture can be performed in culture plates, or preferably in Teflon bags.

[0042] In a preferred embodiment, the cells contained in the composition of the application are obtained by culturing PBMCs under suitable conditions allowing their differentiation. For example, human blood monocytes can be induced to differentiate into macrophages in vitro using three different methods, i.e. 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).

[0043] In a particular embodiment, monocytes purified from PBMC cells are transduced ex vivo with the recombinant nucleic acid, and the transduced cells are then differentiated / cultured into macrophages ex vivo. In this embodiment, the cellular composition of the application contains differentiated macrophages overexpressing the p21 protein.

[0044] In another particular embodiment, the cells are transduced with the recombinant nucleic acid of the application when they are differentiated / cultured into macrophages ex vivo. In this embodiment, the cellular composition of the application also contains differentiated macrophages overexpressing the p21 protein, but the cells are first differentiated into macrophages and then transduced with the recombinant nucleic acid of the application.

[0045] In another particular embodiment, the cells are transduced ex vivo with the recombinant nucleic acid of the application at the monocyte stage of the cells. The cells are then administered to a subject, where they differentiate into macrophages. In this embodiment, the cellular composition of the application contains undifferentiated monocytes overexpressing the p21 protein.

[0046] In another particular embodiment, the recombinant nucleic acid can be administered in vivo to transfect circulating macrophages in situ.

[0047] The terms "in vitro" and "ex vivo" as disclosed herein are equivalent and refer to studies or experiments performed using biological components (e.g. cells or cell populations) isolated from their usual host organism (e.g. an animal or a human). Conversely, the terms "in vivo" or "in situ" refer to studies performed on the whole living organism (e.g. a human) after administration of the composition of the application to a living subject.

[0048] In another embodiment, the cell composition of the application contains monocytes purified from PBMCs which have been transformed ex vivo but not yet differentiated into macrophages ex vivo. Their differentiation will occur in vivo, in the host. In this case, the composition of the application has more than 80% monocytes, or more preferably more than 90% monocytes, still more preferably more than 99% monocytes. This means that the cell composition of the application contains very few other cells, if any.

[0049] The monocytes purified from PBMCs contained in the cell composition of the application are monocytes recovered from the peripheral blood of an individual by conventional methods. These monocytes purified from PBMCs are positive for the following markers: CD14, CD1 lb and CD16, but negative for the following markers: CD56 (NK cell marker), CD3 (T cell marker) and CD20 (B cell marker). Preferably, the cell composition of the application contains more than 90%, preferably more than 95%, ideally more than 99% of such cells. The presence of these markers can be assessed by any conventional means, for example by cytofluorometry (FACS).

[0050] The macrophages differentiated in the tissues after intravenous injection of the cell composition of the application are monocyte-derived cells which are positive for the following markers: CD14, CD1 lb, CD71, CD163 and CD206, but negative for the following markers: CD56 (NK cell marker), CD3 (T cell marker) and CD20 (B cell marker). Preferably, the cell composition of the application contains more than 90%, preferably more than 95%, ideally more than 99% of such cells. The presence of these markers can be assessed by any conventional means, for example by cytofluorometry (FACS).

[0051] The cells to be included in the composition of the application will be transformed with the recombinant nucleic acid of the application in sterile medium under conditions adjusted by the person skilled in the art. In particular, the multiplicity of infection must be adjusted according to the vector used. An example using an SIV virus is given in the following example.

[0052] When using lentiviral vectors, the cells comprised in the composition of the application are contacted with purified virus, for example 50 to 250 pfu per cell, 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 be between 30% and 95%.

[0053] The modified cells thus obtained can then be packaged for immediate use and / or stored for subsequent use. For immediate re-administration, the cells are generally suspended in a phosphate buffer or in physiological saline, at a concentration of between 30 x 10 6 and 10 9 cells per dose. For storing the cells, the cells can be frozen, preferably in the presence of a preservative such as glycerol, DMSO, etc.

[0054] The cells transformed ex vivo according to the application, in particular by using the recombinant viral vectors disclosed above, are tools chosen for the preparation of pharmaceutical compositions, in particular compositions intended to boost the immune and hematopoietic system of a patient.

[0055] The cells of the composition of the application can be derived from the patient himself (the composition thus containing autologous cells) or from a donor (the composition thus containing allogeneic cells). For allogeneic cells, HLA compatibility and matching between the donor and the patient receiving the cells is required.

[0056] The pharmaceutical composition contains the transformed cells described above as active ingredient. It also contains pharmaceutically acceptable excipients.

[0057] The term "pharmaceutically acceptable excipient" refers to an excipient that is useful in preparing a pharmaceutical composition, generally safe, non-toxic and desirable, and includes excipients that are acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. The compositions for treating cancer can generally be administered parenterally, topically, intravenously, intratumorally, orally, subcutaneously, intra-arterially, intracranially, intraperitoneally, intranasally, or intramuscularly. The typical route of administration is intravenously or intratumorally, although other routes can be equally effective.

[0058] For intravenous administration, the composition of the application will be in liquid form. Thus, in addition to the cells, it will contain a pharmaceutically acceptable diluent that does not affect the biological activity of the cells of the application. Examples of such diluents are physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can also contain other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0059] In a preferred embodiment, the composition of the application is in liquid form.

[0060] The pharmaceutical composition of the application can be administered alone or in combination with another pharmaceutical composition or another active ingredient. In particular, the pharmaceutical composition of the application can contain the cell composition of the application in combination with another active ingredient in the same container.

[0061] Such active ingredient 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 TM ), Pirfenidone, Prochlorperazine, Rituximab, Saproin, Tamoxifen, Taxol, Topotecan hydrochloride, Trastuzumab, Vinblastine, Vincristine, and Vinorelbine tartrate.

[0062] The term "combination" as used herein does not imply that the cells of the application and the other active ingredient must be administered at the same time. The term also extends to any use or display involving their administration at different time intervals or in separate containers.

[0063] In another embodiment, the cell composition of the application is combined with, for example, a chemotherapeutic agent as defined above.

[0064] In another embodiment, the cell composition of the application is combined with (or contains) an agent that increases the patient's hematocrit at an effective dose, such as an erythropoiesis stimulating agent (ESA). Such agents are well known and used in the art, including, for example, Epogen® (darbepoetin alfa), Epogen® (epoietin alfa), Epogen® (epoietin alfa), PromegP® (peginesatide), and the like.

[0065] Other combination therapies include administration in combination with a cell-specific antibody (e.g., an antibody selective for a tumor cell marker), radiation, surgery, and / or hormone deprivation.

[0066] Thus, the cell composition of the application is combined with (or contains) an effective dose of said cell-specific antibody.

[0067] Many antibodies are currently used in the clinic for the treatment of cancer, while others are in various stages of clinical development. For example, there are a number of antigens and corresponding monoclonal antibodies used to treat B-cell malignancies. One target antigen is CD20. Rituximab is a chimeric, non-conjugated monoclonal antibody directed against the CD20 antigen. CD20 has an important functional role in the activation, proliferation, and differentiation of B-cells. The monoclonal antibody alemtuzumab targets the CD52 antigen and is used to treat chronic lymphocytic leukemia. A number of antibodies target CD22, which has recently been shown to be effective in treating chemotherapy-resistant hairy cell leukemia in combination with a toxin. Two novel monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the U.S. Food and Drug Administration (FDA). These antibodies are conjugated to a radioisotope. Alemtuzumab (Campath) is used to treat chronic lymphocytic leukemia; gemtuzumab (Mylotarg) is used to treat acute myeloid leukemia; ibritumomab (Zevalin) is used to treat non-Hodgkin's lymphoma; panitumumab (Vectibix) is used to treat colon cancer.

[0068] Angiogenesis inhibitors can also be combined with (or contained in) the compositions of the application.

[0069] In another embodiment, the cell compositions of the application are combined with (or contain) an effective dose of an immune checkpoint modulator, in particular an immune checkpoint inhibitor (ICI).

[0070] "Immune checkpoint inhibitors" (ICIs) include anti-PDl antibodies (such as Nivolumab or Pembrolizumab or Pidilizumab), anti-PD-Ll antibodies (such as Atezolizumab or Durvalumab), anti-CTLA-4 antibodies (such as Ipilimumab or Tremelimumab) and anti-PD-L2 antibodies.

[0071] "Concomitant administration" of the active ingredient and the pharmaceutical composition of the application means administration with the recombinant cells at a time when both the active ingredient and the composition of the application will have a therapeutic effect. This concomitant administration can involve administration of the active ingredient concurrently (i.e. simultaneously), before or after administration of the compound of the application. The appropriate administration times, sequences and dosages for a particular drug and composition of the application will be readily determined by one of ordinary skill in the art.

[0072] It is proposed herein to deliver the cell composition of the application (containing the obtained monocytes expressing p21) in a subject in need thereof as a simple and effective method of treating cancer, in particular leukemia.

[0073] Typically, the subject is a human, but non-human mammals can also be treated, such as companion animals (e.g. dogs, cats, horses, etc.), laboratory mammals (e.g. rabbits, mice, rats, etc.), etc.

[0074] Thus, a "subject in need thereof" as referred to herein is a mammal, preferably a human, suffering from cancer. The cancer can be a liquid or solid cancer, such as, but not limited to, lymphoma, leukemia, carcinoma, melanoma, glioblastoma, sarcoma, myeloma, colorectal tumor, etc., as primary or metastatic cancer. In a particular embodiment, the "subject in need thereof is a human or another mammal suffering from leukemia.

[0075] The present application encompasses a method of treatment by which the cell composition of the application is administered to the subject in need thereof by injection, preferably by intravenous injection. Systemic injection can also be performed by perfusion. These injections are not harmful to the treated subject. Intravenous administration of the cell composition of the application enables increased in vivo phagocytosis of tumor cells present in the blood of the subject, thereby reducing the amount of tumor cells in the subject.

[0076] A particular method of treatment according to the present application comprises:

[0077] 1. Extracting and isolating monocytes or their precursors or pluripotent stem cells from blood or bone marrow or umbilical cord from a subject in need thereof or from a healthy donor,

[0078] 2. Culturing these cells to obtain / isolate a population of monocytes as disclosed above,

[0079] 3. Transforming these cells with a recombinant nucleic acid as defined above,

[0080] 4. Optionally, packaging and / or storing the cells thus obtained, and then

[0081] 5. Administering them to the patient.

[0082] The treatment of the application offers many advantages with respect to other treatments with LAK, TIL or NK (natural killer cells) in adoptive immunotherapy, for example the absence of toxic mediators released by the cells, the fact that the ratio of effector cells to target cells required for cytotoxicity is lower than in other treatments, and the fact that it is not necessary to inject simultaneously a cytokine such as IL-2, the side effects of which are deleterious. Moreover, this treatment can infect only a defined and controlled population of cells, making it possible to select the multiplicity of infection (number of viral particles per cell), to enable irreversible access of the tissue from the blood circulation, and to take full advantage of the central role of macrophages in the body, as described above, by their anti-tumour or anti-infectious activity and their activity in stimulating or modulating the immune system. Furthermore, the pro-inflammatory reprogramming of PrCR+ macrophages enhances innate and adaptive anti-cancer immune responses, which in turn establish a persistent anti-tumour growth microenvironment. Moreover, the considerable longevity of macrophages makes it possible to avoid the obstacles of repeated treatments for the patient.

[0083] The application thus offers new possibilities for more effective treatment, which is less demanding for the patient, cheaper and more repeatable.

[0084] The terms "treat", "treating", "treatment" and the like as used herein refer to reducing or ameliorating the symptoms and / or symptoms associated with a disorder (e.g., leukemia). It will be appreciated that, unless otherwise indicated, treatment of a disorder or condition does not require complete eradication of the disorder, condition or symptoms associated therewith, although such a result is not excluded.

[0085] The effective dosage of the therapeutic entity of the application (e.g., for treating cancer) depends on a variety of factors including the mode of administration, the target site, the physiological condition of the patient, the patient being a human or an animal, other medications with which it is administered, and whether the treatment is prophylactic or therapeutic. The dosage can be titrated to optimize safety and efficacy.

[0086] For prophylactic use, the pharmaceutical composition or drug is administered to a patient susceptible to or otherwise at risk of a disease in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of a disease including biochemical, histological and / or behavioral symptoms of the disease, complications, and intermediate pathological phenotypes that occur during development of the disease. In these prophylactic applications, relatively low dosages are normally administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0087] In contrast, for therapeutic use, a relatively high dosage (50 x 10 6 mononuclear cells per injection per patient) is normally administered at relatively short intervals (usually weekly) until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete improvement. Attached Figure Description

[0088] Figure 1 This demonstrates that p21-dependent PrCR triggers pro-inflammatory reprogramming in macrophages. Figure 1 a to Figure 1 (k) and is beneficial for leukemia remission ( Figure 1 l to Figure 1 (r).

[0089] The attached figure shows that p21 determines programmed cell clearance of macrophages and leukemia regression by regulating SIRPa, and confirms that p21 is beneficial to leukemia regression. Detailed Implementation

[0090] Examples

[0091] The examples given below are not limiting, but rather illustrate the feasibility of expressing p21 protein in macrophages and its therapeutic value in vivo.

[0092] 1. Materials and Methods

[0093] Culture of primary macrophages and leukemia cell lines

[0094] Monocyte-derived macrophages (MDMs) were obtained by differentiating monocytes from the erythrocyte sedimentation rate (ESR) amber layer into macrophages. In accordance with French law, as part of the EFS-INSERM convention, these MDMs were obtained through the French blood bank (Etablissement). Mononuclear cells were isolated from peripheral blood mononuclear cells (PBMC) by adherence to plastic, then cultured in hydrophobic Teflon dishes (Lumox; Duthsher) in macrophage medium (RPMI 1640 supplemented with 200 mM L-glutamine, 100 U penicillin, 100 μg streptomycin, 10 mM HEPES, 10 mM sodium pyruvate, 50 μM β-mercaptoethanol, 1% minimum essential medium vitamins, 1% non-essential amino acids) containing 15% heat-inactivated human serum AB for 6 to 7 days. MDM were then harvested and suspended in macrophage medium containing 10% heat-inactivated fetal bovine serum (FBS). When examined by flow cytometry, MDM obtained by this method had a CD14 positivity rate of 91 to 96%, expressed differentiation markers (C11b and CD71) and M2 macrophage polarization markers (CD163 and CD206). Purity of MDM was also controlled by negative staining for CD56 (NK cells), CD3 (T cells) and CD20 (B cells). Primary blood lymphocytes (PBL) were isolated from the non-adherent PBMC fraction using a T cell negative selection kit (STEM CELL). Lymphocytes obtained by this method were T cells with a CD3 expression rate of 90 to 97% and were cultured in RPMI medium containing 10% FBS. Differentiated MDM (0.125 x 10 6 live leukemia cells (0.125 x 10 6 Jurkat, MT4, CEM, THP1, HEL-5320, K562 or CD34 + acute myeloid leukemia blast (CD34 + AML)) were pre-labeled with a cell tracker for one hour, labeled green (CMFDA) or red (CMTMR). PBL obtained from healthy donors were used as controls. After thorough washing, MDM and leukemia cells were co-cultured in 8x chambered slides in macrophage medium supplemented with 10% FBS in the presence or absence of ZVAD-fmk pan caspase inhibitor (100 μM) for 8 hours in a total volume of 250 μl. After thorough washing, macrophages were fixed (using 2% PFA) and co-localization microscopy analysis was performed. The percentage of PrCR+ macrophages was determined by macrophages (CMFDA + ) internalizing leukemia cells (CMTMR +The number of macrophages determines the total number of macrophages. Time-lapse microscopy was performed directly after co-culturing primary macrophages with leukemia cells. 24 hours after silencing the cyclin-dependent kinase inhibitor p21 in MDM, p21-silenced primary macrophages and leukemia cells were co-cultured. The p21 gene in differentiated MDM was silenced by transfecting 50 nM of an on-target plus siRNA (siRNA p21), a targeted plus siRNA p21 n.12 (SEQ ID NO: 6: 5′AGA CCA GCA UGA CAG AUU U 3′) obtained from Dharmacon. siRNA transfection was performed using the INTERFERin kit (PolyplusTransfection). An equal volume of targeted plus non-targeted siRNA (siCo.) (Dharmacon) was added to control MDM. The efficiency of p21 gene knockdown in MDM was assessed by Western blotting 24 hours after silencing (corresponding to the time of co-culturing with leukemia cells). Two hours after co-culturing macrophages and leukemia cells, PrCR cells were sorted by flow cytometry. + Macrophages. Then sorted PrCR+ macrophages (CMFDA) + CMTMR + ) and PrCR - Macrophages (CMFDA) + The cells were cultured for another 96 hours, and then the membrane expression of scavenger receptor CD163 (by flow cytometry), the expression of IRF5 (by Western blotting), and the secretion of cytokines in the cell supernatant were analyzed (by cytokine microarray analysis).

[0095] Engineered human primary monocytes overexpressing p21

[0096] Primary monocytes (15 × 10⁻⁶ cells) purified were transduced with 150 μg of CAP24 (encoding the p21 gene under the control of the SFFVp promoter, AIP-p21) in the HIV-1-based self-inactivated (SIN) lentiviral vector and virus-like particles (VLPs) containing SIVmac-VPX. 6 (15 × 10⁶ cells) were used to induce the degradation of the myeloid restriction factor SAMHD1 protein by lentiviral infection. Control monocytes were transduced with equal volumes of AIP vector and VLPs-SIV-mac-VPX. Transduction was performed by centrifugation (1200 g, 22 °C) for 1 h and at 37 °C for 1 h. Transduced monocytes (15 × 10⁶ cells) were then labeled with Cell Trace CFSE dye. 6After 1 hour and thorough washing, the transduced monocytes were intravenously injected into male or female NSG mice (6 to 8 weeks old) irradiated with 1 Gy 24 hours after X-ray irradiation. Seven days after p21-monocyte transfer, MT4mCherry+ T cells (1 × 10⁻⁶) were intravenously injected. 6 The overall survival of mice was monitored until 15 days after leukemia transplantation, at which point some mice were sacrificed to monitor PrCR+ macrophages (CFSE) in the bone marrow via confocal microscopy. + mCherry + The presence of PrCR was analyzed by flow cytometry from the spleen and bone marrow of sacrificed mice. + macrophages (CFSE) + mCherry + ) and PrCR - CD163 membrane expression on CFSE macrophages was used to determine PrCR. + Pro-inflammatory activation of macrophages.

[0097] During the in vivo model establishment, CFSE from bone marrow, spleen, and blood was purified 7 days after monocyte transfer. + Cells were used to validate the differentiation of monocytes into macrophages in an NSG mouse model by assessing the expression of differentiation markers (CD71, CD163, and CD14) relative to autologous in vitro differentiated macrophages. Parallel AIP-p21 transduced monocytes were cultured in vitro to assess the upregulation of p21 expression by Western blotting 7 days post-differentiation.

[0098] Mouse treatment studies

[0099] NSG mice were housed and maintained under pathogen-free conditions in the animal facility of the Gustave Roussy Institute. Animal experiments were conducted according to guidelines established by the French Institutional Animal Committee. MT4 leukemia T cells (1 × 10⁻⁶) stably expressing the mCherry fluorescent gene were obtained by lentiviral vector transduction and flow cytometry sorting. 6 (Number of mice) were intravenously injected into female or male mice (6-8 weeks old). mCherry was detected in the bone marrow, spleen, liver, and blood. + The presence of MT4 T ​​cells and disease progression (characterized by weight loss, bone marrow invasion, and significant splenomegaly and overall survival) were assessed for leukemia transplantation 4 weeks after infusion.

[0100] Cloning of SIRPa cDNA

[0101] A cDNA expressing the homo sapiens SIRPa phagocytosis inhibitor factor was cloned into a HIV-1 -based self-inactivating (SIN) lentiviral vector (pRRLEF1-PGK-GFP) between the restriction sites MluI and NheI.

[0102] The sequence of the hsSIRPa cDNA is shown in SEQ ID NO: 7.

[0103] Transduction of monocytes with lentiviral vectors expressing p21 and SIRPa

[0104] Purified primary monocytes (10 7 ) were transduced with 100 μg of CAp24 of a HIV-1 -based self-inactivating (SIN) lentiviral vector encoding the p21 gene under the control of the SFFVp promoter (AIP-p21) and / or 100 μg of CAp24 of a HIV-1 -based self-inactivating (SIN) lentiviral vector encoding the SIRPa gene under the control of the EF1 promoter (PRRL-SIRPa) in the presence of virus-like particles (VLPs) containing SIVmac-VPX to induce degradation of the lentivirus-infected myeloid restriction factor SAMHD1 protein. Control monocytes were transduced with an equivalent amount of AIP and / or PRRL vectors and VLPs-SIV-mac-VPX. Transductions were performed in the presence of polybrene (10 μg / ml) for 3 hours at 37°C. Transduced monocytes (10 7 ) were then labeled with the Cell Trace CFSE dye for 1 hour and washed thoroughly before being injected intravenously in male or female NSG mice (6 to 8 weeks old) at 1 Gray of radiation at the time of 24 hours after X-ray irradiation of the mice or differentiated in vitro into macrophages as described in the material and methods of the main patent.

[0105] 2. Results

[0106] The inventors investigated the molecular mechanisms by which macrophages can regulate PrCR. They evaluated a) phagocytosis of a panel of different live leukemic cells (labeled with the red fluorescent cell tracer CMTMR) by primary anti-inflammatory pro-tumoral human monocyte-derived macrophages (MDMs) (labeled with the green fluorescent cell tracer CMFDA) Figure 1

[0107] They observed, using a confocal microscope, that in the absence of MIC inhibitors, human primary macrophages phagocytosed acute T lymphoblast cells (Jurkat, CEM or MT4 cells, Figure 1 b and Figure 1 c) acute myeloid cells (THP1 cells,​Figure 1 c) Acute megakaryocytes (UT-7 cells, not shown), erythroblasts (HEL-5320, Figure 1 c) Chronic myeloid lymphocytes (K562 cells, Figure 1 c) and primary transformed CD34 purified from the blood of patients with acute myeloid leukemia. + Mother cell ( Figure 1 During co-culture (d), phagocytosis of live tumor cells significantly increased, while live autologous or xenogeneic untransformed peripheral blood lymphocytes (PBLs) remained unaffected. Figure 1 c). Pan-cysteine ​​inhibitor (ZVAD) did not reduce phagocytosis of target cells. Figure 1 (c). These results indicate that macrophages may spontaneously develop PrCR even in the absence of MIC blockade.

[0108] The inventors then observed that once MT4 cells were internalized, they were rapidly degraded by lysosomes. Figure 1 e and Figure 1 They also revealed that PrCR can induce functional reprogramming of phagocytic macrophages, shifting them from an anti-inflammatory phenotype to a pro-inflammatory phenotype (as revealed below: increased expression of the IRF5 transcription factor). Figure 1 Decreased expression of CD163 scavenger receptor membrane (g), CD163 scavenger receptor membrane ( Figure 1 h), and cell sorting "PrCR" + Phagocytic macrophages release pro-inflammatory cytokines (such as MCP-1, serine protease inhibitor (Serpin), and IL-8). Figure 1 (i)).

[0109] Furthermore, the inventors demonstrated that p21, a cyclin-dependent kinase inhibitor overexpressed in primary human macrophages, is a major regulator of PrCR. Phagocytosis of live MT4 cells by human primary macrophages deprived of p21 was inhibited. Figure 1 j and Figure 1 The results (k) reveal this fact. In summary, these results indicate that p21 expression determines the pro-inflammatory reprogramming of macrophages by inducing PrCR.

[0110] The therapeutic potential of manipulating PrCR by adoptive transfer of engineered human primary monocytes overexpressing p21 (p21EHM) was then praised. The biological effects of adoptive transfer of p21EHM into NOD / SCID mice were determined prior to transplantation of HTLV-1-transformed MT4 cells. Control mice developed leukemia (as revealed below: weight loss) Figure 1 L), bone marrow invasion ( Figure 1 (m) and significant splenomegaly in transplanted mice (m) Figure 1of n). After transfer of CFSE-labeled p21EHMs into engrafted mice, it was observed in vivo that they differentiated into macrophages Figure 1 of o and not shown) and the presence of MT4-phagocytizing macrophages was detected in engrafted mice: in bone marrow Figure 1 of p), in liver and in spleen (not shown).

[0111] It was also observed that MT4-phagocytizing macrophages underwent a shift from an anti-inflammatory to a pro-inflammatory phenotype (as revealed by a decrease in membrane expression of CD163 and an increase in secretion of IFNy and IL-1b; Bibliography of q and not shown). Interestingly, p21EHM-based cell therapy led to a delay in disease progression and a significant improvement in overall survival of treated mice ​ of r).

[0112] In conclusion, these data suggest that adoptive transfer of p21EHMs represents a new therapeutic strategy to treat hematological malignancies by inducing macrophage PrCR.

[0113] To further characterize the molecular link between p21 and programmed cell clearance, the effect of p21 expression on the expression of the phagocytosis inhibitor SIRPa was determined. Primary human monocytes were transduced with lentiviral vectors expressing p21 and / or SIRPa, in combination with their respective control empty vectors pCo. (pAIP and / or pRRL) or not. A fraction of monocytes was differentiated into macrophages in vitro for 7 days and the expression of p21 and SIRPa was determined by Western blot. The efficiency of transduction was verified in each case. Overexpression of p21 was detected to inhibit SIRPa expression, revealing that p21 expression negatively regulates SIRPa expression. To determine the effect of these transductions on programmed cell clearance, the transduced macrophages were then co-cultured with leukemia MT4 cells expressing the mCherry fluorescent protein and the phagocytosis of mCherry + MT4 cells by macrophages (PrCR + macrophages). It was observed that the increase in p21 and SIRPa expression respectively enhanced and attenuated the phagocytosis of mCherry + MT4 cells by macrophages. Moreover, the increase in SIRPa expression in p21-transduced macrophages inhibited this process, demonstrating that p21 determines programmed cell clearance of macrophages by regulating SIRPa expression. The effect of these regulations on leukemia progression was then analyzed. Transduced monocytes were adoptively transferred into NOD / SCID mice, which were injected with mCherry +MT4 cells, and overall survival of the transplanted mice was analyzed (n=5 mice per group). P values were calculated using the Mantel-Cox test and revealed statistical significance between analyzed groups *** p<0.001). Altogether, these results demonstrate that adoptive transfer of monocytes overexpressing p21 (p21 EHM) can delay leukemia progression, possibly by modulating SIRPa-dependent programmed cell clearance of macrophages.

[0114] ​

[0115] Advani R. et al., N Engl J Med (2018) Nov 1; 379(18): 1711-1721

[0116] Andressen R. et al., Cancer Res. (1990) 50:7450

[0117] Arandjelovic S and Ravichandran KS, Nat Immunol (2015) Sep; 16(9):907-17

[0118] Aronovich EL et al., Human. Molecular. Genetics (2011) Vol. 20, Review Issue 1 R14-R20

[0119] Bartholeyns J et al., Anticancer Res. 11 (1991) 1201-1204

[0120] Berger G., Gene Therapy (2009) pages 16, 159-163

[0121] Chao MP et al., Nat Rev Cancer (2011) Dec 8; 12(1):58-67

[0122] Feng et al., Nat Comm (2018) Aug 10; 9(1):3194.

[0123] Haddada H. et al., Biochem. Biophys. Res. Commun (1993) Sep 30; 195(3):1174-83.

[0124] Jaiswal S., Trends Immunol. (2010) Jun; 31(6):212-9

[0125] Jaiswal et al., Cell (2009) Jul 23; 138(2): 271-85

[0126] Leyva F. et al., BMC biotechnology (2011); 11: 13

[0127] Majeti et al., Cell (2009) Jul 23; 138(2): 286-99

[0128] Neschadim A. et al., Biol Blood Marrow Transplant. 2007 Dec; 13(12): 1407-16.

[0129] Pardoll DM., Nat Rev Cancer (2012) Mar 22; 12(4): 252-64.

[0130] Sharma SH et al., Cell (2015) Apr 9; 161(2): 205-14

[0131] Singh G. et al., F1000 research 2015, 4:495

[0132] Weiskopf K, Eur J Cancer (2017) May; 76: 100-109. SEQUENCE LISTING <110> Gustaf Ruse Institute <120> P21 expressing monocytes for treatment of cancer cells <130> B378361PCT D39471 <150> EP19305963.1 <151> 2019-07-19 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 2122 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Homo p21 mRNA transcript variant 2 <400> 1 ggtggctatt ttgtccttgg gctgcctgtt ttcagctgct gcaaccacag ggatttcttc 60 tgttcaggcg ccatgtcaga accggctggg gatgtccgtc agaacccatg cggcagcaag 120 gcctgccgcc gcctcttcgg cccagtggac agcgagcagc tgagccgcga ctgtgatgcg 180 ctaatggcgg gctgcatcca ggaggcccgt gagcgatgga acttcgactt tgtcaccgag 240 acaccactgg agggtgactt cgcctgggag cgtgtgcggg gccttggcct gcccaagctc 300 taccttccca cggggccccg gcgaggccgg gatgagttgg gaggaggcag gcggcctggc 360 acctcacctg ctctgctgca ggggacagca gaggaagacc atgtggacct gtcactgtct 420 tgtacccttg tgcctcgctc aggggagcag gctgaagggt ccccaggtgg acctggagac 480 tctcagggtc gaaaacggcg gcagaccagc atgacagatt tctaccactc caaacgccgg 540 ctgatcttct ccaagaggaa gccctaatcc gcccacagga agcctgcagt cctggaagcg 600 cgagggcctc aaaggcccgc tctacatctt ctgccttagt ctcagtttgt gtgtcttaat 660 tattatttgt gttttaattt aaacacctcc tcatgtacat accctggccg ccccctgccc 720 CCCAGCCTCT GGCATTAGAA TTATT TAAAC AAAA ACTAGGCGGTT GAATGAGAGG TTCCT 780 AAGAGTGCTG GGCATTTTTA TTT TATGAAA TACTATT TAAAGCCTCCT CATCCC GTGTT C 840 TCCTTTTCCT CTCT CCCGGAGGTT GGGTGGGCCG GCTTCATGCC AGCTACTTCCT CTCCTC 900 CCACTTGTCCT GCTGGGTGGT ACCCTCTGGAG GGTGTGGCTCCTTCCC ATCGCTGTCACA 960 GGCGGTTATG AAATT CACCCCCTTT CCTGGACACT CAGACCTGAA TTCTTTTTCA TTTGA 1020 GAAGTAAACA GATGGCACCT TGAAGGGGCCT CACC GAGTGGGGCATCAT CAAAAACTTT 1080 GGAGTCCCCT CACCTCCTCT AAGGTTGGGC AGGTTGACCC TGAAGTGAGC ACAGCCTAGG 1140 GCTGAGCTGG GGACCTGGTA CCCTCCTGGC TCTTGATACC CCCCTCTGTC TTGTGAAGGC 1200 AGGGGGAAAGT GGGGTCCTGG AGCAGACCAC CCCGCCTGCC CTCATGGCCC CTCTGACCT 1260 GC ACTGGGGAG CCCGTCTCAGT GTTGAGCCTT TTCCCTCTTT GGCTCCCTGT ACCTTTT 1320 GAGGAGCCCC AGCTACCCCT TCTTCTCCAG CTGGGCTCTG CAATTCCCCT CTGCTGCTGT 1380 CCTCCCCCTT GTCCTTTCCC TTCAGTACCC TCTCAGCTCC AGGTGGCTCT GAGGTGCCTG 1440 tcccaccccc acccccagct caatggactg gaaggggaag ggacacacaa gaagaagggc 1500 accctagttc tacctcaggc agctcaagca gcgaccgccc cctcctctag ctgtgggggt 1560 gagggtccca tgtggtggca caggccccct tgagtggggt tatctctgtg ttaggggtat 1620 atgatggggg agtagatctt tctaggaggg agacactggc ccctcaaatc gtccagcgac 1680 cttcctcatc caccccatcc ctccccagtt cattgcactt tgattagcag cggaacaagg 1740 agtcagacat tttaagatgg tggcagtaga ggctatggac agggcatgcc acgtgggctc 1800 atatggggct gggagtagtt gtctttcctg gcactaacgt tgagcccctg gaggcactga 1860 agtgcttagt gtacttggag tattggggtc tgaccccaaa caccttccag ctcctgtaac 1920 atactggcct ggactgtttt ctctcggctc cccatgtgtc ctggttcccg tttctccacc 1980 tagactgtaa acctctcgag ggcagggacc acaccctgta ctgttctgtg tctttcacag 2040 ctcctcccac aatgctgaat atacagcagg tgctcaataa atgattctta gtgactttac 2100 ttgtaaaaaa aaaaaaaaaa aa 2122 <210> 2 <211> 164 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Human p21 protein isoform 1 <400> 2 Met Ser Glu Pro Ala Gly Asp Val Arg Gin Asn Pro Cys Gly Ser Lys 1 5 10 15 Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gin Leu Ser Arg 20 25 30 Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gin Glu Ala Arg Glu Arg 35 40 45 Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80 Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser Pro Ala Leu Leu Gin Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gin Ala Glu 115 120 125 Gly Ser Pro Gly Gly Pro Gly Asp Ser Gin Gly Arg Lys Arg Arg Gin 130 135 140 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu He Phe Ser 145 150 155 160 Lys Arg Lys Pro <210> 3 <211> 1913 <212> DNA <213> Mus musculus <220> <221> misc_feature <223> p21 Mus mRNA Transcript Variant 1 <400> 3 tgcagcagcc gagaggtgtg agccgccgcg gtgtcagagt ctaggggaat tggagtcagg 60 cgcagatcca cagcgatatc cagacattca gagccacagg caccatgtcc aatcctggtg 120 atgtccgacc tgttccgcac aggagcaaag tgtgccgttg tctcttcggt cccgtggaca 180 gtgagcagtt gcgccgtgat tgcgatgcgc tcatggcggg ctgtctccag gaggcccgag 240 aacggtggaa ctttgacttc gtcacggaga cgccgctgga gggcaacttc gtctgggagc 300 gcgttcggag cctagggctg cccaaggtct acctgagccc tgggtcccgc agccgtgacg 360 acctgggagg ggacaagagg cccagtactt cctctgccct gctgcagggg ccagctccgg 420 aggaccacgt ggccttgtcg ctgtcttgca ctctggtgtc tgagcggcct gaagattccc 480 cgggtgggcc cggaacatct cagggccgaa aacggaggca gaccagcctg acagatttct 540 atcactccaa gcgcagattg gtcttctgca agagaaaacc ctgaagtgcc cacgggagcc 600 ccgccctctt ctgctgtggg tcaggaggcc tcttccccat cttcggcctt agccctcact 660 ctgtgtgtct taattattat ttgtgtttta atttaaacgt ctcctgtata tacgctgcct 720 gccctctccc agtctccaaa cttaaagtta tttaaaaaaa gaacaaaaca aaacaaaaaa 780 aaccaaaaca aaacaaacct aaattagtag gacggtaggg cccttagtgt gggggatttc 840 tattatgtag attattatta tttaagcccc tcccaaccca agctctgtgt ttcctatacc 900 ggaggaacag tcctactgat atcaacccat ctgcatccgt ttcacccaac ccccctcccc 960 ccattccctg cctggttcct tgccacttct tacctggggg tgatcctcag acctgaatag 1020 cactttggaa aaatgagtag gactttgggg tctccttgtc acctctaagg ccagctagga 1080 tgacagtgaa gcagtcacag cctagaacag ggatggcagt taggactcaa ccgtaatatc 1140 ccgactcttg acattgctca gacctgtgaa gacaggaatg gtccccactc tggatcccct 1200 ttgccactcc tggggagccc acctctcctg tgggtctctg ccagctgccc ctctattttg 1260 gagggttaat ctggtgatct gctgctcttt tcccccaccc catacttccc cttctgcagg 1320 tcggcaggag gcatatctag gcacttgccc cacagctcag tggactggaa gggaatgtat 1380 atgcagggta cactaagtgg gattccctgg tcttacctta ggcagctcca gtggcaaccc 1440 cctgcattgt gggtctaggg tgggtccttg gtggtgagac aggcctccca gagcattcta 1500 tggtgtgtgg tggtgggggt gggcttatct gggatgggga ccccagttgg ggttctcagt 1560 gacttctccc atttcttagt agcagttgta caaggagcca ggccaagatg gtgtcttggg 1620 ggctaaggga gctcacagga cactgagcaa tggctgatcc tttctcagtg ttgaataccg 1680 tgggtgtcaa agcacttagt gggtctgact ccagccccaa acatccctgt ttctgtaaca 1740 tcctggtctg gactgtctac ccttagcccg caccccaaga acatgtattg tggctccctc 1800 cctgtctcca ctcagattgt aagcgtctca cgagaaggga cagcaccctg cattgtcccg 1860 agtcctcaca cccgacccca aagctggtgc tcaataaata cttctcgatg att 1913 <210> 4 <211> 159 <212> PRT <213> Mouse (Mus musculus) <220> <221> MISC_FEATURE <223> p21 mouse protein <400> 4 Met Ser Asn Pro Gly Asp Val Arg Pro Val Pro His Arg Ser Lys Val 1 5 10 15 Cys Arg Cys Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Arg Arg Asp 20 25 30 Cys Asp Ala Leu Met Ala Gly Cys Leu Gln Glu Ala Arg Glu Arg Trp 35 40 45 Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asn Phe Val Trp 50 55 60 Glu Arg Val Arg Ser Leu Gly Leu Pro Lys Val Tyr Leu Ser Pro Gly 65 70 75 80 Ser Arg Ser Arg Asp Asp Leu Gly Gly Asp Lys Arg Pro Ser Thr Ser 85 90 95 Ser Ala Leu Leu Gln Gly Pro Ala Pro Glu Asp His Val Ala Leu Ser 100 105 110 Leu Ser Cys Thr Leu Val Ser Glu Arg Pro Glu Asp Ser Pro Gly Gly 115 120 125 Pro Gly Thr Ser Gln Gly Arg Lys Arg Arg Gln Thr Ser Leu Thr Asp 130 135 140 Phe Tyr His Ser Lys Arg Arg Leu Val Phe Cys Lys Arg Lys Pro 145 150 155 <210> 5 <211> 495 <212> DNA <213> Mus musculus <220> <221> misc_feature <223> Nucleic acid sequence of p21 in SIN lentivirus vector in example <400> 5 atgtcagaac cggctgggga tgtccgtcag aacccatgcg gcagcaaggc ctgccgccgc 60 ctcttcggcc cagtggacag cgagcagctg agccgcgact gtgatgcgct aatggcgggc 120 tgcatccagg aggcccgtga gcgatggaac ttcgactttg tcaccgagac accactggag 180 ggtgacttcg cctgggagcg tgtgcggggc cttggcctgc ccaagctcta ccttcccacg 240 gggccccggc gaggccggga tgagttggga ggaggcaggc ggcctggcac ctcacctgct 300 ctgctgcagg ggacagcaga ggaagaccat gtggacctgt cactgtcttg tacccttgtg 360 cctcgctcag gggagcaggc tgaagggtcc ccaggtggac ctggagactc tcagggtcga 420 aaacggcggc agaccagcat gacagatttc taccactcca aacgccggct gatcttctcc 480 aagaggaagc cctaa 495 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA to p21 <400> 6 agaccagcau gacagauuu 19 <210> 7 <211> 1515 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> hsSIRPa cDNA <400> 7 atggagcccg ccggcccggc ccccggccgc ctcgggccgc tgctctgcct gctgctcgcc 60 gcgtcctgcg cctggtcagg agtggcgggt gaggaggagc tgcaggtgat tcagcctgac 120 aagtccgtgt tggttgcagc tggagagaca gccactctgc gctgcactgc gacctctctg 180 atccctgtgg ggcccatcca gtggttcaga ggagctggac caggccggga attaatctac 240 aatcaaaaag aaggccactt cccccgggta acaactgttt cagacctcac aaagagaaac 300 aacatggact tttccatccg catcggtaac atcaccccag cagatgccgg cacctactac 360 tgtgtgaagt tccggaaagg gagccccgat gacgtggagt ttaagtctgg agcaggcact 420 gagctgtctg tgcgcgccaa accctctgcc cccgtggtat cgggccctgc ggcgagggcc 480 acacctcagc acacagtgag cttcacctgc gagtcccacg gcttctcacc cagagacatc 540 accctgaaat ggttcaaaaa tgggaatgag ctctcagact tccagaccaa cgtggacccc 600 gtaggagaga gcgtgtccta cagcatccac agcacagcca aggtggtgct gacccgcgag 660 gacgttcact ctcaagtcat ctgcgaggtg gcccacgtca ccttgcaggg ggaccctctt 720 cgtgggactg ccaacttgtc tgagaccatc cgagttccac ccaccttgga ggttactcaa 780 cagcccgtga gggcagagaa ccaggtgaat gtcacctgcc aggtgaggaa gttctacccc 840 cagagactac agctgacctg gttggagaat ggaaacgtgt cccggacaga aacggcctca 900 accgttacag agaacaagga tggtacctac aactggatga gctggctcct ggtgaatgta 960 tctgcccaca gggatgatgt gaagctcacc tgccaggtgg agcatgacgg gcagccagcg 1020 gtcagcaaaa gccatgacct gaaggtctca gcccacccga aggagcaggg ctcaaatacc 1080 gccgctgaga acactggatc taatgaacgg aacatctata ttgtggtggg tgtggtgtgc 1140 accttgctgg tggccctact gatggcggcc ctctacctcg tccgaatcag acagaagaaa 1200 gcccagggct ccacttcttc tacaaggttg catgagcccg agaagaatgc cagagaaata 1260 acacaggaca caaatgatat cacatatgca gacctgaacc tgcccaaggg gaagaagcct 1320 gctccccagg ctgcggagcc caacaaccac acggagtatg ccagcattca gaccagcccg 1380 cagcccgcgt cggaggacac cctcacctat gctgacctgg acatggtcca cctcaaccgg 1440 acccccaagc agccggcccc caagcctgag ccgtccttct cagagtacgc cagcgtccag 1500 gtcccgagga agtga 1515

Claims

1. A pharmaceutical composition comprising genetically modified monocytes containing a vector encoding a cyclin-dependent kinase inhibitor p21 protein and a pharmaceutically acceptable excipient.

2. The pharmaceutical composition of claim 1, wherein the monocytes contain a replication-deficient recombinant virus encoding a cyclin-dependent kinase inhibitor p21 under the control of regulatory elements that allow its expression.

3. The pharmaceutical composition of claim 2, wherein the virus is a replication-deficient lentivirus.

4. The pharmaceutical composition of claim 3, wherein the replication-deficient lentivirus is a self-inactivating (SIN) lentiviral vector based on HIV-1.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated in an intravenously injectable form or a perfusion form.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains 30 x 10 6 to 10 9 transduced mononuclear cells per mL.

7. The pharmaceutical composition of claim 1, wherein the monocytes contain a Sleeping Beauty transposition system encoding a cyclin-dependent kinase inhibitor p21 under the control of regulatory elements that allow its expression.

8. The pharmaceutical composition of claim 1, wherein the p21 protein is SEQ ID NO: 2 or a functional variant or fragment thereof.

9. The pharmaceutical composition of claim 2, wherein the virus contains the nucleic acid of SEQ ID NO:

5.

10. The pharmaceutical composition of claim 9, wherein the nucleic acid of SEQ ID NO: 5 is under the control of the SFFV promoter.

11. The pharmaceutical composition of claim 7, wherein the transposition system contains the nucleic acid of SEQ ID NO:

5.

12. The pharmaceutical composition of claim 11, wherein the nucleic acid of SEQ ID NO: 5 is under the control of the SFFV promoter.

13. The pharmaceutical composition of claim 1, further containing an effective dose of an agent that increases the hematocrit of a patient, a chemotherapeutic agent, a cell-specific antibody, or an immune checkpoint inhibitor (ICI).

14. Use of the pharmaceutical composition of any one of claims 1 to 13 for the manufacture of a medicament for the treatment of a mammal having leukemia.

15. The use according to claim 14, wherein the composition contains 50 x 10 6 6 mononuclear cells per injection dose and is administered weekly until disease progression is slowed.

16. The use of claim 14, wherein the mammal is a human.

17. A combination product comprising a pharmaceutical composition as defined in any one of claims 1 to 13, and an effective dose of an agent that increases the hematocrit, a chemotherapeutic agent, a cell-specific antibody, or an immune checkpoint inhibitor (ICI), for simultaneous or sequential use in the treatment of a mammal having leukemia.

18. The combination product of claim 17, wherein the mammal is a human.

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