Mononuclear cells expressing p21 for cancer cell therapy

Abstract

The present application relates to P21-expressing monocytes for cancer cell therapy. Identification of effective targets that mitigate programmed cell clearance (PrCR) of tumor cells by macrophages has a very high interest. The present inventors have determined that the cyclin-dependent kinase inhibitor p21 protein is an emphasized regulatory factor of macrophage mediated PrCR. In addition, they also show that mononuclear cells that adoptively transfer overexpression p21 induce in vivo macrophages PrCR and transitions from anti-inflammatory phenotypes to pro-inflammatory phenotypes, delay cancer progression and significantly increase the overall survival rate of mice in which cancer cells are transplanted. Thus, the present invention relates to therapeutic compositions comprising monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein, and their use for treating mammals suffering from cancer, in particular leukemia.

Description

[0001] This application is a divisional application of Chinese patent application No. 2020800519604, filed on July 17, 2020, entitled "P21-expressing mononuclear cells for cancer cell therapy". Technical Field

[0002] Identifying effective targets for mitigating programmed cell clearance (PrCR) of tumor cells by macrophages is of great interest. The inventors have identified the cyclin-dependent kinase inhibitor p21 protein as a key regulator of macrophage-mediated PrCR. Furthermore, they have demonstrated 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 overall survival in mice with transplanted cancer cells. Therefore, this invention relates to therapeutic compositions comprising monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein, and their use in treating mammals with cancer, particularly leukemia. Background Technology

[0003] When properly activated, effector cells of both the innate and adaptive immune systems possess the ability to successfully attack cancer cells. In the past, it has been proposed to enhance the normal capacity of a patient's immune response by inhibiting negative adaptive immunomodulatory factors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), or by using adopted transfer of modified patient-derived autoimmune cells such as chimeric antigen receptor (CAR) T cells. This approach has led to significant advances in the treatment of solid tumors and hematological disorders (Pardoll DM., Nat Rev Cancer (2012) and Sharma SH et al., Cell 2015).

[0004] However, a more comprehensive understanding of immune regulation is needed to develop new cancer treatment strategies.

[0005] Programmed cell clearance (PrCR) is a macrophage-mediated immune surveillance process through which target cells are recognized and phagocytosed (Jaiswal et al., Trends Immunol (2010)). PrCR was initially described as a key mechanism linking programmed cell death with the removal of dead cells (Arandjelovic and Ravichandran, Nat Immunol (2015)), PrCR also participates in the clearance of living tumor cells (Majeti et al., Cell (2009)).

[0006] The efficacy of PrCR depends on the balance between macrophages recognizing pro-phagocytic ("eat me") signals and target cancer cells inhibiting macrophages by activating anti-phagocytic ("don't eat me") pathways. (Chao et al., Nat Rev Cancer (2011)). Recently, the "eat me" signal calreticulin (CRT) secreted by macrophages has been identified as the first signal that favors PrCR by binding to target cancer cells through desialoglycan (Feng et al., Nat Comm (2018). Conversely, the transmembrane protein CD47 was identified as a "don't eat me" signal that inhibits PrCR by binding to and activating signal regulatory protein α (SIRPα), a phagocytic inhibitory receptor expressed on the macrophage membrane (Jaiswal et al., 2018). Cell (2009)). A study showed that blocking the CD47-SIRPα axis with a monoclonal antibody that blocks CD47 selectively induces phagocytosis of tumor cells and demonstrated efficacy in various preclinical models, including 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 CD47 blocking and rituximab has shown promising activity in patients with relapsed or refractory non-Hodgkin's lymphoma (Advani R. et al., 2017). N Engl J Med (2018) highlights the fact that targeting macrophage immune checkpoints (MICs) to remove PrCR limitations represents a significant therapeutic opportunity.

[0007] Therefore, identifying effective targets for mitigating programmed cell clearance (PrCR) of tumor cells by macrophages is of great interest. This invention addresses this need. Summary of the Invention

[0008] As disclosed in the following examples, the inventors discovered that consuming the cyclin-dependent kinase inhibitor p21 in primary human macrophages blocks the phagocytic capacity of said cells, thereby reducing their immune surveillance. Figure 1 j and Figure 1 They identified p21 as a key regulator of macrophage-mediated pro-inflammatory cytokinesis (PrCR). They showed that overexpression of p21 can act as an inhibitor of macrophage immune checkpoints (MICs), thus ultimately leading to the recognition, phagocytosis, and destruction of tumor cells. Accordingly, they demonstrated that adoptive transfer of p21-overexpressing monocytes induced macrophage PrCR and a shift from an anti-inflammatory to a pro-inflammatory phenotype in vivo, delayed cancer progression, and significantly improved overall survival in mice with transplanted cancer cells. Figure 1(r).

[0009] This invention relates to cell compositions and their incorporation into pharmaceutical compositions for cancer treatment, and more specifically, for cancer immunotherapy. Specifically, this invention relates to the isolation, culture, activation, and genetic modification of cells of a mononuclear phagocytic system, and the use of said cells in cell therapy, such as in adoptive immunotherapy.

[0010] The mononuclear phagocytic system comprises peripheral blood monocytes, their bone marrow or blood precursors, and tissue macrophages. Monocytes form in the bone marrow, mature, leave the bone marrow, and migrate to tissues via the peripheral blood. The half-life of human monocytes circulating in the blood is approximately 3 days. When monocytes reach tissues, they are called macrophages. The total number of tissue macrophages far exceeds the number of circulating monocytes, approximately 400 times greater. Macrophages are present throughout the body, but are particularly abundant in the liver (Kupffer cells), lymph nodes, lungs, peritoneum, and skin (Langerhans cells). The migration of monocytes from the systemic circulation to tissues is irreversible.

[0011] Monocytes and macrophages are known to have many important functions, including inducing immune responses in the acute phase, regulating hematopoietic function, activating the immune system, blood clotting, destroying organisms and tumor cells, as well as tissue repair and scar formation.

[0012] Monocytes and macrophages can also be used in adoptive immunotherapy to treat some types of cancer in humans. Typically, these cells are purified from a patient's circulating blood, cultured in vitro, and activated with interferon-gamma to induce differentiation and increase their tumor-killing ability before being injected into the patient. Genes can also be transferred in vitro into monocyte-derived macrophages using suitable vectors, thereby giving them superior properties in terms of cytotoxicity and stimulation of the immune system.

[0013] Therefore, 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.

[0014] This pharmaceutical composition is hereinafter referred to as "the composition of the present invention", "the pharmaceutical composition of the present invention" or "the cell composition of the present invention".

[0015] In particular, the present invention relates to the use of monocytes overexpressing the cyclin-dependent kinase inhibitor p21 protein in the preparation of medicaments designed to enhance the immune and hematopoietic systems of patients to treat cancer (especially leukemia).

[0016] As used herein, the term "p21 protein" can be used interchangeably to refer to cyclin-dependent kinase inhibitor 1, which is also known as "p21 Cip1 "p21" Waf1 The protein contains "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 functioning as a regulator of cell cycle progression during G1 and S phases. The binding of p21 to the CDK complex occurs through the N-terminal domain of p21, which is homologous to other CIP / CDK repressors p27 and p57. As a major target of p53 activity, it is commonly associated with linking DNA damage and cell cycle arrest. This protein is represented by SEQ ID NO: 1 (NM_078467) located on human chromosome 6 (6p21.2). CDKN1A The gene encodes this protein. In mice, this protein is represented by SEQ ID NO: 3 (NM_007669). CDKN1A Gene encoding. 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).

[0017] The term "p21 protein" in this article also covers the functional variants and / or fragments of the aforementioned p21 protein.

[0018] "Functional variants" are wild-type p21 proteins from animal species other than humans or mice (e.g., horses, dogs, cats, or cattle). These proteins are now well-characterized, and their sequences can be readily retrieved from general databases. "Functional variants" are also mutant forms of the natural p21 protein whose amino acid sequence has at least 75%, preferably at least 80%, and more preferably at least 90% identity with the wild-type protein of the corresponding species (SEQ ID NO: 2 for human treatment, SEQ ID NO: 4 for mouse treatment, etc.).

[0019] In the context of this invention, the percentage of identity between the two homologous sequences is determined by a global alignment of the entire sequence, performed using an algorithm well known to those skilled in the art, such as the one disclosed in Needleman and Wunsch (1970). Accordingly, sequence alignment between two amino acid sequences or two nucleotide sequences can be performed, for example, using any software known to those skilled in the art, such as the "needle" software, with a "Gap open" parameter of 10, a "Gapextend" parameter of 0.5, and a "Blosum 62" matrix.

[0020] A “functional fragment” of the p21 protein is any fragment of the wild-type p21 protein or a functional variant thereof that preserves the function of the p21 protein to enhance the programmed cellular clearance (PrCR) of tumor cells by macrophages.

[0021] In a preferred embodiment, the monocytes included in the pharmaceutical composition of the present invention contain a replication-deficient recombinant virus or a non-viral recombinant nucleic acid, wherein the replication-deficient recombinant virus encodes the cyclin-dependent kinase inhibitor p21, and the non-viral recombinant nucleic acid contains a gene encoding p21, which is placed under the control of a regulatory element to allow its expression. This recombinant virus or nucleic acid allows for the overexpression of 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 transposon of the p21 gene derived from a supercoiled minimal DNA vector (called a small loop). Genetic engineering of monocytes can also be performed by non-viral transfer of p21 mRNA transcribed in vitro into monocytes.

[0022] "Overexpression of the cyclin-dependent kinase inhibitor p21 protein" herein refers to the overall expression level of p21 protein in macrophages contained in the compositions of the present invention being higher than that in conventional untreated macrophages. This overexpression can be detected by any conventional method capable of measuring protein levels, such as Western blotting. For use in the compositions of the present invention, macrophages are genetically modified such that the final expression of p21 is at least two to three times higher than that of untreated control macrophages. Stable integration of the p21 gene into the monocyte genome ensures the sustained expression of the cyclin-dependent kinase inhibitor p21 protein in differentiated macrophages transplanted into tissues. In addition to considering the long lifespan of macrophages in tissues, the persistence of p21 protein expression is further guaranteed.

[0023] Using vectors (plasmids, viruses, or in vitro transcribed mRNA) can improve the delivery of nucleic acids encoding p21 to target cells and increase the stability of the nucleic acids entering the cells, thereby enabling a lasting effect.

[0024] In a preferred embodiment, the vector is selected from adenovirus, adeno-associated virus (AAV), herpesvirus, lentivirus, vaccinia virus, cytomegalovirus (CMV), etc., and these vectors have been shown to effectively transfect macrophages (Singh G. et al., F1000 research 2015).

[0025] Advantageously, the virus is a defective virus. The term "defective virus" means a virus that cannot replicate in target cells. Typically, the genome of a defective virus used in the context of this invention therefore lacks at least the sequences required for the virus to replicate in infected cells. These regions may be removed (in whole or in part) or rendered nonfunctional, or replaced with other sequences, particularly recombinant nucleic acids. Preferably, the defective virus still retains its genomic sequences required to encapsulate viral particles.

[0026] In particular, AAV vectors exhibit several advantages, such as i) persistent expression of the synthetic gene, ii) low risk of pathogenicity (because they are artificially created and non-toxic), iii) low immunogenicity, and iv) non-integration into the human genome. AAVs can be genetically modified to improve gene expression efficiency and prevent accidental viral transmission. These modifications include deletion of the E1 region, deletion of the E2 or E4 region, or deletion of the entire adenovirus genome except for cis-acting inverted terminal repeats and packaging signals. Such vectors are advantageously covered in this invention. We can also use non-viral Sleeping Beauty stable transposons of the p21 gene derived from supercoiled minimal DNA vectors (called small loops). Genetic engineering of monocytes can also be performed by non-viral transfer of p21 mRNA transcribed in vitro into monocytes. In both methods, plasmids are delivered by electroporation transfection of monocytes.

[0027] Another advantageous vector for preparing the cell compositions according to the invention is an adenovirus vector. In fact, Haddadah H. et al., Biochem.Biophys.Res.Commun (1993) demonstrated that adenoviruses can very effectively infect, stably maintain, and express therapeutic genes in mononuclear-macrophage cell lines. Different serotypes of adenoviruses exist, differing in structure and properties, but none are pathogenic to humans, particularly to non-immunosuppressed subjects. Furthermore, 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, adenovirus type 2 or type 5 (Ad 2 or Ad 5) is preferred in the context of this invention. In the case of Ad 5 adenovirus, the sequences required for replication are the E1A and E1B regions. These sequences are preferably deleted in the recombinant nucleic acids used in this invention.

[0028] Another advantageous vector for preparing the cell compositions according to the invention is a lentivirus. Lentivirals, such as HIV, have the ability to infect both non-dividing and dividing cells and integrate into the host cell genome. Due to these properties, HIV-based lentiviral vectors have been proposed as good candidates for gene therapy delivery systems, but attempts to use them in clinical trials have raised concerns about their safety, including the risk of genetic recombination leading to the generation of capable-replicating retroviruses in humans. Further modifications have been made to the packaging and genetic components of viral genes to develop safer HIV-based lentiviral vector systems. Today, many safe HIV-based lentiviral vectors have been designed for the efficient transduction of target genes into differentiated monocyte-derived macrophages (Leyva F. et al., BMC biotechnology (2011)). Any of these carriers can be used in the context of this invention.

[0029] Preferred lentiviral vectors are those modified for safe administration to mammals. These vectors are, for example, HIV / SIV vectors known for use in gene therapy in humans or mammals, such as those by Neschadim A. et al. Biol Blood Marrow Transplant. The vectors most interested in are lentiviral self-inactivated vectors based on HIV and SIV (Neschadim A. et al., 2007 Dec;13(12):1407-16). Biol Blood Marrow Transplant. 2007 Dec;13(12):1407-16.), adenovirus vector (Haddada H. et al., Biochem.Biophys.Res.Commun (1993)) and Sleeping Beauty transposon nonviral vectors (Aronovich et al., Human Molecular Genetics (2011)).

[0030] The vector used in the examples, namely an HIV-1-based self-inactivated (SIN) lentiviral vector encoding the p21 protein, can be used. This vector can encode the p21 protein alone, or be fused with another protein such as AIP (aromatic hydrocarbon receptor interacting protein), or fused with a small protein tag such as a Flag tag or a hemagglutinin (HA) tag.

[0031] In a particularly preferred embodiment, the composition of the present invention further comprises virus-like particles (VLPs) containing SIVmac-VPX to induce the degradation of factors that impair lentiviral infection (Berger G., Gene Therapy (2009)). SIVmac-VPX degrades SAMHD1, identified as an HIV-1 restriction factor, and SAMHD1 hydrolyzes dNTPs required for retroviral replication (Lahouassa et al., 2009). Nat Immunol(2012)).

[0032] In one, or even more particularly preferred, embodiment of the cell composition of the present invention contains mononuclear cells transduced with a SIN lentiviral vector, the vector containing the nucleic acid sequence encoding the p21 protein SEQ ID NO: 5: As described above, the gene encoding p21 is placed under the control of regulatory elements that allow its expression. These regulatory elements typically consist of transcription promoter sequences. When these sequences are able to function in monocytes / macrophages, they can be sequences naturally responsible for p21 expression. They can also be sequences from different sources (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 monocyte to be infected. Similarly, they can be promoter sequences derived from viral genomes. In this regard, examples include: promoter E2F1 (E2 promoter binding factor 1) or promoter EFS (elongation factor 1α short), SFFV (splenic silencing lesion-forming virus), CMV (cytomegalovirus), RSV (Raoult's sarcoma virus), etc. Furthermore, these expression sequences can be modified by adding activating sequences, regulatory sequences, etc.

[0033] Technicians should select the vector and regulatory sequence, while keeping in mind that the final expression of p21 in the macrophages of the present invention should be enhanced by at least 2 or 3 times compared with control macrophages that have undergone simulated transfection.

[0034] Methods for constructing expression vectors containing coding sequences and appropriate transcription / translation control signals are well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination. Nucleic acids can be isolated and obtained to a basic degree of purity, and then introduced into suitable host cells using a variety of techniques available in the art.

[0035] All techniques for constructing vectors derived from adenovirus, lentivirus, or AAV, and for incorporating heterologous nucleic acid sequences therein, have been described in the literature and can be used in the context of this invention. Methods conventionally used in molecular biology, such as preparation of extracted plasmid DNA, centrifugation of plasmid DNA in a cesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, extraction of proteins by phenol or phenol-chloroform, precipitation of DNA in saline medium with ethanol or isopropanol, transformation in E. coli, etc., are well known to those skilled in the art and are well described in the literature [Maniatis T. et al., “Molecular Cloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM et al., eds., “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

[0036] After genetically modifying the viral genome, the virus is then propagated, recovered, and purified according to standard molecular biology techniques.

[0037] The pharmaceutical composition of the present invention contains monocytes overexpressing p21 protein.

[0038] As used herein, the term "monocyte-derived macrophage" refers to a mononuclear cell cultured from peripheral blood mononuclear cells (PBMCs) or their bone marrow or blood precursors and differentiated into macrophages 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, monocytes, or premonocytes can also be used as precursors. Intravenous injection of mononuclear cells induces their transplantation into the bone marrow, spleen, and liver, where they differentiate into macrophages.

[0039] Since PBMCs are present in the blood, PBMC samples can be obtained by collecting blood from the subject in a completely harmless and non-invasive manner.

[0040] PBMCs / monocyte-macrophages or their precursors can be extracted and isolated using any techniques known to those skilled in the art. These different techniques may involve physical separation steps (centrifugation, cell sorting (FACS), etc.) and selection using immune compounds (specific antibodies to cell markers, etc.) or biochemical compounds (membrane receptor ligands), etc. The isolated cells can be cultured in various culture media known to those skilled in the art (e.g., RPMI, IMDM), especially those supplemented with serum and amino acids. As shown in the examples, the cells are cultured under sterile conditions, preferably at 37°C. Culture can be performed in culture plates, or preferably in Teflon bags.

[0041] In a preferred embodiment, the cells contained in the composition of the present invention are obtained by culturing PBMCs under suitable conditions that allow for PBMC differentiation. For example, human blood mononuclear cells can be induced to differentiate into macrophages in vitro using three different methods, namely by culturing PBMCs in: 1) human serum (HS), 2) fetal bovine serum (FBS) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), or 3) FBS containing macrophage colony-stimulating factor (M-CSF).

[0042] In a specific embodiment, monocytes purified from PBMCs are transduced in vitro with recombinant nucleic acids, and then the transduced cells are differentiated / cultured into macrophages in vitro. In this embodiment, the cell composition of the present invention contains differentiated macrophages overexpressing p21 protein.

[0043] In another specific embodiment, when differentiating / culturing cells into macrophages in vitro, the cells are transduced using the recombinant nucleic acid of the present invention. In this embodiment, the cell composition of the present invention also contains differentiated macrophages overexpressing p21 protein, but the cells are first differentiated into macrophages before being transduced using the recombinant nucleic acid of the present invention.

[0044] In another specific embodiment, the recombinant nucleic acid of the present invention is transduced in vitro at the monocyte stage of the cells. The cells are then administered to a subject, where they differentiate into macrophages. In this embodiment, the cell composition of the present invention contains undifferentiated monocytes overexpressing the p21 protein.

[0045] In another specific implementation, recombinant nucleic acids can be administered in vivo to transfect circulating macrophages in situ.

[0046] As disclosed in this article, the term "in vitro ( in vitro ")" and "extracorporeal ( ex vivo "In vivo" is equivalent to "in vivo" and refers to research or experiments using biological components (e.g., cells or cell populations) isolated from their usual host organism (e.g., animals or humans). Conversely, the term "in vivo" is... in vivo ")" or "in situ ( in situ ")" refers to a study conducted on a whole living organism (e.g., a human) after the composition of the present invention has been administered to a living subject.

[0047] In another embodiment, the cell composition of the present invention contains monocytes purified from PBMCs, which have been transformed in vitro but have not yet differentiated into macrophages in vitro. Their differentiation will occur in vivo, in the host. In this case, the composition of the present invention has more than 80% monocytes, or more preferably more than 90% monocytes, and even more preferably more than 99% monocytes. This means that the cell composition of the present invention contains very few other cells, if any.

[0048] The monocytes purified from PBMCs contained in the cell composition of the present invention are monocytes recovered from an individual's peripheral blood using conventional methods. These PBMC-purified monocytes are positive for the following markers: CD14, CD11b, 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 present 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, such as by fibrillation cytology (FACS).

[0049] Following intravenous injection of the cell composition of the present invention, the macrophages differentiated in the tissue are monocyte-derived cells that are positive for the following markers: CD14, CD11b, 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 present 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, such as by fibrillation cytology (FACS).

[0050] Cells containing the composition of the present invention will be transformed with the recombinant nucleic acid of the present invention in a sterile culture medium under conditions adjusted by those skilled in the art. In particular, the multiplicity of infection must be adjusted according to the vector used. An example using SIV virus is given in the following examples.

[0051] When using a lentiviral vector, cells containing the composition of the present invention are contacted to purify the virus, for example, at a concentration of 50 to 250 pfu per cell, more preferably 50 to 100 pfu / cell. Depending on the transformation conditions, the percentage of cells modified by inserting recombinant nucleic acids can be between 30% and 95%.

[0052] The modified cells thus obtained can then be packaged for immediate use and / or stored for subsequent use. For immediate reapplication, the cells are typically suspended in phosphate-buffered saline or physiological saline at a concentration of 30 × 10⁻⁶ per dose. 6 Up to 10 9 Between individual cells. For cell storage, cells can be frozen, preferably in the presence of preservatives such as glycerol, DMSO, etc.

[0053] According to the present invention, cells transformed in vitro using the recombinant viral vector disclosed above are the tool of choice for preparing pharmaceutical compositions (particularly compositions designed to enhance the immune and hematopoietic systems of patients).

[0054] The cells in the compositions of the present invention can be derived from the patient themselves (thus the composition contains autologous cells) or from a donor (thus the composition contains allogeneic cells). For allogeneic cells, HLA compatibility and matching between the donor and recipient patients are required.

[0055] The pharmaceutical composition contains the aforementioned transformed cells as the active ingredient. It also contains pharmaceutically acceptable excipients.

[0056] The term "pharmaceutically acceptable excipient" refers to an excipient that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and desirable, and includes excipients acceptable for both veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Compositions for treating cancer can typically be administered via parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal, or intramuscular routes. Typical routes of administration are intravenous or intratumoral, although other routes may be equally effective.

[0057] For intravenous administration, the compositions of the present invention will be in liquid form. Therefore, in addition to cells, it will also contain a pharmaceutically acceptable diluent that does not affect the bioactivity of the cells of the present invention. Examples of such diluents are physiological phosphate-buffered saline, Ringer's solution, glucose solution, and Hank's solution. Furthermore, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0058] In a preferred embodiment, the composition of the present invention is in liquid form.

[0059] The pharmaceutical compositions of the present invention can be administered alone or in combination with another pharmaceutical composition or another active ingredient. In particular, the pharmaceutical compositions of the present invention may contain the cellular composition of the present invention and another active ingredient, all contained in the same container.

[0060] This active ingredient can be, for example, a chemotherapy agent. Exemplary chemotherapy agents include, but are not limited to: interleukin, hexamethasone, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), actinomycin D, docetaxel, doxorubicin, dronabinol, pyruvic acid, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon-alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, and paclitaxel. TM ), pilucarpine, prochlorperazine, rituximab, saproin, tamoxifen, paclitaxel, topotecan hydrochloride, trastuzumab, vincristine, vinorelbine tartrate.

[0061] As used herein, the term "combination" does not mean that the cells of the present invention and another active ingredient must be applied simultaneously. The term also extends to any use or demonstration involving the application of them at different time intervals or in separate containers.

[0062] In another embodiment, the cell composition of the present invention is combined with, for example, a chemotherapeutic agent as defined above.

[0063] In another embodiment, the cellular composition of the present invention is combined with (or contains) an effective dose of an agent that increases the patient's hematocrit, such as an erythropoietin stimulant (ESA). Such agents are known and used in the art, including, for example, Aranesp® (dapoxetine α), Epogen® NF / Procrit® NF (dapoxetine α), Omontys® (peginesatide), Procrit®, etc.

[0064] Other combination therapies include administration in combination with cell-specific antibodies (e.g., antibodies selective for tumor cell markers), radiation, surgery, and / or hormone deprivation.

[0065] Therefore, the cell composition of the present invention is combined with an effective dose of the cell-specific antibody (or contains the antibody).

[0066] Many antibodies are currently used clinically to treat cancer, while others are in various stages of clinical development. For example, there are many antigens and corresponding monoclonal antibodies for treating B-cell malignancies. One target antigen is CD20. Rituximab is a chimeric, unconjugated monoclonal antibody targeting the CD20 antigen. CD20 plays 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. Many antibodies target CD22, and their efficacy in combination with toxins for treating chemotherapy-resistant hairy cell leukemia has recently been demonstrated. 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 with radioisotopes. alemtuzumab (Campath) is used to treat chronic lymphocytic leukemia; gemtuzumab (Mylotarg) is used to treat acute myeloid leukemia; tiimumab (Zevalin) is used to treat non-Hodgkin's lymphoma; and panitumumab (Vectibix) is used to treat colon cancer.

[0067] Angiogenesis inhibitors can also be combined with the compositions of the present invention (or the compositions of the present invention contain the inhibitors).

[0068] In another embodiment, the cell composition of the present invention is combined with an effective dose of an immune checkpoint modulator (or contains said modulator), said modulator being particularly an immune checkpoint inhibitor (ICI).

[0069] "Immune checkpoint inhibitors" (ICIs) include anti-PD1 antibodies (such as nivolumab, pembrolizumab, or pitilizumab), anti-PD-L1 antibodies (such as atezolizumab or durvalumab), anti-CTLA-4 antibodies (such as ipilimumab or tremelimumab), and anti-PD-L2 antibodies.

[0070] The term "concomitant administration" refers to the administration of the active ingredient and the pharmaceutical composition of the present invention together with recombinant cells when both the active ingredient and the composition of the present invention will have a therapeutic effect. Such concomitant administration can involve administering the active ingredient in parallel (i.e., simultaneously), before, or after the administration of the compound of the present invention. Those skilled in the art will readily determine the appropriate timing, sequence, and dosage of specific pharmaceuticals and compositions of the present invention.

[0071] This document proposes to deliver the cell composition of the present invention (containing obtained p21-expressing monocytes) into subjects in need as a simple and effective method for treating cancer, particularly leukemia.

[0072] Typically, the subjects are humans, but non-human mammals can also be treated, such as companion animals (dogs, cats, horses, etc.), laboratory mammals (rabbits, mice, rats, etc.), etc.

[0073] Therefore, as referred to herein, "subjects in need" are mammals with cancer, preferably humans. The cancer can be liquid or solid, such as, but not limited to, lymphoma, leukemia, carcinoma, melanoma, glioblastoma, sarcoma, myeloma, colorectal tumors, etc., as primary or metastatic cancers. In a specific implementation, the "subjects in need" are humans with leukemia or another mammal.

[0074] This invention covers a treatment method in which the cell composition of the invention is administered to a subject in need by injection, preferably by intravenous injection. Systemic injection via perfusion is also possible. These injections are harmless to the treated subject. Intravenous administration of the cell composition of the invention can increase the phagocytosis of tumor cells present in the subject's blood, thereby reducing the amount of tumor cells in the subject.

[0075] The specific treatment methods according to the present invention include: 1. Extraction and isolation of monocytes or their precursors or pluripotent stem cells from blood, bone marrow, or umbilical cord from eligible subjects or from healthy donors. 2. As disclosed above, these cells are cultured to obtain / isolate a population of monocytes. 3. Transform these cells with the recombinant nucleic acids defined above. 4. Optionally, package and / or store the cells thus obtained, and then 5. Apply it to the patient.

[0076] Compared to other adoptive immunotherapy treatments using LAK, TIL, or NK (natural killer cells), the treatment of this invention offers numerous advantages, such as the absence of cellularly released toxic mediators, a lower effector-to-target cell ratio required for cytotoxicity compared to other treatments, and the elimination of the need for simultaneous injection of cytokines such as IL-2 (whose side effects are harmful). Furthermore, this treatment can infect only a defined and controlled population of cells, allowing for selective infection multiplicity (the number of viral particles per cell), enabling irreversible delivery to tissues via blood circulation, and, as mentioned above, fully leveraging the central role of macrophages in vivo through their antitumor or anti-infective activity and their stimulation or modulation of the immune system. Moreover, the pro-inflammatory reprogramming of PrCR+ macrophages enhances both innate and adaptive anticancer immune responses, thereby establishing a durable antitumor growth microenvironment. Additionally, considering the relatively long lifespan of macrophages, the barrier to repeat treatment for patients can be avoided.

[0077] This invention therefore offers new possibilities for more effective treatments that are less demanding on patients, cheaper, and more repeatable.

[0078] As used herein, the terms “treat,” “treating,” and “treatment” refer to reducing or improving the symptoms of a disease (such as leukemia) and / or related symptoms. It should be understood that treating a disease or condition does not require the complete elimination of the disease, condition, or related symptoms, although this is not excluded.

[0079] The effective dose of the therapeutic entity of the present invention (e.g., for treating cancer) depends on many different factors, including the method of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. The therapeutic dose can be titrated to optimize safety and efficacy.

[0080] For prophylactic use, the pharmaceutical composition or drug is administered to patients susceptible to the disease or at risk of disease in an amount sufficient to eliminate or reduce the risk, alleviate the severity, or delay the onset of the disease (including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during the development of the disease). In these prophylactic uses, relatively low doses may be administered at relatively infrequent intervals over a prolonged period. Some patients continue to receive treatment for the remainder of their lives.

[0081] Conversely, in therapeutic applications, it is sometimes necessary to use relatively high doses (50 × 10⁻⁶ per patient per injection) at relatively short intervals (usually weekly). 6 (monocytes), until disease progression slows or stops, and preferably until the patient shows partial or complete improvement in disease symptoms.

[0082] The present invention is further defined by the following embodiments: Implementation Scheme 1. A pharmaceutical composition comprising a genetically modified mononuclear cell and a pharmaceutically acceptable excipient, said genetically modified mononuclear cell containing a vector encoding a cyclin-dependent kinase inhibitor p21 protein.

[0083] Implementation Scheme 2. The pharmaceutical composition according to Implementation Scheme 1, wherein the monocyte contains a replication-deficient recombinant virus encoding a cyclin-dependent kinase inhibitor p21, said p21 being under the control of regulatory elements that allow its expression.

[0084] Implementation Scheme 3. The pharmaceutical composition according to Implementation Scheme 2, wherein the virus is a replication-defective lentivirus.

[0085] Implementation Scheme 4. The pharmaceutical composition according to Implementation Scheme 3, wherein the replication-defective lentivirus is a self-inactivated (SIN) lentiviral vector based on HIV-1.

[0086] Implementation Scheme 5. The pharmaceutical composition according to any one of Implementation Schemes 1 to 4, wherein the pharmaceutical composition is formulated into an intravenous injection form or an infusion form.

[0087] Implementation Scheme 6. A pharmaceutical composition according to any one of Implementation Schemes 1 to 5, wherein the pharmaceutical composition contains 30 × 10 per mL. 6 Up to 10 9 One transduced monocyte.

[0088] Implementation Scheme 7. The pharmaceutical composition according to Implementation Scheme 1, wherein the monocyte contains a Sleeping Beauty transposable system encoding a cyclin-dependent kinase inhibitor p21, said p21 being under the control of a regulatory element that allows its expression.

[0089] Implementation Scheme 8. The pharmaceutical composition according to any one of Implementation Schemes 1 to 7, wherein the p21 protein is SEQ ID NO: 2 or a functional variant or fragment thereof.

[0090] Implementation Scheme 9. The pharmaceutical composition according to any one of Implementation Schemes 2 to 8, wherein the virus or the transposable system contains the nucleic acid of SEQ ID NO: 5, preferably under the control of the SFFV promoter.

[0091] Implementation Scheme 10. The pharmaceutical composition according to any one of Implementation Schemes 1 to 9, further comprising 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).

[0092] Implementation Scheme 11. Use of the pharmaceutical composition of any one of Implementation Schemes 1 to 10 for the treatment of mammals suffering from lymphoma or medullary carcinoma or solid carcinoma, preferably leukemia.

[0093] Implementation Scheme 12. Use of the pharmaceutical composition according to Implementation Scheme 11, wherein each injection dose contains 50 × 10 6 One mononuclear cell and administer weekly until disease progression slows.

[0094] Implementation Scheme 13. Use of the pharmaceutical composition according to Implementation Scheme 11 or 12, wherein the mammal is a human.

[0095] Implementation Scheme 14. A combination product comprising a pharmaceutical composition as defined in Implementation Schemes 1 to 9, and an effective dose of an agent that increases hematocrit, a chemotherapeutic agent, a cell-specific antibody, or an immune checkpoint inhibitor (ICI), for use concurrently or sequentially in the treatment of mammals with lymphoma, medullary carcinoma, or solid carcinoma.

[0096] Implementation Scheme 15. The combined product according to Implementation Scheme 14, wherein the mammal is a human. Attached Figure Description

[0097] Figure 1 It showed that p21-dependent PrCR triggered pro-inflammatory reprogramming of macrophages. Figure 1 a to Figure 1 (k) and is beneficial for leukemia remission ( Figure 1 l to Figure 1 (r).

[0098] Figure 2 This demonstrates that p21 determines programmed cell clearance of macrophages and leukemia regression by regulating SIRPa. Figure 2 a and Figure 2 (b), and confirmed that p21 is beneficial for leukemia remission ( Figure 2 c and Figure 2 d). Detailed Implementation

[0099] Example The examples given below are not limiting, but rather illustrate the feasibility of expressing p21 protein in macrophages and its therapeutic value in vivo.

[0100] 1. Materials and methods Culture of primary macrophages and leukemia cell lines Monocyte-derived macrophages (MDMs) were obtained by differentiating monocytes derived from the erythrocyte sedimentation rate (ESR) amber layer into macrophages. In accordance with French law and as part of the EFS-INSERM convention, ESR amber layers from healthy donors were obtained from the French blood bank (Etablissement Français du sang (EFS)). Monocytes were first isolated from peripheral blood mononuclear cells (PBMCs) by adhering to plastic plates, and then cultured for 6 to 7 days in hydrophage-containing 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 vitamins, and 1% non-essential amino acids) in Lumox (Duthsher) dishes containing 15% heat-inactivated human serum AB. MDMs were then harvested and suspended in macrophage culture medium containing 10% heat-inactivated fetal bovine serum (FBS). When examined by flow cytometry, MDMs obtained by this method showed a CD14 positivity rate of 91% to 96%, expressing differentiation markers (C11b and CD71) and M2 macrophage polarization markers (CD163 and CD206). MDM purity was also controlled by negative staining for CD56 (NK cells), CD3 (T cells), and CD20 (B cells). Primary blood lymphocytes (PBLs) were isolated from non-adherent PBMC fractions using a T-cell negative selection kit (STEM CELL). Lymphocytes obtained by this method were T cells with 90% to 97% CD3 expression and cultured in RPMI medium containing 10% FBS. Differentiated MDMs (0.125 × 10⁻⁶) were then cultured. 6 (number) and live leukemia cells (0.125 × 10⁻⁶) 6 (Jurkat, MT4, CEM, THP1, HEL-5320, K562 or CD34) + Acute myeloid leukemia blast (CD34) + AML cells were pre-labeled with cell tracers for one hour, marked as green (CMFDA) or red (CMTMR). PBL obtained from healthy donors was used as a control. After thorough washing, MDM and leukemia cells were co-cultured for 8 hours in macrophage medium supplemented with 10% FBS (with or without ZVAD-fmk pan-cysteine ​​inhibitor (100 μM), total volume 250 µl) on 8× well slides. After thorough washing, unphagocytosed leukemia cells were removed, macrophages were fixed (using 2% PFA), and then analyzed by confocal microscopy. The percentage of PrCR+ macrophages was determined by internalized leukemia cells (CMTMR). +) macrophages (CMFDA) + 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 amount 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).

[0101] Engineered human primary monocytes overexpressing p21 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) based on the HIV-1 self-inactivated (SIN) lentiviral vector and virus-like particles (VLPs) containing SIVmac-VPX. 6 (15 × 10⁻⁶ 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 × 10⁻⁶ 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, MT4 mCherry+ 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.

[0102] 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.

[0103] Mouse treatment studies 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 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.

[0104] Cloning of SIRPa cDNA The person expressing ( homo sapiensThe cDNA of the SIRPa phagocytic inhibitor factor was cloned into the restriction sites between MluI and NheI of the HIV-1-based self-inactivating (SIN) lentiviral vector (pRRLEF1-PGK-GFP).

[0105] The sequence of hsSIRPa cDNA is shown in SEQ ID NO: 7.

[0106] Monocytes were transduced with a lentiviral vector expressing p21 and SIRPa. In the presence of virus-like particles (VLPs) containing SIVmac-VPX, purified primary monocytes (10⁶ cells / years) were transduced with 100 µg of CAP24 (encoding the p21 gene under the control of the SFFVp promoter, AIP-p21) from an HIV-1-based self-inactivated (SIN) lentiviral vector and / or 100 µg of CAP24 (encoding the SIRPa gene under the control of the EF1 promoter, PRRL-SIRPa) from an HIV-1-based self-inactivated (SIN) lentiviral vector, PRRL-SIRPa. 7 (10 μg / ml) to induce degradation of the myeloid restriction factor SAMHD1 protein by lentiviral infection. Control monocytes were transduced with equal amounts of AIP and / or PRRL vector and VLPs-SIV-mac-VPX. Transduction was performed at 37°C for 3 h in the presence of polybrene (10 µg / ml). Transduced monocytes (10 μg / ml) were then labeled with Cell Trace CFSE dye. 7 After 1 hour and thorough washing, the transduced mononuclear cells were intravenously injected into male or female NSG mice (6 to 8 weeks old) irradiated with 1 Gy of X-rays 24 hours after the mice were irradiated, or the transduced mononuclear cells were differentiated into macrophages in vitro as described in the materials and methods of the main patent.

[0107] 2. result The inventors investigated the molecular mechanism by which macrophages regulate PrCR. They used primary anti-inflammatory, pro-tumor human monocyte-derived macrophages (MDM) (labeled with the green fluorescent cell tracer CMFDA) and a different group of live leukemia cells (labeled with the red fluorescent cell tracer CMTMR)... Figure 1 a) The phagocytic effect was assessed.

[0108] Using confocal microscopy, they observed that in the absence of MIC inhibitors, human primary macrophages and acute T lymphoblasts (Jurkat, CEM, or MT4 cells) showed [significant differences]. 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, 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.

[0109] 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)

[0110] 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.

[0111] 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 1 After CFSE-labeled p21EHMs were transferred into transplanted mice, they were observed to differentiate into macrophages in vivo. Figure 1(o and not shown), and the presence of macrophages that engulfed MT4 was detected in transplanted mice: in the bone marrow ( Figure 1 p), in the liver and in the spleen (not shown).

[0112] Macrophages that engulfed MT4 were also observed to undergo a shift from an anti-inflammatory to a pro-inflammatory phenotype (as revealed below: decreased membrane expression of CD163 and increased secretion of IFNγ and IL-1β); Figure 1 (q and not shown). Interestingly, p21EHM-based cell therapy delayed disease progression in treated mice and significantly improved overall survival (q and not shown). Figure 1 (r).

[0113] In summary, these data indicate that adoptive transfer of p21EHM represents a novel therapeutic strategy for treating hematologic malignancies by inducing macrophage PrCR.

[0114] To further characterize the molecular link between p21 and programmed cell clearance, the effect of p21 expression on the expression of the phagocytic inhibitor SIRPa was determined. Primary human monocytes were transduced with lentiviral vectors expressing p21 and / or SIRPa, along with their respective control empty vectors pCo. (pAIP and / or pRRL), in combination or without combination. A subset of monocytes differentiated into macrophages in vitro for 7 days, and the expression of p21 and SIRPa was measured by Western blotting. Figure 2 (a). The efficiency of transduction in each case was verified. Overexpression of p21 was detected to inhibit SIRPa expression, thus revealing that p21 expression negatively regulates SIRPa expression. Figure 2 (a). To determine the effect of these transductions on programmed cell clearance, transduced macrophages were co-cultured with leukemia MT4 cells expressing mCherry fluorescent protein, and the phagocytosis of mCherry was analyzed using fluorescence microscopy. + MT4 cells are macrophages (PrCR) + The frequency of macrophages Figure 2 (b). Increased expression of p21 and SIRPa was observed to enhance and attenuate mCherry, respectively. + Phagocytosis by MT4 cells. Furthermore, increased expression of SIRPa in p21-transduced macrophages inhibited this process, demonstrating that p21 determines programmed macrophage clearance by regulating SIRPa expression. The effects of these regulations on leukemia progression were then analyzed. Transduced monocytes (such as...) Figure 2 (as shown in a) was adopted into NOD / SCID mice, and mCherry was injected one week later. + MT4 cells ( Figure 2c), and analyzed the overall survival rate of transplanted mice (n=5 mice per group) ( Figure 2 The p-values ​​were calculated using the Mantel-Cox test to reveal statistical significance between the analysis groups (d). *** (p<0.001). In summary, these results confirm that adoptive transfer of p21-overexpressing monocytes (p21EHM) may delay leukemia progression by regulating SIRPa-dependent programmed cell clearance by macrophages.

[0115] bibliography Advani R. et al., N Engl J Med (2018) Nov 1;379(18):1711-1721 Andressen R. et al., Cancer Res. (1990) 50:7450 Arandjelovic S and Ravichandran KS, Nat Immunol (2015) Sep; 16(9):907-17 Aronovich EL et al. Human Molecular Genetics (2011) Vol. 20, Review Issue1 R14-R20 Bartholeyns J et al., Anticancer Res 11 (1991) 1201-1204 Berger G., Gene Therapy (2009) 16, 159-163 Chao MP et al. Nat Rev Cancer (2011) Dec 8; 12(1):58-67 Feng and others Nat Comm (2018) Aug 10;9(1):3194. Haddada H. et al., Biochem.Biophys.Res.Commun (1993) Sep 30; 195(3):1174-83. Jaiswal S., Trends Immunol. 2010) Jun; 31(6):212-9 Jaiswal et al. Cell (2009) Jul 23; 138(2):271-85 Leyva F. et al., BMC biotechnology (2011); 11: 13 Majeti et al. Cell(2009) Jul 23;138(2):286-99 Neschadim A. et al. Biol Blood Marrow Transplant . 2007 Dec; 13(12):1407-16. Pardoll DM. Nat Rev Cancer (2012) Mar 22; 12(4):252-64. Sharma SH et al. Cell (2015)Apr 9; 161(2):205-14 Singh G. et al. F1000 research 2015, 4:495 Weiskopf K, Eur J Cancer (2017) May; 76:100-109.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for treating mammals with solid tumors, the pharmaceutical composition comprising a genetically modified monocyte containing a vector encoding a cyclin-dependent kinase inhibitor p21 protein.

2. The use according to claim 1, wherein each injection dose of the composition contains 50 × 10 6 One mononuclear cell and administer weekly until the progression of solid tumor slows.

3. The use according to claim 1 or 2, wherein the mammal is a human.

4. The use according to any one of claims 1 to 3, wherein the monocyte contains a replication-deficient recombinant virus encoding a cyclin-dependent kinase inhibitor p21, said p21 being under the control of regulatory elements that allow its expression.

5. The use according to any one of claims 1 to 4, wherein the virus is a replication-defective lentivirus.

6. The use according to claim 5, wherein the replication-defective lentivirus is a self-inactivating (SIN) lentiviral vector based on HIV-1.

7. The use according to any one of claims 1 to 6, wherein the pharmaceutical composition contains 30 × 10⁻⁶ per mL. 6 Up to 10 9 One transduced monocyte.

8. The use according to any one of claims 1 to 3, wherein the monocyte contains a Sleeping Beauty transposition system encoding a cyclin-dependent kinase inhibitor p21, said p21 being under the control of a regulatory element that allows its expression.

9. The use according to any one of claims 1 to 8, wherein the p21 protein is SEQ ID NO: 2 or a functional variant or fragment thereof.

10. The use according to claim 5, wherein the virus contains the nucleic acid of SEQ ID NO:

5.

11. The use according to claim 10, wherein the nucleic acid of SEQ ID NO: 5 is under the control of the SFFV promoter.

12. The use according to claim 8, wherein the transposable system contains the nucleic acid of SEQ ID NO:

5.

13. The use according to claim 12, wherein the nucleic acid of SEQ ID NO: 5 is under the control of the SFFV promoter.

14. The use according to any one of claims 1 to 3, wherein the monocytes are genetically modified by a non-viral transfer method of p21-encoded mRNA transcribed in vitro.

15. The use according to any one of claims 1 to 14, wherein the pharmaceutical composition is formulated as an intravenous injection dosage form or an infusion dosage form.

16. The use according to any one of claims 1 to 15, further comprising an effective dose of an agent, chemotherapeutic agent, cell-specific antibody or immune checkpoint inhibitor (ICI) that increases the hematocrit of a patient.

17. A combination product comprising a pharmaceutical composition as defined in claims 1 to 14, and an effective dose of a hematocrit agent, chemotherapeutic agent, cell-specific antibody, or immune checkpoint inhibitor (ICI) for use concurrently or sequentially in the treatment of mammals with solid tumors.

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

19. Use of in vitro transcribed mRNA encoding the cyclin-dependent kinase inhibitor p21 protein in the preparation of a medicament for the treatment of mammals with lymphoma, myeloid cancer, or solid tumors, wherein the medicament comprises monocytes genetically modified via a non-viral transfer pathway through in vitro transcription of p21-encoded mRNA.

20. Use of in vitro transcribed mRNA encoding the cyclin-dependent kinase inhibitor p21 protein in the preparation of a medicament for the genetic modification of monocytes to treat mammals with lymphoma, myeloid cancer, or solid tumors.

21. The use according to claim 19 or 20, wherein the cancer is leukemia.

22. The use according to any one of claims 19 to 21, wherein the cancer is a solid cancer.

23. The use according to any one of claims 19 to 21, wherein the mammal is human.

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