P21 Expressing Monocytes for Cancer Cell Therapy

Abstract

Identification of effective targets alleviating the programmed cell removal (PrCR) of tumor cells by macrophages is of very high interest. The present inventors have identified that the cyclin-dependent kinase inhibitor p21 protein is a strong regulator of the macrophage-mediated PrCR. Also, they showed that the adoptive transfer of p21 overexpressing monocytes induces macrophage PrCR and transition from an anti-inflammatory to a pro-inflammatory phenotype in vivo, delays cancer progression and increases significantly the overall survival of mice engrafted with cancer cells. The present invention therefore concerns therapeutic compositions comprising monocytes that over-express the cyclin-dependent kinase inhibitor p21 protein, and their use for treating mammals suffering from cancer, especially leukemia.

Claims

1.-15. (canceled)

16. A pharmaceutical composition, comprising genetically modified monocytes comprising a vector coding for cyclin-dependent kinase inhibitor p21 protein, and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of claim 16, wherein said monocytes comprise a replication defective recombinant virus encoding cyclin-dependent kinase inhibitor p21 under control of regulatory elements permitting its expression.

18. The pharmaceutical composition of claim 16, wherein said monocytes comprise a replication defective recombinant lentivirus encoding cyclin-dependent kinase inhibitor p21 under control of regulatory elements permitting its expression.

19. The pharmaceutical composition of claim 16, wherein said monocytes comprise a HIV-1 based self inactivated (SIN) lentiviral vector encoding cyclin-dependent kinase inhibitor p21 under control of regulatory elements permitting its expression.

20. The pharmaceutical composition of claim 16, wherein the composition is formulated in an intravenous injectable form or in a perfusion form.

21. The pharmaceutical composition of claim 16, wherein the composition contains 30×106 to 109 of transduced monocytes per mL.

22. The pharmaceutical composition of claim 16, wherein said monocytes comprise a Sleeping Beauty transposon system encoding cyclin-dependent kinase inhibitor p21 under control of regulatory elements permitting its expression.

23. The pharmaceutical composition of claim 16, wherein said p21 protein is SEQ ID NO:2, or a functional variant or fragment thereof.

24. The pharmaceutical composition of claim 16, wherein said monocytes comprise a replication defective recombinant virus or a transposon system comprising the nucleic acid SEQ ID NO:5.

25. The pharmaceutical composition of claim 16, wherein said monocytes comprise a replication defective recombinant virus or a transposon system comprising the nucleic acid SEQ ID NO:5 under control of the SFFV promoter.

26. The pharmaceutical composition of claim 16, further comprising an effective dose of an agent that increases patient haematocrit, of a chemotherapeutic agent, of a cell-specific antibody, or of an immune checkpoint inhibitor (ICI).

27. A method for preventing or treating a mammal suffering from a lymphoid or from a myeloid cancer or from a solid cancer, said method comprising the step of administering a pharmaceutical composition as defined in claim 16 to a mammal in need thereof.

28. The method of claim 27, wherein said lymphoid cancer is leukemia.

29. The method of claim 27, wherein said composition comprises 50×106 monocytes per injection dose and is administered each week until progression of the disease is reduced.

30. The method of claim 27, wherein said mammal is a human.

31. The method of claim 27, further comprising the step of administering an effective dose of an agent that increases haematocrit, of a chemotherapeutic agent, of a cell-specific antibody, or of an immune checkpoint inhibitor (ICI).

32. The method of claim 31, wherein said mammal is a human.

Description

FIGURE LEGENDS

[0090] FIG. 1 shows that p21-dependent PrCR triggers pro-inflammatory reprograming of macrophages (FIGS. 1a to k) and favors leukemia regression (FIGS. 1l to r).

[0091] FIG. 2 shows that p21 dictates programmed cell removal of macrophages and leukemia regression through the modulation of SIRPa (FIGS. 2a and 2b) and confirms that p21 favors leukemia regression (FIGS. 2c and 2d).

EXAMPLES

[0092] The examples given below are not limiting but are an illustration of the feasibility of the expression of the p21 protein into the macrophages, and of their therapeutic value in vivo.

[0093] 1. Material and Methods

[0094] Culture of Primary Macrophages with Leukemic Cell Lines

[0095] Monocytes derived macrophages (MDM) were obtained through the differentiation of monocytes from buffy coats into macrophages. Buffy coats from healthy donors were obtained through the French blood bank (Etablissement Francais du sang (EFS)) as part of EFS-INSERM Convention in accordance with French law. Monocytes were first separated from peripheral blood mononuclear cells (PBMCs) by adherence to the plastic and then cultured for 6 to 7 days in hydrophobic Teflon dishes (Lumox; Duthsher) in macrophages medium (RPMI 1640 supplemented with 200 mM L-glutamine, 100 U of 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% of heat inactivated human serum AB. MDMs were then harvested and suspended in macrophage medium containing 10% of heat inactivated fetal bovine serum (FBS). MDMs obtained with this method are 91 to 96% CD14 positive expressing the differentiation markers (C11b and CD71) and the M2 macrophage polarization markers (CD163 and CD206) when checked by Flow cytometer. The purity of MDMs was also controlled by the negative staining for CD56 (NK cells), CD3 (T cells) and CD20 (B cells). The primary blood lymphocytes (PBLs) were isolated from the non-adherent PBMCs fraction using T cells negative selection kit (STEM CELL). Lymphocytes obtained by this method were 90 to 97% CD3 expressing T cells and were cultured in RPMI medium containing 10% FBS. The differentiated MDMs (0.125×10.sup.6) and the viable leukemic cells (0.125×10.sup.6) (Jurkat, MT4, CEM, THP1, HEL-5320, K562 or CD34.sup.+ acute myeloid leukaemia blasts (CD34.sup.+AML)) were pre-labelled respectively with cell tracker green (CMFDA) or red (CMTMR) for one hour. The PBLs obtained from healthy donors were used as controls. After extensive washings, MDMs and leukemic cells were co-cultured in 8× well chamber slides in macrophage medium supplemented with 10% FBS in the presence or absence of ZVAD-fmk pan-caspase inhibitor (100 μM) in 250 μl total volume for 8 hours. After extensive washings non-engulfed leukemic cells by macrophages were removed and macrophages were fixed (with 2% PFA) before confocal microscopy analysis. The percentage of PrCR+ macrophages was determined by the number macrophages (CMFDA.sup.+) that internalized leukemic cells (CMTMR.sup.+) on the total number of macrophages. Time lapse video microscopy were performed directly after the co-culture of primary macrophages with leukemic cells. The co-culture of primary macrophages that were silenced for the cyclin-dependent kinase inhibitor p21 with leukemic cells were performed at 24 hours after p21 silencing of MDMs. Differentiated MDMs were silenced for p21 gene through the transfection of 50 nM of the siRNA against the p21 gene (siRNA p21) which is the on-target plus siRNA p21 n.12 (SEQ ID NO:6: 5′ AGA CCA GCA UGA CAG AUU U 3′) obtained from Dharmacon. The siRNAs transfection was performed using INTERFERin kits (Polyplus Transfection). Equal amounts of on-target plus non-targeting siRNAs (siCo.) (Dharmacon) were added to control MDMs. The efficiency of p21 gene knockdown in MDMs was evaluated by western blot at 24 hours after silencing that corresponded to the time of co-culture with leukemic cells. The sorting of PrCR.sup.+ macrophages by flow cytometer was performed after two hours of co-culture of macrophages with leukemic cells. Sorted PrCR.sup.+ macrophages (CMFDA.sup.+CMTMR.sup.+) and the PrCR.sup.− macrophages (CMFDA.sup.+) were then cultured for further 96 hours before the analysis of the scavenger receptor CD163 membrane expression (by flow cytometer), the IRF5 expression (by western blot) and the analysis of cytokine secretion in the cell supernatant (by the cytokine microarray profiling).

[0096] p21-Overexpressing Engineered Human Primary Monocytes

[0097] Purified primary monocytes (15×10.sup.6) were transduced with 150 μg CAp24 of a HIV-1-based Self Inactivated (SIN) lentiviral vector encoding to p21 gene under SFFVp promoter (AIP-p21) and the Viral Like Particles (VLPs) containing SIVmac-VPX to induce the degradation of the myeloid restriction factor of lentiviral infections SAMHD1 protein. The control monocytes were transduced with equal amounts of AIP vectors and VLPs-SIV-mac-VPX. The transductions were performed by 1-hour spinoculation (at 1200 g, 22 C) and one hour at 37 C. The transduced monocytes (15×10.sup.6) were then labelled with the Cell Trace CFSE dye for 1 hour and extensively washed prior to their intravenous injection to 1 Gray (1 Gy) irradiated male or female NSG mouse (6 to 8 weeks old) at 24 hours after X-Ray irradiation. MT4 mCherry.sup.+ T cells (1×10.sup.6) were intravenously injected at 7 days after p21-monocyte transfer. The overall survival of the mice was monitored, until 15 days after leukemic engraftment when some mice were sacrificed to monitor the presence of PrCR+ macrophages (CFSE.sup.+mCherry.sup.+) in the bone marrow by confocal microscopy. The pro-inflammatory activation of PrCR+ macrophages was determined by flow cytometer analysis of CD163 membrane expression on the PrCR.sup.+ macrophages (CFSE.sup.+mCherry.sup.+) and PrCR.sup.− macrophages (CFSE) sorted from the spleen and bone marrow of the scarified mice.

[0098] During the in vivo set up model, the differentiation of the monocytes into macrophages in the NSG mice model was verified by purifying, 7 days after the monocyte transfer, the CFSE.sup.+ cells from bone marrow, spleen, and blood and by evaluating the expressions of the differentiation markers (CD71, CD163 and CD14) with respect to the autologous in vitro differentiated macrophages. Some of the AIP-p21 transduced monocytes were cultured in parallel in vitro to evaluate the upregulation of p21 expression by western blot 7 days after differentiation.

[0099] Mouse Treatment Studies

[0100] NSG mice were bred and maintained under pathogen free conditions at the animal facility of Gustave Roussy Institute. Animal experiments were performed in accordance with guidelines established by the French Institutional Animal Committee. MT4 leukemic T cells (1×10.sup.6) that express stably mCherry fluorescent gene, through lentiviral vectors transductions and flow cytometer sorting for mCherry expression, were intravenously injected to female or male mouse (6-8 weeks old). The leukemic engraftment was evaluated until four weeks after injections through the presence of mCherry.sup.−MT4 T cells in the bone marrow, spleen, liver and blood and trough the disease progression characterized by the loss of weight, the invasion of the bone marrow and the marked splenomegaly and the overall survival.

[0101] SIRPa cDNA Cloning

[0102] The cDNA expressing Homo sapiens SIRPa phagocytosis inhibitor was cloned in a HIV-1-based Self Inactivated (SIN) lentiviral vector (pRRLEF1-PGK-GFP) between the restriction sites MluI and NheI.

[0103] The sequence of hsSIRPa cDNA is indicated in SEQ ID NO:7.

[0104] Transduction of Monocytes with Lentiviral Vectors Expressing p21 and SIRPa

[0105] Purified primary monocytes (10.sup.7) were transduced with 100 μg CAp24 of a HIV-1-based Self Inactivated (SIN) lentiviral vector encoding to p21 gene under SFFVp promoter (AIP-p21) and/or and 100 μg CAp24 of a HIV-1-based Self Inactivated (SIN) lentiviral vector encoding to SIRPa gene under EF1 promoter (PRRL-SIRPa) in the presence of Viral Like Particles (VLPs) containing SIVmac-VPX to induce the degradation of the myeloid restriction factor of lentiviral infections SAMHD1 protein. The control monocytes were transduced with equal amounts of AIP and/or PRRL vectors and VLPs-SIV-mac-VPX. The transductions were performed for three hours at 37° C. in the presence of polybrene (10 μg/ml). The transduced monocytes (10.sup.7) were then labelled with the Cell Trace CFSE dye for 1 hour and extensively washed prior to their intravenous injection to 1 Gray irradiated male or female NSG mouse (6 to 8 weeks old) at 24 hours after X-Ray irradiation or in vitro differentiated into macrophages as described before in the main patent materials and methods.

[0106] 2. Results

[0107] The present inventors investigated the molecular mechanisms by which macrophages may regulate PrCR. They assessed the phagocytosis, by primary anti-inflammatory pro-tumorigenic human monocyte-derived macrophages (MDMs) (labeled with green fluorescent cell tracker CMFDA) of a panel of diverse living leukemic cells (labeled with red fluorescent cell tracker CMTMR) (FIG. 1a).

[0108] Using confocal microscopy, they observed that in absence of MIC inhibitors, phagocytosis of living tumor cells was already significantly increased during the co-cultures of human primary macrophages with acute T lymphoblasts (Jurkat, CEM or MT4 cells, FIGS. 1b and 1c), acute myeloid cells (THP1 cells, FIG. 1c), acute megakaryoblastic cells (UT-7 cells, not shown), erythroblasts (HEL-5320, FIG. 1c), chronic myeloid lymphoblasts (K562 cells, FIG. 1c) and primary transformed CD34.sup.+ blasts purified from blood of patients with acute myeloid leukemia (FIG. 1d), while living autologous or heterologous non-transformed peripheral blood lymphoblasts (PBLs) were not affected (FIG. 1c). The pan-caspase inhibitor (ZVAD) did not reduce the engulfment of target cells (FIG. 1c). These results indicate that macrophages may also spontaneously develop PrCR in absence of MIC blockade.

[0109] The inventors then observed that, once internalized, MT4 cells are rapidly degraded by lysosomes (FIGS. 1e and 1f). They also revealed that PrCR can induce the functional reprogramming of engulfing macrophages from an anti-inflammatory to pro-inflammatory phenotype (as revealed by the increased expression of IRF5 transcription factor (FIG. 1g), the decreased membrane expression of CD163 scavenger receptor (FIG. 1h) and the release of pro-inflammatory cytokines (such as MCP-1, Serpin and IL-8) (FIG. 1i) from cell-sorted engulfing “PrCR.sup.+” macrophages.

[0110] The inventors moreover showed that the cyclin-dependent kinase inhibitor p21, which is overexpressed in primary human macrophages, is a master regulator of PrCR. This has been revealed by the inhibition of the engulfment of living MT4 cells by p21-depleted human primary macrophages (FIGS. 1j and 1k). Altogether, these results demonstrated that p21 expression dictates the pro-inflammatory reprogramming of macrophages through the induction of PrCR.

[0111] The curative therapeutic potential of manipulating PrCR through the adoptive transfer of p21-overexpressing Engineered Human primary Monocytes (p21 EHM) was then appreciated. The biological effects of the adoptive transfer of p21 EHMs into NOD/SCID mice were determined before the implantation of HTLV-1 transformed MT4 cells. Control mice developed leukemia (as revealed by the weight loss (FIG. 1l), the bone marrow invasion (FIG. 1m) and the marked splenomegaly (FIG. 1n) of engrafted mice). After the transfer of CFSE-labeled p21 EHMs into engrafted mice, their differentiation was observed in vivo into macrophages (FIG. 1o and not shown) and the presence of MT4-engulfing macrophages was detected in the engrafted mice: in the bone marrow (FIG. 1p), in the liver and in the spleen (not shown).

[0112] It was also observed that MT4-engulfing macrophages underwent transition from an anti-inflammatory to a pro-inflammatory phenotype (as revealed decreased the membrane expression of CD163 and the increased secretion of IFNγ and IL-1β; FIG. 1q and not shown). Interestingly, p21EHM-based cell therapy led to the delay of disease progression in treated mice and increased significantly the overall survival (FIG. 1r).

[0113] Altogether, these data demonstrated that the adoptive transfer of p21 EHM represent a novel therapeutic strategy to treat hematological malignancies through macrophage PrCR induction.

[0114] To further characterize the molecular link between p21 and programmed cell removal, the impact of p21 expression was determined on the expression of the phagocytosis inhibitor SIRPa. Primary human monocytes were simultaneously transduced with lentiviral vectors expressing p21 and/or SIRPa in combination or/not with their respective control empty vectors pCo. (pAIP and/or pRRL). A fraction of monocytes was differentiated in vitro into macrophages for seven days and p21 and SIRPa expressions were determined by western blot (FIG. 2a). The efficiency of transduction was validated in each condition. It was detected that the overexpression of p21 repressed SIRPa expression, thus revealing that p21 expression regulates negatively SIRPa expression (FIG. 2a). To determine the impact of these transduction on programmed cell removal, cocultures of transduced macrophages were then performed with leukemic MT4 cells that expressed mCherry fluorescent protein and the frequency of macrophages that engulfed mCherry.sup.+ MT4 cells (PrCR.sup.+ macrophages) was analyzed using fluorescence microscopy (FIG. 2b). It was observed that the increased expression of p21 and SIRPa enhanced and reduced the phagocytosis of mCherry.sup.+ MT4 cells, respectively. In addition, the increased expression of SIRPa in macrophages that were transduced for p21 inhibited this process, thus demonstrating that p21 dictates programmed cell removal of macrophages through the modulation of SIRPa expression. The impact of these modulations was then analyzed on leukemia progression. Transduced monocytes (shown in FIG. 2a) were adoptively transferred to NOD/SCID mice one week before the injection of mCherry.sup.+ MT4 cells (FIG. 2c) and the overall survival of the engrafted mice was analyzed (n=5 mice in each group) (FIG. 2d). P values were calculated using Mantel-Cox test and revealed statistical significance between analyzed groups (***p<0.001). Altogether, these results confirmed that the adoptive transfer of p21 overexpressing monocytes (p21 EHM) may delay leukemia progression through the modulation of SIRPa-dependent programmed cell removal of macrophages.

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