IMMUNE CELLS DEFECTIVE FOR SUV39H1

Abstract

The present invention relates to an engineered immune cell defective for Suv39h1. Preferably, said engineered immune cell further comprises a genetically engineered antigen receptor that specifically binds a target antigen. The present invention also relates to a method for obtaining a genetically engineered immune cell comprising a step consisting in inhibiting the expression and/or activity of Suv39h1 in the immune cell; and further optionally comprising a step consisting in introducing in the said immune cell a genetically engineered antigen receptor that specifically binds to a target antigen. The invention also encompasses said engineered immune cell for their use in adoptive therapy, notably for the treatment of cancer.

Claims

1-15. (canceled)

16. A method for treating a subject suffering from cancer comprising: administering to said subject an engineered immune cell comprising a genetically engineered antigen receptor, wherein inactivation or disruption of the SUV39H1 gene results in enhanced anti-cancer activity of said immune cell.

17. The method of claim 16, wherein the engineered immune cell is a T cell, or NK cell or T cell progenitor.

18. The method of claim 16, wherein the immune cell is a CD4+ T cell, or a CD8+ T cell, or a CD4+ and CD8+ T cell.

19. The method of claim 16 wherein the cell comprises a second genetically engineered antigen receptor that recognizes a different antigen.

20. The method of claim 16, wherein the genetically engineered antigen receptor is a T cell receptor (TCR).

21. The method of claim 17, wherein the genetically engineered antigen receptor is a T cell receptor (TCR).

22. The method of claim 16, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR).

23. The method of claim 17, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR).

24. The method of claim 16, wherein the genetically engineered antigen receptor is a CAR comprising (a) an intracellular signaling domain from CD3 zeta chain and (b) one or more costimulatory domains of 4-1BB, CD28, ICOS, OX40 or DAP10.

25. The method of claim 17, wherein the genetically engineered antigen receptor is a CAR comprising (a) an intracellular signaling domain from CD3 zeta chain and (b) one or more costimulatory domains of 4-1BB, CD28, ICOS, OX40 or DAP10.

26. The method of claim 16, wherein the engineered immune cell is autologous.

27. The method of claim 16, wherein the engineered immune cell is allogeneic.

28. A method for treating a subject suffering from cancer comprising administering to said subject: (1) an engineered immune cell comprising a genetically engineered antigen receptor, wherein inactivation or disruption of the SUV39H1 gene results in enhanced anti-cancer activity of said immune cell; and (2) a second cancer therapeutic agent.

29. The method of claim 28, wherein the second cancer therapeutic agent is an immune checkpoint modulator, cancer vaccine, chemotherapeutic or anti-angiogen.

30. A method for treating a subject suffering from cancer comprising administering to said subject: (1) an engineered immune cell comprising a genetically engineered antigen receptor, wherein inactivation or disruption of the SUV39H1 gene results in enhanced anti-cancer activity of said immune cell; and (2) an immune checkpoint modulator.

31. The method of claim 30 wherein the immune checkpoint modulator is an inhibitor of PD1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2/4 adenosine receptor, or A2AR.

32. The method of claim 30 wherein the immune checkpoint modulator is an anti-PD-1 inhibitor or anti-PDL-1 inhibitor.

Description

FIGURES

[0221] FIG. 1: The number of central memory CD8+ T cells is increased in Suv39h1-deficient mice. A. Representative FACS dot plots of gated splenic CD8+ T cells. B. The percentage of central memory CD8+ T cells was measured in different hematopoietic compartments (black circles, Suv39h1 KO; white circles: WT littermates).

[0222] FIG. 2: Stemness and memory signature in endogenous antigen specific Suv39h1 KO CD8+ T cells. Suv39h1-deficient Kb-OVA+ CD8+ T cells have a stem cell-like and memory gene expression signature. Gene set enrichment analysis (GSEA) is shown.

[0223] FIG. 3: Increased survival and self-renewal of Suv39h1 deficient CD8T cells. A. Experimental set up showing the transfer of congenic CD45.2 Kb-OVA pentamers+CD8+ T cells, isolated from WT and Suv39h1 KO LM-OVA infected mice, into na?ve recipient CD45.1 mice (1: CD8+ T subsets sort and transfer (DO); 2: analysis of self-renewal and differentiation (D40-44). 40 days after the adoptive transfer the na?ve recipients mice were challenge with LM-OVA, and 4 days later the Kb-OVA pentamers+CD8+ T cells were analyzed by FACS. B. Representative FACS plots of donor and recipient CD8+ T cells are shown. C. The total number of recovered CD8+ T cells was measured. The results are combined data from 2 independent experiments.

[0224] FIG. 4: Suv39h1 deficient CD8+OT-1 cells control tumor growth. (A) Treated IL-2/OVA Suv39h1-KO CD45.2 OT-1 and littermate WT cells were transferred i.v. into recipient bearing tumor mice after 7 days of inoculation and were intraperitoneally injected with anti-PD-1 antibody (Bio X Cell, RMP-14). Tumor growth curves of treated IL-2/OVA littermate WT (B) and Suv39h1-KO OT-1 cells (C) adoptively transferred to recipient bearing tumor mice.

RESULTS

[0225] Material and Methods:

[0226] Littermate and Suv39h1-KO mice were infected i.v. with 5?10.sup.3 CFU and challenged 40 days later with 2?10.sup.6 CFU recombinant Listeria monocytogenes expressing OVA (LM-OVA, derived from wild type strain 140403s). The bacteria were grown in TSB medium (BD Bioscience) till early log phase and their growth was assessed with a photometer at OD.sub.600. Na?ve, dump.sup.? CD44.sup.high K.sup.b-OVA.sup.+ CD8.sup.+ T cells and related subsets were FACS-sorted. For each subset analysed, we have collected 3 or 4 biological replicates. RNA was extracted using Rneasy Micro Kit (QUIGEN) according to the manufacturer protocol. A column DNAse treatment was included (QUIGEN). For each condition, RNA was employed to synthetize cDNA according to the standard Affymetrix protocol. Labelled DNA was hybridized on the Affymetrix mouse Gene 2.1 ST, and processed on an Affymetrix GeneTitan device.

[0227] Microarray Data Analysis:

[0228] Microarray data were processed into R (version 3.0.0) using packages from the Bioconductor. Raw data CEL files were used and the quality control analysis was performed using ArrayQualityMetrics package. The raw data were preprocessed using the RMA method available in oligo package. Probes with no annotation were removed from analysis. Moderated t-tests were performed using the limma package and the p-values were adjusted using the multiple testing with the Benjamini Hochberg method. Finally, we considered as statistically significant if adjusted p-value is lower than 5%.

[0229] Gene Set Enrichment Analysis.

[0230] Gene Set Enrichment Analysis (GSEA) was performed with gene with the immunologic signatures (C7) and C2 (curated) gene sets from Molecular Signatures DataBase (MSigDB database v5.1, http://software.broadinstitute.org/gse/msigdb/indexjsp). GMT file was downloaded with the gene symbol information. The GCT file was composed of a total of 41345 probes and was imported into GSEA. GSEA was running with default parameters except the number of permutation (n=10000). The BubbleGUM analysis has been done as previously described.

[0231] Analysis of Tumor Growth inSuv39h1Deficient CD8+OT-1 Cells were Performed as Follow:

[0232] Total spleen and lymph node from male Suv39h1-KO CD45.2 OT-1 (specific for OVA257-264 peptide SIINFEKL in a H2-Kb MHC class I context) and littermate WT mice were cultured in complete medium (RPMI 1640 supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine) for 3 days and then activated with 100 UI/ml IL-2 and 1 ug/ml OVA257-264 peptide for other 3 days.

[0233] For tumor inoculation, 1?10.sup.6 EL4-OVA lymphoma cells were injected subcutaneously into the right flank of CD45.1/C57BL/6 male mice and after 7 days, mice were injected i.v. with 2?10.sup.6 IL-2/OVA treated Suv39h1-KO CD45.2 OT-1 or littermate WT cells.

[0234] Mice were intraperitoneally injected with anti-PD1 (Bio X Cell, RMP-14) administrated at a dose of 7.5 mg/Kg body weight per dose twice/week during 2 weeks. Tumor growth was measured using a manual calliper.

[0235] Results:

[0236] 1) Our results show that in the cell population obtained from Suv39h1-KO mice, early progenitors with stem cell-like phenotype accumulate and re-program with increased efficiency into longed-lived central memory T cells expressing both CD44 and CD62L as compared to the cell population obtained from wild-type mice. As illustrated in FIG. 1, the proportion and numbers of central memory T cells (expressing both CD44 and CD62L) are both increased in Suv39h1-defective mice.

[0237] 2) Transcriptomic analysis was used to understand the mechanism of this peculiar phenotype:

[0238] Wild-type and Suv39h1-deficient mice were immunized with OVA-expressing Listeria m. and OVA-specific T cells were isolated at day 7 after immunization using sorting flow cytometry.

[0239] After Affymetrix analysis of the cells, we found that effector T cells from Suv39h1 express higher levels of mRNAs coding for stem-cell cell-related proteins (CD8+ T stem cell-like memory signature (86 genes): Abcb1a Irf8 Rest Abcb1b Jarid2 Rif1 Alpl Kat6a Rnf138 Antxr2 Klf4 Rras Arl4c Ldha Sall4 Atr Ldhb Satb1 Baalc Ldhc Setbpl Basp1 Ldhd Setdbl Bcl6 Lect1 Skil Bub1 Lpin1 Smarcad1 Ccr7 Ly6a Sox2 Ccr9 Ly6e Spon1 Cd27 Map3k8 Stat3 Cxcr6 Mapk12 Tbx3 Dock9 Mcm3ap Tcf3 Dusp9 Mcoln2 Tcl1 Eomes Myc Tdgf1 Esrrb Nanog Tert Evl Ncor2 Tigit Fas Nr0b1 Tnfaip2 Fgf2 Nr1d2 Tnfrsf1b Fut4 P2ry14 Traf1 Gzmk Pax6 Traf4 Handl Pcgf2 Trib2 Hesx1 Plekha5 Txnip Ier3 Podxl Zfp42 Il2rb Pou5f1 Zfx 117r Pou6f1 Zic3 Irf4 Prkce).

[0240] We concluded that OVA-specific T cells from Suv39h1-deficient mice stimulated after Listeria m. infection express a stem cell-like mRNA signature.

[0241] 3) Suv39h1 Deficient CD8+ T Cells Show Increased Survival and Self-Renewal In Vivo.

[0242] Congenic CD45.2 Kb-OVA pentamers+CD8+ T cells, isolated from WT and Suv39h1 KO LM-OVA infected mice were transferred into na?ve recipient CD45.1 mice. 40 days after the adoptive transfer the na?ve recipients mice were challenge with LM-OVA, and 4 days later the Kb-OVA pentamers+CD8+ T cells were analyzed by FACS.

[0243] As illustrated in FIG. 3, the result show that Suv39h1-deficient cell express increased levels of a stem cell-like signature. They also survive better and re-populate more efficiently wild type mice after adoptive transfer.

[0244] 4) Suv39h1 Deficient CD8+OT-1 Cells Control Tumor Growth In Vivo

[0245] FIGS. 4 B and C show that mice adoptively transferred with Suv39h1-KO OT-1 cells and treated with anti-PD1 monoclonal antibody controlled tumor growth better than mice that received a similar treatment but with WT OT-1 cells. Of note, by day 30 after tumor injection 4 out of 5 WT OT-1-transferred mice showed growing tumor, compared to only 1 out of 5 mice injected with the Suv39h1-KO OT-1 cells.

CONCLUSIONS

[0246] Absence of Suv39h1 activity in CD8+ T cells is associated to a better anti-tumor efficacy in an adoptive T cell transfer-based therapeutic approach.