T CELLS MODIFIED TO EXPRESS MUTATED CXCR4 OR PARTIALLY DELETED AND USES THEREOF

Abstract

The present invention relates to modified T cells, and in particular to CD8 T cells, for use in therapy. The current inventors investigates the effect of CXCR4.sup.Whim mutation on CD8 effector and memory responses. By analysing the lymphoid organs, including the BM compartment, in a mouse model of Whim syndrome and in mice where only CD8 T-cells carry the mutation, this study shows that CXCR4.sup.Whim mutation only partially affect CD8 primary responses. By contrast, CXCR4.sup.Whim mutation considerably improve the long-term maintenance and magnitude of CD8 memory responses by increasing the pool size of Antigen specific CD8 T-cells, first in the BM and then in other lymphoid organs, bringing new insight into the current discrepancy regarding the role of the BM in the maintenance CD8 memory cells. In particular, the present invention relates to T cell, and more particular to CD8 T cell, characterized in that it expresses CXCR4.sup.Whim mutation or a CXCR4 with the deletion of the C-terminal domain between and 20 amino acid residues.

Claims

1-8. (canceled)

9. A method of producing a T cell which expresses a chimeric antigen receptor that recognizes and binds to an antigen, comprising transfecting or transducing, in vitro or ex vivo, a T cell that expresses a CXCR4.sup.Whim mutation or that has a deletion of from 10 to 20 amino acid residues within a C-terminal domain of CXCR4, in with a vector encoding for the chimeric antigen receptor.

10. (canceled)

11. A pharmaceutical composition comprising a population of T cells that express a CXCR4.sup.Whim mutation or that have a deletion of from 10 to 20 amino acid residues within a C-terminal domain of CXCR4, and a pharmaceutically acceptable carrier.

12. A method of treating an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of T cells which express a CXCR4.sup.Whim mutation or which have a deletion of 10-20 amino acid residues in a C-terminal domain of CXCR4.

13. The method according to claim 12 wherein the infectious disease is caused by a virus or a bacteria that causes lung infection.

14. A method of treating cancer in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of T cells which express a CXCR4.sup.Whim mutation or which have a deletion of 10-20 amino acid residues in a C-terminal domain of CXCR4.

15. The method according to claim 14 wherein the cancer is a hematopoietic malignancy selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic myeloid leukemia, lymphoid leukemia, lymphoma and myelodysplastic syndrome.

16. The method of claim 12, wherein the CXCR4.sup.Whim mutation is selected from the group consisting of: R334X, G336X, E343X, S341fs, S339fs342X, S338X and E343K.

17. The method of claim 12 wherein the deletion is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 16, 17, 18, 19 or 20 amino acid residue deletion.

18. The method of claim 12, wherein the T cells are CD8+ T cells.

19. The method of claim 12, wherein the T cells express a chimeric antigen receptor which recognizes and binds to an antigen.

20. The method of claim 14, wherein the CXCR4.sup.Whim mutation is selected from the group consisting of: R334X, G336X, E343X, S341fs, S339fs342X, S338X and E343K.

21. The method of claim 14 wherein the deletion is a 10, 11, 12, 13, 14, 15, 16, 17, 18, 16, 17, 18, 19 or 20 amino acid residue deletion.

22. The method of claim 14, wherein the T cells are CD8+ T cells.

23. The method of claim 14, wherein the T cells express a chimeric antigen receptor which recognizes and binds to an antigen.

24. The method of claim 14, wherein the T cells are administered in adoptive cell therapy.

Description

FIGURES

[0130] FIG. 1: Schematic View of the Materiel and Method

[0131] Schematic view of the experimental system used in the study. Left: WT mice and Whim mice (CXCR4.sup.1013/+) were infected intranasally by 2.Math.10.sup.5 PFU of Vaccinia Virus (VV). Right: nave (CD44.sup.low) CD8 T-cells were FACS-sorted from F5-WT (expressing CD45.1. congenic marker) and FS-Whim (expressing CD45.2 congenic marker) mice and were mixed in equal numbers in PBS. The equivalent of 1.Math.10.sup.5 F5-WT and 1.Math.10.sup.5 FS-Whim were injected intravenously into C57Bl/6 WT recipient (expressing both CD45.1 and CD45.2 congenic marker). 1 to 2 days following cells transfer, mice were infected intranasally by 2.Math.10.sup.5 PFU of Vaccinia Virus expressing NP68 peptide (VV-NP68, that is specific for F5-TCR)

[0132] (FIG. 2 Delayed recirculation pattern of CD8 T-cells in Whim mice following infection. (a, b) WT and Whim mice were infected intranasally by Vaccinia Virus and absolute Numbers of total CD8 T-cells (gated as CD8.sup.+ CD44.sup.hi) (a) or antigen-specific CD8 T-cells (gated as CD8.sup.+ CD44.sup.hi B8R.sup.+ except for day 0, gated on total CD8.sup.+ B8R.sup.+) (b) were followed in the blood of infected animals, at indicated time points. (c) Absolute numbers of Antigen-specific CD8 T-cells (gated as CD8.sup.+ CD44.sup.hi B8R.sup.+) in lung draining LN. White and black dots show WT and CXCR4 Whim mice, respectively. Data represent a pool of 3 (a, b) or 2 (c) independent experiments.

[0133] FIG. 3: WT and Whim mice were infected intranasally by Vaccinia Virus. (a) Absolute Numbers of total CD8 T-cells (gated as CD8.sup.+ CD44.sup.hi) or antigen-specific CD8 T-cells (gated as CD8.sup.+ CD44.sup.hi B8R.sup.+) were calculated by adding up corresponding cells counted in the medLN, spleen, BM and lung, 6 weeks following infection. (b) Distribution of total CD8 T-cells (top panel) or Antigen specific CD8 T-cells (lower panel) was evaluated as the fraction of total memory cells that is located in indicated organs. (c) Splenocytes from WT and Whim infected animals were stimulated in vitro for 5 h with B8R cognate peptide. % of cells producing indicated cytokines was evaluated by flow-cytometry within CD8.sup.+ CD44.sup.hi gated cells. White and black dots show WT and CXCR4 Whim mice, respectively. Data represent a pool of 4 (a), 3 (b), or 1 (c) independent experiments with 4 (c) to 19 animals (a-b).

[0134] FIG. 4: NP68 specific F5-WT (CD45.1+) and F5-Whim (CD45.2+) nave CD8 T-cells were co-transferred into congenic mice (CD45.1+CD45.2+) and further subjected to intranasal infection by Vaccinia Virus expressing NP68. Mice were analysed two to 3 months post-infection. (a) Absolute Numbers of total F5 CD8 T-cells were calculated by adding up corresponding cells counted in the medLN, spleen, BM and lung. (b) Splenocytes from infected animals containing F5-WT and FS-Whim CD8 T-cells) were stimulated in vitro for 5 h with NP68. % of cells producing IFNg was evaluated by flow-cytometry within F5-WT (CD45.1+ CD45.2) and FS-Whim (CD45.2+ CD45.1) CD8.sup.+ CD44.sup.hi gated cells. (c) Distribution of F5-WT (white dots) and FS-Whim (Black dots) CD8 T-cells was evaluated as the fraction of total F5-WT or FS-Whim memory cells located in indicated organs.

[0135] White and black dots show WT and CXCR4 Whim mice, respectively. Data represent a pool of 3 (a,c) or 2 (b) independent experiments with at least 8 animals in total.

[0136] FIG. 5: Mice co-transferred with F5-WT and F5-Whim nave CD8 T-cells were infected by VV-NP68 as previously described. [0137] (a) F5-WT andWhim CD8 T-cells were followed in the blood of infected animals, at indicated time points. Datas are indicated as the ratio between the number of F5-WT and the number of FS-Whim cells. Dotted line represents a ratio=1, indicating similar numbers of F5-WT and F5-Whim.(b) Absolute numbers of F5-WT and FS-Whim in the blood of infected animals, at indicated time points.(c) Absolute Numbers of total F5 CD8 T-cells were calculated by adding up corresponding cells counted in the medLN, spleen, BM and lung, day 578 post infection. (d) Absolute Numbers of total F5 CD8 T-cells in in the Bone Marrow (top panel) and the spleen (lower panel) at indicated time post infection.

[0138] White and black dots show FS-WT and FS-Whim, respectively. Data are representative of 3 independent experiments with at least 5 mice per experiment.

EXAMPLE

[0139] Material & Methods (see FIG. 1)

[0140] Mice:

[0141] C57BL/6 mice from Charles River were used as wild-type controls. C57BL/6 CXCR4.sup.+/1013 mice (Whim mice) were provided by Dr K. Balabanian.sup.6. F5 TCR-transgenic mice (CD45.1.sup.+) were obtained from Dr D. Kioussis.sup.25. FS-CXCR4.sup.+/1013 (FS-Whim, CD45.2.sup.+) and C57BL/6.Ly5.1 (CD45.1.sup.+.CD45.2.sup.+) were generated in the animal house (PBES). All mice were bred in the PBES (SFR Biosciences animal facility, Lyon, France) under specific pathogen-free conditions. Experiments were done on mice aged from six weeks to 18 months (memory compartment). All animal procedures were approved by our local Animal Evaluation Committee (CECCAPP).

[0142] Virus and Immunization:

[0143] Recombinant Vaccinia Viruses (VV) expressing the NP68 epitope were engineered from VV (strain Western Reserve) by Dr Denise Yu-Lin Teoh from Pr Sir Andrew McMichael's laboratory at the Human Immunology unit, Institute of Molecular Medicine, Oxford, UK.

[0144] For cell transfer, C57BL/6 (CD45.1.sup.+.CD45.2.sup.+) recipients were transferred with 1.Math.10.sup.5 CD8-Ts from F5-Whim and FS-WT donor mice, by intra-venous injection in the retro-orbital sinus. The following day, recipients were infected by intra-nasal injection with VV-NP68 (2.Math.10.sup.5 PFU/mouse).

[0145] Cell Preparation, Culture and Activation:

[0146] Blood was collected from the retro-orbital sinus into 1 mL PBS containing 4 mM EDTA (Gibco). Flow-count fluorospheres (Beckman-Coulter) were then added to each tube and absolute numbers of cells/mL were calculated using the formula: [(Total number of cells/Total number of fluorospheres)fluorosphere concentration]/volume.

[0147] Single cells suspension from mediastinal lymph node and spleen were obtained by mechanical disaggregation on a 100 m cell strainer (BD Falcon). Lungs were flushed with PBS before harvesting from animals. To analyze the lung resident compartment, CD45 (clone 30-F11) was injected intra-venously before killing animals.sup.26. Single cells suspensions were obtained using the lung dissociation kit, according to the manufacturer's instructions (Miltenyi Biotec). Cells from bone marrow were collected by flushing complete medium through tibias and femurs. For all organs, cells were resuspended in complete medium (DMEM, 6% Fetal Calf Serum, 1M hepes, 50 g mL.sup.1 gentamicin, 50 M -mercapto-ethanol). Absolute numbers of lymphocytes were determined using Accuri C6 Flow instrument (BD Biosciences) and Cflow software.IFN- production by CD8-Ts was induced by restimulation with NP68 (10 nM) for five hours, in presence of brefeldin A (1/500 dilution).

[0148] Flow Cytometry:

[0149] Cells were stained for 30 minutes on ice, in PBS 0.5% BSA, 0.01% NaAzide. The following antibodies, coupled to the appropriate fluorochromes, were used: CD8 (clone 53-6.7), CD45.1 (A20), CD44 (IM7.8.1), from BD Biosciences; CD45 (30-F11), from Biolegend; CD45.2 (104), CD62L (MeI14), from eBiosciences.

[0150] To evaluate cytokine secretion, cells were permeabilized using Cytofix/cytoperm kit (BD Biosciences), before being incubated with an antibody against IFN- (XMG1.2, BD Biosciences).

[0151] All samples were acquired on LSR Fortessa flow cytometer (BD Biosciences) and analysed using FlowJo software (TreeStar).

[0152] Statistical Analysis:

[0153] Results were analysed with Prism software. Data are expressed as means+/SEM. Statistical analyses used the unpaired two-tailed t-test and one-way or two-ways Anova. A p-value<0.05 was considered to be statistically significant.

[0154] Results

[0155] CD8 Responses to Lung Viral Infection are Slightly Delayed in Whim Mice

[0156] Upon infection, CD8 responses encompass substantial trafficking between SLO and infected tissue. Nave CD8 T-cells are activated in the mediastinal LN (medLN, lungs draining LN) before re-entering blood circulation in order to migrate back to the lung. In order to analyse the role of CXCR4 on CD8 trafficking, we made use of a mouse model of Whim syndrome, carrying CXCR4+/1013 gain-of-function mutation (referred as Whim mice).sup.6. Importantly and as described previously, this mouse model recapitulates immune defects observed in Whim patients, including decreased numbers of CD8 T-cells in the spleen and the blood of nave animals, as compared to wild type (WT) animals, reflecting lymphopenia described in patients (.sup.6 and data not shown). In order to evaluate CD8 T-cells responses following lung viral infection, we performed intranasal infection of WT and Whim mice with Vaccinia Virus (VV). Blood analysis of WT infected animals indicate that absolute numbers of total activated CD8 T-cells (as characterized by the expression of CD44 activation marker) was increased in this compartment at day 7 post infection, probably reflecting their release from the draining LN (FIG. 2a). Interestingly, when similar analysis was made in the blood of Whim mice, we could not observe such an increase at day 7 post infection (FIG. 2a). However, comparable numbers of cells could be detected in the blood of both WT and Whim mice 15 days post-infection (although with a slight advantage for Whim animals) suggesting a delayed circulation pattern of activated CD8 T-cells, in Whim animals (FIG. 2a). In order to specifically assess the recirculation pattern of Antigen specific CD8 T-cells, we made use of tetramers that recognise B8R specific CD8 T-cells, since B8R is a dominant epitope of Vaccinia Virus in C57Bl/6 animals. FIG. 1b shows that the delayed recirculation pattern observed for total activated CD8 T-cells in Whim mice was also true for B8R specific CD8 T-cells, with similar kinetics. Since CXCL12, the major ligand for CXCR4, is largely secreted in the LNs, we hypothesised that CD8 T-cells could be sequestered in this compartment, in Whim animals. As anticipated, the delayed appearance of CD8 T-cells in the blood of Whim animals was associated with a corresponding retention of Antigen-specific cells in the mediastinal LN, at day 7 post infection (FIG. 2c). Altogether, those results suggest that CD8 responses can be generated in Whim mice, with a minor defect during the initial activation phase that delay the release of CD8 T-cells from the draining LN.

[0157] To further investigate the degree and nature of this delay, we performed mathematical modelling that estimated approximatively 1 to 2-day delay (data not shown).

[0158] CD8Whim Memory Cells are Preferentially Localised in the BM

[0159] We next thought to analyze the capacity of Whim mice to generate CD8 memory T-cells following lung infection. To that aim, we performed intranasal infection of WT and Whim animals with Vaccinia virus, and studied CD8 T-cells numbers and phenotype in the Lung, medLN and spleen, six weeks following infection. Since memory CD8 T-cells can home in the bone marrow (BM) and because this compartment is enriched for CXCL12, we included it in our analysis. By evaluating the sum of memory CD8 T-cells harvested from the spleen, the lung, the mediastinal LN and BM, we could show that comparable numbers of total and Ag-specific CD8 T-cells (specific for B8R) were generated in WT and Whim mice following infection (FIG. 3a, left and right panel, respectively). In addition, no differences could be observed with regards to their capacity to produce IFN, IL-2 and TNF- (typical cytokines produced by memory CD8) when memory CD8 splenocytes were re-stimulated ex vivo with their cognate antigen (here B8R) (FIG. 3c). We next seek to understand whether CXCR4-Whim mutation could affect the localization of memory CD8 T-cells amongst the different organs. To that aim, we evaluate the fraction of total memory CD8 T-cells that could be harvested from the spleen, the lung, mediastinal LN and BM, in WT and Whim animals. Remarkably, distinct distribution was observed, with a highly preferential localization of CXCR4.sup.Whim CD8 memory cells in the BM of infected animals, as compared to WT, and a respective decrease of cells in the spleen (FIG. 3b).

[0160] In order to assess whether this preferential localization of memory CD8 T-cells is the BM was CD8-cells intrinsic, we made use of F5 TCR transgenic mice whose TCR specifically recognise NP68 peptide.sup.25. WT (FS-WT) expressing CD45.1 congenic marker and CXCR4+/1013 (FS-Whim, CD45.2+) nave CD8 T-cells expressing F5 transgene were co-transferred in congenic host (CD45.1+CD45.2+) before intranasal infection with a recombinant Vaccinia Virus expressing NP-68 (VV-NP68).sup.16. 60 days post infection, analysis of medLN, Lung, Spleen and BM compartment showed neither differences in absolute numbers between FS-WT and FS-Whim (FIG. 4a), nor in the capacity of spleen memory cells to respond to NP68 peptide ex vivo and produce IFNg (FIG. 4b).

[0161] Nevertheless and similar to Whim mice, FS-Whim memory cells showed a modified distribution as compared to FS-WT, with a substantial increase of cells in the BM (FIG. 4c). Of note this was also observed when FS-WT or FS-Whim were transferred in separate hosts. Altogether, those results suggest that CXCR4 Whim mutation within CD8 T-cells confers their preferential localisation in the BM, a site that is enriched for CXCL12.

[0162] CD8whim Memory Cells Outnumber WT Memory Cells in Lymphoid Organs Over Time

[0163] BM has been suggested as a preferential site for CD8 memory maintenance, but discrepancies exist regarding the effect of specific bone marrow niches on CD8 memory cells outcome.

[0164] Since we observed a preferential localization of CD8-whim in the BM, we next thought to investigate whether this could impact long-term maintenance and/or accumulation of CD8 memory cells. To that aim, we co-transferred FS-WT and FS-CXCR4 in nave WT host and subsequently performed intranasal infection with VV-NP68 virus. Blood kinetics of FS-WT and FS-CXCR4 indicate comparable numbers of both cell types in the blood (ratio FS-WT/F5-CXCR4 close to 1), during early memory phase (FIG. 5A et 5B, until day 100). However, over time, blood was gradually populated by F5-CXCR4 and the ratio became in favour of F5-CXCR4 (FIG. 5A). Remarkably, analyses of cell numbers in the blood over time indicate that F5-WT numbers were maintained throughout time, while F5-CXCR4 numbers increased when the ratio started to be reverted (FIG. 5C). Even more interestingly, analyses of cell numbers in Spleen, Lungs and LN at very late time points (from day 300 post infection) showed a dramatic increase in F5-CXCR4 absolute numbers in all organs, while F5-WT CD8 numbers remain similar to early memory stage (FIG. 5 C). More specifically, numbers of FS-Whim cells gradually increased in the BM between 3 and 15 months post infection (FIG. 5D, top pannel), while their number started increasing in the spleen only between 6 and 15 months post infection (FIG. 5D, lower panel)

[0165] Molecular and Cellular Mechanism Underlying the Increase Number of CD8.sup.Whim Memory Cells

[0166] To apprehend the relative contribution of BM localization in CD8 proliferation and/or survival of memory CD8 T-cells, current analysis includes: [0167] BrDU assay (Chaix:2014gp) and cell cycle analysis combined with mathematical modelling; [0168] scRNA sequencing.sup.27 comparing F5-WT and FS-Whim isolated from the bone marrow, >10 months post infection with VV-NP68

[0169] Role of CXCR4.sup.Whim in the Long-Term Maintenance of Anti-Tumor Responses.

[0170] Innovating immunotherapy approaches are currently developed and include Chimeric Antigen Receptor (CAR) that are engineered and transferred into T-cells to reprogram their cytotoxicity toward an antigen expressed by a given tumour.sup.28. However, and although CAR-T cells therapies have given promising results thus far on Multiple Myeloma and Acute Lymphoblastic Leukemia, some patients still relapse.sup.29,30, emphasizing the need for a better maintenance of CAR-T cells. Interestingly, CD8 T-cells over-expressing CXCR4 have shown a better protection in mice models of lymphoma, as compared to WT.sup.31. Since much insight related to the feature of current immunotherapies targeting CD8 and memory CD8 T-cells has come from the study of virus infection of mice (e.g. the role of PD1 in regulating CD8 T cells post LCMV infection.sup.32), we study the impact of CXCR4 Whim-mutation on the long-term maintenance and pool size of tumor-specific CD8 T-cells responses.

[0171] This analysis include: [0172] investigating anti-tumoral responses of Whim mice (as compared to WT) and mice transferred with F5-WT and F5-CXCR4.sup.Whim CD8 T-cells following immunization with a tumor cell line expressing NP68. This study include the analysis of the long term maintenance of tumor-specific memory CD8 T-cells. [0173] proof of concept of the benefit of CAR-T cells expressing CXCR4.sup.Whim for the long-term maintenance of CD8 responses against a tumor

TABLE-US-00002 TABLE2 Usefulaminoacidsequencesfor practicingtheinvention SEQIDNO Aminoacidsequence 1.Cterm KFKTSAQHALTSVSRGSSLK domainWT ILSKGKRGGHSSVST CKCR4 ESESSSFHSS 2.Cterm KFKTSAQHALTSVSRGSSLK domainof ILSKGKRGGHSSVST E343K KSESSSFHSS 3.Cterm KFKTSAQHALTSVSRGSSL domainof KILSKGKRGGHSSVST E343X 4.Cterm KFKTSAQHALTSVSRGSS domainof LKILSKGKRGGHSSVPL E341fs SLSLQVFTPA...* 5.Cterm KFKTSAQHALTSVSRGSS domainof LKILSKGKRGGHSCFH E339fs 6Cterm KFKTSAQHALTSVSRGS domainof SLKILSKGKRGGH E338X 7.Cterm KFKTSAQHALTSVSRGS domainof SLKILSKGKRG E336X 8.Cterm KFKTSAQHALTSVSRGS domainof SLKILSKGK E334X

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