Prevention or Treatment of Hematologic Malignancy Relapse Using a TNFR2 Antagonist

Abstract

The present disclosure relates to the in vivo prevention or treatment of hematologic malignancy relapse using a TNFR2 antagonist (an anti TNFR2 antagonist antibody) (i) for use in the prevention or treatment of hematologic malignancy relapse after allogeneic hematopoietic stem cell transplantation (AHCT) or after a treatment with lymphocytes and (ii) for use in enhancing the graft-versus-leukemia-activity (GVL activity) of a hematopoietic stem cell transplantation (HCT) or a treatment with lymphocytes.

Claims

1. A TNFR2 antagonist for use in the prevention or treatment of hematologic malignancy relapse after allogeneic hematopoietic stem cell transplantation (AHCT) or after a treatment with lymphocytes, wherein said TNFR2 antagonist is to be administered to the subjects during or after the allogeneic hematopoietic stem cell transplantation (AHCT) or the treatment with lymphocytes.

2. The TNFR2 antagonist for use according to claim 1, wherein the TNFR2 antagonist is selected from the group consisting of an anti-TNFR2 antibody, a peptide, a small molecule and a protein, preferably an anti-TNFR2 monoclonal antibody.

3. The TNFR2 antagonist for use according to claim 1 or 2, wherein the hematologic malignancy is selected from the group consisting of acute myeloid leukemia, myeloproliferative disorders, myelodysplasia and lymphoproliferative syndromes, and is preferably acute myeloid leukemia or acute lymphoblastic leukemia.

4. The TNFR2 antagonist for use according to any of claims 1 to 3, wherein the TNFR2 antagonist is to be administered less than 2 hours after the allogeneic hematopoietic stem cell transplantation (AHCT), preferably less than 1 hour, more preferably simultaneously to AHCT or the TNFR2 antagonist is to be administered after diagnosis with a hematologic malignancy relapse.

5. The TNFR2 antagonist for use according to any of claims 1 to 4, wherein said TNFR2 antagonist is to be administered in the form of a pharmaceutical composition.

6. The TNFR2 antagonist for use according to any of claims 1 to 5, wherein said TNFR2 antagonist is to be administered in an amount from 0.001 mg/kg to 10 mg/kg of body weight per day.

7. A TNFR2 antagonist for use in enhancing the graft versus leukemia activity (GVL activity) of an allogeneic hematopoietic stem cell transplantation (AHCT) or a treatment with lymphocytes, wherein said TNFR2 antagonist is to be administered to the subjects during or after the allogeneic hematopoietic stem cell transplantation (AHCT) or the treatment with lymphocytes.

8. The TNFR2 antagonist for use according to claim 7, wherein the TNFR2 antagonist is selected from the group consisting of an anti-TNFR2 antibody, a peptide, a small molecule and a protein, preferably an anti-TNFR2 monoclonal antibody.

9. The TNFR2 antagonist for use according to claim 7 or 8, wherein the hematologic malignancy is selected from the group consisting of acute myeloid leukemia, myeloproliferative disorders, myelodysplasia and lymphoproliferative syndromes, and is preferably acute myeloid leukemia or acute lymphoblastic leukemia.

10. The TNFR2 antagonist for use according to any of claims 7 to 9, wherein the TNFR2 antagonist is to be administered less than 2 hours after the allogeneic hematopoietic stem cell transplantation (AHCT), preferably less than 1 hour, more preferably simultaneously to AHCT or the TNFR2 antagonist is to be administered after diagnosis with a hematologic malignancy relapse.

11. The TNFR2 antagonist for use according to any of claims 7 to 10, wherein said TNFR2 antagonist is to be administered in the form of a pharmaceutical composition.

12. The TNFR2 antagonist for use according to any of claims 7 to 11, wherein said TNFR2 antagonist is to be administered in an amount of from 0.001 mg/kg to 10 mg/kg of body weight per day.

Description

FIGURES

[0063] FIG. 1 is a set of graphs showing that TNF?/TNFR2 disruption using anti-TNFR2 blocking mAb abolishes the protective effect of Treg after AHCT (A and B): [B6?C3H]F1 female mice underwent TBI (Total Body Irradiation) followed by transplantation with B6 BM (Bone Marrow) cells plus T cells or with B6 BM cells plus T cells supplemented with HY-Tregs. HY peptide was administered at day 0, 1, 3 and 6 and mice were treated or not with blocking anti-TNFR2 mAb administered at day, 0, 2 and 4. The experiment was performed twice and the resulting survival (A) and clinical score (B) data were pooled and compared among the three groups of mice. (C and D) Experimental groups consisted of mice grafted with B6 BM cells plus T cells treated or not with anti-TNFR2 administered on days, 0, 2 and 4. The experiment was performed twice and the resulting survival (C) and clinical score data (D) were pooled. Mice were sacrificed in case of weight loss>30% of initial weight or maximal clinical grade (i.e. 5/5). Kaplan-Meier survival curves were compared using log-rank test. For analysis of GVHD clinical grading curves, Area Under Curve (AUC) was calculated for each mouse then T-test or one-way ANOVA with post-Hoc analysis were performed depending on number of comparatives. ns: non-significant; *: P<0.05; **: p<0.01; ***: p<0.001.

[0064] FIG. 2 is a set of graphs showing that TNF?/TNFR2 disruption using TNFR2-KO Tregs abolishes the protective effect of Treg after AHCT. [B6?C3H]F1 female mice underwent TBI followed by transplantation with B6 BM cells plus T cells or with B6 BM cells plus T cells supplemented with HY-Tregs produced from WT B6 or from TNFR2 deficient mice in order to prevent GVHD. HY peptide was administered on days 0, 1, 3 and 6. The experiment was performed twice and the resulting survival and clinical score data were pooled. (A) Kaplan-Meier survival curves and (B) curves of evolution of GVHD clinical score over time were compared between the three groups of mice. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001.

[0065] FIG. 3 is a set of graphs showing that TNF?/TNFR2 disruption using anti-TNFR2 blocking mAb increases inflammatory cytokine production by donor CD4 and CD8 T cells. [B6?C3H]F1 female mice underwent TBI followed by transplantation with B6 BM cells plus T cells treated or not blocking anti-TNFR2 mAb administered at day, 0, 2 and 4. Mice were sacrificed and donor CD4.sup.+ and CD8.sup.+ T cells were analyzed at day 14 post-transplantation in the spleen of grafted animals. Mean absolute numbers of splenocytes and percentage of CD4.sup.+ and CD8.sup.+ donor T cells are determined (A) as well as intracellular IFN? (B) and TNF? (C) production. Each plot represents a mouse; T-test analysis was performed to compare anti-TNFR2 mAb effect on T cells. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001.

[0066] FIG. 4 is a set of graphs showing that TNF?/TNFR2 disruption and its effect on GVHD does not depend on the antigen specificity of therapeutic Tregs. (A and B): [B6?C3H]F1 female mice underwent TBI followed by transplantation with (i) B6 BM cells plus 2?10.sup.6 T cells or (ii) with B6 BM cells plus 2?10.sup.6 T cells supplemented with 2?10.sup.6 rs-Tregs or (iii) BM cells plus 2?10.sup.6 T cells collected from TNF?-deficient mice supplemented with 2?10.sup.6 rs-Tregs. HY peptide was administered at day 0, 1, 3 and 6 and mice were treated or not with blocking anti-TNFR2 mAb administered at day, 0, 2 and 4. The resulting survival (A) and clinical score (B) data were compared among the three groups of mice. Mice were sacrificed in case of weight loss>30% of initial weight or maximal clinical grade (i.e. 5/5). Kaplan-Meier survival curves were compared using log-rank test. For analysis of GVHD clinical grading curves, Area Under Curve (AUC) was calculated for each mouse then one-way ANOVA with post-Hoc analysis was performed. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001.

[0067] FIG. 5 is a set of graphs showing that TNF?/TNFR2 disruption and its effect on GVHD do not depend on the Treg/Tconv ratio.

[0068] (A and B): [B6?C3H]F1 female mice underwent TBI followed by transplantation with B6 BM cells plus T cells or with B6 BM cells plus 2?10.sup.6 Tcells supplemented with 4?10.sup.6 HY-Tregs. HY peptide was administered at day 0, 1, 3 and 6 and mice were treated or not with anti-TNFR2 administered at day, 0, 2 and 4. The resulting survival (A) and clinical score (B) data were compared among the three groups of mice. Mice were sacrificed in case of weight loss>30% of initial weight or maximal clinical grade (i.e. 5/5). Kaplan-Meier survival curves were compared using log-rank test. For analysis of GVHD clinical grading curves, Area Under Curve (AUC) was calculated for each mouse then oneway ANOVA with post-Hoc analysis was performed. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001.

[0069] FIG. 6 is a set of graphs showing that blockade of the TNF?/TNFR2 interaction reduces Foxp3 expression in Tregs used to prevent GVHD.

[0070] GVHD experiments were reproduced using (up) blocking anti-TNF mAb treatment or (down) T cells collected from TNF?-deficient mice. Splenocytes from grafted animals were harvested at day 13 post-transplantation and enriched in CD4.sup.+ and CD8.sup.+ cells through positive magnetic selection using large selection columns (Miltenyi Biotec). Cells were then gated on CD4.sup.+ cells. MFI values are represented as ratio of the measured value for each sample to the mean value of the control group (i.e. the group of mice receiving BM cells plus T cells and T reg cells). We have normalized the Mean Fluorescence Intensity (MFI) values with Tcell+Treg control group. Then, we compared Foxp3.sup.high, Foxp3.sup.int and Foxp3.sup.low expression on CD4.sup.+ cells. We used unpaired, two-tailed Student's t tests for generation of p-values. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0071] FIG. 7 is a set of graphs showing that blockade of the TNF?/TNFR2 interaction reduces Foxp3 and activation markers expressions in Tregs used to prevent GVHD. GVHD experiments were reproduced using (A) blocking anti-TNFR2 mAb treatment or (B) T cells collected from TNF?-deficient mice. Splenocytes from grafted animals were harvested at day 13 post-transplantation and enriched in CD4.sup.+ and CD8.sup.+ cells through positive magnetic selection using large selection columns (Miltenyi Biotec). Depending on the marker evaluated, Tregs were stained with CD4-FITC, CD4-APC or CD4-Vioblue, Foxp3-PE-Cy5 or Foxp3-V450, and CD25-PE-Cy7, ICOS-PE, CTLA4-biotin. Intracellular Foxp3 staining was performed using the Foxp3 staining buffer set from eBioscience. Cells were gated on CD4+ Foxp3+ cells except for the percentage of Foxp3 (up), which is gated on CD4.sup.+ cells. For each marker, the strategy of gating is indicated on the left of the figure. Each dot represents a single mouse. For each group of mice, horizontal lines represent mean value and SEM. MFI values are represented as ratio of the measured value for each sample to the mean value of the control group (i.e. the group of mice receiving BM cells plus Tcells and Treg cells. We have normalized the Mean Fluorescence Intensity (MFI) values with Tcel+Treg control group. Then we used unpaired, two-tailed Student's t tests for generation of p-values. ns: non-significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0072] FIG. 8 is a set of graphs showing: [0073] FIG. 8A: P815 characterization in blood sample of grafted animals at day 12. 12 days after bone marrow transplantation and P815 administration. p815 cells were detected in blood of grafted mice [0074] FIG. 8B: Tumor incidence in alloSCT grafted mice. Efficiency of anti-TNFR2 blocking mAb treatment was T cells dependent because there is no difference between treated and untreated group when mice were transplanted without T cells. [0075] FIG. 8C: GVHD incidence in alloSCT grafted mice. The GVL effect has linked to GVHD incidence.

EXAMPLES

Materials and Method

[0076] Mice

[0077] Wild-type C57BL/6 (66 H-2b) and B6C3HF1 (H-2kxb) mice were purchased from Harlan Laboratories (Gannat, France) and Charles River Laboratories (Saint-Germain-Nuelles, France). TNFR2?/? (TNFRs1b?/?) mice (i.e. mice KO for TNFR2) were purchased from the Jackson Laboratory (Bar Harbor, Me., USA). All mice were on a C57BL/6 background. Mice were housed under specific pathogen-free conditions. All experimental protocols were approved by the local ethics committee (authorization N? 11/12/12-5B) and are in compliance with European Union guidelines.

[0078] Treg Preparation

[0079] Treg were prepared as previously described (Martin G H, Gregoire S, Landau D A, et al. In vivo activation of transferred regulatory T cells specific for third-party exogenous antigen controls GVH disease in mice. Eur J Immunol. 2013; 43(9):2263-2272). Briefly, spleens and lymph nodes from C57BL/6 female mice were collected and mechanically dilacerated. Cell suspension was stained with biotin-coupled anti-CD25 monoclonal antibody (mAb) (7D4, BD Biosciences, San Diego, Calif., USA), followed by anti-biotin microbeads (Miltenyi Biotec, Paris, France) and CD25+ cells were positively selected through magnetic large selection column (Miltenyi Biotec). Selected cells were stained with the following mAbs: CD4-FITC (eBioscience, San Diego, Calif., USA), CD62L-PE (eBioscience), CD25-biotin (BD Biosciences) and streptavidin-PE-Cy5 (eBioscience). CD4.sup.+ CD25.sup.high CD62L.sup.high cells (i.e. Treg cells) were then sorted using a MoFlo Legacy (Beckman Coulter, Villepinte, France), with a purity of 99%.

[0080] For HY-Treg preparation, purified Treg cells were cultured for 3 to 4 weeks in the presence of recombinant murine IL-2 (long/mL; PeproTech, Neuilly-sur-Seine, France) and weekly stimulated with CD8.sup.+ dendritic cells (DCs) previously loaded with the HY peptide (10 ?g/mL, N-15-S, NY, PolyPeptide, Strasbourg, France) in the presence of GM-CSF (20 ng/mL; PeproTech). CD8+ DCs were isolated from splenocytes of C57BL/6 mice, as previously described (Maury S, Lemoine F M, Hicheri Y, et al. CD4+CD25+ regulatory T cell depletion improves the graft-versus-tumor effect of donor lymphocytes after allogeneic hematopoietic stem cell transplantation. Sci Trans/Med. 2010;2(41):41ra52). For recipient-specific (rs)Treg preparation, purified Treg cells were cultured for 3 to 4 weeks in the presence of recombinant murine IL-2 and weekly stimulated with irradiated total splenocytes from C3H female mice, as previously described (Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD -and promote immune reconstitution in HLA-haploidentical transplantation. Blood. 2011;117(14):3921-3928andGaidot A, Landau D A, Martin G H, et al. Immune reconstitution is preserved in hematopoietic stem cell transplant co-administered with regulatory T cells for GVHD prevention. Blood. 2011;117 (10):2975-2983).

[0081] GVHD and Transplantation Models

[0082] Eight-to-twelve weeks-old recipient B6C3HF1 female mice received a 10 Gy irradiation followed by retro-orbital infusion of 10.106 bone marrow cells+2.106 CD3.sup.+ T cells, with or without HY-Treg cells in a 1:1 ratio (i.e. 2.106 HY-Treg cells). Bone marrow and T cell suspensions were prepared using leg bones and splenocytes respectively, as previously described (Cohen J L; Boyer O, Salomon B et al. Blood 1997). All infused cells (Bone Marrow, T lymphocytes T and Treg) were isolated from female C57BL/6 mice (semi-allogeneic model). As recipient and donor mice were females, HY-Treg cells were activated in vivo by repeated retro-orbital infusions of 100 g of the HY peptide (at Day 0, Day 1, Day 3 and Day 6), as previously described (Martin G H, Gregoire S, Landau D A, et al. In vivo activation of transferred regulatory T cells specific for third-party exogenous antigen controls GVH disease in mice. Eur J Immunol. 2013;43(9):2263-2272). For rs-Treg experiments, mice were transferred with rs-Treg cells in a 1:1 ratio.

[0083] Antibody Treatment

[0084] Anti-TNFR2 (TR75-54.7) mAb was purchased from Bio X Cell (West Lebanon, N.H., USA). Recipient mice were treated with 3 intra-peritoneal injections of 500 g of the antibody on days 0, 2 and 4. GVHD clinical grading GVHD clinical score was calculated 2 to 3 times per week. Each of the 5 following parameters was scored 0 (if absent) or 1 (if present): weight loss>10% of initial weight, hunching posture, skin lesions, dull fur and diarrhea. Dead mice received a global score of 5. Mice were sacrificed in case of weight loss>30% of initial weight or maximal clinical grade (i.e. 5/5).

[0085] It has to be understood from the examples that a high GVL activity is correlated with a high GVHD clinical score. Therefore, the increase of GVHD clinical score reflects an enhancement of the GVL activity and a GVHD protection reflects a decrease of GVL activity.

[0086] Histopathological Examination.

[0087] Livers, lungs, skin, small and large bowels samples were preserved in Bouin's fixative and embedded in paraffin. For these organs, 5-?m-thick sections were stained with hematoxylin and eosin for histological examination as previously described (Trenado A, Sudres M, Tang Q, et al. Ex Vivo-Expanded CD4+ CD25+ Immunoregulatory T Cells Prevent Graft-versus-Host-Disease by Inhibiting Activation/Differentiation of Pathogenic T Cells. J Immunol. 2006;176(2):1266-1273). Briefly, one pathologist analyzed slides in a blinded fashion to assess the intensity of GVHD. GVHD lesions in each sample were scored according to a semi-quantitative scoring system described by Hill et al. with minor modifications (Hill G R, Cooke K R, Teshima T, et al. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J Clin Invest. 1998; 102(1): 115-123).

[0088] Flow Cytometry

[0089] Two weeks after transplantation (i.e. at Day 13, Day 0 being the date of transplantation and Treg cell injection), recipient mice were sacrificed and their spleens collected. Because of the low proportion of Treg cells among splenocytes and the low overall spleen cellularity at Day 13, cell suspensions obtained for each spleen were enriched in CD4.sup.+ and CD8.sup.+ cells after labeling with anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec) and positive magnetic selection through large selection columns (Miltenyi Biotec). Selected cells were then stained with the following mAbs: CD4-FITC, CD4-APC and CD4-Vioblue (Miltenyi Biotec), Foxp3-PE-Cy5 and Foxp3-V450 (eBioscience), CD25-PE-Cy7 (eBioscience), CD62L-PE (eBioscience), ICOS-PE (eBioscience), CTLA4-biotin (followed by streptavidin-PE-Cy7; eBioscience), IFN?-PE (Miltenyi Biotec), TNF?-FITC (Miltenyi Biotec), CD8?-FITC (eBioscience). Intracellular Foxp3 staining was performed according to the manufacturer's instructions, using the Foxp3 staining buffer set from eBioscience. For intracellular cytokine staining, cells were re-stimulated with 1 ?g/mL PMA (Sigma Aldrich, Saint Quentin Fallavier, France) and 0.5 ?g/mL Ionomicyn (Sigma Aldrich) for 5 h, in the presence of GolgiPlug (1 ?L/mL; BD Biosciences). Events were acquired on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software vX.0.7 (FlowJo, LLC, Ashland, Oreg., USA).

[0090] Statistical Analysis

[0091] Prism (GraphPad Software) was used for statistical analysis. Kaplan meier survival curves were compared using log-rank test. For analysis of GVHD clinical grading curves, Area under curve (AUC) were calculated for each mouse then T-test or one-way ANOVA with post-Hoc analysis were performed depending on number of comparatives. For cytometry analysis, we have normalized the Mean fluorescence intensity (MFI) values with T cell+Treg cell control group. Then we used unpaired, two-tailed Student's t tests for generation of p-values.

Example 1

TNFR2 Plays a Pivotal Role in Treg-Mediated Prevention of GVHD and Anti-TNFR2 Antagonist Enhancement of GVL

[0092] To assess the role of TNF on GVHD and GVL by Treg administration, we first used our recently described model in which the disease was prevented by transfer in females recipients of donor Tregs specific for the exogenous (i.e non-donor, non-recipient) HY antigen at time of AHCT, followed by their in vivo re-activation by HY peptide immunization (Martin G H, Gregoire S, Landau D A, et al. In vivo activation of transferred regulatory T cells specific for third-party exogenous antigen controls GVH disease in mice. Eur J Immunol. 2013;43(9):2263-2272). In a semi-allogeneic condition C57BL/6>[B6?C3H]F1 of bone marrow transplantation, GVHD protection at 1/1 Treg/Tconv ratio strictly depends on HY immunization.

[0093] We evaluated the role of TNFR2 in Treg-mediated GVHD and GVL using a anti-TNFR2 antagonist mAb (hereafter blocking anti-TNFR2 mAb) (FIGS. 1A and B). Mice transferred with Tconvs developed severe GVHD that was prevented by the co-transfer of HY-Tregs. The GVHD protection of Treg administration was fully abolished in mice that were treated with the blocking anti-TNFR2 mAb. These latter mice displayed high clinical GVHD scores and decreased survival, as compared to HY-Treg-treated control mice. The decrease of GVHD protection suggests that blocking anti-TNFR2 mAb can enhance the GVL activity of AHCT.

[0094] In order to assess whether a higher Treg:Tconv ratio could overcome the effect due to TNFR2 blockade, we reproduced the same experiment doubling the number of HY-Treg infused in recipient mice (2/1 Treg/Tconv ratio). Even with this increased numbers of therapeutic Tregs, blocking TNFR2 fully abolished the Treg-dependent GVHD protection (FIG. 5). This demonstrates the specificity of blocking anti-TNFR2 mAb to enhance the GVL activity of AHCT.

[0095] We then used TNFR2-deficient HY-specific Tregs, obtained from TNFR2KO mice, to confirm that the Treg control of GVHD and GVL by TNF was mediated by TNFR2 expression by Tregs. Whereas TNFR2-sufficient control Tregs fully protected from GVHD, TNFR2-deficient Tregs completely failed to protect mice from GVHD (FIG. 2). Survival of the mice and clinical scores of GVHD were identical in mice receiving donor Tconvs alone and mice receiving donor Tconvs and TNFR2-deficient Tregs. These results suggest that TNFR2 is key for GVL activity and that blocking anti-TNFR2 mAb is sufficient to enhance GVL activity.

[0096] We then assessed the role of TNFR2 in Treg-mediated protection in another transplant setting. Tregs naturally present in the donor T-cell inoculum were present in sufficient number to attenuate GVHD since their depletion accelerated the disease (Cohen J L, Trenado A, Vasey D, Klatzmann D, Salomon B L. CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease. J Exp Med. 2002;196(3):401-406). Here, we observed that blocking TNFR2 with blocking anti-TNFR2 mAb led to a similar high GVHD clinical score, which reflects an increase of GVL activity. Indeed, in mice grafted with bone marrow cells (AHCT) and whole T cells containing Tregs at physiological level, administration of the blocking anti-TNFR2 mAb induced an accelerated GVHD (FIGS. 1 C and D). These results suggest that blocking anti-TNFR2 mAb can enhance the GVL activity of AHCT. The number of splenocytes collected at day 14 importantly varied between the two groups of mice. Whereas spleens of mice grafted with T cells contain 65.4?106?2.2 cells, this number fell sharply to 12.3?106?9.6 in mice treated with blocking anti-TNFR2 mAb, probably reflecting an accelerated GVHD and an increased GVL. We next evaluated the effect of blocking TNFR2 mAb on cytokine production in the spleen of mice developing GVHD after transfer of WT donor T cells. Mice treated with the blocking anti-TNFR2 mAb had an increase in IFN? and TNF?-production in both CD4 and CD8 donor T cells (FIG. 3). In order to reinforce the robustness of our observations, we used a third transplant setting consisting in infusing Tregs that were rendered specific for recipient-type allo-Ag, (namely rs-Treg) instead of HY-Treg to prevent GVHD. Whereas GVHD was prevented by rs-Treg administration, this protective effect was fully abolished when using blocking anti-TNFR2 mAb (FIG. 4). Thus, using 2 different approaches (anti-TNFR2 mAb and TNFR2-deficient Tregs) and different types of Tregs (Tregs of the T cell inoculum, therapeutic HY-Tregs or rs-Tregs), we demonstrate that the control of GVHD by Tregs is TNFR2 dependent. Thus, we have demonstrated that blocking anti-TNFR2 mAb can accelerate GVHD, suggesting that blocking anti-TNFR2 mAb can enhance GVL activity of an allogeneic hematopoietic stem cell transplantation (AHCT) or a treatment with lymphocytes.

[0097] Conclusion: The enhancement of GVL activity of AHCT could be mediated through TNFR2 expressed by Tregs. TNF-TNFR2 interaction is critical in the regulation of GVL activity by Tregs in AHCT performed either in routine or in clinical trials when therapeutic Tregs are injected. A TNFR2 antagonist can enhance the GVL activity of a AHCT or a treatment with lymphocytes.

Example 2

After AHCT, TNF/TNFR2 Blockade Reduces Foxp3 and Activation Markers Expression on Tregs

[0098] To analyze by what mechanism the control of GVHD by Tregs and enhancement of the GVL activity depends on TNF/TNFR2 interaction, we measured the proportion and activation markers of Tregs in the spleen collected at day 13 in mice grafted with HY-Tregs and either WT Tcells and treated with blocking anti-TNFR2 mAb or TNF-deficient Tcells. First, the expression level of Foxp3 was significantly reduced when TNF/TNFR2 interaction was inhibited in both settings, whereas Treg proportions remained unchanged (FIG. 7 A, B). This lower Foxp3 expression among whole Tregs was characterized by a reduced proportion of Foxp3.sup.high expressing cells and an increased proportion of Foxp3.sup.int expressing cells in both experimental models (FIG. 6). A likely explanation would be that, in the absence of TNFR2 signaling in Tregs, Foxp3 would be down-modulated, suggesting that TNF stabilized Foxp3 expression in Tregs. In the same line, in the absence of TNFR2 signaling, Treg could be less stable and could convert into pro-inflammatory T cells.

[0099] We further analyzed the expression of CD25, the a chain of the IL-2 receptor constitutively expressed at the Treg cell surface membrane. We observed a dramatic decrease of the percentage of CD25+ cells and CD25 expression level among Tregs when blocking anti-TNFR2 mAb was administered to grafted mice. Since in experimental AHCT CD25 expression is up-regulated by IL-2, these results suggest that TNF? increased IL-2 responsiveness of Tregs in this context.

[0100] Finally, we evaluated the expression of ICOS and CTLA4, which are important molecules in Treg biology. In both models of inhibition of TNF/TNFR2 interaction (i.e. blocking anti-TNFR2 mAb or TNF-deficient Tcells), ICOS and CTLA-4 expressions were reduced compared to controls, with a more pronounced effect in mice treated with the blocking anti-TNFR2 mAb than mice grafted with TNF?-deficient T cells (FIG. 7), probably reflecting the more complete abrogation of TNF signaling in the presence of the monoclonal antibody.

[0101] Conclusion: TNF activates Treg via stabilization of Foxp3. The anti-TNFR2 reduces Foxp3 and thus blocks Treg and therefore increases alloreactivity. This suggests an enhancement of the GVL activity.

Example 3

Improved Anti-Tumor Effect of T Cells by Anti-TNFR2 Treatment

[0102] Eight to 12-week-old recipient B6D2F1 female mice received a 10 Gy irradiation followed by retro-orbital infusion of 5?10.sup.6 bone marrow (BM) cells+2?10.sup.4 host type P815 (DBA/2-derived, H-2Dd, GFP) tumor cells, with (Tumour+T cells group; n=8) or without (Tumor group n=7) 1?10.sup.6 T cells. BM and T cells were isolated from female C57BL/6 mice. Tumor (Tumor+TNFR2 group; n=11) and Tumor+Tcells group (Tumor+Tcells+?TNFR2 group; n=11) were treated with 3 intraperitoneal injections of 500 ?g of the anti-TNFR2 blocking mAb (TR75-54.7) on days 0, 2, and 4.

[0103] The results are shown in FIG. 8: (FIG. 8A) P815 characterization in blood sample of grafted animals at day 12. Tumoral (FIG. 8B) and GVHD (FIG. 8C) incidence in alloSCT grafted mice.

[0104] Results: 12 days after bone marrow transplantation, p815 cells were detected in blood of grafted mice (FIG. 8A). When mice were transplanted with T cells then treated with the blocking anti-TNFR2 mAb, tumor incidence decrease compared to untreated mice (36% vs 91% respectively). Efficiency of blocking anti-TNFR2 mAb treatment was T cells dependent because there is no difference between treated and untreated group when mice were transplanted without T cells (FIG. 8B). As expected, this GVL effect has linked to GVHD incidence (FIG. 8C) and score (data no shown).

[0105] Conclusion: These results demonstrate that mice grafted with suboptimal numbers of T cells were capable to reject tumor cells when Treg functionality was abolished by anti-TNFR2 blocking treatment.