Antibodies against human CD39 and use thereof for inhibiting T regulatory cells activity

Abstract

The invention relates to antibodies against human CD39 and use thereof for inhibiting T regulatory cells (Treg) activity. More particularly CD39 antibodies may be used for the treatment or prevention of cancers and infectious diseases.

Claims

1. An anti-CD39 antibody or antigen binding fragment thereof comprising: a heavy chain wherein the variable domain comprises the sequence of SEQ ID NO:2 for CDR-H1, SEQ ID NO:3 for CDR-H2 and SEQ ID NO:4 for CDR-H3; and a light chain wherein the variable domain comprises the sequence of SEQ ID NO:6 for CDR-L1, SEQ ID NO:7 for CDR-L2 and SEQ ID NO:8 for CDR-L3.

2. The anti-CD39 antibody or antigen binding fragment thereof of claim 1, which comprises: a heavy chain variable region having the amino acid sequence set forth as SEQ ID NO: 1 and a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.

3. The anti-CD39 antibody or antigen binding fragment thereof of claim 1, wherein said anti-CD39 antibody or antigen binding fragment thereof is selected from the group consisting of a Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, a diabody, and multispecific antibodies formed from antibody fragments.

4. The anti-CD39 antibody or antigen binding fragment thereof according to claim 3 that is a humanized antibody or antigen binding fragment.

5. A pharmaceutical composition comprising the anti-CD39 antibody or antigen binding fragment thereof according to any one of claims 1, 2, and 3-4 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises a therapeutically effective amount of the antibody or antigen binding fragment thereof for treating cancer or an infectious disease associated with an increased T regulatory cell (Treg) activity.

7. The pharmaceutical composition of claim 6, wherein the cancer is selected from the group consisting in lung cancer, breast cancer, ovarian cancer, melanoma, liver cancer, gastric cancer and lymphoma.

8. A method of treating a subject for a cancer or an infectious disease associated with an increased T regulatory cell (Treg) activity comprising administering the pharmaceutical composition of claim 6 to a subject in need of treatment for a cancer or an infectious disease associated with an increased T regulatory cell (Treg) activity.

9. The method of claim 8, wherein the cancer is selected from the group consisting in lung cancer, breast cancer, ovarian cancer, melanoma, liver cancer, gastric cancer and lymphoma.

Description

FIGURES

(1) FIG. 1: Expression of CD39 on CD4.sup.+CD25.sup.low and CD4.sup.+CD25.sup.high T cells from HIV.sup.− controls and HIV.sup.+ patients. Peripheral blood lymphocytes from HIV.sup.− (□∇) controls and HIV.sup.+ (.square-solid..Math.) patients were stained with anti-CD39 BY40, anti-CD4 and anti-CD25 mAbs and percentages of CD39.sup.+ cells within CD4.sup.+CD25.sup.low and CD4.sup.+CD25.sup.high populations (A) as well as mean fluorescence intensity (MFI) for CD39 expression in both subpopulations (B) were determined by flow cytometry. Representative overlays comparing the levels of CD39 expression on CD4.sup.+CD25.sup.low(left) and CD4.sup.+CD25.sup.high (right) T cells from HIV.sup.+ (empty histograms) and HIV donors (filled histograms) are represented in C. In A and B, p values assessed by Wilcoxon's and Man-Witney's non-parametric tests are indicated.

(2) FIG. 2: Effect of the CD39 mAb, BY40, on the immunosuppressive activity of CD4.sup.+CD25.sup.high regulatory T cells. A. CFSE-labeled CD8 T cells from HIV.sup.+ patients were incubated in the presence of immobilized CD3 mAb (5 μg/ml) and CD39 mAb (upper histogram) or in the presence of autologous CD4.sup.+CD25.sup.high Tregs (4:1 ratio CD8:Tregs) (middle histogram) or in the presence of autologous CD4.sup.+CD25.sup.high Treg pre-incubated with CD39 mAb (bottom histogram). After 72 h, percentages of divided cells (CFSE low) were determined by flow cytometry. B. Percentages of inhibition of CD8 T cell proliferation in the presence of autologous CD4.sup.+CD25.sup.high cells (upper panel) are calculated as (% of CFSE.sup.low CD8.sup.+ T cells in the presence of autologous CD4.sup.+CD25.sup.high cells)×100 (% Of CFSE.sup.low CD8.sup.+ T cells in the absence of CD4.sup.+CD25.sup.high cells). Percentages of restoration of CD8 T cell proliferation in the presence of anti-CD39 mAb (lower panel) are calculated as 100−(% CFSE.sup.low CD8.sup.+ T cells in the presence of autologous CD4.sup.+CD25.sup.high cells pre-treated with CD39)×100/(% CFSE.sup.low in the presence of autologous CD4.sup.+CD25.sup.high cells). p values assessed by Wilcoxon's and Man-Witney's non-parametric tests are indicated.

(3) FIG. 3: Effect of the CD39 mAb, BA54g, on the immunosuppressive activity of CD4.sup.+CD25.sup.high regulatory T cells. A. CFSE-labeled CD8 T cells from HIV.sup.+ patients were incubated in the presence of immobilized CD3 mAb (5 μg/ml) and BA54g mAb (upper histogram) or in the presence of autologous CD4.sup.+CD25.sup.high Tregs (4:1 ratio CD8:Tregs) (middle histogram) or in the presence of autologous CD4.sup.+CD25.sup.high Treg pre-incubated with BA54g mAb (bottom histogram). After 72 h, percentages of divided cells (CFSE low) were determined by flow cytometry.

(4) FIG. 4: Modulation of CD39 expression by several anti-CD39 mAbs. YT2C2 NK cells (1×10.sup.6 cells/ml) were cultured in a 96 wells plate in presence of BY12, BY40, BA54g, A1 (Biolegend), BU61 (Santa Cruz Biotech) or AC2 (Immunotech) (10 μg/ml) for 2 h at 37° C. Then cells were washed and incubated for 20 min at 4° C. with the same antibody (10 μg/ml) that had been used for the culture. Antibody fixation was revealed with APC-coupled goat anti-mouse Ab and measured by flow cytometry. Percentage of inhibition of antibody fixation was calculated according to the following formula: 100−(MFI of mAb-treated cells/MFI of control isotype-treated cells)*100.

EXAMPLE

(5) Expression of CD39 on CD4+CD25low and CD4+CD25high T cells from HIV− controls and HIV+ patients: It has been recently reported that mouse and human CD4+CD25high regulatory T cells constitutively express CD39, an ectonucleotidase that converts extra-cellular ATP generated in sites of immune activation, leading to adenosine generation an inhibitor of cell proliferation, Here we used the anti-CD39 mAb BY40 to examine the expression of CD39 on CD4+CD25low and CD4+CD25high peripheral blood lymphocyte (PBL) populations from both HIV− controls and HIV+ patients. Results from FIG. 1, indicate that the percentage of CD39+ cells within CD4+CD25high subpopulation is significantly higher (p<0.01) than the percentage of CD39+ cells within CD4+CD25low subpopulation in PBL from both HIV- and HIV+ individuals (FIG. 1A). Means are 16% versus 46% (HIV−) and 17% versus 42% (HIV+). No statistical differences were observed between the percentage of CD39+ cells when compare each of the same CD4+ the population between HIV- and HIV+ groups.

(6) Next we analyzed the level of expression of CD39 on CD4+ cells from both HIV+ and HIV− individual (FIGS. 1B and 1C). We observed that CD39 expression on CD4+CD25low and CD4+CD25high of HIV+ patients is significantly higher as compared to the level of CD39 expression on cells of HIV− controls (means 84 versus 45 and 136 versus 93 respectively).

(7) These results indicate that a higher percentage of CD4+CD25high stained positive for CD39 as compared to CD4+CD25low population and that both CD4+CD25high and CD4+CD25low regulatory T cells from HIV+ patients expressed a higher level of CD39.

(8) Blocking CD39 on CD4+CD25high regulatory T cells reverts their immunosuppressive effect towards autologous CD8 effector T cells: Having demonstrated that CD4+CD25high regulatory T cells from HIV+ patients expressed a high level of CD39, we examined the possible role of CD39 in HIV+ regulatory T cells-mediated inhibition of CD8 T cell proliferation.

(9) As expected we observed, in co-cultures experiments, that autologous untreated CD4+CD25high regulatory T cells efficiently inhibit CD8+ T cell proliferation, an effect that is not observed when CD4+CD25high regulatory T cells have previously been incubated in the presence of the CD39 mAb BY40 (FIG. 2A, bottom histogram). The CD39 mAb has no direct effect on proliferation of CD8+ T cells (FIG. 2A, upper histogram). The capacity of BY40 mAb to reverse immunosuppressive effect of CD4+CD25high regulatory T cells, is not limited to CD4+CD25high regulatory T cells from HIV+ patients. A similar effect has been observed in co-culture experiments done with T cells from HIV− individuals. However BY40 mAb was more efficient to inhibit immunosuppressive activity of CD4+CD25high regulatory T cells and to restore proliferation of autologous CD8 T cells from HIV+ patients (FIG. 2B).

(10) These results indicate that triggering of CD39 molecule on CD4+CD25high regulatory T cells with BY40 mAb can revert their immunosuppressive activity and restore proliferation of autologuous CD8 T cells induced by CD3 mAb.

(11) The effect observed with the BY40 mAb has been extended to another anti-CD39, the BA54g mAb (FIG. 3).

(12) Down-regulation of CD39 molecule induced by CD39 mAbs. As a potential mechanism of action for BY40 and BA54g mAbs, we measured their capacity to down-modulate the expression of CD39 on cell surface. As shown in FIG. 4, we demonstrated that incubation of YT2C2 cells for 2 h in the presence of BA54g or BY40 mAb led to a potent inhibition of CD39 expression (>70% of inhibition). We did not observed significant modulation of CD39 expression with BU61 mAb, whereas BY12, A1 and AC2 mAb induced an intermediate down-modulation of CD39 cell surface expression.

(13) Pre-treatment of CD4+CD25high regulatory T cells with BY40 mAb partially reverts their immunosuppressive effect towards the generation of tumor-specific cytotoxic effector CD4 T cells. We next measured the capacity of the BY40 mAb to sustained generation of tumor-specific T cells with effector functions. As demonstrated in Table 3, co-culture of PBL with CD4+CD25high Tregs and the melanoma cell line HM11 led to the generation of low level of cytotoxic effector CD4 T cells as revealed by the acquisition of the CD107 expression, a degranulation specific marker. Preliminary experiments showed that pre-treatment of CD4+CD25high Tregs with the BY40 mAb slightly increased percentage CD4+CD107+ effector T cells (Table 3), suggesting that BY40 mAb partially reverts Tregs immunosuppressive activity and enables generation of cytotoxic T cells.

(14) TABLE-US-00003 TABLE 3 Effect of the CD39 mAb, BY40, on the generation of tumor-specific cytotoxic CD4 T cells. % expression increase CTL CD107a activity % CD4 + CD25high + IgG1 4.21% CD4 + CD25high + BY40 6.56% 56%
2×10.sup.5 peripheral blood mononuclear cells and 1×10.sup.5 HM11 irradiated tumor cells (60 Gray) supplemented with 5×10.sup.4 purified CD4.sup.+CD25.sup.high cells (Treg), that have been previously incubated for 1 h with either IgG1 control or BY40 mAb (10 μg/ml) were co-cultured for 6 days. Every 48 h, 0.5 μg of IgG1 control or BY40 mAb were added to the culture. Then lymphocytes were harvested and incubated in a 96 wells plate with HM11 cells (1/1 ratio) in the presence of either IgG1 control or BY40 mAb (10 μg/ml). Percentage of tumor-specific effector cytotoxic CD4 T cells was determined by labeling with anti-CD107a-PC5 antibody in the presence of Monensin (2 μM final concentration). After 4 h at 37° C. 5% C02, cells were washed and stained with anti-CD4-FITC antibody. The dual expression of CD4 and CD107a was measured by flow cytometry. The percentage of CD107a expression restoration was measured according to the following formula: (% of CD4.sup.+CD107a.sup.+ cells following BY40 treatment)−(% of CD4.sup.+CD107a.sup.+ cells following control IgG1 treatment)/% of CD4.sup.+CD107a.sup.+ cells following control IgG1 treatment)*100.

(15) BA54g mAb inhibits ATPase activity of human PBMC. CD39 has been previously described as an integral component of the suppressive machinery of Tregs, acting at least in part through the modulation of pericellular levels of adenosine. We analyzed here the effect of BA54g CD39 mAb on the spontaneous ATPase activity of human PBMC. We observed that spontaneous ATPase activity of human PBMC following 24 h culture in the presence of BA54g was decreased by 29% compared to PBMC cultured in the presence of control IgG1 (Table 4). The effect of others CD39 mAb and in particular BY40 is currently under investigation.

(16) TABLE-US-00004 TABLE 4 Effect of the CD39 mAb, BA54g, on ATPase activity of human peripheral blood mononuclear cells (PBMC). OD 620 nM % inhibition control IgG1 0.215 BA54g 0.151 29%
4×10.sup.5 PBMC were cultured in complete RPMI medium in the presence of BA54g mAb or IgG1 control (10 μg/ml). After 24 h, cells were washed three times in phosphate-free buffer (10 mM glucose, 20 mM Hepes, 5 mM KCL, 120 mM NaCl, 2 mM CaCl2)) and resuspended in 400 μl of incubation buffer supplemented with 2 mM ATP. After 10 min at 37° C. cells were centrifuged, phosphate concentration in the supernatants was measured by spectrophotometer (620 nM) after addition of Malachite green/polyvinylalcohol/ammonium molybate solution for 20 min.

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