METHODS AND PHARMACEUTICAL COMPOSITIONS FOR ENHANCING CD8+ T CELL-DEPENDENT IMMUNE RESPONSES IN SUBJECTS SUFFERING FROM CANCER

Abstract

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by unleashing exhausted CD8+ T-cell thereby restoring anti-tumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Here, the inventors show that NRP1 is recruited in the cytolytic synapse of PD1+CD8+ T-cells, interacts and enhances PD-1 activity. In mice, CD8+ T-cell specific deletion of Nrp1 improves spontaneous and anti PD1 antibody anti-tumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8+ T-cells predicts poor outcome of patients treated with anti-PD1 (e.g. pembrolizumab). Finally, the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in human, specifically in CD8+ T-cells anti-tumor response. Thus the therapeutic inhibition of NRP1 alone or combined with an immune checkpoint inhibitor (e.g. anti-PD1 antibody) could efficiently repress tumor growth in human cancer. The present invention also relates to multispecific antibodies comprising at least one binding site that specifically binds to an immune checkpoint molecule (e.g. PD-1), and at least one binding site that specifically binds to NRP-1. The present invention also relates to a population of cells engineered to express a chimeric antigen receptor (CAR) and wherein the expression of NRP-1 in said cells is repressed.

Claims

1. A multispecific antibody comprising at least one binding site that specifically binds to PD-1, and at least one binding site that specifically binds to NRP-1.

2. The multispecific antibody of claim 1, which is a bispecific antibody.

3. The multispecific antibody of claim 1, comprising a first binding site that specifically binds to NRP-1 that comprises a light chain variable domain comprising the following Complementary Determining Region (CDR) amino acid sequences: VL-CDR1 (RASQSISSYLA; SEQ ID NO:3), VL-CDR2 (GASSRAS; SEQ ID NO:4) and VL-CDR3 (QQYMSVPIT; SEQ ID NO:5) and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO:6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO:7) and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8).

4. The multispecific antibody of claim 1 comprising a first binding site that specifically binds to NRP-1 that comprises the light chain variable domain (VL) sequence of SEQ ID NO:9 and the heavy chain variable domain (VH) sequence of SEQ ID NO:10.

5. The multispecific antibody of claim 1 comprising a second binding site that specifically binds to PD-1 and that comprises the VH domain of SEQ ID NO:11 and the VL domain of SEQ ID NO: 12.

6. The multispecific antibody of claim 1 comprising a second binding site that specifically binds to PD-1 and that comprises the VH domain of SEQ ID NO:15 and the VL domain of SEQ ID NO: 16.

7. The multispecific antibody of claim 1 comprising: a first binding site that specifically binds to NRP-1 and that comprises the light chain variable domain (VL) sequence of SEQ ID NO:9 and the heavy chain variable domain (VH) sequence of SEQ ID NO:10 and, a second binding site that specifically binds to PD-1 and that comprises the VH domain of SEQ ID NO:11 and the VL domain of SEQ ID NO: 12.

8. The multispecific antibody of claim 1 comprising: a first binding site that specifically binds to NRP-1 and that comprises the light chain variable domain (VL) sequence of SEQ ID NO:9 and the heavy chain variable domain (VH) sequence of SEQ ID NO:10 and, a second binding site that specifically binds to PD-1 and that comprises the VH domain of SEQ ID NO:15 and the VL domain of SEQ ID NO: 16.

9. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount a multispecific antibody according to claim 1.

10. The method of treating cancer in a patient in need thereof according to claim 9, wherein administration of the multispecific antibody results in enhanced therapeutic efficacy relative to the administration of the antibody comprising said at least one binding site that specifically binds to PD-1 alone.

11. The method of claim 9, wherein the at least one binding site that specifically binds to NRP-1 binds to the domain c of NRP-1, and/or to the region of NRP-1 which binds to Semaphorin 3A and/or for the amino acid sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 280 in SEQ ID NO:1.

12. The method of claim 9, wherein the multispecific antibody does not inhibit the binding of VEGF to NRP-1.

13. The method of claim 9, wherein the multispecific antibody cross-competes for binding to the NRP-1 isoform with the antibody that comprises: a light chain variable domain comprising the following Complementary Determining Region (CDR) amino acid sequences: VL-CDR1 (RASQSISSYLA; SEQ ID NO:3), VL-CDR2 (GASSRAS; SEQ ID NO:4) and VL-CDR3 (QQYMSVPIT; SEQ ID NO:5) and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO:6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO:7) and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8).

14. The method of claim 9, wherein the multispecific antibody is a bispecific antibody.

15. The method of claim 9, which further comprises determining the expression level of CD8.

16. The method of claim 9, comprising i) quantifying the density of CD8+ T cells in a tumor tissue sample obtained from the patient ii) comparing the density quantified at step i) with a predetermined reference value and iii) administering to the patient a therapeutically effective amount of the multispecific antibody.

Description

FIGURES

[0184] FIG. 1: Nrp1 expression in tumor infiltrating Tetramer H2kb/SIINFEKL mice CD8+ T cells. B16F10-OVA tumor cells were subcutaneously injected on the right flank of WT C57BL/6 mice. Seven and 14 days after tumor injection, mice were immunized with combined Poly-IC/ovalbumine subcutaneously. Tumors were harvested 21 days after the injection and tumor infiltrating CD8+ T cells were stained using Tetramer H2kb-SIINFEKL, anti-CD8 and anti-Nrp1, and analyzed by flow cytometry. Data are presented in Flow cytometry graph with anti-CD8 antibody and Tetramer H2kb-OVA. Tetramer H2kb-OVA positive population is presented in histogram of Nrp1 expression normalized to mode. Data are representative of 3 independent experiments

[0185] FIG. 2: NRP1 expression profiles in H2-Db GP33-specific CD8.sup.+ T-cells according to in vivo infection in mice with LCMV Armstrong (n=16), LCMV clone 13 (n=16) or nave CD44.sup.lowCD8.sup.+ T-cells from controls (n=4) at days 6, 8, 15 and 30. Data from transcriptomics analysis were available from Doering et al. Immunity, 2012. P value was determined by two-way ANOVA (p=0.0008).

[0186] FIG. 3: B16F10 Tumor volume follow up in a model of anti-tumoral immune response. CD8+ Nrp1 KO (KO) mice or CD8 CRE (WT) mice received 1 million B16F10 in the right flank at day 0, followed by an immunization at day 7 and day 14 with Poly-IC/Ovalbumine injected subcutaneously at 40 g of PolyIC and 400 g Ovalbumine per mice. Data are presented as mean of tumor volume+/SEM at day 0, 8, 11, 14, 18, 21 after injection. Data are representative of 3 independent experiments

[0187] FIG. 4: Percentages of Tetramer/PE-H-2 Kb OVA CD8.sup.+ TILs in B16-OVA tumors of four different mice group assessed at day 14 post-immunization by flow cytometry from CD8Nrp1KO (KO) and control (WT) mice immunized or not immunized (control) with ovalbumine and poly-IC. Data are presented as mean percentage of CD8.sup.+ TILs Tetramer positiveSEM. P values were determined by student T test **p<0.01, *p<0.05. Data are representative of 3 independent experiments

[0188] FIG. 5: Quantification by ImageStream of NRP1 expression (mean pixel intensity/MPI) in an allogeneic synapse model between activated CD8.sup.+ T-cells and cell tracer violet labeled A20 cells. NRP1 expression was analysed in activated CD8.sup.+ T-cells at the synapse junction (high phalloidin labelling zone). Data are presented as mean MPISEM. P value (p<0.0001) was determined by Wilcoxon matched pairs test. Data are representative of 4 independent experiments from 2 synapse models.

[0189] FIG. 6: PD1 is recruited within the synapse between activated CD8.sup.+ T-cells and tumor cells. Analysis by Imagestream of PD1 expression (mean pixel intensity/MPI) in a synapse model between activated CD8.sup.+ T-cells and allogeneic A20 tumor cells: PD1 expression was analysed in phalloidine high area between activated CD8.sup.+ T-cells and tumor cells (A20). Data are presented as mean MPISEM. P value was determined by student T test. Data arc representative of 5 experiments.

[0190] FIG. 7: Flow cytometry analysis of NRP1 and PD1 expression in human CD8.sup.+ TILs. Data are representative of 3 independent experiments in human endometrial, kidney and ovarian cancer.

[0191] FIG. 8: Quantification by Imagestream of PD1 expression (MPI) in the synapse junction (high phalloidin labelling zone) between activated CD8.sup.+ T-cells from CD8Nrp1KO mice (KO) or controls (WT), and allogeneic A20 tumor cells. Data are presented as mean MPISEM. P value (p<0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.

[0192] FIG. 9: Quantification by Image stream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labelling zone) between activated CD8.sup.+ T-cells from CD8Nrp1KO mice (KO) or control mice (WT), and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPISEM. P value (p<0.0001) was determined by Mann Whitney test. Data are representative of 3 independent experiments

[0193] FIG. 10: Flow cytometry analysis of phospho-ZAP70 in human PD1.sup.+CD8.sup.+ TILs according to NRP1 expression. Data are representative of one experiment in human endometrial cancer.

[0194] FIG. 11: Flow cytometry analysis of CD25 expression in CD8.sup.+ T-cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N=5), respective to SEB superantigen concentration (0, 1, 10 or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 hours. Data are presented as mean % of CD25 expressionSEM. Human anti-PD1 antibody (Pembrolizumab, Merck).

[0195] FIG. 12: Flow cytometry analysis of percentage of divided CD8.sup.+ T-cells from a patient bearing an NRP1 haploinsufficiency (patient) or from controls (N=5), respective to SEB superantigen concentration (0, 1, 10 or 100 ng/mL), in the presence or not of anti-PD1 antibody. Activation was performed during 72 hours. Data are presented as meanSEM. Human anti-PD1 antibody (Pembrolizumab, Merck).

[0196] FIG. 13: CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumine and poly-IC and treated or not with anti-PD1 antibody in vivo. Overall survival was assessed until 50 days after immunization. Data are presented as meanSEM and as Kaplan Meyer curve. P values were determined by Log rank test. Data are representative of 5 experiments. Mouse anti-PD1 antibody from Bio X Cell (J43 clone).

[0197] FIG. 14: Analysis of overall survival of patients with metastatic melanoma treated with anti-PD1, according to RNA NRP1 expression (low or high expression) assessed in the tumor before anti-PD1 treatment. Data from transcriptomics analysis of metastatic melanoma tumors were available from Hugo et al. Cell, 2016. Data are presented as Kaplan Meyer curve. P value (p=0.03) was determined by Log rank test (n=25 patients).

[0198] FIG. 15: Analysis of relapse free survival of patients with metastatic melanoma treated with anti-PD1 and reached at least a partial response, according to NRP1 expression (NRP1.sup./low compared with NRP1.sup.+/high) in CD8.sup.+ TILs assessed by immunohistochemistry before starting therapy. Blind analysis has been performed to assess NRP1 expression. Data are presented as Kaplan Meyer curve. P value (p=0.042) was determined by Log rank test (n=15 patients).

[0199] FIG. 16: Quantification by Image stream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in human activated CD8.sup.+ T-cells in synapse with tumor cells (Raji). Data are presented as mean MPISEM. P value was determined by Mann Whitney test. Human anti-NRP1 antibody (AF3870, R&D systems), Human anti-PD1 antibody (Pembrolizumab, Merck).

EXAMPLE

Summary

[0200] Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by unleashing exhausted CD8.sup.+ T-cell thereby restoring anti-tumor immune responses.sup.1,2. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development.sup.3,4. NRP1 is also expressed in several types of immune cells and is involved in immunological synapse formation, activation and termination.sup.5-7. NRP1 impairs anti-tumor immune response by modulating macrophages and Treg activities.sup.8-10. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1.sup.+CD8.sup.+ T-cells, interacts and enhances PD-1 activity. In mice, CD8.sup.+ T-cell specific deletion of Nrp1 improves spontaneous and anti PD1 antibody anti-tumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8.sup.+ T-cells predicts poor outcome of patients treated with anti-PD1. Finally, the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in human, specifically in CD8.sup.+ T-cells anti-tumor response by increasing TCR signaling in CD8.sup.+ T-cells in synapse with tumor cells.

Results

[0201] Although PD1 is a key factor of exhaustion, its expression is not sufficient to induce an exhaustion profile in CD8.sup.+ T-cells. For example, in the mice LCMV clone 13 infection model, most antigen-specific CD8.sup.+ T-cells that have been induced, while maintaining PD1 expression after antigen withdrawal a fraction of these CD8.sup.+ T-cells retain their ability to produce cytokines upon new LCMV antigen challenge.sup.11. This observation suggests the involvement of a potential additional partner. Because NRP1 is unable to signal autonomously.sup.12, and is also expressed in activated T-cells at the synapse level, we hypothesized that NRP1 may be involved in PD1 inhibitory activity. In vitro NRP1 was expressed on murine CD8.sup.+ T-cells after activation driven by OVA peptide, and the intensity of its expression correlated positively with antigen availability. To investigate in vivo the expression of NRP1 on CD8.sup.+ T-cells we studied 3 models of acute or persistent antigen specific immunization. As previously reported.sup.13,14, NRP1 was not expressed on naive CD8.sup.+ T-cells (data not shown). In contrast activated specific CD8.sup.+ T-cells expressed NRP1 after intramuscular adeno-associated virus-OVA immunization (AAV-OVA), with a peak of expression at day 21 post-immunization. NRP1 was highly expressed in mice specific anti-OVA.sub.257 CD8.sup.+ TILs in a model of B16-OVA tumor progression (FIG. 1) and by specific CD8.sup.+ T-cells in the exhaustion model of LCMV clone 13 viral infection when compared with LCMV Armstrong infection (FIG. 2).

[0202] In order to further study the role of NRP1 expression in CD8.sup.+ T-cells in vivo, we generated a mouse model in which CD8.sup.+ T-cells were specifically invalidated for Nrp1 (CD8Nrp1KO), by breeding Nrp1flox/flox mice with CD8CreTg mice. At steady state, the CD8Nrp1KO mouse harbored no immunological phenotype, and as expected, CD8.sup.+ T-cells did not express NRP1 upon activation. In an antigen-specific anti-tumor immune response, tumor growth was significantly decreased in CD8Nrp1KO mice as compared to the control (FIG. 3). Accordingly, analysis of the tumor immune microenvironment in CD8Nrp1KO and control mice showed an increase in CD8.sup.+ TILs frequency in CD8Nrp1KO mice (FIG. 4). These results suggest that NRP1 expression on CD8.sup.+ TILs might be involved in the negative regulation of anti-tumor immune responses.

[0203] Since we previously reported that NRP1 was involved in the immunological synapse between T-cells and dendritic cells.sup.5, we then investigated whether NRP1 could be localized in the synapse between T-cells and tumor cells, and could thereby be involved in the effector function of CD8.sup.+ TILs in this specific context. To address this question, we developed a synapse model between transgenic TCR OT1 T-cells and tumor cells (EL4-CFP cells) bearing the cognate antigen (OVA.sub.257) and between activated CD8.sup.+ T-cells from CD8Nrp1KO mice or littermate and allogeneic tumor cells (A20 cells). In these models, imaging flow cytometry analysis of cell conjugates showed that NRP1 and PD1 were recruited together to the synapse between activated CD8.sup.+ T-cells and tumor cells (FIGS. 5-6).

[0204] Since it has been previously reported that the clustering and co-localization of PD1 and TCR is critical in inducing low level of phospho-ZAP70 in the synapse junction in response to the binding of PD-L1 to PD-1.sup.15,16, which characterize the exhaustion synapse, we then investigated whether NRP1 was involved in PD1 recruitment and function at the synapse. First, by immunofluorescence, in vivo we showed that PD1 and NRP1 were co-localized in CD8.sup.+ TILs from mice (data not shown) and NRP1 was specifically expressed on human PD1.sup.+CD8+ TILs (FIG. 7). An interaction between NRP1 and PD1 was demonstrated on activated mice CD8.sup.+ T-cells by a proximity ligation assay (Duolink) in vitro (data not shown). Performing co-immunoprecipitation experiments, we provided additional evidence for this interaction in a protein complex (data not shown). In CD8Nrp1KO CD8.sup.+ T-cells, although PD1 was expressed, its localization within the synapse with tumor cells was significantly reduced as compared with CD8.sup.+ T-cells from WT mice (FIG. 8). Thus, phospho-ZAP70 was increased in CD8.sup.+ T-cells from CD8Nrp1KO in synapse with tumor cells compared with controls (FIG. 9). Taken together, our data suggest that NRP1 is a partner of PD1 enhancing its recruitment and activity at the synapse between CD8.sup.+ T-cells and tumor cells.

[0205] We next investigated whether the role in exhaustion of NRP1 in mice held truc in human CD8.sup.+ T-cells. Within the human tumor microenvironment, NRP1 expression was found on CD8.sup.+ TILs, specifically on PD1.sup.+CD8.sup.+ T-cells and identified a subset of PD1.sup.+CD8.sup.+ TILs with low phospho-ZAP70 expression (phospho-ZAP70.sup.lowNRP1.sup.+PD1.sup.+CD8.sup.+ TILs) (FIG. 10). No patient bearing a homozygous NRP1 mutation had been described so far, potentially due to the lethality of homozygous NRP1 deletion in utero.sup.17. However, we could identified a unique patient with NRP1 haploinsufficiency caused by a heterozygous deletion of the chromosomal region (10p11.22) including the NRP1 gene.sup.18. CD8.sup.+ T-cells from the NRP1.sup.+/ patient have an increased ability to proliferate and to express CD25 after in vitro activation by the staphylococcal enterotoxin b (SEB) superantigen (FIGS. 11-12). In addition, the increase of patient's CD8.sup.+ T-cells activation was synergistic in combination with an anti-PD1 antibody.

[0206] To address this synergistic effect between PD1 and NRP1, we evaluated the in vivo efficacy of anti-PD1 antibody in the B16-OVA tumor growth mouse model. As previously reported in this model, anti-PD1 treatment had no effect on overall survival in WT mice. In constrast, a significant increase in mouse survival was observed in the CD8Nrp1KO, which was more pronounced upon anti-PD1 treatment indicating a strong synergistic effect (FIG. 13).

[0207] To assess the role of NRP1 in humans cancer, we next performed an in silico study analyzing micro-array data from metastatic melanoma cancer treated in clinical trial with anti-PD1 therapy.sup.19 (FIG. 14). In accordance with our hypothesis, a low expression of NRP1 in tumor before therapy was associated with improved patients' overall survival (p=0.040). Since NRP1 might be expressed in other cells than CD8.sup.+ T-cells, we then investigated the outcome of 28 patients with metastatic melanoma treated with anti-PD1 therapy, depending on the expression of NRP1 on CD8.sup.+ TILs before starting therapy. Following our hypothesis, we found a trend for highest complete response rate (data not shown) and a significant increase of relapse-free survival in patients with NRP1.sup./lowCD8.sup.+ TILs compared with NRP1.sup.+/highCD8.sup.+ TILs (p=0.042, FIG. 15). Taken together, our data demonstrate that NRP1 should be considered as a new actor of exhaustion by enhancing PD1 activity on CD8.sup.+ TILs.

[0208] At last, we showed that the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in human anti-tumor immune response. Indeed, in an in vivo synapse model between human activated CD8.sup.+ T-cells and tumor cells (Raji), the combination induced an increase of phospho-ZAP70 expression (and thus TCR signaling) in CD8.sup.+ T-cells compared with anti-PD1 antibody alone (FIG. 16).

Discussion

[0209] NRP1 has already been implicated in the immune response against tumors.sup.8-10, by acting as a break on both innate and adaptive immunity. Immune checkpoint therapies have led to multiple successes in patients with cancer.sup.1,2. Unfortunately, most patients relapse or are refractory even with a combination of immune checkpoints inhibitors.sup.20. Data from our observations in human suggest that NRP1 inhibition could be a potential therapeutic strategy to improve anti-PD1 efficacy. With respect to safety, no side effect was reported in the experiments in mice evaluating the association with anti-PD1 therapy (data not shown). Furthermore, CD8Nrp1KO mice that were cured of B16-OVA tumor cells with anti-PD1 did not exhibit any autoimmune or inflammatory phenotype. This observation argues for the potential safety of using either a drug able to reduce NRP1 expression, or an antibody blocking both NRP1 and PD1 on CD8.sup.+ T-cells.

[0210] Here, we report that specific deletion of Nrp1 on CD8.sup.+ T-cells dramatically enhances survival of mice bearing B16-OVA tumors, with potential cure with the addition of anti-PD1 therapy. Moreover, we showed that a combination of anti-NRP1 and anti-PD1 antibodies is synergistic in human CD8+ T-cells anti-tumor immune response. Thus, our data suggest that strategies using NRP1-deleted CD8.sup.+ CAR-T-cells alone or combined with immune checkpoint inhibitor (e.g anti-PD1 antibody) could be a way to improve efficacy of CAR-T-cells and. In addition, our data suggest that bispecific anti-NRP1/PD1 antibodies could be a way to improve efficacy of immune checkpoint inhibitor (e.g. anti-PD1 antibody).

[0211] In conclusion, we have identified NRP1 as a new immune checkpoint, which acts through an original mechanism by enhancing PD-1 inhibitory effect at the synapse level, and our data strongly suggest that a therapeutic inhibition of NRP1 alone, or combined with an immune checkpoint inhibitor (e.g. anti-PD1 antibody) could efficiently repress tumor growth in human cancer.

REFERENCES

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