METHODS FOR DISCONTINUING A TREATMENT WITH A TYROSINE KINASE INHIBITOR (TKI)

Abstract

The present invention relates to a method for discontinuing a treatment with a TKI by determining the number and/or frequency of innate CD8(+) T-cells in subject and concluding that the treatment with a TKI should be discontinued when the number and/or frequency of innate CD8 T-cells is higher than the predetermined reference value. Inventors evaluated whether innate CD8(+) T-cells are an early target of CML therapy success. Among peripheral blood effector CD8(+) T-cells, inventors shown that both number nd/or frequency and functional signatures of innate CD8(+) T-cells are enhanced as early as 3 months of therapy. Strikingly, they observe that patients with high innate CD8(+) T-cell number and/or frequency at 3 months and/or diagnosis achieve a DMR earlier than patients with low innate CD8(+) T-cell number. Furthermore, a higher number and/or frequency of high innate CD8(+) T-cell patients achieved a stable DMR for over 2 years. They have also observed that the success of TKI therapy cessation is associated with higher proportion of innate CD8 T-cells expressing perforin. Collectively, these findings highlight innate CD8(+) T-cells as a potential marker for both CML therapy success and successful long-term treatment-free remission (TFR), and thus therapy discontinuation eligibility.

Claims

1-9. (canceled)

10. A method for treating a subject suffering from a cancer comprising the following steps: i) determining the number and/or frequency of innate CD8(+) T-cells in a biological sample obtained from the subject, ii) determining that the number and/or frequency of innate CD8(+) T-cells in the biological sample is higher than a reference value, and iii) treating the subject determined to have a higher number and/or frequency of innate CD8(+) T-cells with a TKI.

11. The method according to claim 10, wherein the subject is treated with i) a TKI and ii) IFN.sub.α, IFN-α2a, IFN-α2b, an allosteric inhibitor of BCR-ABL or an immune checkpoint inhibitor as a combined preparation when: the number and/or frequency of innate CD8(+) T-cells is higher than the reference value; the number and/or frequency of innate CD8(+) T-cells is higher than the reference value and/or a level of NK cells is higher than a reference value and/or a level of non-conventional T lymphocytes is higher than a reference value; or the number and/or frequency of innate CD8(+) T-cells is higher than the reference value and/or a level of perforin is higher than a reference value, and/or a level of granzymes is higher than a reference value and/or a level of IFNγ among innate CD8(+) T-cells is higher than a reference value.

12. The method according to claim 10, wherein the TKI is dasatinib.

13. The method according to claim 10, wherein the biological sample is a blood sample.

14. The method according to claim 10, wherein the innate CD8 T-cells are as KIR2D+KIR3DL1/KIR3DL2+NKG2A cells.

15. The method according to claim 10, wherein the subject is suffering or susceptible to suffer from a cancer.

16. The method according to claim 10, wherein the subject is first treated with dasatinib for three months.

17. The method according to claim 16, wherein the subject is subsequently treated with i) a TKI and ii) IFN.sub.α, IFN-α 2a, IFN-α 2b, an allosteric inhibitor of BCR-ABL or an immune checkpoint inhibitor, as a combined preparation.

Description

FIGURES

[0242] FIG. 1. Frequency and functional signatures of innate CD8(+) T-cells are impaired in patients at CML diagnosis. (A) Innate CD8(+) T-cell (Eomes+ panKIR/NKG2A+) frequency in CML patients at diagnosis (Diag, n=38) or in healthy donors (HD, n=21) among total CD8+TCR-αβ+ cells. Representative histograms of one HD and one CML patient are shown. (B) Eomes MFI (upper panel; Diag n=36; HD n=21) and CD49d MFI (lower panel; Diag n=32; HD n=20) in innate CD8 T-cells from CML patients at diagnosis or from HD. Representatives histograms are shown. Data are expressed as Box and whiskers (5 percentile, median and 95 percentile). Statistical analysis: unpaired two-tailed Mann-Whitney test.

[0243] FIG. 2. Innate CD8(+) T-cells are enhanced after 3 months of CML therapy. (A) Kinetics of innate CD8(+) T-cell frequencies in CML patients analyzed from diagnosis and up to 24 months of treatment (Diag: n=38; M3: n=34; M6: n=34; M12: n=33; M24: n=31). (B) Frequencies of innate CD8(+) T-cells at diagnosis (Diag, n=38) and after 3 months of CML therapy (M3, n=34). (C) Eomes MFI (upper panel; Diag n=36; M3 n=32) and CD49d MFI (lower panel; Diag n=32; M3 n=29) in innate CD8 T-cells at diagnosis (Diag) and after 3 months of CML therapy (M3). Data are expressed as curve of mean±SEM (panel A), as symbol and line (panel B) or as Box and whiskers (5 percentile, median and 95 percentile, panel C). Statistical analysis: unpaired two-tailed Mann-Whitney test (panel A) or paired two-tailed Wilcoxon test (panel B and C) were used.

[0244] FIG. 3. Early shift in CD8(+) T-cells after 3 months of therapy in CML patients. Frequencies of conventional-memory CD8(+) T-cells (Eomes.sup.+ panKIR/NKG2A.sup.−) (A) and naive CD8(+) T-cells (Eomes.sup.− panKIR/NKG2A.sup.− cells) (B) among total CD8.sup.+TCR-αβ.sup.+ cells in CML patients at diagnosis (Diag, n=38) or after 3 months of therapy (M3, n=34). Data are expressed as Box and whiskers (5 percentile, median and 95 percentile). Statistical analysis: a paired two-tailed Wilcoxon test was used.

[0245] FIG. 4. Achievement of a deep molecular response (DMR) at 12 months of CML therapy is associated with a high innate CD8(+) T-cell frequency at 3 months of treatment. (A) Frequencies of innate CD8(+) T-cells after 3 months of dasatinib treatment in patients having achieved DMR (M12-DMR, n=15) or not (M12-noDMR, n=12) after 12 months of therapy. (B) Eomes (upper panel, noDMR n=18; DMR n=14) and CD49d (lower panel, noDMR n=17; DMR n=12) MFI after 3 months of dasatinib treatment in patients having achieved DMR (M12-DMR) or not (M12-noDMR) after 12 months of therapy. (C) Kinetics of innate CD8(+) T-cell frequencies in CML patients having achieved DMR (M12-DMR, green line) or not (noDMR, blue line) after 12 months of therapy. Patients were analyzed from diagnosis and up to 24 months of treatment (Diag: noDMR n=22; DMR n=16; M3: noDMR n=19; DMR n=15; M6: noDMR n=20; DMR n=14; M12: noDMR n=18; DMR n=15; M24: noDMR n=17; DMR n=14). Data are expressed as Box and whiskers (5 percentile, median and 95 percentile, panel A and B) or as curve of mean±SEM (panel C). Statistical analysis: paired two-tailed Wilcoxon test (panel A) or unpaired two-tailed Mann-Whitney test (panel B and C). ns: not significative.

[0246] FIG. 5. High innate CD8(+) T-cell frequency in CML patients after 3 months of treatment is preferentially associated with an early and sustained DMR during CML therapy. (A) Cumulative incidence of the deep molecular response (DMR) over the first 24 months of therapy in patients with low (green line) or high (red line) innate CD8 T-cell frequency after 3 months of treatment. The number of subjects at risk are shown below the curve. (B) Frequencies of innate CD8(+) T-cells after 3 months of treatment in patients achieving a stable DMR over 2 years or not (<2-year DMR n=16; >2-year DMR n=18). (C) Conventional-memory (left) and naive (right) CD8(+) T-cell frequencies after 3 months of treatment in patients achieving a stable DMR over 2 years or not (<2-year DMR n=16; ≥2-year DMR n=18). Data are expressed with Kaplan-Meier curves (panel A) or as Box and whiskers (5 percentile, median and 95 percentile, panels B and C). Statistical analysis: Mantel-cox test was performed for cumulative incidence curve (panel A) and an unpaired two-tailed Mann-Whitney test for panels B and C. ns: not significative.

[0247] FIG. 6. High innate CD8(+) T-cell frequency in CML patients at diagnosis is preferentially associated with an early and sustained DMR. (A) Cumulative incidence of the deep molecular response (DMR) over the first 24 months of CML therapy in patients (the numbers of subjects at risk are shown below the curve) with low (green line) or high (red line) innate CD8(+) T-cell frequency at diagnosis. (B) Frequencies of innate CD8(+) T-cells at diagnosis in patients achieving a stable DMR over 2 years or not (<2-year DMR n=19; ≥2-year DMR n=18). (C) Conventional-memory (left) and naive (right) CD8(+) T-cell frequencies at diagnosis in patients achieving a stable DMR over 2 years or not (<2-year DMR n=19; ≥2-year DMR n=18). Data are expressed with Kaplan-Meier curves (panel A) or as Box and whiskers (5 percentile, median and 95 percentile, panels B and C). Statistical analysis: Mantel-cox test was performed for cumulative incidence curve (panel A) and an unpaired two-tailed Mann-Whitney test for panels B and C. ns: not significative.

[0248] FIG. 7: Successful TKI therapy cessation is associated with higher proportion of innate CD8 T-cells expressing perforin. Quantitative and functional analysis of innate CD8 LTs (Eomes+KIR/NKG2A+) in the prospective treatment discontinuation cohort. Proportion of innate CD8 LT expressing perforin was measured. Each symbol represents a healthy subject or a patient. Results are expressed as a percentage of total innate CD8 LT with representation of the median. Statistical analysis: Mann-Whitney test. HD (for a healthy donor): healthy donors (n=9), “relapse”: patients with molecular recurrence and resuming treatment during the first 6 months of stopping treatment (n=7), “no relapse”: patients in remission without treatment beyond 12 months of discontinuation (n=13).

EXAMPLE 1

Material & Methods

1. Patient and Healthy Donor Characteristics

[0249] The phase II DASA-PEGIFN clinical trial was registered with EudraCT number 2012-003389-42. Briefly, newly diagnosed Ph.sup.+ CP-CML patients were treated first-line with dasatinib at 100 mg/day. At 3 months, they were assigned to receive Peg-IFNα2b associated to dasatinib when platelets>100×109/L, neutrophils>1.5×109/L and lymphocytes<4.0×109/L counts were achieved. Venous blood was collected on heparin at several time points: diagnosis, 3, 6, 12 and 24 months after initiation of treatment Among the 61 patients receiving Dasatinib+Peg-IFNα2b therapy, we analysed samples from 40 patients. Two out of them were excluded for technical issues. See Table 1 for detailed patient's characteristics. Patients response to treatment were classified conforming to 2013 ELN criteria. Major molecular response was defined as a ratio of BCR-ABL1/ABL1≤0.1% on the international scale (IS). A ratio of BCR-ABL1/ABL1IS≤0.01% defined a molecular response of 4-log reduction or in our work a deep molecular response (DMR). All patients gave informed consent in accordance with the Declaration of Helsinki for participation in the study, which was approved by the scientific committee of the INSERM CIC-1402 (Poitiers, France) and Comité Protection Personnes Recherche Biomédicale Région Poitou Charentes (protocol number 12.10.31).

[0250] Frozen PBMC from healthy donors (HD) were obtained from the French Blood Institute (EFS, Lyon, France) (see Table 2).

2. PBMC Isolation and Cryopreservation

[0251] Peripheral blood mononuclear cells (PBMC) were isolated from blood samples by density gradient centrifugation (Histopaque®-1077, Sigma-Aldrich, St Louis, Mo., US), resuspended in 90% Fetal Bovine Serum (10270106, Gibco®, Thermo Fisher Scientific, Waltham, Mass., US) with 10% DMSO (D2650, Sigma-Aldrich), and cryopreserved at −80° C. or in liquid nitrogen until use.

3. Flow Cytometry

[0252] Phenotypic analysis of cells from HD and CML patients was performed by ex vivo flow cytometry. All monoclonal antibodies (mAbs) used in this study are listed in Supplementary Table 2. The expression of the different markers was assessed by staining PBMC with appropriate combinations of mAbs. panKIR/NKG2A referred to staining with the mix of the three following antibodies from Miltenyi Biotec (Bergisch Gladbach, Germany) KIR2D, KIR3DL1/KIR3DL2 (CD158e/k) and NKG2A (CD159a). Dead cells were excluded using the Live/Dead® Fixable NearIR Dead Cell Stain kit (L10119, Invitrogen™, Thermo Fisher Scientific). For intranuclear Eomes staining, cells were permeabilized with anti-human Foxp3 staining kit according to the manufacturer's protocol (73-5776-40, eBioscience™, Thermo Fisher Scientific). Flow data were acquired on a FACSVerse flow cytometer (Becton, Dickinson & Compagny, Franklin Lakes, N.J., US) with FACSuite™ software (Becton, Dickinson & Compagny) and analyzed using FlowJo™ v10 (Becton, Dickinson & Compagny). Results are expressed as either percent positive cells or as mean fluorescence intensity (MFI).

3. Statistical Analysis

[0253] Results are shown as box and whiskers with 5 percentile, median and 95 percentile unless indicated otherwise. All statistical data analyses were performed using GraphPad Prism v7.0 (GraphPad software, Inc). Wilcoxon and Mann-Whitney two-tailed test were used for paired and unpaired data analysis, respectively. A p value<0.05 was considered significant. Cumulative response rates were calculated using the cumulative incidence approach and Mantle-Cox method. The cut-off point for innate CD8(+) T-cells was optimized by receiver operating characteristics (ROC) curves and the Youden index using the logarithm of innate CD8(+) T-cell frequency at diagnosis and after 3 months of therapy. Patients were dichotomized according to their low or high percentage of innate CD8(+) T-cells. Data are presented as Kaplan-Meier curves.

TABLE-US-00001 TABLE 1 Dasa-PegIFN patient's characteristics Patients eligible Selected patients for to Peg-IFN after 3- immunological month dasatinib analysis of this article n = 61 n = 40 Median age, (range) 45 (20-65) 48 (20-65) Pts <45 y, n (%) 29 (48) 17 (43) Gender Male/Female, n (%) 34 (56) 27 (44) 26 (65)/14 (35) Sokal, n (%) Low 30 (51) 22 (55) Int 21 (36) 13 (33) High 8 (14) 5 (12)

TABLE-US-00002 TABLE 2 Healthy donor's characteristics HEALTHY DONORS n = 21 Median age, (range) 28 (22-65) Pts <45 y, n (%) 16 (76) Gender Male/Female, n (%) 7 (33)/14 (66)

Results

[0254] We analyzed innate CD8(+) T-cells (defined as Eomes+panKIR/NKG2A+ cells among TCR-αβ+ CD8+ cells) by flow cytometry from diagnosis and at several time points up to 24 months of DasaPegIFN therapy in 38 CML patients from the phase II clinical trial DASA-PEGIFN (see Methods, Table 1). In this group of patients, we found a lower frequency of innate CD8(+) T-cells at diagnosis as compared to the healthy donor (HD) group (FIG. 1A and data not shown). Moreover, innate CD8(+) T-cells showed a lower expression level of both the transcription factor Eomes and of the integrin CD49d (FIG. 1B and data not shown), two markers of the functional signature of these cells.sup.21. By contrast, frequencies of naive CD8(+) T-cells (defined as Eomes− panKIR/NKG2A−) and of conventional-memory CD8(+) T-cells (defined as Eomes+ panKIR/NKG2A−) in CML patients at diagnosis did not differ from those measured in HD (data not shown). These findings lead to definitively conclude that innate CD8(+) T-cells are specifically impaired in number and function at CML diagnosis.

[0255] Knowing that innate CD8(+) T-cell deficiencies have been at least partially reversed in patients with TKI treatment over two years and having achieved a deep molecular response (DMR).sup.22, we first analyzed whether normalization of this particular pool of cells could be reached in the first 24 months of therapy. The kinetics of innate CD8(+) T-cell frequencies from diagnosis to 24 months of therapy showed an increased frequency of innate CD8(+) T-cells as early as 3 months of therapy in the whole group (FIG. 2A). Focusing on this time point, we found a significant increase of the frequency of innate CD8(+) T-cells (5.98±5.43% vs 3.78±3.39%, mean±SD) (FIG. 2B) and of their Eomes and CD49d expression levels (FIG. 2C), as compared to diagnosis. Early effects of TKI therapy on the pool of CD8(+) T-cells were not restricted to innate CD8(+) T-cells, as conventional-memory CD8(+) T-cell frequency was concomitantly increased whereas naive CD8(+) T-cell frequency was decreased (FIG. 3A et B). Taken together, these findings strongly suggest an early shift within the pool of CD8(+) T-cells favoring an innate/memory phenotype in the first 3 months of therapy.

[0256] As innate CD8(+) T-cells were early impacted by CML therapy, we next searched for a link between their frequency and early DMR. To this end, we separated CML patients achieving DMR at 12 months (M12-DMR) or not (M12-noDMR). Remarkably, at 3 months after initiation of CML therapy, innate CD8(+) T-cell frequency in the M12-DMR group was nearly three-fold higher than in the M12-noDMR group (9.14±6.54% vs 3.48±2.43%, mean±SD) (FIG. 4A), even though it trended to be already higher than in the M12-noDMR group at CML diagnosis (FIG. 3A et B). At the 3-month time point, the higher frequency of innate CD8(+) T-cells in the M12-DMR group was not accompanied by a distinct functional status, as reflected by similar Eomes and CD49d expression levels in the two groups (FIG. 4B). This phenomenon was specific to innate CD8(+) T-cells, as conventional-memory CD8(+) T-cell frequency did not differ between the two groups (data not shown). Overall, the higher frequency of innate CD8 T-cells in the M12-DMR group was at the expense of their naive T-cell counterparts (data not shown), thereby suggesting a shift within the CD8(+) T-cell pool in favor of innate CD8(+) T-cells. Taking into account the whole kinetics, we evidenced that innate CD8(+) T-cell frequencies in M12-DMR patients were maintained at a higher level than in their M12-noDMR counterparts over the 24 months of therapy (FIG. 4C). Taken together, these results show for the first time that innate CD8(+) T-cells are associated with treatment success in patients with CML diagnosis.

[0257] As innate CD8(+) T-cell frequency seemed to be an early target of CML therapy, we hypothesized that these cells are an indicator of early DMR achievement. Cumulative response rates of DMR occurred significantly earlier and at higher rates in innate CD8(+) T-cell high patients after 3-months therapy. Indeed, more than 50% of innate CD8(+) T-cell high patients achieved DMR at 12 months while less than 10% of innate CD8(+) T-cell low patients reached it (FIG. 5A). Interestingly, at 12-months the number of innate CD8(+) T-cell high patients reaching DMR remained stable up to 24-months of therapy whereas innate CD8(+) T-cell low patients continuously achieved DMR without reaching 50%. Moreover, taking into account innate CD8(+) T-cell frequency at CML diagnosis, cumulative response rates of DMR also occurred significantly earlier and at higher rates in innate CD8(+) T-cell high patients (FIG. 6A). These data lead to conclude that elevated innate CD8(+) T-cell frequency in peripheral blood both at diagnosis and at 3 months of therapy is an indicator of early DMR achievement.

[0258] Alongside DMR, its stability over time is another important criteria for CML patients. Indeed, patients achieving a stable DMR for two or more years are eligible to treatment discontinuation.sup.4. To test whether innate CD8(+) T-cells were associated with the stability of the treatment response, we separated patients having a stable DMR for over 2 years to patients without a stable DMR. Remarkably, after 3 months of therapy innate CD8(+) T-cell frequency was higher in patients having a stable DMR for over 2 years than in patients without a stable DMR (FIG. 5B). Once again, the same conclusion could be applied when considering the frequency of innate CD8(+) T-cells at CML diagnosis (FIG. 6B), thereby reinforcing the notion that the status of innate CD8(+) T-cells was closely associated with a stable DMR in CML patients. Finally, this effect was specific to innate CD8(+) T-cells, as conventional-memory and naive CD8(+) T-cell frequencies were similar between the two groups regardless of the time point (at diagnosis or after 3 months of therapy) (FIG. 5C and FIG. 6C). Collectively, our results show that innate CD8(+) T-cell numbers are associated with an early, deep and stable response to CML therapy.

EXAMPLE 2

[0259] We prospectively investigated the quantitative and functional features of innate CD8 T-cells in patients who had stopped TKI according to the French group expert recommendations. The proportions of innate CD8 LTs (Eomes+MR/NKG2A+) expressing perforin were measured.

[0260] Our preliminary results (n=20) showed a significantly higher proportion of innate CD8 T-cells and of innate CD8 T-cells expressing perforin in TFR patients at M12 versus relapsing patients during the same period (FIG. 7). This result is in favour of the notion that the success of TKI therapy cessation is associated with higher proportion of innate CD8 T-cells expressing perforin.

[0261] Collectively, our findings highlight innate CD8 T-cells as a potential marker for both DMR achievement and successful long-term treatment-free remission (TFR).

REFERENCES

[0262] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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