Methods for predicting the sensitivity of a subject to immunotherapy

Abstract

The present invention relates to a method of assessing, predicting or monitoring the sensitivity of a subject having a tumor or cancer to an immunotherapeutic molecule acting on the subject's T cells, to a method of selecting an appropriate treatment of cancer, to a method of screening or identifying a compound suitable for improving the treatment of a cancer, and to the use of corresponding kits. The method of predicting or monitoring the sensitivity of a subject having a tumor to an immunotherapeutic molecule acting on the subject's T cells typically comprises a step a) of determining, in a biological sample from said subject, the expression level of soluble CD25 (sCD25) and, when the expression level is determined, a step b) of comparing at least said expression level to a reference expression level, thereby assessing whether the subject having a tumor is responsive or resistant to the immunotherapeutic molecule.

Claims

1. An in vitro method of assessing the sensitivity of a subject having melanoma to an immunotherapeutic molecule acting on the subject's T cells for treating melanoma, which method comprises a step a) of determining, in a biological sample from said subject selected from a blood, a serum, a plasma sample or a derivative thereof, the respective basal expression levels of soluble CD25 (sCD25) and of lactic acid dehydrogenase (LDH), and, when the sCD25 and LDH expression levels are determined, a step b) of comparing said sCD25 and LDH basal levels of expression to sCD25 and LDH, respectively, thereby assessing whether the subject having a tumor is sensitive or resistant to the immunotherapeutic molecule, wherein the immunotherapeutic molecule is an anti-CTLA-4 antibody and: when the LDH reference expression level is about 500 IU, the step of assessing whether the subject having melanoma is sensitive or resistant to the immunotherapeutic molecule comprises: i) identifying the subject as having melanoma that is resistant to the immunotherapeutic molecule based on the sCD25 basal expression level being above the sCD25 reference expression level and/or the LDH basal expression level being above the LDH reference expression level of about 500 IU; or ii) identifying the subject as having melanoma that is sensitive to the immunotherapeutic molecule based on the sCD25 basal expression level being below the sCD25 reference expression level together with the LDH basal expression level being below the LDH reference expression level of about 500 IU; and iii) administering therapeutic doses of an anti-CTLA-4 antibody to the subject identified as having a melanoma that is sensitive to the immunotherapeutic molecule, or iv) administering therapeutic doses of an anti-CTLA-4 antibody in combination with therapeutic doses of a compensatory molecule selected from interleukin-2 (IL-2), interleukin-15 (IL-15), IL-2 superkine, sushi IL-15, radioimmunoconjugate of anti-CD25 and immunotoxin or a combination of the compensatory molecules to the subject identified as having a melanoma that is resistant to the immunotherapeutic molecule.

2. The method according to claim 1, wherein the sCD25 and LDH expression levels determined in step a) are the sCD25 and LDH basal expression levels in the subject, and wherein step b) further comprises comparing said sCD25 and LDH basal expression levels, used as reference expression levels, to sCD25 and LDH response expression levels in the subject determined after administration to said subject of the immunotherapeutic molecule.

3. The method according to claim 1, wherein the melanoma is a metastatic or an unresectable melanoma.

4. The method according to claim 2, wherein the method comprises a step a) of determining the sCD25 and LDH basal expression levels, in a biological sample of the subject having melanoma, before any administration to the subject of the immunotherapeutic molecule for treating the subject's melanoma, a step a′) of determining, in a biological sample of the subject having melanoma, the respective sCD25 and LDH response expression levels after the administration to said subject of at least one therapeutic dose of the immunotherapeutic molecule for treating melanoma, and a step b) of comparing said sCD25 and LDH response expression levels to said sCD25 and LDH basal expression levels and to reference expression levels in a control population, a sCD25 response expression level identical to the sCD25 basal expression level or below the sCD25 reference expression level, together with a LDH basal expression level below the LDH basal expression level and reference expression level being indicative of the sensitivity of the subject to the immunotherapeutic molecule.

5. The method according to claim 2, wherein the method comprises a step a) of determining the sCD25 and LDH basal expression levels, in a biological sample of the subject having melanoma, before any administration to the subject of the immunotherapeutic molecule for treating the subject's melanoma, a step a′) of determining, in a biological sample of the subject having a tumor, the respective sCD25 and LDH response expression levels after the administration to said subject of at least one therapeutic dose of the immunotherapeutic molecule for treating melanoma, and a step b) of comparing said sCD25 and LDH response expression levels to said sCD25 and LDH basal expression levels and to reference expression levels in a control population, a sCD25 response expression level above the sCD25 basal expression level or the reference expression level, together with a LDH basal expression level above the LDH basal expression level and reference expression level, being indicative of a resistance of the subject to the immunotherapeutic molecule.

6. The method according to claim 1, wherein the LDH reference expression level is about 500 IU and the sCD25 reference expression level is 1260 pg/ml, and wherein the step of assessing whether the subject having the tumor is sensitive or resistant to the immunotherapeutic molecule comprises: i) identifying the subject as having the tumor that is resistant to the immunotherapeutic molecule based on the sCD25 basal expression level being above the sCD25 reference expression level of 1260 pg/ml and/or the LDH basal expression level being above the LDH reference expression level of about 500 IU; or ii) identifying the subject as having the tumor that is sensitive to the immunotherapeutic molecule based on the sCD25 basal expression level being below the sCD25 reference expression level of 1260 pg/ml and the LDH basal expression level being below the LDH reference expression level of about 500 IU.

7. The method of claim 1, the method comprising administering therapeutic doses of an anti-CTLA-4 antibody to the subject identified as having a melanoma that is sensitive to the immunotherapeutic molecule.

8. The method of claim 7, wherein the anti-CTLA-4 antibody is Ipilimumab or Tremelimumab.

9. A method of treating a melanoma in a subject, the method comprising the steps of: a) determining, in a biological sample from said subject selected from a blood, a serum, a plasma sample or a derivative thereof, the basal expression levels for sCD25 and LDH; b) comparing said basal expression levels of sCD25 and LDH in the biological sample of the subject to reference expression levels for sCD25 and LDH; and c) treating a melanoma in the subject by administering to the subject: i) therapeutic doses of an anti-CTLA-4 antibody alone when the basal expression level of sCD25 in the biological sample of the subject is below the reference expression level for sCD25 of 1260 pg/ml and the basal expression level of LDH in the biological sample of the subject is below the reference expression level for LDH of about 500 IU, or ii) therapeutic doses of an anti-CTLA-4 antibody in combination with therapeutic doses of a compensatory molecule selected from interleukin-2 (IL-2), interleukin-15 (IL-15), IL-2 superkine, sushi IL-15, radioimmunoconjugate of anti-CD25 and immunotoxin or a combination of the compensatory molecules when the expression level of sCD25 in the biological sample of the subject is above the reference expression level for sCD25 of 1260 pg/ml and/or the expression level of LDH in the biological sample of the subject is above the reference expression level for LDH of about 500 IU.

10. The method of claim 9, wherein the anti-CTLA-4 antibody is Ipilimumab or Tremelimumab.

11. The method of claim 1, said method comprising administering therapeutic doses of an anti-CTLA-4 antibody in combination with therapeutic doses of a compensatory molecule selected from interleukin-2 (IL-2), interleukin-15 (IL-15), IL-2 superkine, sushi IL-15, radioimmunoconjugate of anti-CD25 and immunotoxin or a combination of the compensatory molecules to the subject identified as having a melanoma that is resistant to the immunotherapeutic molecule.

12. The method of claim 7, wherein the anti-CTLA-4 antibody is Ipilimumab or Tremelimumab.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Murine anti-CTLA4 induces the control of tumor growth.

(2) MCA 205 OVA (A) or MC38-OVA.sup.dim (B) tumor cell lines have been inoculated subcutaneously in C57/B16 mice. When tumor sizes reach 30-40 mm.sup.2, mice receive 100 μg of anti-CTLA4 every 3 days during 12 days, as indicated. Tumor sizes are monitored every 2-3 days until mice are sacrificed. **p<0.05; ***p<0.01 T test. (n=5 mice/group).

(3) FIG. 2: IL-15Rα is up regulated post-anti-CTLA4 by both tumoral and intratumoral leukocytic fractions.

(4) MCA 205 OVA tumors have been harvested from mice that have received 3 anti-CTLA4 injections (as described in FIG. 1), 2 days after the last injection. Tumors have been digested and a CD45 magnetic cell separation has been performed to separate CD45− (Tumor) and CD45+(Leukocytes). The expression of IL-15Rα has been assessed by qRT-PCR. Results are expressed relative to β2 microglobuline expression. (n=5 mice/group).

(5) FIG. 3: IL-2/CD122 axis is critical for anti-CTLA4 efficacy.

(6) MCA 205 OVA (A) or MC38-OVA.sup.dim (B) tumor cell lines have been inoculated subcutaneously in C57/B16 mice. When tumor sizes reach 30-40 mm.sup.2, mice receive 100 μg of anti-CTLA4 every 3 days for 12 days, as indicated. Anti-CD122 (100 μg/mouse, i.p. injected) or anti-IL-2 (50 μg/mouse, i.t. injected) neutralizing antibodies have been injected every 3 days throughout the experiment to block either the receptor or the cytokine. Tumor sizes are monitored every 2-3 days until mice sacrifice. ***p<0.01 T test. (n=5 mice/group).

(7) FIG. 4: IL-2Rα (CD25) axis is critical for anti-CTLA4 efficacy.

(8) MCA 205 OVA tumors have been inoculated subcutaneously in C57/B16 mice. When tumor sizes reach 30-40 mm.sup.2, mice receive 100 μg of anti-CTLA4 every 3 days for 12 days, as indicated. Anti-CD25 neutralizing Ab or IgG control Ab (500 μg/mouse, i.p. injected) have been injected 1 day before tumor inoculation and 1 day before anti-CTLA4 treatment. Tumor sizes are monitored every 2-3 days until mice sacrifice. ***p<0.01 T test. (n=5 mice/group).

(9) FIG. 5: IL-15 (recognized by CD122) is also critical for anti-CTLA4 efficacy.

(10) MCA 205 OVA (A) or MC38-OVA.sup.dim (B) tumor cell lines have been inoculated subcutaneously in C57/B16 mice. When tumor sizes reach 30-40 mm.sup.2, mice receive 100 μg of anti-CTLA4 Ab every 3 days for 12 days, as indicated. Anti-IL-15 (50 μg/mouse, i.t. injected) neutralizing Ab have been injected every 3 days throughout the experiment to neutralize the cytokine. Tumor sizes are monitored every 2-3 days until mice sacrifice. ***p<0.01 T test. (n=5 mice/group).

(11) FIG. 6: Mice exhibiting a significant anticancer response to anti-CTLA4 Ab display a loss of ICOSL.sup.+CD4+ T cells and an accumulation of LAG-3+CD4+ T cells among Tumor Infiltrating Leukocytes.

(12) MCA 205 OVA tumors have been harvested from mice that have received 3 anti-CTLA4 Ab injections, 2 days after the last injection. Flow cytometry analyses have been performed. ICOSL (A) and LAG-3 (B) expression by CD4.sup.+ T cells are presented. Panel C shows the cytokine production (IFNγ and TNFα) by CD4+ T cells following PMA/ionomycin stimulation, followed by intracellular staining for flow cytometry analysis. (n=5 PBS group; n=3 anti-CTLA4 Ab responders; n=2 anti-CTLA4 Ab non responders).

(13) FIG. 7: Synergistic effect between anti-CTLA4 Ab and rIL-2.

(14) MCA 205 OVA tumors have been inoculated subcutaneously in C57/B16 mice. When tumor sizes reach 30-40 mm.sup.2, mice receive 100 μg of anti-CTLA4 Ab every 3 days for 12 days, as indicated. 100,000 U of rIL-2 have been injected twice a day every day throughout the experiment, as indicated. (A) Means of tumor growth over time. (B) Tumor growth curves of each mouse treated by anti-CTLA4Ab alone or in combination with IL-2. (n=5 mice/group).

(15) FIG. 8: Follow-up of sCD25 overtime in metastatic melanoma patients treated by 10 mg/kg of Ipilimumab, associated with radiotherapy.

(16) (A) Responding patients; (B) non-responding patients. Dashed line: Positivity threshold 80 pMol/L.

(17) FIG. 9: Follow-up of sCD25 overtime in metastatic melanoma patients treated by 3 mg/kg of Ipilimumab.

(18) (A) Responding patients; (B) non-responding patients. Dashed line: Positivity threshold 80 pMol/L.

(19) FIG. 10: Continuous increase of sCD25 in metastatic melanoma patients that have developed severe Ipilimumab-related toxicity.

(20) Serum level of sCD25 post-C2 is superior to post-C1 which is superior to baseline (before treatment). All these patients are from cohort #1. HALRA and BAYJE experienced grade III colitis and DURHE experienced a DRESS syndrome.

(21) FIGS. 11A-11I: sCD25 serum level seems to inversely correlate with PSA serum level in hormono-resistant prostate cancer patients treated by 10 mg/kg Ipilimumab.

(22) Each graph shows 1 patient. pPanel B shows the responding patient.

(23) FIG. 12: Follow-up of sCD25 serum level overtime in hormonoresistant prostate cancer patients treated by 10 mg/kg of Ipilimumab.

(24) The responding patient is indicated. Dashed line: Positivity threshold 2000 pg/ml.

(25) FIG. 13: High serum levels of soluble CD25 compromise the efficacy of anti-CTLA4 blockade in mice.

(26) a-d. Serum levels of sCD25 and sLAG3 in patients. Eight independent cohorts of 262 MM were analyzed and compared with one cohort of 9 patients treated with rIL-2 for an autoimmune disorder (Saadoun et al.). Graphs depict the ELISA-determined concentrations of sCD25 (a-b) or sLAG3 (c-d) in the serum prior to and 3 weeks after ipilimumab (a-c) or rIL-2 (b-d), respectively. Each dot represents one patient. Paired Student's t-test:* p<0.05, ***p<0.001, ns: not significant. e. Inhibitory effects of high concentrations of sCD25 on the efficacy of ipilimumab in mice. 2 mg of sCD25 was administered systemically i.v. prior to and following the two first injections of anti-CTLA4 Ab in MCA205-OVA tumor bearers (left panel). Tumor sizes (one dot/tumor) were monitored for the first 8 days of sCD25 administration at three time points (day 0, at start, day+3 and day+6) and compared with untreated tumors (empty dots). Groups comprised 5 mice and three experiments were performed and concatenated. Mean tumor sizes of 15 mice/group are shown at day 3 and day 6 post-first ipilimumab injection (right panel) while each tumor is depicted at various kinetics in FIG. 14a. f-g. The effects of sCD25 on the composition of TILs were examined in each group: treated with ipilimumab, responding (R) or not (NR), and coadministered with sCD25. Flow cytometry analyses of CD45.sup.+ cells among live cells (f) and CD4.sup.+Lag3.sup.+Foxp3.sup.+ TILs (g), each dot representing one tumor specimen. Paired Student's t-test or ANOVA:* p<0.05, **p<0.01, ***p<0.001, ns: not significant.

(27) FIG. 14: Inhibitory effects of high concentrations of sCD25 on the efficacy of ipilimumab in mice.

(28) a. Lack of measurable sCD25 in mice receiving anti-mCTLA-4 Ab. Kinetic study of sCD25 concentrations in the serum of tumor-bearing mice treated with anti-mCTLA-4 or mice that did not receive tumors, treated with high dosing of rIL-2 (100,000 IU twice a day for 4 days) using commercial ELISA. b. 2 mg of sCD25 was administered systemically i.v. prior to and following the two first injections of anti-CTLA-4 Ab in MCA205-OVA tumor bearers (FIG. 6e, left). Tumor sizes (one dot/tumor) were monitored for the first 8 days of sCD25 administration at three time points (day 0, at start, day+3 and day+6) to ipilimumab-treated (black dots) or PBS-treated (empty dots) tumors and compared with mice which did not receive sCD25. Groups comprised 5 mice and three experiments were performed and concatenated. Each tumor is depicted at various kinetics with one dot. c-d. The effects of sCD25 on the composition of TILs were examined in each group: treated with ipilimumab, responding (R) or not (NR), and coadministered with sCD25. Flow cytometry analyses of ICOS expression on CD4.sup.+Lag3.sup.+ TILs (b) and CD8.sup.+TILs (c), each dot representing one tumor specimen. Paired Student's t-test or ANOVA:* p<0.05, **p<0.01, ***p<0.001, ns: not significant.

(29) FIG. 15: Tremelimumab induced a rise in the levels of serum sCD25.

(30) A cohort of 20 MM patients described in Ribas et al. received 15 mg/kg of Tremelimumab. Serum levels of sCD25 have been evaluated in ELISA before and 3 weeks after the first injection (C1). Paired Student's t test:* p<0.05.

(31) FIGS. 16a-f: Univariate analyses of multiple parameters of clinical significance for OS of MM patients.

(32) According to the data presented in Table 2, each parameter (indicated above each graph) that was statistically associated with overall survival (except sCD25 and LDH featuring in FIG. 17) has been plotted over time (Log Rank survival curves). Statistical methods and results are either shown or explained in the Materials & Methods section.

(33) FIG. 17: Baseline sCD25 serum levels predict resistance to ipilimumab in humans.

(34) a-b. Baseline serum levels of sCD25 and overall survival (OS). Kaplan-Meier curves in all of the 8 cohorts gathering 262 patients segregated into two groups according to the median LRT=4.69, p<0.030 (a) and the tercile LRT=9.17, p<0.010 (b) c. Baseline serum levels of sCD25 and LDH and OS. Kaplan-Meier curves of 249 MM patients segregated into 6 groups according to the median value of LDH (with 500 as the threshold, also herein identified as the cut-off value) and the tercile of sCD25 LRT=5.76 p=0.056 (c). d. Post-first ipilimumab serum levels of sCD25 and OS. Kaplan-Meier curves of 98 MM patients segregated into two groups according to the median value of sCD25 post C1 (3 weeks post-first ipilimumab injection). LRT=6.01, p<0.014.

(35) FIG. 18: sCD25 serum levels at T0 versus T21 and OS in MM patients.

(36) a. Baseline serum levels of sCD25 and OS in three cohorts shown in details. Kaplan-Meier curves of 3 representative cohorts segregated into 3 groups according to the terciles. b. Baseline serum levels of sLAG3 and OS. Kaplan-Meier curves of 194 MM patients segregated into two groups according to the median LRT=1.28, p=0.257. c. Analysis of the ratio between post-C1 sCD25/baseline sCD25 and OS. A subgroup of 98 patients in the 262 aforementioned patients for whom samples at 1 injection were available was analyzed for the correlation between sCD25 post-C1/sCD25 baseline ratio and OS. LRT=1.04, p<0.007.

(37) FIG. 19. Baseline sCD25 serum levels and overall survival in Tremelimumab-treated patients.

(38) Baseline serum levels of sCD25 and overall survival (OS). Kaplan-Meier curves in a cohort of 20 patients segregated into two groups according to the median (490 pg/ml; HR: 1.58 [0.52; 4.82], p<0.3999).

(39) Other characteristics and advantages of the invention are given in the following experimental section (with reference to FIGS. 1 to 19), which should be regarded as illustrative and not limiting the scope of the present application.

EXPERIMENTAL PART

Example 1—Prospective Study Using Ipilimumab

(40) In a prospective study conducted by the inventors, patients were treated using Ipilimumab.

(41) Inclusion criteria were: a diagnosis of unresectable stage III or IV melanoma, at least one previous line of chemotherapy, and survival 12 weeks after the first perfusion.

(42) Four courses of Ipilimumab were administered at the dose of 3 mg/kg every 3 weeks. 3 patients were included. The median overall survival was 9.1 months (95% CI: 6.4-11.3) from the start of Ipilimumab. Immune-related adverse events were noted in 45 patients (62%), including 20 grade 3-4 events (27.4%). No drug-related death occurred. In univariate analysis, factors associated with improved overall survival were the number of Ipilimumab infusions (≧4), the lymphocyte count at the start of the second course (cut off level at 1000/mm.sup.3), and an eosinophil count increase of more than 100>mm.sup.3 between the first two infusions.

(43) In small trials, it was shown that Ipilimumab results in the accumulation of ICOS-expressing CD4+ T cells (and to a lesser extent of CD8+ T cells) and TH17 cells. Inventors concluded that these criteria do not adequately predict the clinical benefit of Ipilimumab in Phase III trials.

Example 2—Soluble CD25 as a Predictive Marker of Response to Anti-CTLA4 Antibodies

(44) Material & Methods

(45) Patient Cohorts

(46) Cohort 1: Ipilimumab associated with radiotherapy

(47) 7 metastatic melanoma patients (4M and 3F, age: 45-80 years) received Ipilimumab (10 mg/kg, BMS) every 3 weeks for a total of 4 injections. Between the second and third injection, patients received fractioned radiotherapy (3×3Gy i.e., 9Gy in total over 1 week).

(48) Toxicities reported are mainly Grade II-III colitis.

(49) Cohort 2: Ipilimumab as monotherapy

(50) 8 metastatic melanoma patients (age: 39-84 years) received Ipilimumab (3 mg/kg, BMS) every 3 weeks for a total of 4 injections

(51) Toxicities reported are diarrhea and hypophysitis.

(52) Cohort 3: Ipilimumab associated with radiotherapy

(53) 9 hormonoresistant prostate cancer patients have been treated for bone metastases with the combination of local radiotherapy followed by 4 injections of Ipilimumab at 10 mg/kg, every 3 weeks.

(54) Cohort 4: 12 metastatic melanoma (MM) patients treated with 3 or 10 mg/kg

(55) Twelve MM patients have been treated either with 3 or 10 mg/kg of Ipilimumab 4 times, every 3 weeks.

(56) Mice Tumor Models and Anti-CTLA4 Ab Treatment

(57) C57/B16 WT mice are provided by Harlan, France. For tumor inoculation: 1 million tumor cells (the fibrocarcoma cell line MCA 205 OVA or the colon carcinoma cell line MC38 OVA.sup.dim) in 100 μl of PBS, in the right flank. When tumor size reaches 30-40 mm.sup.2, mice are treated intraperitoneally (i.p.) with 100 μg of anti-CTLA4 Ab (Bio X Cell, Clone 9D9), 3 times every 3 days.

(58) Neutralizing Antibodies

(59) Anti-IL-2 Ab (eBioscience, Clone: JES6-1A12) and anti-IL-15 Ab (eBioscience, Clone: AIO.3) or IgG control Ab (eBioscience, Rat IgG2a) have been injected intratumorally the same day as anti-CTLA4 treatment (20 μg in 50 μl of PBS). Anti-CD122 Ab (Bio X Cell, Clone: TM-beta1) have been injected every 3 days throughout the experiment starting from day 1 of anti-CTLA4 treatment to sacrifice.

(60) Anti-CD25 Ab (Bio X Cell, Clone: PC-61.5.3) or IgG control Ab (Bio X Cell, Rat IgG1) have been injected 1 day before and 1 day after the tumor inoculation (250 μl g/mouse/time point, i.p.).

(61) Soluble CD25 Dosage

(62) sCD25 serum levels have been performed using the ELISA kit from Beckman Coulter (capture mAb: Clone 24204, detection Ab: clone 33b3) for cohorts 1 and 2, and by using Quantikine IL-2sRa from R&D Systems (capture mAb: clone 24204, detection Ab: clone MAB623) for cohorts 3 and 4. The experiments have been performed according to the manufacturers' instructions.

(63) Quantitative RT-PCR:

(64) Tumor-bearing mice have been treated (or not) by 3 injections of anti-CTLA4 Ab as described above. Two days after the last injections, tumors have been harvested and digested into a cell suspension (5 mice/group). CD45 positive and negative fractions have been purified using anti-CD45 Ab coated magnetic beads (Miltenyi) followed by Automacs cell separation (Miltenyi) according to the manufacturer's instructions. RNAs have been extracted and retro-transcripted. Then a quantitative PCR has been performed using a Taqman procedure (Invitrogen). A commercial IL-15Ra probe (Applied Biosystems) has been used.

(65) Flow Cytometry

(66) Cells have been stained in PBS 2% FCS in presence of FcRBlocking and fluorochrome-coupled antibodies and viability marker for 15 min at 4° C. Then cells have been washed and resuspended in PBS for acquisition. For intracellular detection of cytokines, cells have been stimulated with PMA/ionomycin for 4 h in the presence of GolgiStop. After the membrane staining, cells have been permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences) accordingly to the manufacturer's instructions. Samples have been acquired and analyzed on a FACSCanto II (BD Biosciences) using FACSDiva Software (BD Biosciences). All the antibodies (Anti-ICOSL, -LAG-3, -IFNg, -TNFa, -CD4, -CD3, -CD45) come from eBioscience. For viability, Vivid Yellow has been used (Molecular Probes).

(67) Results

(68) The Efficacy of Mouse Ipilimumab Relies on the IL-2R13/IL-2 Signaling Pathway and IL-15 and IL-2R Alpha Chain.

(69) Two transplantable mouse tumor models syngeneic with C57BL/6 mice, i.e., MC38-OVA.sup.dim (colon cancer) and MCA205 sarcoma, were established for 10 days and then treated (when reaching a size of 40 mm.sup.2) with systemic (i.v.) anti-CTLA4 antibodies (mouse Ipilimumab) every 3-4 days for 12 days. Tumor size was monitored until sacrifize. Ipilimumab mediated transient tumor control in both tumor models (FIG. 1A, FIG. 1B) in a CD4+ and CD8+ T cell-dependent manner, since antibodies depleting CD4 or CD8+ T cells abrogated Ipilimumab-mediated antitumor effects (not shown). The transcription profile of cytokine and cytokine receptor-encoding gene products revealed a markedly enhanced expression of IL-15Rα in both CD45+ and CD45− fractions of tumor beds post-anti-CTLA4 Ab (FIG. 2). The inventors then neutralize IL-15, IL-15 related cytokines (IL-2) and the corresponding receptors (CD25, CD122) during mouse Ipilimumab therapy using specific neutralizing antibodies directed against such molecules. Surprisingly neutralization of IL-2 and CD122 completely abolished the anti-CTLA4 Ab-mediated tumoricidal activity, in both MCA205 and MC38-OVA.sup.dim tumor models (FIG. 3A, 3B). Blockade of CD25 (IL-2Rα chain) prior to tumor inoculation and Ipilimumab therapy also reduced the antitumor effects of Ipilimumab (FIG. 4). Similarly, neutralization of rIL-15 using specific antibodies targeting this cytokine abolished Ipilimumab-induced tumor stabilization (FIG. 5).

(70) Altogether, these data indicate that the efficacy of mouse Ipilimumab relies, at least in part, on the bioactivity of the IL-2/IL-2Rαβ signalling pathway.

(71) Loss of ICOSL+CD4+ T Cells and Accumulation of Lag3+CD4+ T Cells in TILs of Responders.

(72) The inventors investigated which T cell subsets accumulated in tumor beds post-Ipilimumab (after 3 injections), discriminating cancers responding from those progressing despite the treatment with anti-CTLA4 Abs. Flow cytometry analyses of the CD45+ fraction of tumor beds revealed that the efficacy of Ipilimumab relied upon the loss of ICOS ligand and the gain of Lag3 and TNFα/IFNγ-producing CD4+ T cells residing in regressing tumors (FIG. 6A, 6B, 6C).

(73) There were no significant differences for IL-2-producing CD4+ T cells between the regressors and progressors (not shown).

(74) The Combination of Mouse Ipilimumab and rIL-2 is Synergistic.

(75) Since MCA205 sarcomas failed to respond to high doses of daily rIL-2 and were only partially sensitive to anti-CTLA4 Ab (transient and partial effect), the inventors combined anti-CTLA4 Ab (same protocol as described above) with rIL-2 (200,000 IU daily or 100,000 IU bid, daily). This combination therapy induced strong synergistic antitumor effects in 60% of mice (FIG. 7). These data suggest that tumor-bearing animals that failed to respond to anti-CTLA4 Ab because of a lack of bioactivity of IL-2 are advantageously compensated by a combination of both compounds.

(76) In Metastatic Melanoma (MM), Low Basal sCD25 Serum Levels as Well as >1.5-Fold Increase of sCD25 after One Injection of 3-10 mg/kg of Anti-CTLA4 Ab/Human Ipilimumab Predict Clinical Benefit Observed at 4 Injections.

(77) Since the bioactivity of IL-2 can be monitored by a serum surrogate marker of T cell activation, i.e., soluble CD25, the inventors undertook the dosing of sCD25 in the serum of metastatic melanoma (MM) patients included in a Phase II trial or treated on a compassionate basis with human Ipilimumab (3 or 10 mg/kg). They first analyzed, as a positive control, sCD25 in MM treated with rIL-2 at 1, 2, 5, and 9 million IU three times a week for 1 week using a first commercial ELISA kit revealing the normal ranges of sCD25 in normal volunteers and most MM (upper threshold of detection: 80 pMol/l, FIG. 8). In similar conditions, a second commercial ELISA kit revealed a value of 900 pg/ml (not shown). Therefore, to analyze the kinetics of the sCD25 rise in patients undergoing Ipilimumab therapy (FIGS. 8A, 8B and FIGS. 9A, 9B), the inventors defined the following criteria.

(78) Criteria of response using the sCD25 kinetics: Basal levels of sCD25 should be in normal ranges (defined in normal volunteers, i.e., <80 pMol or 900 pg/ml) before Ipilimumab initiation and should increase, preferably by 1.5-fold, after one cycle of therapy.

(79) Out of 28 MM, 7 partial responses (RECIST criteria) were recorded at cycle 4. Seven out 7 responded to the above-mentioned criteria. Out of the 21 non-responders (RECIST criteria), 8/28 were anticipated to respond (false positive). None of the patients whose sCD25 levels did not match the criteria of response developed a response (no false negatives) (Table 1).

(80) TABLE-US-00001 TABLE 1 Prediction of clinical response and toxicity based on sCD25. Prediction Test for Prediction Test for Clinical Response Toxicity Parameters sCD25 under threshold sCD25 under threshold (<80 pM) AND Increase (<80 pM) AND Increase of sCD25 by 1.5 times of sCD25 by 1.5 times post 1.sup.st Ipilimumab post 2nd Ipilimumab injection injection False Positive 8/28 2/8 False Negative 0/28 0/8 Correct Prediction 20/28  6/8 Sensitivity (%) 100 100 Specificity (%) 61.9 33.33 Predictive Positive 46.67 71.43 Value (%) Predictive Negative 100 100 Value (%) False Positive Rate 53.33 28.57 (%) False Negative Rate 0 0 (%)

(81) To predict the efficacy, the sensitivity of the test (dosing of sCD25 in serum) was 100%, the specificity of the test was 61.9%, the positive predictive value (PPV) was 46.67%, the negative predictive value (NPV) was 100%, the false positive rate (FPR) was 53.33%, and the false negative rate (FNR) was 0%.

(82) In Metastatic Melanoma (MM), Low Basal sCD25 Serum Levels as Well as >1.5-Fold Increase of sCD25 after Two Injections of 3-10 mg/kg of Anti-CTLA4 Ab/Human Ipilimumab Predict Toxicity Observed at 4 Injections.

(83) Such a test could also predict toxicity but when used after 2 cycles of Ipilimumab: sensitivity=100%, specificity: 33.33%, PPV=71.43%, NPV=100%, FPR=28.57%, FNR=0%.

(84) Out of 8 MM, 5 Ipilimumab-related toxicity were recorded at cycle 4. Five out of 5 responded to the above-mentioned criteria. Of the 3 patients that did not develop Ipilimumab-related toxicity, 2/3 were anticipated to undergo toxicity (false positive). None of the patients whose sCD25 levels did not match the criteria of toxicity developed toxicity (no false negatives) (Table 1).

(85) Three patients developed severe Ipilimumab-related toxicity. Serum level of sCD25 increased continuously after each Ipilimuab injection (i.e., post-C2>post-C1>baseline; FIG. 10). Thus continuous sCD25 elevation after 2 injections predicts severe Ipilimumab-related toxicity (grade III-IV toxicities). None of the patients whose sCD25 level did not increase continuously developed toxicity.

(86) In Metastatic Hormonoresistant Prostate Cancer, sCD25 Serum Levels Inversely Correlate with PSA Serum Levels.

(87) In a series of 9 hormonoresistant prostate cancers treated for bone metastases with the combination of local radiotherapy and Ipilimumab at 10 mg/kg, sCD25 serum levels were inversely correlated with the PSA serum levels (surrogate marker of tumor burden) (FIG. 11, all 9 panels) in 7 out 9 cases. Interestingly, only one patient exhibited a spectacular complete response under Ipilimumab therapy and this patient, after initial Ipilimumab discontinuation attributed to a concomitant infection, showed a markedly enhanced rise in sCD25 after reintroduction of Ipilimumab (FIG. 12, showing all patients' kinetics of sCD25).

Example 3—High Serum Levels of Soluble CD25 Compromise the Efficacy of Anti-CTLA4 Blockade in Mice/High Soluble CD25 Serum Levels at Baseline are Associated with Resistance to Therapy in Metastatic Melanoma (“MM”) Patients/Baseline sCD25 and Lactate Dehydrogenase (LDH) Serum Levels Predict Resistance to CTLA4 Blockade

(88) Materials & Methods

(89) Patients and Specimens.

(90) The study was carried out on a total of 282 metastatic melanoma patients. Patient characteristics are depicted in Table 2 and Table 3. The immunomonitoring studies on fresh peripheral blood mononuclear cells (PBMC) were prospectively conducted on 10 patients. Patient samples (plasmas or sera) were provided by the Gustave Roussy Institute (Villejuif, France), the Memorial Sloan-Kettering Cancer Center (New York, USA), the Siena University Hospital (Siena, Italy), the German Cancer Research Center (Heidelberg, Germany), the Poznan University of Medical Sciences (Poznan, Poland) and the Essen University Hospital (Essen, Germany). Clinical responses were classified according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria and the immune-related response criteria IrRC.

(91) Serum Levels of the Soluble Form of CD25 and LAG3.

(92) Soluble CD25 (sCD25) and LAG3 (sLAG3) levels were determined by ELISA in accordance with the manufacturer's procedure. The Soluble IL-2 Receptor EIA Kit obtained from Beckman Coulter (Miami, Fla., USA) and the human soluble LAG3 Duo Set® from R&D Systems (Minneapolis, Minn., USA) were used for the sCD25 and sLAG3 dosage respectively. Patient sera (or plasmas) were collected before treatment and after the first and the second ipilimumab administrations. Mice: The mouse IL-2Rα Duo Set® from R&D Systems (Minneapolis, Minn., USA) was used for the sCD25 dosage.

(93) Mice.

(94) Mice aged between 7 and 12 weeks were used. Female C57BL/6J mice were obtained from Harlan (Gannat, France). Ifnar1 −/−C57BL/6J mice have been kindly provided by Gilles Uzé (UMR 5235, Montpellier Fance). Animals were maintained in specific pathogen-free conditions at either the Institut Gustave Roussy (IGR, Villejuif, France) or at the Peter MacCallum Cancer Center (East Melbourne, Australia) and housed in a temperature-controlled environment with a 12 h light-dark cycle and received food and water ad libitum. Experiments were carried out in compliance with French and European laws and regulations or with the Peter MacCallum Animal Experimentation Ethics Committee.

(95) Cell Culture and Reagents.

(96) OVA-expressing mouse fibrosarcoma MCA205 cells (class I MHC H-2.sup.b, syngeneic with C57BL/6 mice) were cultured at 37° C. under 5% CO.sub.2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids, all from Gibco-Invitrogen (Carlsbad, Calif., USA). OVA-expressing MCA205 cells selected in complete medium (as above) further supplemented with 50 μg/ml Hygromycin B (Invitrogen, Life Technologies™). Murine colon carcinoma MC38 OVA.sup.dim cells have been cultured in complete DMEM medium.

(97) Tumor Challenge and Treatment.

(98) Mice were subcutaneously injected in the right flank with 1×10.sup.6 MCA205-OVA cells. When tumor sizes reached 25 to 40 mm.sup.2, mice were injected intraperitoneally (i.p.) either with 100 μg of anti-CTLA4 (clone 9D9 (kindly supplied by J. Allison, University of Texas, MD Anderson, Houston, Tex.)) or with 100 μg of anti-PD1 (clone RMP1-14) from Bio X Cell (West Lebanon, N.H., USA). Anti-CTLA4 Ab was a mouse anti-mouse CTLA-4 antibody derived by immunization of human CTLA-4 transgenic mice (Peggs et al.). Mice were injected 5 times at 3-day intervals with 9D9 or 4 times at 4-day intervals with RMP1-14 and tumor sizes were routinely monitored by means of a common caliper until end-point reached.

(99) Anti-CD122 (clone TM-beta1), anti-CD25 (clone PC-61.5.3), anti-IL10R (clone 1B1.3A), anti-LAG3 (clone C9B7W), anti-ICOS (clone 17G9), and rat IgG (LTF-2, HRPN) used in vivo were obtained from Bio X Cell and anti-IL-2 (JES6-1A12) and anti-IL-15 (AIO.3) were provided by eBioscience (San Diego, Calif., USA). Mice received 200 μg of anti-CD122, 250 μg of anti-CD25, 500 μg of anti-IL-10R, 200 μg of anti-LAG3, and 250 μg of anti-ICOS, injected i.p. 1 day before each injection. Anti-IL-15 and anti-IL-2 were administered into the tumor bed at 20 μg on day 0 and day 3 and 10 μg per mouse on day 6 post-anti-CTLA4. sCD25 (Recombinant Human IL-2 Receptor a, Peprotech, Rocky Hill, N.J., USA) was injected i.v. at 25 μg as described in FIG. 13e).

(100) Flow Cytometry.

(101) Mice: Eight-color flow cytometry analysis was performed with fluorescein isothiocyanate, phycoerythrin, phycoerythrin cyanin 7, Peridinin Chlorophyll Protein Cyanin 5.5, allophycocyanin cyanin 7, Pacific blue, and allophycocyanin-conjugated antibodies. The staining was performed on spleens and tumors dissected from mice two days after the third injection of anti-CTLA4. Briefly, excised tumors were cut in small pieces and digested in RPMI medium containing Liberase™ at 25 μg/ml (Roche) and DNase I at 150 UI/ml (Roche) for 30 minutes at 37° C. Single-cell suspension was obtained by crushing the pieces on a 100 μm cell strainer. Two million splenocytes (after red blood cell lysis) or tumor cells were preincubated with purified anti-mouse CD16/CD32 (clone 93, purchased from eBioscience) for 15 minutes at 4° C. before the membrane staining. For intracellular staining, the FoxP3 staining kit (eBioscience) was used according to the manufacturer's instructions. Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit (Life Technologies™). Stained samples were run on a Canto II (BD Biosciences, San Jose, Calif., USA) cytometer and analyses were performed with FlowJo software (Tree Star, Ashland, Oreg., USA). For cytokine staining, cells were stimulated for 4 hours at 37° C. with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA), 1 μg/ml of ionomycin (Calbiochem) and BD Golgi STOP™ (BD Biosciences) according to the manufacturers' instructions. Anti-CD45.2 (104), CD127 (A7R34), FoxP3 (FJK-16s), LAG3 (eBioC9B7W), ICOS (7E17G9), ICOSL (HK5.3), IL17a (eBio17B7), IFN-γ (XMG1.2), and TNF-α (MP6-XT22) and isotype controls rat IgG1 (eBRG1), IgG2a (eBRG2a), and IgG2b (eBRG2b) were purchased from eBioscience. Anti-CD3 (145-2C11), CD25 (PC61.5.3), IL-10 (554467), CD11b (M1/70), and rat IgG1κ were provided by BD Biosciences. Anti-CD4 (GK1.5), CD8β (YTS1567.7), CD11c (N418), and rat IgG2a (RTK2758) were purchased from BioLegend (San Diego, Calif., USA). Anti-CD25 (7D4) was provided by Miltenyi Biotech. Human: Anti-CD3 (BW262/56) and mouse IgG1 (I55-21F5) were purchased from Miltenyi Biotech. Anti-CD4 (SK3), CD8 (SK1), CD25 (M-A251), CD45 (J33), and mouse IgG1 (MOPC-21) were provided by BD Biosciences. Anti-Foxp3 (PCH101) was purchased from eBioscience and anti-LAG3 from R&D Systems.

(102) Bioinformatics and statistical analysis. Data were analyzed with Microsoft Excel (Microsoft Co., Redmont, Wash., USA), Prism 5 (GraphPad, San Diego, Calif., USA) and the environment R (R Foundation for Statistical Computing, Vienna, Austria). Data are presented as mean±SEM and p-values were computed by paired or unpaired t-test where applicable. Tumor growth modeling was carried out with linear mixed effect modeling on log pre-processed tumor volumes (Sugar et al.). Reported p-values are obtained from testing jointly that both tumor growth slopes and intercepts (on log scale) are the same between treatment groups of interest. Overall survivals (OS) determined from the first injection and immune-related Response Criteria (ir-RC) at the date of diagnosis/time of treatment were used as the primary end-points. Likelihood ratio test (LRT) from Cox proportional hazards regression modeling was used for assessing predictors of survival (including patient characteristics) as well as the added prognostic value of sCD25. All reported tests are two-tailed and were considered significant for p-value<0.05. Regression model analyses were conducted on the log basis 2 transformed marker concentration and were evaluated with the use of Schoenfeld residuals. Therefore, hazard ratios and associated 95% confidence intervals are easily interpretable in terms of doubling in concentration. For graphical representations, serum marker levels were partitioned into 2/3 groups based on the observed median/tertile (sCD25, sLAG3) or a threshold of 250 (LDH). For completeness, statistics from both specifications of the marker in the model are presented but statistics resulting from the marker(s) in the continuous scale are used to draw conclusions.

(103) TABLE-US-00002 TABLE 2 Characteristics of Patients Ipilimumab treatment Overall Survival Univariate Analsysis n (%) HR [95% CI], p value Nb of patients 262 Median Survival Time Weeks 41 (3-156) Age Mean (Min-Max) 59.78 [16.37; 91.05] Gender Male 128 (59.8) 1 Female 86 (40.2) 0.90 [0.63; 1.28], p < 0.5501 Metastasis M1a 29 (14.9) 1 M1b 35 (18) 1.99 [0.88; 4.51], p < 0.099  M1c 130 (67) 5.22 [2.52; 10.82], p << 1e−04  Nb of 1 7 (4.6) 1 Ipilimumab 2 18 (11.8) 0.21 [0.08; 0.59], p < 0.0027 Injection 3 21 (13.7) 0.27 [0.10; 0.72], p < 0.0087 4 101 (66) 0.13 [0.05; 0.32], p << 1e−04 >4 6 (3.9) NA Dose of 3 mg/kg 204 (77.9) 1 Ipilimumab 10 mg/kg 16 (6.1) 0.10 [0.01; 0.77], p < 0.0271 3 or 10 (Blinded) 42 (14) Clinical RECIST CR 6 (3.3) 1 response PR 21 (11.7) 1 SD 17 (9.4) 2.83 [0.47; 17.22], p < 0.2585  PD 136 (75.6) 20.18 [4.87; 83.70], p << 1e−04  Unknown 29 (14) IrRC CR 4 (2.9) 1 PR 22 (15.7) 1 SD 26 (18.6) 4.35 [0.91; 20.73], p < 0.0646  PD 88 (62.9) 23.50 [5.66; 97.52], p << 1e−04  Unknown 10 (7) Toxicity Grade 0 .sup.   100 (64.1) 1 Grade I-II 44 (28.2) 0.41 [0.23; 0.73], p < 0.0027    Grade III-IV 12 (7.7) 0.39 [0.14; 1.10], p < 0.0759 Line of 1 50 (21) Ipilimumab 2 130 (55) Treatment 3 36 (15) 4 20 (8) 5 1 (0.5) LDH at IU <250 104 (41.8) 1 Baseline 250-500 94 (37.8) 2.47 [1.68; 3.62], p << 1e−04 >500 51 (20.5) 5.08 [3.25; 7.95], p << 1e−04 sCD25 at pg/ml <1260 (Low) 130 (49.6) 1 baseline >1260 (High) 132 (50.4) 1.39 [1.02; 1.90], p < 0.0346 sLAG3 at pg/ml <849 (Low) 96 (49.5) 1 baseline >849 (High) 98 (50.5) 1.21 [0.84; 1.75], p < 0.3093

(104) Table 2 summarizes the characteristics of the patients. Hazard ratios were estimated with the use of unstratified Cox proportional hazards models. Lines represent 95% confidence. The metastasis (M) stage was classified according to the tumor-node-metastasis (TNM) categorization for melanoma. LDH, Lactate dehydrogenase. IU: International Unit.

(105) TABLE-US-00003 TABLE 3 Characteristics of Patients Tremelimumab treatment Overall Survival Univariate Analsysis n (%) HR [95% CI], p value Nb of patients 20 Median Survival Time Weeks (Min-Max) 32 [12; 132] Age Mean (Min-Max) 56.55 [27; 81] 0.99 [0.95; 1.03], p < 0.6822 Gender Male 16 (80) 1 Female 4 (20) 0.39 [0.07; 2.26], p < 0.2167 Metastasis M1a 10 (50) 1 M1b 4 (20) 2.46 [0.61; 9.95], p < 0.2053 M1c 6 (30) 0.97 [0.27; 3.47], p < 0.9649 Nb of 1 12 (60) 1 Tremelimumab 2 4 (20) 0.21 [0.03; 1.38], p < 0.0386 Injection 3 3 (15) 0.33 [0.05; 2.09], p < 0.1498 4 1 (5)  0.8 [0.12; 5.19], p < 0.7971 Clinical RECIST CR 3 (15) 1 response PD 17 (85) 8.18 [0.43; 155.99], p < 0.0403  Toxicity No Toxicity 8 (40) 1 Toxicity 12 (60) 0.39 [0.13; 1.18], p < 0.0867 LDH at Normal 11 (55) 1 Baseline High 9 (45) 6.76 [1.97; 23.23], p < 0.0011  sCD25 at pg/ml <490 (Low) 10 (50) 1 baseline >490 (High) 10 (50) 1.58 [0.52; 4.82], p < 0.3999 sCD25 pg/ml <921 (Low) 10 (50) 1 Post C1 >921 (High) 10 (50) 1.88 [0.62; 5.74], p < 0.2440

(106) Table 3 summarizes the characteristics of the patients. Hazard ratios were estimated with the use of unstratified Cox proportional hazards models. Lines represent 95% confidence. The metastasis (M) stage was classified according to the tumor-node-metastasis (TNM) categorization for melanoma. LDH, Lactate dehydrogenase.

(107) Results

(108) Soluble CD25 Inhibits the Efficacy of Ipilimumab

(109) The inventors monitored surrogate markers of lymphocyte activation such as soluble CD25 and Lag3 in the serum of cancer patients (MM) treated with ipilimumab. Like patients harbouring autoimmune vasculitis receiving a low dosage of rIL-2 (Saadoun et al.), MM patients (N=262) being administered ipilimumab (most of them receiving 3 mg/kg on a compassionate basis, Table 2) exhibited a significant rise in the serum levels of sCD25 (FIG. 13 a-b). Intriguingly, a proportion of MM patients presented with high baseline levels of sCD25 (above the median of normal volunteers: 330-1650 pg/ml (Bien, E. & Balcerska, A.)), a finding reminiscent of the high IL-2/IL-10 ratio found in circulating CD4.sup.+ T cells (data not shown). Of note, the concentrations of sLAG3 were barely augmented post-ipilimumab or post-rIL-2 (FIG. 13 c-d).

(110) To investigate the negative impact of high sCD25 baseline levels on the efficacy of anti-CTLA4 blockade, the inventors experimentally raised the serum concentrations of sCD25 by iterative systemic infusions of high doses of recombinant sCD25 in mice during ipilimumab administrations (FIG. 13e, left panel). Indeed, it is noteworthy that mouse ipilimumab failed to induce shedding of sCD25 at this dose/scheduling, in contrast to human settings (FIG. 13a, FIG. 14a). This maneuver significantly compromised the antitumor effects observed right after starting CTLA4 blockade (FIG. 13e, right panel and FIG. 14b). In the presence of high concentrations of soluble CD25, the ipilimumab-induced influx of CD45.sup.+ leukocytes and the downregulation of Foxp3 and ICOS expression on CD4.sup.+Lag3.sup.+ TILs associated with tumor regression were significantly impaired (FIG. 14 c-d, FIG. 13 f-g) while CD8.sup.+ ICOS.sup.+TILs that were CD122-independent were not affected by sCD25 (FIG. 14d).

(111) Altogether, these preclinical data indicate that high concentrations of the receptor sCD25 in the serum prior to therapy are detrimental for the efficacy of a CTLA4 blockade immunotherapy.

(112) Soluble CD25 Inhibits the Efficacy of Tremelimumab

(113) MM patients (N=20) (Table 3) treated with an alternative anti-CTLA-4 mAb, Tremelimumab (3 weeks after a single dose of 15 mg/kg) exhibited a significant rise in the serum levels of sCD25 (FIG. 15).

(114) Baseline sCD25 and LDH Serum Levels Predict Resistance to CTLA4 Blockade Immunotherapy

(115) The inventors analyzed the potential negative impact of high baseline levels of sCD25 on the clinical outcome of metastatic melanoma (“MM”) patients receiving ipilimumab in a retrospective study encompassing 9 independent cohorts from 5 different countries (Table 2). Considering the median value of sCD25 in the whole cohort of 262 patients, they showed that sCD25 as well as LDH levels, immune-related response criteria (irRC) at 4 weeks, and M1a/1b/1c dissemination status were all parameters statistically associated with overall survival (OS) in univariate analyses (Table 2, FIG. 16, HR=1.29 [1.06; 1.57], p<0.00827, FIG. 17 a-b, focus on distinct cohorts in FIG. 18a) whereas levels of sLAG3 and lymphocyte counts at diagnosis did not correlate with OS (HRc=1.02 [0.94; 1.11], p<0.65, FIG. 18b and not shown). The prognostic value of sCD25 remained significant (HR=1.26 [1.04; 1.54], p<0.0165) after including LDH in the model pointing to an independent contribution of sCD25 to the OS (FIG. 17c). sCD25 basal concentrations above a reference expression level at diagnosis correlated with poor outcomes for patients thereafter treated with ipilimumab, specifically in the subset presenting with high basal LDH levels, i.e., above about 500 IU (FIG. 17c). Similarly, sCD25 levels above a reference expression level at 3 weeks (T21) post-ipilimumab negatively predicted OS (FIG. 17d) while the ratio between sCD25 at T21/T0 failed to do so (FIG. 18c). The inventors conclude from their experiments that combined with LDH values, the basal sCD25 serum level represents a valuable predictor of resistance to ipilimumab or Tremelimumab.

(116) The inventors analyzed the potential negative impact of levels of sCD25 above a reference expression level on the clinical outcomes of MM patients receiving Tremelimumab (another anti-CTLA4 Ab, MedImmune) in a retrospective study (FIG. 19, Table 3). Considering the continuous value of sCD25 in the cohort of 20 patients, the inventors show that sCD25 has the same trend of association with overall survival (OS) in univariate analysis (2.03 [0.77; 5.36], p<0.1503). LDH at baseline (i.e., before any treatment) and RECIST criteria were statistically associated with overall survival (OS) in univariate analysis (Table 3).

CONCLUSIONS

(117) Both preclinical (FIG. 13e) and clinical data (FIG. 17 a-b) indicate that the antitumor efficacy of anti-CTLA4 blockade immunotherapy is compromised in patients presenting with basal serum levels of sCD25 above a reference expression level at diagnosis. In MM presenting with LDH>500 IU, sCD25 serum concentration added a negative prognostic value, indicating a subset of about 25% patients doomed to fail ipilimumab therapy (FIG. 17c).

(118) Detecting high sCD25 basal concentrations at diagnosis is an indication to use humanized anti-CD25 therapeutic strategies (radioimmunoconjugates of anti-CD25 and immunotoxins) in MM prior to ipilimumab therapy.

REFERENCES

(119) Kaehler K C, Piel S, Livingstone E, Schilling B, Hauschild A, Schadendorf D. Update on immunologic therapy with anti-CTLA-4 antibodies in melanoma: identification of clinical and biological response patterns, immune-related adverse events, and their management. Semin. Oncol. 2010 October; 37(5):485-98. Hodi F S, O′Day S J, McDermott D F, Weber R W, Sosman J A, Haanen J B, et al. Improved survival with Ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 2010 Aug. 19; 363(8):711-23. Robert C, Thomas L, Bondarenko I, O′Day S, Weber J, Garbe C, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N. Engl. J. Med. 2011 Jun. 30; 364(26):2517-26. Weber J. Ipilimumab: controversies in its development, utility and autoimmune adverse events. Cancer Immunol. Immunother. 2009 May; 58(5): 823-30. Di Giacomo A M, Biagioli M, Maio M. The emerging toxicity profiles of anti-CTLA-4 antibodies across clinical indications. Semin. Oncol. 2010 October; 37(5):499-507. Wolchok J D, Neyns B, Linette G, Negrier S, Lutzky J, Thomas L, et al. Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol. 2010 February; 11(2):155-64. Weber J S, Kahler K C, Hauschild A. Management of Immune-Related Adverse Events and Kinetics of Response With Ipilimumab. Journal of Clinical Oncology. 2012 May 21; 30(21):2691-7. Wolchok J D, Hoos A, O′Day S, Weber J S, Hamid O, Lebbé C, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin. Cancer Res. 2009 Dec. 1; 15(23):7412-20. Peggs, K. S., Quezada, S. A., Chambers, C. A., Korman, A. J. & Allison, J. P. Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti-CTLA-4 antibodies. J Exp Med 206, 1717-1725 (2009). Sugar, E., Pascoe, A. J. & Azad, N. Reporting of preclinical tumor-graft cancer therapeutic studies. Cancer Biol Ther 13, 1262-1268 (2012). Hodi, F. S., et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363, 711-723 (2010). Robert, C., et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 364, 2517-2526 (2011). Topalian, S. L., et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443-2454 (2012). Saadoun, D., et al. Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. N Engl J Med 365, 2067-2077 (2011). Bien, E. & Balcerska, A. Serum soluble interleukin 2 receptor alpha in human cancer of adults and children: a review. Biomarkers 13, 1-26 (2008). Ribas, A., et al. Phase III randomized clinical trial comparing tremelimumab with standard-of-care chemotherapy in patients with advanced melanoma. J Clin Oncol 31, 616-622 (2013).