Universal cancer peptides derived from telomerase

Abstract

The invention relates to a peptide of 15 to 20 amino acids deriving from TERT protein, which peptide is capable of (i) binding to HLA class II and (ii) stimulating a CD4 Th response. These universal cancer peptides are especially useful in anti-tumor immunotherapy and immunomonitoring.

Claims

1. A method for treating a tumor in a patient in need thereof, wherein the method comprises administering to the patient (i) a first peptide consisting of the sequence SLCYSILKAKNAGMS (SEQ ID NO: 3), and (ii) a second peptide consisting of the sequence KSVWSKLQSIGIRQH (SEQ ID NO: 1).

2. The method of claim 1, wherein the method further comprises administering to the patient (iii) a third peptide consisting of the sequence GTAFVQMPAHGLFPW (SEQ ID NO: 2) and (iv) a fourth peptide consisting of the sequence PAAFRALVAQCLVCV (SEQ ID NO: 4).

3. The method of claim 1, wherein the method further comprises administering an immunogenic tumor antigen.

4. The method of claim 1, wherein the tumor is a cancer.

5. The method of claim 4, wherein the cancer is selected from the group consisting of chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, malignant myeloma, Hodgkin's disease, melanoma, brain cancer, carcinomas of the bladder, breast, cervix, colon, lung, pancreas, prostate, head and neck, and stomach.

6. The method of claim 4, wherein the cancer is a lung cancer.

7. The method of claim 2, wherein the tumor is a lung cancer and the method comprises administering peptides KSVWSKLQSIGIRQH (SEQ ID NO: 1), GTAFVQMPAHGLFPW (SEQ ID NO: 2), SLCYSILKAKNAGMS (SEQ ID NO: 3), and PAAFRALVAQCLVCV (SEQ ID NO: 4).

Description

LEGENDS TO THE FIGURES

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1: UCP-specific T cell lines obtained from healthy donors.

(3) CD4 T cell lines were obtained from PBMCs of healthy donors after three rounds of stimulation with a mixture of the four UCP and IFN--producing CD4 T cells were assessed by ELISPOT. (A) Responses against individual UCPs are shown for six healthy donors. (B) UCP-specific T cell lines were stimulate with the relevant peptide in presence of anti-HLA class I (W6.32), HLA-DR (L243) or HLA-DP (B7/21) blocking antibodies (C) Responses against individual UCPs for three healthy donors with various HLA-DR genotype.

(4) FIG. 2: Functional characterization of UCP-specific CD4 T cell clones.

(5) T cell clones were obtained by limiting dilution of cancer patients T cell lines stimulated one time with the pool of UCPs. (A and C) Percentage of TNF-producing T cells and of T cell clones isolated from patients GE001 in response to 10 M of the relevant UCP; 10.sup.5 T cells were incubated for 5 h in the presence of Brefeldin A, stained with CD4 antibody, fixed, and stained with anti-TNF antibody in a permeabilization buffer; 10.sup.4 T cells were then analyzed in flow cytometry. (B and D) Reactivity of the CD4 T cell clones in response to relevant UCP. CD4 T cell clones were culture with a range of the indicated peptide concentration. TNF secretion was assessed 5 h in the presence of Brefeldin A, by flow cytometry. (E) Detection of cytokines produced by GE001.36 T cell clone in response to 10 M of UCP4 using human ten-plex cytokines assay.

(6) FIG. 3: Naturally occurring UCPs specific response in metastatic NSCLC patients.

(7) (A) Spontaneous UCP specific-T cell responses were assessed in 84 NSCLC patients and 22 healthy donors as control. After short time stimulation (one week) with a mixture of the four UCPs the presence of specific-T cells was detected using IFN- ELISPOT assay. The results represented specific IFN- spots after subtraction of background. Responses were positive when IFN- spots were >10 and more than two fold the background

(8) (B) Frequency of individual UCP-specific T cell responses in 12 NSCLC patients was shown.

(9) (C) Illustration of UCPs versus viral-specific immune responses in eight NSCLC patients after one week in vitro stimulation. (D) Baseline Neutrophils on Lymphocytes Ratio (NLR) and CD4+ Foxp3+ T cell frequency in patients according to the UCP-specific immune status.

(10) FIG. 4: Impact of spontaneous UCPs CD4 T cell response in metastatic NSCLC patients. (A) UCPs responder and non-responder frequencies in patients with progressive disease (PD) or control disease (CD).

(11) (B) Kaplan-Meier estimates of overall survival (OS) and

(12) (C) progression free survival (PFS) of CD patients.

(13) (D) OS and

(14) (E) PFS of CD patients treated with platinum-based first line chemotherapy.

(15) FIG. 5: UCPs vaccinations stimulate high avidity Th1 polarized CD4 T cell responses.

(16) (A-B), A2/DR1 mice (n=8) were immunized twice with a DNA encoding TERT.

(17) (A), Proliferation of spleen lymphocytes in presence of UCPs.

(18) (B), CD8 depleted spleen lymphocytes from DNA-immunized mice were assayed in ex vivo IFN- ELISPOT. Columns mean of triplicate from 4 mice; bars, SD.

(19) (C-D), Mice (3-4/group) were immunized once with each UCP in IFA.

(20) (C), Ten days later, spleen-isolated CD4 T cells were cultured overnight in presence of DC loaded with UCP. The cytokines production was measured in the supernatant by Luminex assay. Columns, mean of cytokine levels; bars, SD.

(21) (D), Isolated CD4 T cells were cultured ex vivo with increasing concentrations of peptide as indicated. IFN- production was measured by ELISPOT. Curves, mean responses from 3 mice, bars, SD.

(22) (E), Mice were vaccinated once with low dose of UCP as indicated. UCP-specific T cell responses were evaluated in spleen by ex vivo IFN- ELISPOT.

(23) FIG. 6: CD4 helper role of UCPs vaccinations on the self/TERT-specific CTL responses

(24) Mice (3/group) were immunized either with pY988 plus each UCP in IFA or with pY988/IFA alone and the immune responses were monitored ten days later in the spleen.

(25) A, freshly isolated CD8 T cells were stained with TERT pY988/A2+ pentamer. Representative flow cytometry dot plots (upper panel) and mean percentages of pY988/A2+ CD8 T cells (lower panel) are shown.

(26) B, Ex vivo detection of anti-pY988 CD8 T cells by IFN- ELISPOT.

(27) C-D, simultaneous UCP-specific CD4 T cell responses were assessed in CD8-depleted fraction by IFN- (C) and interleukine-2 (D) ELISPOT assays. Columns, mean of spots from 3 mice; bars, SD.

(28) Data are representative of three independent experiments.

(29) FIG. 7: Immunization in presence of UCP2 enhances the quality of self pY988-specific CTL responses.

(30) A-C, Mice (3-4/group) were immunized once either with pY988 plus UCP2 (UCP2+pY988/IFA) or with pY988/IFA alone.

(31) A, Ten days later, freshly isolated spleen CD8 T cells were cultured with increasing pY988 peptide concentration and IFN--secreting CD8 T cells were detected by ex vivo ELISPOT.

(32) B, In vivo cytototoxic assay. Representative flow cytometry histograms showing lysis of CFSE-labeled pY988-loaded target cells compared to unpulsed (UP) and the mean of in vivo percentage lysis are shown.

(33) C-D, Long-term T cell responses were evaluated 30 days after immunization.

(34) C, Frequencies of pY988/A2 pentamer+ CD8 T cells gated on CD44hiCD62lo cells (left) and by IFN- secretion assay (right).

(35) D, UCP2-specific CD4 T cell response measured in CD8-depleted fraction by ex vivo IFN- ELISPOT.

(36) E, Mice (4/group) were treated either with anti-CD4 mAb (GK1.5) (CD4 depleted, white bars) or with saline (non depleted, black bar) 3 days before immunization with DNA/TERT.

(37) The self/TERT-specific CTLs (left) and UCP-specific CD4 T cell responses (right) were measured in spleen by ex vivo IFN- ELISPOT.

(38) Data are representative of three independent experiments.

(39) FIG. 8: UCP2-specific CD4 Th1 cells active dendritic cells.

(40) A, Mice (3/group) were immunized once either with UCP2+pY988/IFA or pY988/IFA alone. Ten days later, the expression of activation markers CD80, CD86 and HLA-DR were analyzed on lymph nodes CD11c+ DC by flow cytometry. Representative Flow cytometry histograms (upper panels) and the mean of MFI (lower panels) are shown. Columns, mean of MFI; bars, SD. B-E: Analysis of DC and CD4 T cells cross talk.

(41) B, Schema of the in vitro DC-CD4 T cell co-culture.

(42) C, IFN- and GM-CSF production measured by ELISA in the supernatant.

(43) D, Expression of CD86 and HLA-DR on CD11c+ DC.

(44) E, Interleukin 12 production measured in supernatant by ELISA.

(45) Data are representative of two independent experiments.

(46) FIG. 9: Therapeutic antitumor effect of UCP-based vaccination.

(47) A, TERT expression by western-blot (left) and activity by TRAP-ELISA assay (right) in B16-A2 melanoma.

(48) B-E. Tumor-bearing mice (6-8 mice/group) were therapeutically vaccinated with peptides as described (materials and methods).

(49) B, Follow-up of tumor size. The numbers in parentheses indicate mice with tumor regression per group.

(50) C, Survival curves recorded until 50 days.

(51) D, Detection of anti-TERT immune responses in the spleen of tumor free mice from UCP2-vaccinated group by IFN- ELISPOT.

(52) E, In this experiment, tumor-bearing mice (n=4/group) were vaccinated as above and tumor-infiltrating immune cells were analyzed at day 25 by flow cytometry. Columns, mean of percentages of cells; bars, SD.

(53) Data are representative of two independent experiments.

(54) FIG. 10: Analysis of UCP-specific T cell responses in human.

(55) A, Blood lymphocytes from cancer patients were directly cultured with pool of UCPs during five days and specific proliferation was measured by 3H thymidine incorporation. Representative data from nine responding patients are shown.

(56) B-D, Lymphocytes were cultured in vitro with pool of UCPs for one week.

(57) B, Detection of UCP-specific T cell by IFN- ELISPOT. Representative data from nine responding patients are shown. Columns, mean of triplicate; bars, SD.

(58) C, Detection of cytokine production by DIAplex assay in supernatant after 15 h of culture in presence of UCPs. Columns, mean cytokine levels from three patients; bars, SD. D, T cell responses against individual UCP for six responding patients.

(59) FIG. 11: Isolation of UCP4-specific CD4 Th1 cell clone from a cancer patient.

(60) A, IFN- producing UCP4-specific T CD4 cell clone was isolated from a colorectal cancer patient.

(61) B, Evaluation of mRNA expression of UCP4-specific T CD4 clone by real-time RT-PCR.

(62) Positive and negative fold change values indicate the up- or down-regulation of mRNA expression compared to CD4 T cells from a healthy volunteer.

(63) C, Analysis of cytokines production by intracellular staining.

EXAMPLES

Example 1

Identification of Promiscuous HLA-DR Telomerase-Derived Epitopes and Analysis of Spontaneous Tumor-Specific CD4 T Cell Immunity in Lung Cancer Patients

(64) 1.1 Materials and Methods

(65) Patients

(66) Patients were enrolled at the university hospital Georges Pompidou (Paris, France) and university Hospital Jean Minjoz (Besancon, France) from January 2009 to February 2011. Tumor stage and grading were determined according to the International Union against Cancer (UICC) classification. After informed consent, patients with histologically proven NSCLC were prospectively included in the clinical trial. This study was conducted in accordance with French laws and after approval by the local ethics committee. Blood cells were collected from anonymous healthy donors at the Etablissement Francais du Sang (EFS, Besancon, France) as apheresis kit preparations after informed consent and following EFS guidelines. HLA-DR genotyping was performed by using the Olerup SSP DRB1 typing kit (Olerup, Sweden).

(67) Telomerase-Derived CD4 T Epitopes Selection and Binding Assay

(68) The four peptides derived from TERT referred as Universal Cancer Peptide (UCP1 (PAAFRALVAQCLVCV, SEQ ID NO:4), UCP2 (KSVWSKLQSIGIRQH, SEQ ID NO:1), UCP3 (GTAFVQMPAHGLFPW, SEQ ID NO: 2) and UCP4 (SLCYSILKAKNAGMS, SEQ ID NO:3) were predicted in order to bind multiples HLA-DR molecules by using SIFPETHI (accessible via World Wide Web at syfpeithi.de), NetMHCpan 2.1 (accessible via World Wide Web at cbs.dtu.dk/services/NetMHCII/) and NetMHCII 2.2 (accessible via World Wide Web at cbs.dtu.dk/services/NetMHCII/) softwares (Kobayashi et al, 2008). Synthetic peptides (>80% purity) were purchased from Activotec (Cambridge, United Kingdom). The binding capacity to HLA-DR molecules was assessed by competitive ELISA as previously reported (Wang et al).

(69) Generation of UCP-Specific T Cell Lines from Healthy Donors

(70) Peripheral Blood Mononuclear Cells (PBMC) were isolated by density centrifugation on Ficoll-Hyperpaque gradients (Sigma-Aldrich, France) and plated at 4.10.sup.6 cells per well in a 24-well plate in RPMI 5% human serum with 10 M of pool of the four UCPs. Recombinants interleukin 7 (5 ng/mL) (Peprotech, France) and interleukin 2 (20 UI/mL) (Novartis, Switzerland) were added day 1 and day 3 respectively. At day 7 and 14, cells were stimulated with -irradiated autologous PBMC pulsed with 10 M of UCPs and 20 UI/mL IL-2 was added at day 8 and 15 as previously reported (Wang et al, Adotevi et al, 2006). At day 21, CD4 T cells were purified (Miltenyi, France) and the specificity of T cell lines was investigated by IFN- ELISPOT. Briefly, CD4 T cells (10.sup.5/well) were cultured in anti-human IFN- mAb precoated ELISPOT plate with each UCP (5 M) in AIM V medium (Invitrogen, United Kingdom) for 18 h at 37 C. Cells cultured with medium alone or PMA (100 ng/ml) (Sigma-Aldrich) and ionomycin (10 M) (Sigma-Aldrich) were used as negative and positive controls, respectively. The IFN- spots were revealed following the manufacturer's instructions (Gene Probe, France). The number of specific T cells expressed as spot-forming cells/10.sup.5 cells was calculated after subtracting negative control values (background). Spot-forming cells were counted using the C.T.L. Immunospot system (Cellular Technology Ltd., USA). For HLA-DR-restriction, the following blocking antibodies anti-HLA-class 1 (clone W6.32), HLA-DR (clone L243) and HLA-DP (clone B7/21) (10 g/ml) were added in cell culture during the ELISPOT. All the experiments were performed in triplicates.

(71) CD4 T Cell Clones Isolation and Amplification

(72) T Cells Clones were Isolated by Limiting Dilution and Amplified after Stimulation by PHA in presence of irradiated allogenic PBMC, B-EBV cell line and 150 UI of interleukin 2 according to previously described procedure (Godet et al). Functional analyses of UCP-specific CD4 T cell clones were performed by using intracytoplasmic TNF- staining and Human Ten-plex cytokines assay (Human Th1/Th2/Inflammation Diaplex, Diaclone, France).

(73) Assessment of Spontaneous UCP-Specific CD4 T Cell Responses

(74) Ficoll-isolated PBMC from cancer patients or healthy volunteers were cultured with 10 M of pool of UCPs in a 24-well plate (4.10.sup.6 cells per well) in RPMI 5% human serum and interleukin 7 (5 ng/mL) and interleukin 2 (20 UI/mL) were added day 1 and day 3 respectively. For the recall response against viruses, cells were similarly cultured with the mix of 32 peptides from cytomegalovirus, influenza virus and Epstein Barr virus (CTL, Germany). After one week cell culture, the presence of UCP-specific T cells was measured by IFN- ELISPOT as detailed above.

(75) Flow Cytometry

(76) For intracytoplasmic cytokine staining, after a 5-h stimulation period with or without 10 M peptide, T cells were labeled with anti-CD4 (BD Bioscience, USA) and anti-TNF- (ebioscience, USA) using Cytofix/Cytoperm KIT (BD Bioscience). For flow cytometry Treg analysis, PBMC were first stained with surface antibodies (anti-CD4, anti-CD25), fixed, permeabilized, and then stained with anti-hFoxp3 (PCH101; eBioscience). Samples were acquired on a FACS Canto II (BD Biosciences) and analyzed with the DIVA software. NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count (Suzuki et al).

(77) Statistics

(78) Statistical analyses were performed with NCSS 2007 software (Number Cruncher Statistical Systems, Kaysville, USA). The level of significance was set at p<0.05 for all tests. Variables were expressed as a meanSD or median, and tested with the Wilcoxon Rank-Sun test when suited. Survival curves were calculated with the Klapan-Meier method and compared with the Log-rank test.

(79) 1.2. Results

(80) Identification of Universal HLA-DR-Restricted CD4 T Cell Epitopes from TERT

(81) To predict the existence of CD4 epitopes within the amino acid sequence of TERT capable of binding to multiple HLA-DR molecules, the inventors have combined results from three algorithms Syfpeithi, NetMHCpan-2.1 and NetMHC2.2. They have selected four 15-mers peptide sequences referred as UCP1 to UCP4 that scored high in the probability scale for their binding capacity to the commonly found human HLA-DR alleles (Table 1). To confirm this result, the inventors have performed an in vitro binding assay based on competitive ELISA as previously reported. The data have been presented as relative affinities (RA) to easily compare their binding properties to high-binder peptides that the inventors have used as references and the strong binders have a relative affinity <100. Results confirm the ability of all the peptides to effectively bind to the most common alleles encoded by the HLA-DR (Table 5). Data are expressed as relative activity RA (ratio of the IC50 of UCPs to the IC50 of the reference peptide) and are the means of three experiments. Good binders have a RA<100 and weak binder are RA>50.

(82) TABLE-US-00006 TABLE 5 Relative affinities of the peptides towards the most common alleles encoded by the HLA-DR locus SEQ HLA-DR alleles Peptides ID NO: DR1 DR3 DR4 DR7 DR11 DR13 DR15 DRB3 DRB4 DRB5 UCP44 4 3 0.4 50 3 25 1 4 30 4 1 (UCP1) UCP578 1 0.2 144 112 1 4 231 8 154 229 1 (UCP2) UCP916 2 0.1 173 2 2 0.2 134 0.2 53 >500 0.2 (UCP3) UCP1041 3 0.3 >500 34 8 0.3 >500 3 154 >500 0.5 (UCP4)

(83) The four peptides exhibited a strong capacity to bind seven different HLA-DR molecules including DR1, DR4, DR7, DR11, DR15, DRB3 and DRB5. Particularly, UCP1 and UCP2 were able to bind every HLA-DR molecules tested with RA from intermediate (100-500) to low RA (<100). Thus, according to phenotypic frequencies of the 10 prevalent HLA-DR antigens, these peptides could cover more than 90% of population (Wang et al). Furthermore, CD4 T cell responses against UCPs have been induced in humanized HLA-DR1*0101/HLA-A2 transgenic mouse model following immunization with a DNA plasmid encoding the full length TERT protein (Adotevi et al, 2006, Pajot et al) and indicating that they are endogenously processed and presented to CD4 T cell in vivo.

(84) Then, the ability of UCP to stimulate human CD4 T cells has been tested. For this purpose, lymphocytes isolated from peripheral blood of healthy volunteers have been in vitro stimulated using a pool of UCP and the generation of UCP-specific CD4 T cell lines has been screened using ELISPOT assay. As shown in FIG. 1A, CD4 T cells were able to recognized at least one UCP. The HLA-DR restriction of the UCPs specific CD4 T cell response has been confirmed with the inhibition of IFN- secretion in presence of pan HLA-DR blocking antibody (FIG. 1B). The HLA-DR spectra-typing reveals that the HLA-DR alleles of normal individual were not shared supporting the promiscuous nature of the UCPs (FIG. 1C). Thus, these results imply that precursor CD4 T cells against UCPs are present in human peripheral T repertoire and they recognize these peptides in multiple HLA-DR contexts. To further characterize these responses the inventors have isolated CD4 T cell clones specific for UCP2 and UCP4 from cancer patient. All the UCP4-specific CD4 T cell clones were strongly reactive in presence of cognate peptide, and showed a half-maximal TNF secretion observed at very low peptide concentration (<0.1 M) (FIG. 2A, B). Similar results have been obtained for UCP2 specific clones with a half-maximal TNF secretion observed at 4 M (FIG. 2C, D). In addition, the inventors have showed after peptide stimulation that the clones mainly produced IFN- and TNF- but not IL-4 nor IL-17A in agreement with a Th1 polarization (FIG. 2E). The reactivity of these CD4 T cell clones are inhibited by HLA-DR blocking antibody indicating their HLA-DR restriction.

(85) Thus, these results showed that high avidity UCP-specific CD4 T cell clones can be generated from cancer patients and were Th1 polarized. They also demonstrate that these UCPs are naturally processed and presented to CD4 T cell in the context of malignancies.

(86) Presence of Naturally Occurring CD4 T Cells Against UCPs in NSCLC Patients

(87) Telomerase gene polymorphisms have been associated with lung cancer susceptibility and TERT expression is found in all types of NSCLC (Non small Lung Carcinoma) (Lantuejoul et al, Rafnar et al). Therefore the inventors have performed a comprehensive analysis of spontaneous UCP-specific CD4 T cell responses in a NSCLC. Ficoll-isolated blood lymphocytes from eighty-four advanced NSCLC patients have been collected prior first line chemotherapy and cultured shortly (one week) with the pool of UCPs and the specific T cells have been measured by IFN-. ELISPOT. Blood lymphocytes from 22 healthy volunteers have been used as control. Responses have been considered positive when the number of INF- secreting cells was at least two-fold above the negative control. This experimental design enables the inventors to measure specific CD4 T cell memory responses. As shown in FIG. 3A, UCP-specific memory immune responses has been found in 32 out of 84 patients (38%) whereas no specific IFN- responses against UCPs have been detected in healthy individuals. Analysis of the cytokine secretion profile of these responses reveals high production of TNF- and IFN- in absence of IL-4, IL-17 and IL-10 indicating a Th1 polarization (data not shown). Analyzed individually, each of the four UCPs is able to generate a CD4 T cell response in patients. However, the frequency of T cell responses to UCP-2 and UCP-4 suggest that these peptides are more immunogenic (FIG. 3B). The absence of UCPs specific immune responses in patients could not be related to a global T cell anergy as illustrated by the presence of effective antiviral recall responses in patients without UCPs specific response (FIG. 3C). To exclude the influence of a number of immune parameters that have been reported to decrease antitumor response in NSCLC (Suzuki et al), the inventors have measured circulating CD4+ Foxp3+ regulatory T cells (Tregs) and the plasmatic IL-10 in the patients with or without UCP-specific immune response. The inventors have showed similar level of circulating Tregs in the two groups (FIG. 3D) and absence of plasmatic IL-10 detection by ELISA has been observed regardless the UCP-immune status (data not shown). In addition the total lymphocyte counts and neutrophil-lymphocyte ratio (NLR) are quite similar in these two groups (FIG. 3D).

(88) The results indicate that patients with NSCLC are able to spontaneously mount TAA-specific CD4 T cell responses and that UCPs are prototypic peptides to monitor antitumor immune response in NSCLC.

(89) Spontaneous UCP-Specific T Cell Immune Response Increase Overall Survival of Patients Responding to Chemotherapy

(90) The impact of the UCP-specific CD4 immune response on clinical outcome was analyzed in patients that responded or progressed after first line chemotherapy (CT). For this purpose, the inventors have focused on 55 out of 84 advanced NSCLC patients with a median follow-up of ten months.

(91) All the patients included have been classified as metastatic stage IV. T-cell responses against TERT were not correlated with clinical or prognostic variables such as age, tobacco, ECOG PS status or histological subtype. Except six patients who received Erlotinib therapy, all patients were treated with platinum doublet. After first line, control disease (CD) based on RECIST criteria have been achieved in 36 out of 55 (65%), 25% of them achieved a partial response (PR) (14 out of 55) and 40% a stable disease (SD) (22 out of 55). Progressive disease (PD) has been observed in 19 out of 55 (35%). The frequency of spontaneous TERT-specific CD4 immune response was similar in patient with CD or PD after CT (FIG. 4A). In contrast patients displaying a TERT-specific immunity prior CT had an increased overall survival (OS) in the CD group compared to patients with no TERT-specific immunity (Median OS: 53 vs 40 weeks, p=0.034, HR=0.54, 95% CI [0.3-1]). The preexistence of UCP-specific immune response non-significantly increased the progression free survival (PFS) of CD patients (Median PFS: 33 vs 24 weeks, p=0.391, HR=0.76, 95% CI [0.4-0.7]) (FIG. 4B). Similar results have been observed when the inventors focused on patients that received platinum-based CT, after excluding the Erlotinib-treated patients (Median OS: 53 vs 40 weeks, p=0.049, HR=0.52 95% CI [0.3-0.9]) (FIG. 4C, D). By contrast, in patients with PD after first line CT, the inventors have found no survival difference regardless UCP-specific immune status (data not shown). Thus, the presence of natural TERT-specific CD4 Th1 responses in patients whose tumors are sensitive to chemotherapy is correlated to a higher OS.

Example 2

The Potent CD4 Helper Activity of Novel UCPs Derived from Telomerase on Self Antitumor CD8 T Cell Responses

(92) 2.1. Materials and Methods

(93) Synthetic Peptides.

(94) The four peptides derived from TERT called universal cancer peptides (UCPs): UCP1 (TERT44-58: PAAFRALVAQCLVCV, SEQ ID NO:4), UCP2 (TERT578-592: KSVWSKLQSIGIRQH, SEQ ID NO: 1), UCP3 (TERT916-930: GTAFVQMPAHGLFPW, SEQ ID NO:2) and UCP4 (TERT1041-1055: SLCYSILKAKNAGMS, SEQ ID NO:3) and the HLA-A2-restricted pY988 (YLQVNSLQTV, SEQ ID NO: 5) and pY572 (YLFFYRKSV, SEQ ID NO: 6) peptides derived from TERT have been used by the inventors. The native forms of the two cryptic HLA-A2 TERT peptides are fully conserved in human and mouse TERT (Hernandez et al, 2002). Synthetic peptides (>80% purity) were purchased from Activotec (UK).

(95) Mouse.

(96) The HLA-DRB1*0101/HLA-A*0201-transgenic mice (A2/DR1 mice) have been previously described (Pajot et al, 2004). These mice are H-2 class I and IA class II knockout, and their CD8 T and CD4 T cells are restricted by the sole HLA-A*0201 and HLA-DR1*0101 molecules respectively.

(97) Immunizations.

(98) To study the processing of UCP, A2/DR1 mice were immunized with a pTrip-TERT DNA (100 g) at days 0 and 14 as previously reported (Adotevi et al, 2010). In some experiment CD4 T cells were depleted with anti-CD4 mAb treatment (clone GK1.5) prior DNA immunization. For UCP immunization, mice were injected with 100 g of each UCP emulsified in incomplete Freund adjuvant (IFA, Sigma-Aldrich, France). In some experiments, 50 g of pY988 peptide was co-injected with 100 g of each UCP in IFA. All peptide vaccinations were done subcutaneously (s.c) at the right abdominal flank. Eight to ten week-old mice were bred and maintained in our animal facilities. All experiments were done according to the good laboratory practices defined by animal experimentation rules in France.

(99) Mouse Proliferation Assay.

(100) Proliferation assays were performed ten days after the last DNA immunization as previously described (Pajot et al, 2004). Results are given as stimulation index=(cpm with specific peptide)/(cpm with irrelevant peptide).

(101) Pentamer Staining and ELISPOT Assays.

(102) Ex vivo pentamer staining was performed as previously described (Adotevi et al, 2010; Adotevi et al, 2006). Cells were stained with PE-conjugated pY988 and pY572 HLA-A2.1 pentamer (Prolmmune, UK). After cell staining, samples were analyzed by flow cytometry on a FACS Canto II (BD Biosciences, France) and using Diva software. Ex vivo ELISPOT was performed as previously described and following manufacturers instructions (GenProbe, France) (Adotevi et al, 2010; Adotevi et al, 2006)

(103) Dendritic Cells Activation.

(104) Spleen or lymph nodes CD11c+ DCs from peptide-immunized mice were directly analyzed for co-stimulatory receptor expression. In some experiments, immature bone marrow-derived DCs (iDC) from A2/DR1 mice were cultured 15-h with CD4 T cells from mice immunized with UCP or IFA alone and then stained for cell surface expression of co-stimulatory receptors and cytokines production.

(105) Tumor Challenge.

(106) The HLA-A2.1-positive B16F10 murine tumor cell line (referred as B16-A2) was previously shown to express high amounts of TERT (Adotevi et al, 2010). A2/DR1 mice were s.c. injected with 2.105 B16-A2 cells in 100 l of saline buffer on the abdominal flank. At day 5, groups of mice were immunized with either the mix of pY988 and pY572 peptides (100 g) with or without UCP2 (100 g). A boost injection was done at day 17. Control mice were treated with adjuvant IFA in saline buffer. Tumor growth was monitored every 2-3 days using a calliper and mice were euthanized when the tumor mass reached a surface>200 mm2. The mice survival was assessed using the Kaplan-Meier model.

(107) Detection of UCP-Specific T Cell Responses in Cancer Patients.

(108) Blood was collected from cancer patients at the university hospital of Besancon (France) after informed consent. The study was conducted in accordance with French laws and after approval by the local ethics committee. Ficoll-isolated lymphocytes were analyzed by 3H thymidine incorporation as previously described (Pajot et al, 2004). After a short in vitro stimulation of lymphocytes with UCPs, UCP-specific immune response was analyzed by human ELISPOT assay (GenProbe). Concomitantly, cytokines production were measured after a 15H-culture with or without UCPs, using DIAplex Human Th1/Th2 kit (GenProbe) according to the manufacturers instructions,

(109) Statistics.

(110) Data are presented as meansSD. Statistical comparison between groups was based on Student t test using Prism 4 GraphPad Software. Mouse survival time was estimated using the Kaplan-Meier method, and the log-rank test was used. P values less than 0.05 (*) were considered significant.

(111) 2.2. Results

(112) Immunization with UCP Induces High Avidity Th1 Polarized CD4 T Cell Responses In Vivo

(113) The inventors and others have previously reported that the use of humanized HLA transgenic mice models to screen for human tumor antigens represents a potent alternative to optimize reverse immunology approaches for epitope identification (Adotevi et al, 2006; Osen et al, 2010). Here the inventors have used A2/DR1 mice to study the in vivo immunogenicity of UCPs, based on to their binding capacity to HLA-DRB1*0101 molecules. To assess whether UCPs can be endogenously processed from the TERT protein, the inventors performed immunizations with a plasmid DNA encoding the full length TERT sequence and the UCP-specific CD4 splenocytes were monitored by a five-day 3H-thymidine incorporation assay. As shown in FIG. 5A, all the UCPs differentially stimulate proliferation of spleen lymphocytes from DNA-immunized mice. Especially, high T cell proliferation was measured in response to UCP2 and 3 as compared to UCP1 or UCP4. The inventors have confirmed these results by using ex vivo IFN- ELISPOT assay and found strong UCP-specific CD4 T cell responses (FIG. 5B). Contrary to UCP1, specific CD4 T cell responses were detected against UCP2, 3 and 4 in all immunized mice (FIG. 5B). These data clearly indicate that UCPs are efficiently processed and presented to CD4 T cells in vivo in the context of DRB1*0101 restriction. Different populations of CD4 helper T cells control the antitumor immune responses (Pardoll et al, 1998), thus the inventors have studied the polarization of the UCP-specific CD4 T cell responses in vivo. To this end, freshly isolated CD4 T cells from UCP-vaccinated mice were cultured in the presence of syngenic iDC pulsed or not with UCP and cytokines production was measured. In all cases, the inventors have showed that UCP-specific CD4 T cells produce high level of IFN- and IL-2 but not IL-4, IL-5, IL-10 nor IL-17 indicating that UCP immunization preferentially induces a Th1 polarized immune response in vivo (FIG. 5C).

(114) Next, to assess the avidity of UCP-specific CD4 T cell, freshly purified CD4 T cells from UCP-immunized mice were cultured in the presence of increasing concentrations of peptide and the number of specific IFN- producing CD4 T cells was measured by ELISPOT. Results in FIG. 5D showed that mice immunized with UCP2, UCP3 and UCP4 induced high avidity specific CD4 T cells (<10-7 M). By comparison CD4 T cells from mice vaccinated with UCP1 or UCP4 responded to 10-1 and 10-3 M of peptide concentration respectively. Based on this, the inventors have concluded that low doses of UCP2 or UCP3 peptides (1 g) stimulated potent IFN-+ CD4 T cells in vivo (FIG. 5E). Collectively, UCPs are efficiently processed in vivo and stimulate high avidity Th1 polarized CD4 T cells in A2/DR1 mice.
UCP-Specific CD4 T Cells Provide Help for Optimal Anti-Self/TERT CD8 T Cell Responses In Vivo

(115) CD4 T cell helper functions are thought to be important for the generation of potent and sustained CTL responses (Shedlock et al, 2003). To address this question concerning UCP-specific CD4 T cells, the inventors co-immunized mice with self/TERT peptide pY988 in the presence of UCP. This peptide is fully conserved in human and mouse TERT sequences. The pY988-specific CTL response was measured ex vivo by pentamer staining and ELISPOT assays. As shown in FIG. 6A, a higher frequency of functional pY988-specific CD8 T cells was detected in mice immunized with pY988 plus UCP compared to mice vaccinated with pY988 alone. Although UCP1 vaccination had little impact on the frequency of pY988/A2 pentamer+ CD8 T cells-specific response, the four UCPs were able to significantly increase the number of IFN--secreting CD8 T cells against self/TERT peptide (FIG. 6B). The magnitude of the pY988-specific CD8 T cells response was strongly correlated with the intensity of UCP-specific immune responses concomitantly induced in mice (FIGS. 6C, D). Furthermore, these UCPs exerted similar helper effect on the self/TERT pY572-specific CTL responses in vivo. Thus, the addition of UCPs as helper peptides efficiently breaks immune tolerance against self/TERT CD8 epitopes in vivo.

(116) The inventors next have sought out to study the impact of UCPs helper peptides on CTL avidity and memory, two critical functions for tumor eradication. To this end the inventors focused on the UCP2 which induces potent Th1 immune responses in vivo. As shown in FIG. 7A, freshly isolated CD8 T cells from mice immunized with pY988+UCP2 were still reactive against very low concentrations of peptide pY988 (<10-3 M). These cells also recognized the cryptic native counterpart p988, underlining their high avidity (data not shown). Accordingly, mice vaccinated with pY988+UCP2 displayed stronger in vivo cytotoxicity against CFSE-labelled target cells than pY988 group (FIG. 7B). Thus, UCP2 helper immune responses enhance the quality of CTL response in vivo. This result supports a previous report showing that the stimulation of high avidity CD4+ T cells increases antitumor CTL avidity and cytolytic activity (Bandmaier et al, 2009). Furthermore, sustained anti-self/TERT CTL responses were detected in mice co-injected with UCP2 (FIG. 7C). This response was correlated to the long-lasting UCP2-specific CD4 T cell response in vivo (FIG. 7D). To confirm the role of UCP-specific CD4 T cell help, the inventors have showed that anti-self/TERT CD8 T cell response was strongly reduced in mice depleted of CD4 T cells prior to the TERT-DNA immunization as compared to non depleted mice (FIG. 7E). Similar results were obtained in other antigens model using peptides from HPV-16 E7 and NA-17 (data not shown). Thus, simultaneous stimulation of UCP-specific CD4 T cells is required for the optimal priming of tumor specific CTL in vivo.

(117) UCP-Specific CD4 T Cells Promote Dendritic Cell Activation In Vivo

(118) The induction of dendritic cell activation represents one major helper mechanism used by CD4+ Th1 cells to sustain antigen presentation and provide costimulatory signals to the CTLs. This is referred as the mnage trois model (Ridge et al, 1998). To test this mechanism, the inventors have analyzed the expression of co-stimulating receptors on DCs from mice immunized with the mix of pY988+/UCP2. As shown in FIG. 8A, lymph nodes CD11c+ DCs from UCP2/pY988 immunized mice expressed higher level of CD86, CD80, as well as HLA-class II molecules as compared to pY988-immunized mice. In a second set of experiments, isolated CD4 T cells from UCP2/IFA or IFA injected mice were co-cultured with syngenic iDCs as shown in FIG. 8B. As expected, UCP2-specific CD4 T cells produced significant amounts of Th1 cytokines such as IFN- and GM-CSF (FIG. 8C). The inventors have shown that the presence of UCP2-specific CD4 T cells induced potent DCs activation and enhanced their ability to produce high amounts of interleukin-12 (FIGS. 8D, E). Together, these results showed that the stimulation of UCP2-specific T cells shapes the phenotype and function of DCs in vivo.

(119) UCP2 Helper Peptide Enhances the Efficacy of Self/TERT CD8 Peptides Vaccination Against Established 816-A2 Melanoma

(120) To investigate the helper role of UCP in a therapeutic vaccination protocol, the inventors have focused on the UCP2 helper peptide which exhibits potent CTL helper function in vivo. The aggressive and poor immunogenic B16F10-HLA-A*0201 melanoma model was used in A2/DR1 mice. As previously reported this cell line highly expresses functional murine TERT (FIG. 9A) and is recognized by the self/TERT-specific CTLs (Adotevi et al, 2010). Tumor bearing mice were vaccinated twice either with two self/TERT CTL peptides (pY572+pY988/IFA) alone or in presence of the UCP2 helper peptide. As shown in FIG. 9B, the tumor growth reached an area >200 mm.sup.2 at day 25 in the control group injected with the adjuvant IFA alone. In this representative experiment, tumor regression was observed in 1/8 mice vaccinated with pY572+pY988/IFA while two mice achieved a delay in tumor growth. In the group vaccinated with pY988+pY572/IFA combined with UCP2, complete tumor regression was achieved in 5/8 mice. Accordingly, survival analysis out to day 50 after tumor cell injection showed that 63% of mice vaccinated in presence of UCP2 were still alive as compared to 13% in the group of mice injected with pY988+pY572/IFA (p<0.05) (FIG. 9C). Two months later, anti-pY988 effector CTL response was detected in tumor free mice and this was correlated to long term UCP2-specific CD4 T cell response in vivo (FIG. 9D). This sustained T cell immunity provides protection against a second lethal dose of B16/A2 tumors.

(121) The density of tumor-infiltrating CD8 T cells was shown to be critical for tumor control (Galon et al, 2006). Therefore, the inventors have analyzed immune cell infiltration within tumor in mice treated with the same vaccination protocols. Higher total CD3+ CD8+ T cells infiltration was observed in mice that received vaccine plus UCP2 helper peptide as compared to pY988+pY572/IFA group (67% vs 40%, p<0.05) (FIG. 9E). In contrast, UCP vaccination did not influence NK cells or regulatory T cells tumor infiltration (FIG. 9E), suggesting that UCP2-specific immune response mainly drive effector CTLs at the tumor microenvironment.

(122) Together, our results clearly showed that UCP2 specific CD4 T cells exert strong helper activity on tumor-specific CTL responses in vivo. Moreover the addition of UCP2 influences the homing of CD8 T cells to the tumor site. All these data support the use of UCP for antitumor therapeutic vaccination.

(123) Naturally Occurring UCP-Specific CD4 T Cell Responses in Human Cancers

(124) Based on the broad expression of TERT in cancers, the inventors have sought for UCP-specific CD4 T cell responses in patients with cancer of different histological origins. For this purpose, the inventors have measured 3H-thymidine incorporation of blood lymphocytes obtained from cancer patients directly stimulated with UCPs during 6 days. As shown in FIG. 10A, specific T cell proliferation was induced upon UCP stimulation. Next, UCP-specific T cells were measured by IFN- ELISPOT after short-term in vitro stimulation of PBMCs. The inventors have found high numbers of IFN--producing T cells directed against UCP in various cancers such as colon, renal, lung, stomach, and leukaemia supporting the T cell proliferation response (FIG. 10B). The UCP-specific T cells mainly produce Th1 cytokines but no IL-4, IL-10 or IL-17 in agreement with the in vivo studies of the inventors (FIG. 10C). As previously shown, T cell responses against individual UCP were also found in the PBMCs of patients presenting various cancers, supporting the idea that UCP epitopes are promiscuous (FIG. 10D). To study more precisely the polarization of UCP-specific CD4 T cells, the inventors have generated CD4 T cell clones specific for UCP4 derived from one responding colorectal cancer patient (FIG. 11A). Compared to CD4 T cells from healthy donors, these clones expressed a two-fold increased level of T-bet mRNA and lower GATA-3, RORc, and Foxp3 mRNA expressions (FIG. 11B). In addition these clones produced high amounts of IFN-, TNF- and a few IL-10, but no IL-13 nor IL-17, a cytokine pattern related to Th1 (FIG. 11C). Thus, these results indicate that the UCP-specific T cell repertoire is spontaneously stimulated in cancer patients and that these UCP-specific immune responses are Th1 polarized.

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