Use of a Vaccine Targeting a Cryptic Tert Epitope, for Treating Cancer in a HLA-A*0201-Positive Patient Having a Non-Immunogenic Tumor Expressing Tert

Abstract

The invention pertains to the use of a tumor vaccine composed of two peptides of nine amino acidsthe WT cryptic TERT572 (RLFFYRKSV, SEQ ID No: 1) expressed by tumor cells and its optimized variant TERT572Y (YLFFYRKSV, SEQ ID No: 2)for treating cancer in a HLA-A*0201-positive patient having a non-immunogenic tumor expressing TERT.

Claims

1. A method of treating cancer in an HLA-A*0201-positive patient having a tumor expressing TElomerase Reverse Transcriptase (TERT), wherein said tumor is non-immunogenic comprising administering a peptide of SEQ ID No: 2 to the patient, wherein said peptide of SEQ ID No: 2 induces a Cytotoxic T lymphocyte (CTL) response against the cryptic TERT572 peptide of SEQ ID No: 1.

2. A method of treating cancer in a HLA-A*0201-positive patient who has a tumor expressing TERT and who has already been vaccinated with the peptide of SEQ ID No: 2 comprising administering a peptide of SEQ ID No: 1 to the patient, wherein said tumor is non-immunogenic.

3. A method of treating cancer in an HLA-A*0201-positive patient having a tumor expressing TERT comprising administering peptides of SEQ ID No: 1 and SEQ ID No: 2 to the patient, wherein said tumor is non-immunogenic.

4. The method of claim 2, wherein the tumor is considered non-immunogenic if no CTL response specific for the peptide of SEQ ID No: 1 and/or no CTL response specific for the peptide of SEQ ID No: 3 and/or no CTL response specific for the peptide of SEQ ID No: 5 is detectable in a blood sample from the patient before vaccination.

5. The method of claim 4, wherein the blood sample from the patient has been collected less than 2 weeks after the end of a platinum-based first line chemotherapy.

6. The method of claims 1 to 5, wherein said patient has non-small cell lung cancer (NSCLC).

7. The method of claim 6, wherein said patient has non-squamous (NSQ) NSCLC.

8. The method of claim 1, wherein said patient is a never-smoker or a former smoker who had been smoking less than 30 years.

9. The method of claim 1, wherein said patient is a never-smoker or a light-smoker.

10. The method of claim 1, wherein said patient has received a platinum-based chemotherapy and responded thereto prior to vaccination with the peptide of SEQ ID No: 2.

11. The method of claim 1, wherein said patient is more than 65 years old.

12. The method of claim 1, wherein said patient is a male.

13. A method for in vitro determining whether an HLA-A*0201-positive patient having a tumor expressing TERT is likely to be a good responder to an immunotherapy treatment by vaccination with the peptides of SEQ ID Nos: 1 and 2, comprising assessing the immunogenicity of the tumor, wherein if the tumor is non-immunogenic, the patient is likely to be a good responder to said immunotherapy treatment.

14. The method of claim 13, wherein the immunogenicity of the tumor is assessed by measuring CTL response specific for the peptide of SEQ ID No: 1 and/or by measuring CTL response specific for the peptide of SEQ ID No: 3 and/or by measuring CTL response specific for the peptide of SEQ ID No: 5 in a blood sample from said individual, wherein if no such CTL response is detectable, the tumor is non-immunogenic.

15. The method of claim 14, wherein CTL response specific for the peptide of SEQ ID No: 1 is measured by enzyme-linked immunospot (ELISpot) IFNg assay.

16. A kit of parts for performing the method of claims 13 to 15, comprising: (i) reagents and a plate for performing an IFNg ELISPOTS assay, (ii) a peptide selected from the group consisting of SEQ ID No: 1, SEQ ID No: 3 and SEQ ID No: 5 and (iii) an irrelevant peptide as negative control.

Description

LEGENDS TO THE FIGURES

[0019] FIG. 1: Vaccination protocol

[0020] FIG. 2: Response to different tumor antigens of patients with (left) or without (right) natural immunity

[0021] FIG. 3: Clinical outcome (OS and TTF) of all evaluated patients

[0022] FIG. 4: Clinical outcome (OS and TTF) of Vx-001 treated patients who developed a vaccine specific immune response

[0023] FIG. 5: Clinical outcome of patients without natural immunity

[0024] FIG. 6: Clinical outcome of patients with natural immunity

[0025] FIG. 7: Clinical outcome of never-smokers and former smokers without natural immunity. A: Never smokers; 8.6 vs 16.2 mo; p=0.0008; HR=0.2 (0.05-0.79). B: Never smokers and former smokers who have been smoking during less than 25 years; 7.9 vs 20.7 mo; p=0.0007; HR=0.29 (0.13-0.67). C: Never smokers and former smokers who have been smoking during less than 20 years; 7.9 vs 20.2 mo; p=0.0001; HR=0.23 (0.08-0.64).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] According to a first embodiment, the present invention pertains to the use of the optimized peptide TERT572Y (peptide of SEQ ID No: 2), for inducing an anti-tumor CTL response in an HLA-A*0201-positive patient having a non-immunogenic tumor expressing TERT.

[0027] When performing this anticancer immunotherapy, vaccination with TERT572Y induces a CTL response against the cryptic peptide TERT572 (SEQ ID No: 1). The CTL response induced by the initial vaccination with the peptide of SEQ ID No: 2 is then preferably maintained (or amplified) by vaccination with the peptide of SEQ ID No:1.

[0028] In the present text, an immunogenic tumor designates a tumor that elicits a significant CTL response against tumor antigens. When performing the present invention, the immunogenicity of the tumor can be assessed by measuring, in a blood sample from the patient, the number of CTLs specific for TERT572 (SEQ ID No: 1) and/or the number of CTLs specific for TERT540 (SEQ ID No: 3), and/or the number of CTLs specific for another tumor antigen. For example, the immunogenicity of the tumor can be assessed by measuring the CTL response against Survivin, which is a universal tumor antigen expressed by a large majority of tumors (Andersen M H. and Thor S P., 2002). In HLA-A*0201-positive patients considered herein, this response can be detected by measuring, in a blood sample from the patient, the number of CTLs specific for the epitope Survivin96 (SEQ ID No: 5).

[0029] A contrario, the tumor will be considered as non-immunogenic if no CTL response specific for TERT572 (SEQ ID No: 1), TERT540 (SEQ ID No: 3) and/or Survivin96 (SEQ ID No: 5) is detected in a blood sample from the patient. The IFNg ELISpot assay described in the experimental part below can be used to detect T cells specific for the TERT572 peptide or for any other tumor epitope. Of course, as described in the experimental part below, in absence of a significant difference between the response to the tumor antigen tested peptide and the response to an irrelevant peptide, it will be considered that there is no response specific for the tested tumor peptide, i.e., that the tumor is non-immunogenic.

[0030] Alternatively, the immunogenicity of the tumor can be assessed by measuring the level of tumor-infiltrating lymphocytes (TILs) in a biopsy from the tumor such as with immunoscore (Pages F. et al. 2009) or determining the profile of genes expression of the tumor using gene profiling methods (Galon J. at al., 2013, Rizvi N A. et al., 2015, Wang E. et al. 2013).

[0031] As used herein, cancer means all types of cancers. In particular, the cancers can be solid or hematopoietic cancers. Non limitative examples of cancers are squamous carcinomas or adenocarcinomas such as breast, prostate, ovary, lung, pancreas or colon cancer, sarcomas, lymphomas, melanomas, leukemias, germ cell cancers and blastomas.

[0032] As used herein, the terms treat, treatment and treating refer to any delay of the progression, reduction of severity, and/or duration of cancer, particularly a solid tumor; for example, in a non-small cell lung cancer (NSCLC), amelioration of quality of life thereof and/or an increase in survival that results from the administration of one or more therapies.

[0033] The present invention also pertains to the use of the native peptide TERT572 (peptide of SEQ ID No: 1), for treating cancer in an HLA-A*0201-positive patient having a non-immunogenic tumor expressing TERT. In the frame of this immunotherapy treatment, the peptide of SEQ ID No: 1 maintains a CTL immune response initiated by vaccination of the patient with a peptide of SEQ ID No: 2.

[0034] The present invention also relates to the use of Vx-001, i.e., the combination of peptides of SEQ ID No: 1 and SEQ ID No: 2, in the treatment of cancer in an HLA-A*0201-positive patient having a non-immunogenic tumor expressing TERT. As already described, the two peptides of Vx-001 are administered separately. A CTL response against the tumor antigen TERT, more precisely against the cryptic TERT peptide of SEQ ID No: 1 is first induced by vaccinating the patient with the peptide of SEQ ID No: 2 and then maintained by vaccinating the patient with the peptide of SEQ ID No: 1.

[0035] According to a preferred embodiment illustrated in the experimental part below, the patient first receives 2 vaccinations with the peptide of SEQ ID No: 2 and then 4 vaccinations with the peptide of SEQ ID No:1, with a three weeks interval between said vaccinations (inducing phase). Of course, the skilled artisan (physician or clinical investigator) can chose a different vaccination protocol. Possible variations include the number of initial vaccinations with SEQ ID No: 2 (one, two or more) for inducing the CTL response, the interval between the vaccinations (for example, from 1 to 4 weeks or more), the number of vaccinations with SEQ ID No: 1 in this inducing phase (from 1 to 10 or more), and the formulation of the vaccine. In particular, adjuvants different from Montanide can be tested and could possibly necessitate an adaptation of the protocol.

[0036] According to another embodiment, after the inducing phase mentioned above, the patient receives additional vaccinations with the peptide of SEQ ID No:1, to maintain the CTL response against TERT (stabilization phase). These additional vaccinations can for example be administered every 3 months. They can be administered until relapse. Of course, the skilled artisan (physician or clinical investigator) can chose a different protocol for the stabilization phase. Possible variations include those concerning the interval between the vaccinations (for example, vaccinations can be performed every month, every 2 months, or less frequently, for example every 6 months, especially after a long period of remission), as well as the peptide used. Indeed, the clinician can choose to monitor the CTL response regularly and vaccine the patient with the peptide of SEQ ID No: 2 again if a decrease in the response is observed. In other words, the clinician can adapt the stabilization phase depending on the patient's response and status.

[0037] The inventors found that in some patients, a CTL response against tumor antigens is detected after chemotherapy and before vaccination, probably because tumor cell lysis following chemotherapy leads to the release of a large amount of tumor epitopes able to induce an antitumor immune response in these patients. These tumor specific CTLs are highly detectable just after the end of chemotherapy, and the amount of CTLs then decreases in the following weeks. The inventors' hypothesis is that this CTL response appears in patients having an immunogenic tumor, who are those who are fully responsive to other immunotherapies such as anti-PD(L)1 treatment. Therefore, for patients who received a chemotherapy treatment such as a platinum-based first line chemotherapy, the immunogenicity status of the tumor will preferably be assessed less than 2 weeks after the end of said chemotherapy, for example less than 7 days after the end of the chemotherapy.

[0038] According to a particular embodiment of the present invention, the tumor is considered as non-immunogenic if no CTL response specific for TERT572 (SEQ ID No: 1) is detectable in a blood sample from the patient collected before vaccination and preferably less than 2 weeks after the end of a platinum-based first line chemotherapy, more preferably less than 7 days after the end of a platinum-based first line chemotherapy.

[0039] According to another particular embodiment of the present invention, the tumor is considered as non-immunogenic if no CTL response specific for TERT540 (SEQ ID No: 3) is detectable in a blood sample from the patient collected before vaccination and preferably less than 2 weeks after the end of a platinum-based first line chemotherapy, more preferably less than 7 days after the end of a platinum-based first line chemotherapy.

[0040] According to another particular embodiment of the present invention, the tumor is considered as non-immunogenic if no CTL response specific for Survivin96 (SEQ ID No: 5) is detectable in a blood sample from the patient collected before vaccination and preferably less than 2 weeks after the end of a platinum-based first line chemotherapy, more preferably less than 7 days after the end of a platinum-based first line chemotherapy.

[0041] According to another particular embodiment of the present invention, illustrated in the experimental part below, the patient has a non small cell lung cancer (NSCLC).

[0042] As shown in the results of the phase Ilb study disclosed below, vaccination with Vx-001 significantly prolonged survival in patients with non-immunogenic non-squamous NSCLC. Hence, according to another particular embodiment of the present invention, the patient has a non-squamous (NSQ) NSCLC.

[0043] The results of the clinical trial also show that never-smokers and former smokers who have been smoking during less than 30 years respond better to the treatment.

[0044] Hence, according to another particular embodiment of the present invention, the patient is a never-smoker or is a former smoker who smoked during less than 30 years.

[0045] According to another preferred embodiment of the present invention, the patient is a never-smoker or is a former smoker who smoked during less than 25 years.

[0046] According to another preferred embodiment of the present invention, the patient is a never-smoker or is a former smoker who smoked during less than 20 years.

[0047] According to another preferred embodiment of the present invention, the patient is a never-smoker or is a former smoker who smoked during less than 10 years.

[0048] Surprisingly, the inventors observed even more statistically significant results when the light-smokers (i.e., individuals who smoke during at most 25 years), including patients who did not stop smoking but started less than 25 years or cumulated less than 25 years smoking in their life, were selected. Hence, according to yet another preferred embodiment of the present invention, the patient is a never-smoker or a light-smoker.

[0049] According to another particular embodiment of the present invention, illustrated in the experimental part below, the patient has received a platinum-based chemotherapy prior to vaccination with Vx-001, in particular prior to administration of the peptide of SEQ ID No: 2.

[0050] According to another particular embodiment of the present invention, the patient responded (objective response and stable disease) to a platinum-based first line chemotherapy prior to the first vaccination with the peptide of SEQ ID No: 2.

[0051] According to a more particular embodiment of the present invention, illustrated in the experimental part below, the patient has received a cisplatin-based chemotherapy (CDDP) prior to vaccination with Vx-001, in particular prior to administration of the peptide of SEQ ID No: 2.

[0052] According to another more particular embodiment of the present invention, the patient responded (objective response and stable disease) to a cisplatin-based first line chemotherapy prior to the first vaccination with the peptide of SEQ ID No: 2.

[0053] Other therapeutic schemes are envisioned in the frame of the present invention. For example, Vx-001 can be administered in an HLA-A*0201-positive patient having a non-immunogenic tumor expressing TERT either before or during chemotherapy. In this case, immunogenic status of the tumor will be determined before chemotherapy. First-line Vx-001 treatment can also be envisioned for HLA-A*0201-positive patients having a non-immunogenic tumor expressing TERT, especially if they are never-smokers of former smokers who smoked for less than 30 years or light-smokers (individuals who have smoked during less than 25 years and who stopped smoking or not). In this case, immunogenic status of the tumor will be determined at diagnosis. In any case, immunogenic status of the tumor will be determined either by detecting tumor antigen specific CTLs using enzyme-linked immunospot (ELISpot) IFNg assay or by any other method such as TILs detection or gene expression profiling.

[0054] According to another particular embodiment of the present invention, the patient is more than 65 years old. The results of the clinical trial indeed show that patients more than 65 years old respond better to the treatment.

[0055] According to another particular embodiment of the present invention, the patient is a male. The results of the clinical trial indeed show that male patients respond better to the treatment.

[0056] Another aspect of the present invention is a method for in vitro determining whether an HLA-A*0201-positive patient having a tumor expressing TERT is likely to be a good responder to an immunotherapy treatment by vaccination with Vx-001, comprising measuring the immunogenicity of the tumor; the patient is considered as a good candidate for Vx-001 vaccination if the tumor is non-immunogenic.

[0057] According to a particular embodiment of the above method, the tumor immunogenicity of the tumor is assessed by measuring CTL response specific for the peptide of SEQ ID No: 1 in a blood sample from said individual, wherein if no such CTL response is detectable before vaccination, the tumor is considered non-immunogenic.

[0058] According to another particular embodiment of the above method, the immunogenicity of the tumor is assessed by measuring CTL response specific for the peptide of SEQ ID No: 3 in a blood sample from said individual, wherein if no such CTL response is detectable before vaccination, the tumor is considered non-immunogenic.

[0059] According to yet another particular embodiment of the above method, the immunogenicity of the tumor is assessed by measuring CTL response specific for the peptide of SEQ ID No: 5 in a blood sample from said individual, wherein if no such CTL response is detectable before vaccination, the tumor is considered non-immunogenic.

[0060] According to a particular embodiment of the method according to the invention, CTL response specific for the peptide of SEQ ID No: 1 or for the peptide of SEQ ID No: 3 or for the peptide of SEQ ID No: 5 or for any other relevant tumor-specific peptide is detected by enzyme-linked immunospot (ELISpot) IFNg assay.

[0061] As mentioned above, the correlation between natural immunity and immunogenicity is higher when natural immunity is assessed close to the end of chemotherapy. According to another particular embodiment of the method according to the invention, the immunogenicity of the tumor is thus assessed less than two weeks, preferably less than 7 days after the end of chemotherapy.

[0062] Of course, when performing the theranostic method according to the invention, any other method can be used for assessing the immunogenicity of the tumor. In particular, this can be done by measuring the amount of TILs in a biopsy from the tumor or by gene profiling.

[0063] The present invention also pertains to a kit of parts for performing the theranostic method described above, comprising (i) reagents and a plate for performing an ELISpot assay, (ii) a peptide selected amongst SEQ ID No: 1, SEQ ID No: 3 and SEQ ID No: 5 and (iii) an irrelevant peptide as negative control.

[0064] Other characteristics of the invention will also become apparent in the course of the description which follows of the clinical study and biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.

Experimental Results

[0065] Material and Methods

[0066] Study Design and Participants

[0067] The Vx-001-201 study is a randomized, double blind multicenter study done in 70 centers in France, Germany, Spain, Italy, Greece, Poland, Romania and Czech Republic. Key eligibility criteria were a) non small cell lung cancer (NSCLC), b) stage IV or recurrent stage I-III, c) disease control after platinum based first line chemotherapy according to RECIST 1.1 criteria, d) HLA-A*0201 positivity, e) tumors expressing TElomerase Reverse Transcriptase (TERT), f) ECOG 0 or 1 g) absence of brain metastasis.

[0068] The study was conducted according to the Declaration of Helsinki and all applicable regulatory and ethical requirements. The study was approved by the Independent Ethics Committee responsible for each study site in accordance with the local legislation. All patients provided written informed consent.

[0069] Endpoints

[0070] a) Principal endpoint: overall survival (OS) measured from randomization

[0071] b) Secondary endpoints: [0072] Time to Treatment Failure (TTF) measured from randomization [0073] OS at 12 months

[0074] c) Main exploratory objectives [0075] Evaluation of vaccine induced specific immune response [0076] Correlation between vaccine specific immune response and clinical response

[0077] Procedures and Vaccination Protocol

[0078] Patients who fulfilled all inclusion criteria were randomized within 4 weeks after the end of first line chemotherapy.

[0079] The vaccination protocol consisted in six vaccinations at three week interval. The optimized Vx-001/TERT572Y was used in the first two vaccinations and the native Vx-001/TERT572 in the following four vaccinations. Patients who continued controlling their disease after the sixth vaccination received boost vaccination with the Vx-001/TERT572 every three months. Vaccination was stopped at disease progression (FIG. 1).

[0080] Immune response was evaluated before the first vaccination (baseline), before the third vaccination (W6) and three weeks after the sixth vaccination (W18). Patients who received boost vaccinations were monitored for immune response every six months.

[0081] Statistical Analysis

[0082] To calculate the sample size we estimated the median OS of the placebo arm to be 9.8 months and we expected the median OS in the Vx-001 arm to be 15.2 months. Thus, we needed to randomize 220 patients (including 10% of drop out at the final analysis) to achieve a power of 83% and a one-sided alpha of 0.05.

[0083] The placebo: Vx-001 ratio was 1:1.

[0084] The primary and secondary endpoints were analysed in the Full Analysis Set (FAS) composed of all patients who fulfilled the five main criteria a) NSCLC, b) stage IV or recurrent stage I-III, c) disease control after first line chemotherapy, d) HLA-A*0201 positivity and e) TERT expressing tumors.

[0085] We used the Kaplan-Meier method to estimate OS and TTF in each arm and a Cox proportional hazards regression model to estimate Hazard Ratio (HR) relating OS and TTF to the treatment effect.

[0086] Immunomonitoring

[0087] Immune response was measured using IFNg ELISpot assay to detect T cells specific for the TERT572 peptide. Peripheral Blood Mononuclear Cells (PBMC) were isolated from blood samples collected before vaccination, at W6 (before the third vaccination), W18 (three weeks after the 6th vaccination) and every six months thereafter for patients who received boost vaccinations. PBMC were stored at 160 C. and tested when study was unblended. 210.sup.5 PBMC/well were overnight stimulated with TERT572 or an irrelevant peptide as negative control or the CEF peptides pool as positive control or Phyto hemagglutinin (PHA) as a specific positive control in plates coated with anti IFNg antibody (Diaclone) in AIMV serum free medium (in 6-plicate). The presence of alive PBMC was verified using PHA. The quality of the PBMC was evaluated by measuring the response to CEF peptides pool (a mixture of polyallelic epitopic peptides from common viruses, flu, HPV and CMV). Samples were considered when they responded to CEF or when all samples from the same patient were unresponsive to CEF (indicating an absence of CEF reactivity due to patient medical history). Number of spots were quantified using a counter and for each condition, the average of the 6 values was calculated. A blood sample was considered responsive to TERT572 or CEF when a) there was a difference higher than 10 spots between the negative control average value and the TERT572 or CEF group average value and b) there was a statistically significant difference between negative control average value and TERT572 or CEF group average value (p<0.05). A patient without pre-vaccination TERT572 response was considered immune responder when a response to TERT572 was detected during the study protocol. Patients with pre-vaccination TERT572 reactivity were considered as immune responders only if (i) this TERT572 reactivity was amplified (at least two folds) after vaccination or if (ii) the patient first lost the pre-vaccination TERT572 reactivity and a new TERT572 reactivity was detected later in the vaccination protocol.

[0088] Immune responses to other TERT peptides and to other antigens were also measured using IFNg ELISpot assay. The peptides used to detect T cells are described in Table 1 below.

TABLE-US-00001 TABLE1 peptidesusedtodetectspecificCTLs Antigen Peptide Sequence SEQIDNo TERT TERT540 ILAKFLHWL 3 TERT TERT988 DLQVNSLQTV 4 Survivin Survivin-3A96 LTLGEFLKL 5 NY-ESO-1 NY-ESO-1157 SLLMWITQV 6 HER-2/neu HER-2/neu402 TLEEITGYL 7 MAGE-A MAGE-A248D9 YLEYRQVPD 8 MAGE-A MAGE-A24809 YLEYRQVPG 9

[0089] Results

[0090] Patients

[0091] 1407 patients were screened and 221 patients were randomized. The main reasons for screening failure were a) patients were HLA-A*0201 negative, b) there was no biopsy material suitable for TERT expression evaluation and c) disease progressed after first line chemotherapy.

[0092] Thirty-one patients were excluded from the Full Analysis Set (FAS) analysis because they did not fulfil the main inclusion criteria (25 patients entered the study with progressive disease, 2 patients with a tumor other than NSCLC and 4 patients with no metastatic or recurrent disease). Table 2 shows demographics of the 190 patients of the FAS.

TABLE-US-00002 TABLE 2 Demographics of evaluated patients. Placebo Vx-001 All patients 101 89 Sex Male 72 71% 60 67% Female 29 29% 29 33% Age >65 years 54 54% 45 50% <65 years 47 46% 44 50% Histology NSQ 59 59% 55 62% SQ 38 37% 34 38% Mixt 4 4% Response to 1st line chemotherapy OR 52 52% 36 40% SD 49 48% 53 60% ECOG 0 42 42% 33 37% 1 59 58% 56 63% Stage IV 89 89% 73 82% recurrent 12 11% 16 18% Smoking status Never smoker 15 15% 10 11% Former smoker (all) 52 51% 63 71% Former smoker (smoker during 20 years) 10 10% 12 13% Former smoker (smoker during 25 years) 14 14% 16 18% Former smoker (smoker during <30 years) 17 17% 19 21% Former smoker (smoked during >30 years) 35 35% 44 49% Current smoker 34 34% 16 18% NSQ: non-squamous. SQ: squamous. OR: objective response. SD: stable disease ECOG: Eastern Cooperative Oncology Group scale of performance status.

[0093] Patients were randomized within 4 weeks after the end of chemotherapy.

[0094] Immune Response in FAS Patients

[0095] TERT572 specific immune response was detected before vaccination (natural immunity) in 45 out of 166 evaluable patients (27.1%). Percentage of patients with natural immunity was 24.1% and 30.4% in placebo-treated and Vx-001-treated patients respectively.

[0096] Natural immunity was not limited to TERT572 but extended to other tumor antigens. Blood samples of baseline of six patients with natural immunity and three patients without natural immunity were tested against six additional antigens overexpressed in NSCLC. All patients with natural immunity to TERT572 had T cells reactive to other tumor antigens (such as TERT988, TERT540, MAGE248, HER402, Survivin96, NY-E5096) while patients without natural immunity to TERT572 did not respond to other tumor antigens and in the rare case a response was detected, it was very weak (FIG. 2).

[0097] These results strongly suggest that patient with natural immunity against TERT572 have immunogenic tumors while patients without natural immunity against TERT572 have non-immunogenic or poorly immunogenic tumors.

[0098] In Vx-001 treated patients, TERT572 specific immune response was evaluated in 79 patients at baseline, 73 patients at W6, 42 patients at W18 and 16 patients who received boost vaccinations. Overall, immune response was measured at least at W6, W18 or thereafter in 75 patients. Twenty-two patients mounted a TERT572 specific immune response (29.3%). Surprisingly this response was significantly more frequent in non-squamous (NSQ) than in squamous (SQ) NSCLC (36% vs 13.3%, p=0.037).

[0099] The frequency of patients with vaccine-induced immune response was not significantly different between patients with and patients without natural immunity against TERT572 (15% vs 36.2% p=0.14).

[0100] Clinical Response

[0101] Analysis of the FAS patients showed that there was no significant difference in OS between placebo treated and Vx-001 treated patients (11.3 vs 14.3 months, p=0.86, HR=0.97, 95% CI 0.70-1.34). There was significant difference neither in TTF (3.5 vs 3.6 months, p=0.36, HR=0.88, 95% CI 0.66-1.16) nor in 12 months survival (49.5% vs 58%, p=0.24) (FIG. 3).

[0102] Subgroups analysis showed no significant difference in OS and 12 months survival (table 3) in either subgroup tested excepted the 12 months survival that was significantly higher in males (43% vs 61%, p=0.05).

TABLE-US-00003 TABLE 3 Sub-group analysis for OS and 12 months survival Median survival 12 months (months) P OS (%) Sub-group Placebo Vx-001 value HR 95% CI Placebo Vx-001 P value Histology NSQ (n = 118) 11.3 13.4 0.56 0.88 0.58-1.34 50 55 0.58 SQ (n = 72) 11.2 14.3 0.64 1.13 0.66-1.93 46 64 0.16 Response to chemotherapy OR (n = 87) 11.1 15.3 0.64 0.89 0.54-1.45 48 69 0.07 SD (n = 103) 12.4 14.3 0.98 0.99 0.64-1.55 51 52 1 1st line chemotherapy CARBO (n = 119) 12.8 14.3 0.76 1.06 0.69-1.63 50 58.2 0.46 CDDP (n = 69) 9.9 15 0.44 0.81 0.48-1.32 46 59 0.33 Age >65 years (n = 98) 10 15 0.29 0.78 0.50-1.24 42.6 59 0.11 <65 years (n = 92) 15.4 13.4 0.36 1.2 0.77-2.00 55.3 57.7 0.83 Sex Male (n = 131) 9.9 15 0.29 0.81 0.55-1.20 43 61 0.05 Female (n = 59) 15.8 13.2 0.16 1.52 0.82-2.79 62 53.3 0.6

[0103] We then asked whether there is a correlation between the Vx-001 induced immune response and clinical outcome and we compared OS and TTF between Vx-001 responders and Vx-001 non-responders vaccinated patients. There was a very significant difference in OS (13.4 vs 21.3 months, p=0.0042, HR=0.39, 95% CI 0.23-0.68) and TTF (3.6 vs 9.1 months, p=0.0001, HR=0.41, 95% CI 0.26-0.45) (FIG. 4).

[0104] We then analyzed the impact of natural immunity on OS, TTF and 12 months survival in placebo and Vx-001 treated patients, considered separately. Tables 4 and 5 show that in the placebo-treated population, natural immunity was related to a quite significant prolongation of OS (8.6 vs 20.1 months, p=0.057, HR=0.59, 95% CI 0.36-0.98) and TTF (3.1 vs 5.3 months, p=0.056, HR=0.63, 95% CI 0.40-0.98). The difference in 12 months survival was statistically significant (71.4% vs 36.3%, p=0.006).

[0105] In contrast, in Vx-001 treated patients, there was no difference in OS (13.2 vs 15.5 months, 0.73, HR=0.91 95% CI 0.53-1.54) and TTF (4.1 vs 2.9 months, p=0.16, HR=1.4, 95% CI 0.83-2.33). There was no significant difference in 12 months survival either (55% vs. 65%, p=0.46).

TABLE-US-00004 TABLE 4 Clinical outcome of placebo and Vx-001 treated patients with and without natural immunity (OS) Median survival (months) No 12 months OS % natural Natural No immunity immunity P natural Natural P Sub-group (n = 122) (n = 45) value HR 95% CI immunity immunity value Placebo 8.6 21 0.057 0.59 0.36- 36.3 71.4 0.006 treated 0.98 (n = 87) Vx-001 13.2 15.5 0.73 0.91 0.53- 55 65 0.46 treated 1.54 (n = 80)

TABLE-US-00005 TABLE 5 Clinical outcome of placebo and Vx-001 treated patients with and without natural immunity (TTF) Median survival (months) No natural Natural immunity immunity Sub-group (n = 122) (n = 45) P value HR 95% CI Placebo treated 3.1 5.3 0.056 0.63 0.40-0.98 (n = 87) Vx-001 treated 4.1 2.9 1.4 0.83 0.53-2.33 (n = 80)

[0106] These results show that patients with immunogenic tumors have a better clinical outcome than patients with non-immunogenic tumors. Importantly, this difference in clinical outcome between immunogenic and non-immunogenic tumors disappeared in patients treated with Vx-001. In other words, Vx-001 treated patients with non-immunogenic tumors had the same clinical outcome as patients with immunogenic tumors.

[0107] These results led us to focus our attention on patients with non-immunogenic tumors. Table 6 shows demographic data of the population of patients without natural immunity against TERT572. There is no imbalance between placebo and Vx-001-treated patients in terms of histology, response to first-line chemotherapy, first-line chemotherapy, gender, age and smoking history.

TABLE-US-00006 TABLE 6 Demographics of patients without natural immunity Placebo Vx001 All patients 66 56 Sex Male 50 76% 39 70% Female 16 24% 17 30% Age >65 years 35 53% 28 50% <65 years 31 47% 28 50% Histology NSQ 37 56% 37 66% SQ 27 41% 19 34% Mixt 2 3% Response to 1st line chemotherapy OR 36 55% 19 34% SD 30 45% 37 66% 1st line chemotherapy CARBO 42 64% 32 57% CDDP 24 36% 22 39% CARBO/CDDP 2 Stage IV 60 91% 47 84% recurrent 6 9% 9 16% Smoking status Never smoker 7 11% 7 13% Former smoker (all) 33 50% 39 70% Former smoker (20 years) 5 8% 8 14% Former smoker (25 years) 9 14% 10 18% Former smoker (<30 years) 11 17% 11 20% Current smoker, including 5 light- 26 39% 10 18% smokers (25 years)

[0108] There is not a significant difference in OS between these two groups (8.6 vs 13.2 months, p=0.27, HR=0.80 95% CI 0.53-1.9) but a positive trend in TTF (3.1 vs 4.1 months, p=0.10, HR=0.75, 95% CI 0.52-1.07) and a significant difference in 12 months survival (55% vs 36%, p=0.045) (FIG. 5). These differences were increased when natural immunity was assessed less than 26 days after the end of chemotherapy in OS (6.5 vs 11.7 months, p=0.04, HR=0.58 95% CI 0.33-1.0 and TTF (3 vs 4.1 months, p=0.05, HR=0.62 95% CI 0.36-1.0) whereas no significant difference was observed in patients for whom natural immunity was assessed later than 26 days after chemotherapy (OS 10 vs 13.2 months, p=0.91, HR=0.96 95% CI 0.54-1.78) (n.b.: 26 days is the median of the period between the end of chemotherapy and randomization of a patient (inclusion in a group) in our cohort).

[0109] In contrast, there was no difference in OS and 12 months survival between placebo and Vx-001-treated patients with natural immunity (FIG. 6) even if the tumor immunogenicity was assessed before 26 days after the end of chemotherapy (OS (21 vs 15.3 months, p=0.31, HR=1.54 95% CI 0.61-3.88) and TTF (5.4 vs 2.2 months, p=0.08, HR=1.98 95% CI 0.82-4.79)).

[0110] Analysis of different sub-groups showed that Vx-001 significantly prolonged survival in patients with non-immunogenic NSQ tumors (7.7 vs 13.4 months, p=0.034, HR=0.58, 95% CI 0.35-0.97) and in patients with non-immunogenic tumors having been treated with cisplatin-based (CDDP) chemotherapy (7.1 vs 13.4 months, p=0.032, HR=0.51, 95% CI 0.26-0.98). Moreover, there was a significant difference in TTF in patients with non-immunogenic NSQ NSCLC (3.0 vs 4.6 months, p=0.025, HR=0.61, 95% CI 0.39-0.97) and in patients with non-immunogenic tumors and OR after first-line chemotherapy (3.1 vs 5.3 months, p=0.029, HR=0.56, 95% CI 0.33-0.95).

[0111] The smoking history of the patients with non-immunogenic tumors is obviously an important parameter, since Vx-001 significantly prolonged survival in never-smoker patients with non-immunogenic tumors (8.6 vs 16.2 months, p=0.0008, HR=0.2, 95% CI 0.02-0.79) (FIG. 7A), in patients with non-immunogenic tumors who either never smoked or have been smokers during less than 25 years (7.9 vs 20.7 months, p=0.0007, HR=0.29, 95% CI 0.13-0.67) (FIG. 7B), and in patients with non-immunogenic tumors who either never smoked or have been smokers during less than 20 years (7.9 vs 20.2 months, p=0.0001, HR=0.23, 95% CI 0.08-0.64) (FIG. 7C). A significant survival prolongation was also observed in patients with non-immunogenic tumors who either never smoked or have been smokers during less than 25 or 30 years. Moreover, there was a significant difference in TTF in patients with non-immunogenic tumors who either never smoked or have been smokers during less than 20 years (3.3 vs 5.6 months, p=0.005, HR=0.39, 95% CI 0.16-0.95), and in patients with non-immunogenic tumors who either never smoked or have been smokers during less than 25 years (3.1 vs 5.6 months, p=0.006, HR=0.43, 95% CI 0.20-0.92).

[0112] A trend of significativity was observed in male patients with non-immunogenic tumors (3.1 vs 3.7 months, p=0.09, HR=0.7, 95% CI 0.46-1.06) and in elderly patients with non-immunogenic tumors (3.1 vs 4.4 months, p=0.08, HR=0.65, 95% CI 0.40-1.07). Finally, there was a significant difference in 12 months survival in patients with non-immunogenic NSQ tumors (56.7% vs 30.8%, p=0.036), and in patients with non-immunogenic tumors and OR after 1st line chemotherapy (59% vs 25%, p=0.034), in male patients with non-immunogenic tumors (56.4% vs 32%, p=0.03) and in elderly patients with non-immunogenic tumors (57.1% vs 29.4%, p=0.039) (Tables 7 and 8).

TABLE-US-00007 TABLE 7 Clinical outcome of sub-groups of patients without natural immunity (OS) Median survival 12 months (months) OS % Vx- P Vx- P Sub-group Placebo 001 value HR 95% CI Placebo 001 value Histology NSQ (n = 76) 7.7 13.4 0.034 0.58 0.35-0.97 30.8 56.7 0.036 SQ (n = 46) 9.3 13.2 0.28 1.41 0.73-2.73 44.4 52.6 0.76 Response to chemotherapy OR (n = 55) 8.6 13.2 0.3 0.72 0.39-1.32 38.9 57.9 0.25 SD (n = 67) 7.1 13.6 0.48 0.82 0.47-1.42 33.3 54 0.32 1st line chemotherapy CARBO (n = 74) 8.9 13.1 0.89 1.03 0.61-1.74 42.8 53 0.48 CDDP (n = 46) 7.1 13.4 0.032 0.51 0.26-0.98 25 59 0.034 Age >65 years (n = 63) 8.1 14.1 0.11 0.63 0.36-1.11 29.4 57.1 0.039 <65 years (n = 59) 9.3 12.7 0.98 1.00 0.56-1.79 41.9 53.6 0.43 Sex Male (n = 89) 8.1 14.1 0.14 0.70 0.44-1.12 32 56.4 0.030 Female (n = 33) 11.2 12.7 0.6 1.24 0.55-2.77 50 52.9 1 Smoking status Never smoker 8.6 16.2 0.0008 0.2 0.05-0.79 0 85.7 0.004 (NS) NS + Former 7.9 20.2 0.0001 0.23 0.08-0.64 8 80 0.0003 smoker (20 years) NS + Former 7.9 20.7 0.0007 0.29 0.13-0.67 18.7 82.3 0.0004 smoker (25 years) NS + Former 7.9 20.2 0.005 0.37 0.17-0.80 22 78 0.002 smoker (<30 years) Current smoker 8.3 6.4 0.50 1.3 0.56-3.01 42.3 20 0.27 NS or light smoker 9.3 20.7 0.0001 0.27 0.12-0.58 21.0% 83.0% 0.0002 (25 years, former or current smoker) Heavy smoker (>25 8.9 9.9 0.55 1.15 0.71-1.85 42.5% 42.1% 0.99 years, former or current smoker)

TABLE-US-00008 TABLE 8 Clinical outcome of sub-groups of patients without natural immunity (TFF) Median TTF (months) P Sub-group Placebo Vx-001 value HR 95% CI Histology NSQ (n = 78) 3 4.6 0.025 0.61 0.39-0.97 SQ (n = 46) 3.3 3 0.56 1.18 0.65-2.14 Response to chemotherapy OR (n = 55) 3.1 5.3 0.029 0.56 0.33-0.95 SD (n = 67) 3 3.6 0.85 0.95 0.59-1.55 1st line chemotherapy CARBO (n = 74) 3.3 3.7 0.35 0.80 0.51-1.27 CDDP (n = 46) 2.8 4.1 0.15 0.67 0.37-1.20 Age >65 years (n = 63) 3.1 4.4 0.08 0.65 0.40-1.07 <65 years (n = 59) 3 3.5 0.50 0.84 0.5-1.41 Sex Male (n = 89) 3.1 3.7 0.09 0.70 0.46-1.06 Female (n = 33) 3 4.6 0.78 0.91 0.46-1.81 Smoking status Never smoker 3.3 5.6 0.16 0.51 0.17-1.55 Former smoker 3.3 5.6 0.005 0.39 0.16-0.95 (20 years) Former smoker 3.1 5.6 0.006 0.43 0.20-0.92 (25 years) Former smoker 3.1 4.2 0.05 0.55 0.28-1.09 (<30 years) Current 2.9 4.4 0.32 0.70 0.35-140

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