Use of vaccines targeting recessive TERT epitopes for treatment of cancer in HLA-A*0201-positive patients suffering from TERT-expressing non-immunogenic tumors

Abstract

The present invention relates to the use of a tumor vaccine consisting of two peptides of nine amino acids, which are WT recessive TERT572 (RLFFYRKSV, SEQ ID NO: 1) and optimized variants TERT572Y (YLFFYRKSV, SEQ ID NO: 2) expressed by tumor cells, for the treatment of cancer in HLA-A * 0201 positive patients suffering from non-immunogenic tumors expressing TERT.

Description

[0001] This application is a divisional application of the following application: application number 201880030508.2, invention name "Use of a vaccine targeting a recessive TERT epitope for treating cancer in HLA-A*0201-positive patients with non-immunogenic tumors expressing TERT". Field of the Invention

[0002] The present invention relates to the field of cancer immunotherapy, and more particularly to the field of anti-tumor vaccination. Background Art

[0003] Cancer immunotherapy aims to stimulate cytotoxic T lymphocytes (CTLs) that recognize peptides derived from tumor antigens and presented on the surface of tumor cells by HLA class I molecules. CTL-targeting peptides can be dominant or recessive, depending on their affinity for MHC molecules.

[0004] Tumor-associated antigens (TAAs) are often expressed by both tumor cells and normal tissues, in contrast to tumor-specific and most often patient-specific neoantigens. In order to overcome the problem of TAA tolerance while still targeting antigens widely expressed by tumors, the inventors proposed a vaccine (Vx-001) that targets a hidden peptide from telomerase reverse transcriptase (TERT), i.e., a peptide that exhibits low affinity to HLA-A*0201 molecules and forms an unstable peptide / HLA-I complex (Menez-Jamet J. et al., 2016). Given the strong correlation between HLA-I affinity and immunogenicity, the hidden peptide is naturally not immunogenic. Therefore, in order to use it as a cancer vaccine, the hidden peptide is optimized to enhance its immunogenicity.

[0005] Therefore, Vx-001 is composed of two nine-amino acid peptides: WT recessive TERT572 (RLFFYRKSV, SEQ ID NO: 1) expressed by tumor cells and its optimized variant TERT572Y (YLFFYRKSV, SEQ ID NO: 2). These two peptides are combined with the adjuvant Montanide (a mixture of highly purified mineral oil (Drakeol6VR) and surfactant (Mannide Monoleate)) were co-administered. Optimized immunogenic TERT572Y was administered in the first two vaccinations to elicit a large immune response. WT TERT572 was administered in subsequent vaccinations to select from all TERT572Y-stimulated T cells those with the highest specificity for WT TERT572 presented on the surface of tumor cells associated with HLA-A*0201.

[0006] In a randomized Phase IIb clinical trial, Vx-001 was tested in patients with metastatic or recurrent Stage I-III NSCLC who had experienced disease control after four cycles of platinum-based chemotherapy. Patients needed to be HLA-A*0201 positive and have tumors that express TERT. The primary objective of the study was overall survival. The results of the study were not statistically significant globally, but a detailed analysis of the results, along with an analysis of the patient's specific immune response to the vaccine (immune monitoring data), led to a stratification of patients that identified a class of patients who were shown to benefit from Vx-001 vaccination.

[0007] The identification of tumor antigens is the first step towards tumor vaccination. In the blood or tumors, many tumor antigens are identified as targets of naturally activated anti-tumor T lymphocytes (tumor infiltrating lymphocytes, TIL). The idea is to use these antigens to expand the naturally occurring anti-tumor immune response. Speiser et al. proposed adding the presence of activated antigen-specific CD8 T cells as a further criterion for selecting patients to be vaccinated (Speiser DE. et al., 2003).

[0008] Similarly, the results shown by Salazar et al. demonstrated that patients with detectable pre-existing antigen-specific immune responses at the time of initiation of vaccination generally acquired high levels of tumor-specific T cell immunity, most likely due to the enhancement of memory responses. These authors therefore proposed the development of vaccines targeting antigens, which have demonstrated that increased incidence of endogenous immune responses in cancer patients can improve vaccine efficacy and potential therapeutic efficacy (Salazar LG. et al., 2007).

[0009] In another publication, the authors indicated that the overall prevalence of these endogenous responses would provide information to further optimize peptide vaccination clinical trials (Cesson V. et al., 2011).

[0010] Therefore, a widely accepted idea is to use cancer vaccines to amplify pre-existing anti-tumor immune responses.

[0011] However, in many clinical trials, a very weak correlation between vaccine-induced immune responses and clinical responses was observed. For example, a strong immune response is not always accompanied by a strong clinical response. In addition, in most responding patients, the number of vaccine-induced T cells observed in the blood was very small, while in the regressed tumors, an important expansion of anti-tumor T cells against antigens rather than the vaccine itself was detected. These observations have led to new hypotheses about the mechanism of action of cancer vaccines (Ma W. et al., 2011, Coulie PG. et al., 2014). According to this hypothesis, a small number of active anti-vaccine T cells penetrate the tumor and attack tumor cells. As a result of this interaction, these CTLs are restimulated and produce cytokines that reverse the local microenvironment. This reawakens many inactive anti-tumor T cells already present in the tumor. The mobilization of these T cells (which target tumor antigens rather than vaccine antigens) provides the number required to reject the tumor.

[0012] According to this hypothesis, tumor vaccines may have antitumor clinical activity only in patients with immunogenic tumors capable of stimulating multispecific endogenous antitumor immune responses.

[0013] In patients with non-small cell lung cancer, this group with immunogenic tumors corresponds to those with better prognosis. In fact, it is shown that the high density of CD4+ and CD8+T infiltrating lymphocytes (TILs) associated with high mutation load in tumor stroma is an indicator of good prognosis in NSCLC patients (Al-Shibli KI. et al., 2008) and other tumors (Fridman WH. et al., 2011). This observation has led to the development of a method for scoring patient prognosis based on TIL analysis and immune core (Pagès F et al., 2009) or a method for identifying molecular markers related to tumor immune status and prognosis of immunotherapy response (Wang E. et al., 2013).

[0014] Furthermore, immune checkpoint inhibitors, which have enhanced the field of immunotherapy during the past few years and have received marketing approval for several cancer indications, are only effective in patients whose tumors have a high somatic non-synonymous mutation burden (SooRA. 2015) associated with strong infiltration of TILs (Fehrenbacher L. et al., 2016). Finally, good prognosis, immune checkpoint inhibitor efficacy, and high mutational burden associated with immunogenic tumors were all shown to be strongly associated with molecular signatures identified in smoker patients, while never smokers or former light smokers were shown to have poor benefit from immune checkpoint inhibitor treatment (Rizvi NA. et al., 2015). SUMMARY OF THE INVENTION

[0016] The present invention is directed to selecting patients who are more likely to respond to vaccination with Vx-001, which consists of two separately administered peptides (SEQ ID NO: 1 and SEQ ID NO: 2).

[0017] Contrary to what would be expected from the prior art, a detailed analysis of the Vx-001 Phase IIb clinical trial showed that the patients who benefited best from treatment were those with the worst prognosis, who had no detectable pre-existing tumor-specific immune response at the start of vaccination (i.e., patients with non-immunogenic or poorly immunogenic tumors).

[0018] Therefore, the present invention relates to the use of this vaccine and each of its components for treating cancer in HLA-A*0201 positive patients with non-immunogenic tumors expressing TERT.

[0019] The invention also relates to a theranostic method for determining whether an HLA-A*0201 positive patient with a tumor expressing TERT is likely to respond to Vx-001, wherein if the tumor is non-immunogenic, the patient is likely to be a good responder.

[0020] Another aspect of the invention is a kit for performing the theranostic method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Vaccination regimen

[0022] Figure 2 : Responses to different tumor antigens in patients with (left) or without (right) natural immunity

[0023] Figure 3 : Clinical outcomes (OS and TTF) for all evaluated patients

[0024] Figure 4 : Clinical outcomes (OS and TTF) of Vx-001-treated patients who developed a vaccine-specific immune response

[0025] Figure 5 : Clinical outcomes in patients without natural immunity

[0026] Figure 6 : Clinical outcomes in patients with natural immunity

[0027] Figure 7: Clinical outcomes in never smokers and former smokers without natural immunity. A: Never smokers; 8.6 vs 16.2mo; p = 0.0008; HR = 0.2 (0.05-0.79). B: Never smokers and former smokers who smoked for less than 25 years; 7.9 vs 20.7mo; p = 0.0007; HR = 0.29 (0.13-0.67). C: Never smokers and former smokers who smoked for less than 20 years; 7.9 vs 20.2mo; p = 0.0001; HR = 0.23 (0.08-0.64). DETAILED DESCRIPTION OF THE INVENTION

[0029] According to a first embodiment, the present invention relates to the use of the optimized peptide TERT572Y (peptide of SEQ ID NO: 2) for inducing anti-tumor CTL responses in HLA-A*0201 positive patients suffering from non-immunogenic tumors expressing TERT.

[0030] When this anticancer immunotherapy is performed, 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.

[0031] In this context, an "immunogenic tumor" refers to a tumor that elicits a significant CTL response against a tumor antigen. When performing the present invention, the immunogenicity of a tumor can be assessed by measuring 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 in a blood sample from a patient. For example, the immunogenicity of a tumor can be assessed by measuring the CTL response against survivin, which is a universal tumor antigen expressed by most tumors (Andersen MH. and Thor SP., 2002). In HLA-A*0201-positive patients contemplated herein, the response can be detected by measuring the number of CTLs specific for the epitope survivin 96 (SEQ ID NO: 5) in a blood sample from the patient.

[0032] Conversely, if no CTL response specific to TERT572 (SEQ ID NO: 1), TERT540 (SEQ ID NO: 3) and / or survivin 96 (SEQ ID NO: 5) is detected in a blood sample from a patient, the tumor is considered "non-immunogenic". The IFNg ELISpot assay described in the following experimental section can be used to detect T cells specific to the TERT572 peptide or any other tumor epitope. Of course, as described in the following experimental section, in the absence of a significant difference between the response to the tumor antigen test peptide and the response to an unrelated peptide, it can be considered that there is no specific response to the tested tumor peptide, i.e., the tumor is non-immunogenic.

[0033] Alternatively, the immunogenicity of a tumor can be assessed by measuring the levels of tumor infiltrating lymphocytes (TILs) in tissue from a tumor biopsy, for example using immune scoring (Pagès F et al., 2009) or determining the gene expression profile of the tumor using genotyping methods (Galon J. et al., 2013, Rizvi NA. et al., 2015, Wang E. et al., 2013).

[0034] As used herein, "cancer" refers to all types of cancer. In particular, the cancer may be a solid cancer or a hematopoietic cancer. Non-limiting examples of cancer are squamous carcinomas or adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, lung cancer, pancreatic cancer or colon cancer, sarcomas, lymphomas, melanomas, leukemias, germ cell cancers and blastomas.

[0035] As used herein, the terms "treat," "treatment" and "treating" refer to any delay in the progression, reduction in severity and / or duration of cancer, particularly a solid tumor; for example, in non-small cell lung cancer (NSCLC), an improvement in quality of life and / or an increase in survival resulting from the administration of one or more therapies.

[0036] The present invention also relates to the use of the natural peptide TERT572 (peptide of SEQ ID NO: 1) for the treatment of cancer in HLA-A*0201-positive patients with non-immunogenic tumors expressing TERT. In the framework of this immunotherapy treatment, the peptide of SEQ ID No: 1 maintains the CTL immune response elicited by vaccination of the patient with the peptide of SEQ ID No: 2.

[0037] The present invention also relates to the use of Vx-001, a combination of peptides of SEQ ID NO: 1 and SEQ ID NO: 2, in the treatment of cancer in HLA-A*0201-positive patients with non-immunogenic tumors expressing TERT. As already described, the two peptides of Vx-001 are administered separately. First, a CTL response against the tumor antigen TERT, more precisely against the cryptic TERT peptide of SEQ ID No: 1, is induced by vaccinating the patient with the peptide of SEQ ID No: 2, and then this CTL response is maintained by vaccinating the patient with the peptide of SEQ ID No: 1.

[0038] According to the 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 three weeks between vaccinations ("induction period"). Of course, the skilled person (physician or clinical researcher) can choose a different vaccination schedule. Possible variations include the number of initial vaccinations with SEQ ID No: 2 used to induce a CTL response (one, two or more times), the interval between vaccinations (e.g. 1 to 4 weeks or more), the number of vaccinations with SEQ ID No: 1 during the induction period (1 to 10 or more times), and the formulation of the vaccine. In particular, adjuvants other than Montanide can be tested and the schedule may need to be adjusted.

[0039] According to another embodiment, after the above-mentioned induction period, the patient receives additional vaccinations with the peptide of SEQ ID No: 1 to maintain the CTL response against TERT ("stabilization period"). These additional vaccinations can be performed, for example, every 3 months. It can be administered until relapse. Of course, the technician (physician or clinical researcher) can choose a different regimen for the stabilization period. Possible changes include changes related to the vaccination interval (for example, vaccination can be performed monthly, every 2 months, or less frequently (for example, once every 6 months, especially after long-term remission)), as well as the peptide used. In fact, the clinician can choose to monitor the CTL response regularly, and if a decrease in the response is observed, the patient can be vaccinated again with the peptide of SEQ ID NO: 2. In other words, the clinician can adjust the stabilization period according to the patient's response and status.

[0040] The inventors have found that in some patients, CTL responses against tumor antigens were detected after chemotherapy and before vaccination, which may be because tumor cell lysis after chemotherapy leads to the release of a large number of tumor epitopes that can induce anti-tumor immune responses in these patients. These tumor-specific CTLs can be highly detected immediately after the end of chemotherapy, and then the amount of CTLs decreases in the following weeks. The inventors' hypothesis is that this CTL response occurs in patients with immunogenic tumors who are patients who fully respond to other immunotherapies such as anti-PD(L)1 treatment. Therefore, for patients receiving chemotherapy treatment such as first-line platinum-based chemotherapy, it is preferred to assess the immunogenicity status of the tumor less than 2 weeks after the end of chemotherapy, for example, less than 7 days after the end of chemotherapy.

[0041] According to a specific embodiment of the present invention, if no CTL response specific to TERT572 (SEQ ID No: 1) is detected in patient blood samples collected before vaccination and preferably less than 2 weeks after the end of first-line platinum-based chemotherapy, and more preferably less than 7 days after the end of first-line platinum-based chemotherapy, the tumor is considered to be non-immunogenic.

[0042] According to another specific embodiment of the present invention, if no CTL response specific to TERT540 (SEQ ID No: 3) is detected in patient blood samples collected before vaccination and preferably less than 2 weeks after the end of first-line platinum-based chemotherapy, and more preferably less than 7 days after the end of first-line platinum-based chemotherapy, the tumor is considered to be non-immunogenic.

[0043] According to another specific embodiment of the present invention, if no CTL response specific to survivin 96 (SEQ ID No: 5) is detected in patient blood samples collected before vaccination and preferably less than 2 weeks after the end of first-line platinum-based chemotherapy, and more preferably less than 7 days after the end of first-line platinum-based chemotherapy, the tumor is considered to be non-immunogenic.

[0044] According to another specific embodiment of the present invention, as shown in the experimental part below, the patient suffers from non-small cell lung cancer (NSCLC).

[0045] As shown in the Phase IIb study results disclosed below, vaccination with Vx-001 significantly prolonged the survival of patients with non-immunogenic non-squamous NSCLC.Thus, according to another specific embodiment of the present invention, the patient suffers from non-squamous (NSQ) NSCLC.

[0046] Results from clinical trials also show that never smokers and former smokers who have smoked for less than 30 years respond better to treatment.

[0047] Therefore, according to another specific embodiment of the present invention, the patient is a never smoker or a former smoker who has smoked for less than 30 years.

[0048] According to another preferred embodiment of the present invention, the patient is a never smoker or a former smoker who has smoked for less than 25 years.

[0049] According to another preferred embodiment of the present invention, the patient is a never smoker or a former smoker who has smoked for less than 20 years.

[0050] According to another preferred embodiment of the present invention, the patient is a never smoker or a former smoker who has smoked for less than 10 years.

[0051] Surprisingly, when light smokers (i.e., individuals who smoked for a maximum of 25 years) were selected, including patients who did not stop smoking during their lifetime but started smoking less than 25 years ago or accumulated smoking less than 25 years, the inventors observed statistically even more significant results. Therefore, according to another preferred embodiment of the present invention, the patient is a never smoker or a light smoker.

[0052] According to another specific embodiment of the invention, as shown in the experimental part below, the patient has received platinum-based chemotherapy before vaccination with Vx-001, in particular before administration of the peptide of SEQ ID NO: 2.

[0053] According to another specific embodiment of the invention, the patient responded (objective response and stable disease) to first-line platinum-based chemotherapy before the first vaccination with the peptide of SEQ ID NO:2.

[0054] According to a more specific embodiment of the invention, as shown in the experimental part below, the patient had received cisplatin-based chemotherapy (CDDP) before vaccination with Vx-001, in particular before administration of the peptide of SEQ ID No. 2.

[0055] According to another more specific embodiment of the invention, the patient responded (objective response and stable disease) to first-line cisplatin-based chemotherapy before the first vaccination with the peptide of SEQ ID No:2.

[0056] Other treatment options are contemplated within the framework of the present invention. For example, Vx-001 can be administered to HLA-A*0201-positive patients with non-immunogenic tumors expressing TERT before or during chemotherapy. In this case, the immunogenicity status of the tumor will be determined before chemotherapy. First-line Vx-001 treatment may also be considered for HLA-A*0201-positive patients with non-immunogenic tumors expressing TERT, especially if they are never smokers or former smokers who have smoked for less than 30 years or light smokers (individuals who have smoked for less than 25 years and have stopped or not stopped smoking). In this case, the immunogenicity status of the tumor will be determined at diagnosis. In any case, the immunogenicity status of the tumor will be determined by detecting tumor antigen-specific CTLs using an enzyme-linked immunospot (ELISpot) IFNg assay or by any other method (e.g., TIL detection or gene expression typing).

[0057] According to another specific embodiment of the invention, the patient is over 65 years old. Clinical trial results indeed show that patients over 65 years old respond better to treatment.

[0058] According to another specific embodiment of the present invention, the patient is a male. Clinical trial results indeed show that male patients respond better to treatment.

[0059] Another aspect of the invention is an in vitro method for determining whether an HLA-A*0201-positive patient with a TERT-expressing tumor is likely to be a good responder to immunotherapy treatment with Vx-001 vaccination, comprising measuring the immunogenicity of the tumor; if the tumor is non-immunogenic, the patient is considered to be a good candidate for vaccination with Vx-001.

[0060] According to a specific embodiment of the above method, the tumor immunogenicity of the tumor is assessed by measuring CTL responses specific to the peptide of SEQ ID No: 1 in a blood sample from the individual, wherein if no such CTL response is detected prior to vaccination, the tumor is considered non-immunogenic.

[0061] According to another specific embodiment of the above method, the tumor immunogenicity of the tumor is assessed by measuring CTL responses specific for the peptide of SEQ ID No: 3 in a blood sample from the individual, wherein if no such CTL response is detected prior to vaccination, the tumor is considered non-immunogenic.

[0062] According to another specific embodiment of the above method, the tumor immunogenicity of the tumor is assessed by measuring CTL responses specific for the peptide of SEQ ID No: 5 in a blood sample from said individual, wherein if no such CTL response is detected prior to vaccination, the tumor is considered non-immunogenic.

[0063] According to a specific embodiment of the method of the present invention, CTL responses specific to the peptide of SEQ ID NO: 1 or the peptide of SEQ ID NO: 3 or the peptide of SEQ ID NO: 5 or any other relevant tumor-specific peptide are detected by ELISpot IFNg assay.

[0064] As mentioned above, the correlation between innate immunity and immunogenicity is higher when innate immunity is assessed near the end of chemotherapy. According to another specific embodiment of the method of the invention, the immunogenicity of the tumor is assessed less than two weeks, preferably less than 7 days after the end of chemotherapy.

[0065] Of course, when performing the therapeutic diagnostic method according to the invention, any other method can be used to assess the immunogenicity of a tumor. In particular, this can be accomplished by measuring the amount of TILs in a biopsy from the tumor or by genotyping.

[0066] The present invention also relates to a kit of elements for performing the above-mentioned therapeutic diagnostic method, which comprises (i) reagents and plates for performing ELISpot assays, (ii) a peptide selected from SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 and (iii) an irrelevant peptide as a negative control.

[0067] Other features of the invention will also become apparent in the subsequent description of clinical studies and biological assays performed within the framework of the invention and provide it with the necessary experimental support without limiting its scope.

[0068] The present invention relates to the following projects:

[0069] 1. A peptide of SEQ ID NO: 2 for use in treating cancer in HLA-A*0201-positive patients with tumors expressing telomerase reverse transcriptase (TERT), wherein the tumor is non-immunogenic, and wherein the peptide of SEQ ID NO: 2 induces a CTL response against the cryptic TERT572 peptide of SEQ ID No: 1.

[0070] 2. A peptide of SEQ ID NO: 1 for use in treating cancer in an HLA-A*0201 positive patient having a tumor expressing TERT and who has been vaccinated with a peptide of SEQ ID No: 2, wherein the tumor is non-immunogenic.

[0071] 3. A combination of peptides of SEQ ID No: 1 and SEQ ID No: 2 for use in treating cancer in HLA-A*0201 positive patients having tumors expressing TERT, wherein the tumors are non-immunogenic.

[0072] 4. The peptide or combination of peptides according to any one of items 1 to 3, for use according to any one of items 1 to 3, wherein if no CTL response specific to the peptide of SEQ ID NO: 1 and / or no CTL response specific to the peptide of SEQ ID NO: 3 and / or no CTL response specific to the peptide of SEQ ID NO: 5 is detected in a blood sample from the patient before vaccination, the tumor is considered to be non-immunogenic.

[0073] 5. The peptide or combination of peptides according to any one of 1 to 4 above, for use according to item 4, wherein the blood sample from the patient is collected less than 2 weeks after the end of first-line platinum-based chemotherapy.

[0074] 6. The peptide or combination of peptides according to any one of 1 to 5 above, for use in any one of 1 to 5, wherein the patient suffers from non-small cell lung cancer (NSCLC).

[0075] 7. The peptide or combination of peptides according to any one of 1 to 6 above, for use in any one of 1 to 6, wherein the patient suffers from non-squamous (NSQ) NSCLC.

[0076] 8. A peptide or combination of peptides according to any one of 1 to 7 above, for use according to any one of 6 and 7, wherein the patient is a never-smoker or a former smoker who has smoked for less than 30 years, preferably less than 25 years, more preferably less than 20 years, and even more preferably less than 10 years.

[0077] 9. The peptide or combination of peptides according to any one of 1 to 8 above, for use according to any one of 6 to 8, wherein the patient is a never-smoker or a light smoker.

[0078] 10. The peptide or combination of peptides according to any one of 1 to 9 above, for use in any one of 1 to 9, wherein the patient has received and responded to platinum-based chemotherapy, preferably first-line cisplatin-based chemotherapy, before vaccination with the peptide vaccine of SEQ ID No: 2.

[0079] 11. The peptide or combination of peptides according to any one of 1 to 10 above, for use according to any one of 1 to 10, wherein the patient is over 65 years old.

[0080] 12. The peptide or combination of peptides according to any one of 1 to 11 above, for use according to any one of 1 to 11, wherein the patient is a male.

[0081] 13. An in vitro method for determining whether an HLA-A*0201-positive patient with a tumor expressing TERT is likely to be a good responder to immunotherapy treatment with a peptide vaccination of SEQ ID NOs: 1 and 2, the method comprising assessing the immunogenicity of the tumor, wherein if the tumor is non-immunogenic, the patient is likely to be a good responder to the immunotherapy treatment.

[0082] 14. The method according to item 13 above, wherein the immunogenicity of the tumor is assessed by measuring a CTL response specific to a peptide of SEQ ID NO: 1 and / or by measuring a CTL response specific to a peptide of SEQ ID NO: 3 and / or by measuring a CTL response specific to a peptide of SEQ ID NO: 5 in a blood sample from the individual, wherein if no such CTL response is detected, the tumor is non-immunogenic.

[0083] 15. The method according to the above 14, wherein the CTL response specific to the peptide of SEQ ID NO: 1 is measured by ELISpot IFNg assay.

[0084] 16. A kit for performing the elements of the methods of 13 to 15 above, comprising: (i) reagents and plates for performing 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 a negative control.

[0085] Experimental Results

[0086] Materials and methods

[0087] Study Design and Participants

[0088] The Vx-001-201 study is a randomized, double-blind, multicenter study conducted in 70 centers in France, Germany, Spain, Italy, Greece, Poland, Romania, and the Czech Republic. Key inclusion criteria are: a) non-small cell lung cancer (NSCLC), b) stage IV or recurrent stage I-III, c) disease control after first-line platinum-based chemotherapy according to RECIST1.1 criteria, d) HLA-A*0201 positivity, e) tumors expressing telomerase reverse transcriptase (TERT), f) ECOG 0 or 1; g) no brain metastases.

[0089] The study was conducted in accordance with 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 local law. All patients provided written informed consent.

[0090] end

[0091] a) Primary endpoint: Overall survival (OS) measured by randomization

[0092] b) Secondary endpoints:

[0093] - Time to treatment failure (TTF) measured from randomization

[0094] -OS at 12 months

[0095] c) Main exploration objectives

[0096] - Evaluation of vaccine-induced specific immune responses

[0097] - Correlation between vaccine-specific immune responses and clinical responses

[0098] Procedures and vaccination schedules

[0099] Patients who met all inclusion criteria were randomized within four weeks after completion of first-line chemotherapy.

[0100] The vaccination regimen consisted of six vaccinations at three-week intervals. Optimized Vx-001 / TERT572Y was used for the first two vaccinations, while native Vx-001 / TERT572 was used for the subsequent four vaccinations. Patients who continued to control their disease after the sixth vaccination received booster vaccinations with Vx-001 / TERT572 every three months. Vaccinations were discontinued at the time of disease progression ( Figure 1 ).

[0101] Immune responses were assessed before the first vaccination (baseline), before the third vaccination (W6), and three weeks after the sixth vaccination (W18). Immune responses in patients who received booster vaccinations were monitored every six months.

[0102] Statistical analysis

[0103] To calculate the sample size, we estimated that the median OS in the placebo group would be 9.8 months, whereas the median OS in the Vx-001 group was expected to be 15.2 months. Therefore, we needed to randomly assign 220 patients (including 10% who withdrew from the final analysis) to achieve a power of 83% and a one-sided alpha value of 0.05.

[0104] The placebo:Vx-001 ratio was 1:1.

[0105] The primary and secondary endpoints were analyzed in the comprehensive analysis set (FAS), which consisted of all patients who met the following five major 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) tumors expressing TERT.

[0106] We estimated OS and TTF for each group using the Kaplan–Meier method, and we used Cox proportional-hazards regression models to estimate hazard ratios (HRs) associating OS and TTF with treatment effects.

[0107] Immune monitoring

[0108] Immune responses were measured using an IFNg ELISpot assay to detect T cells specific for the TERT572 peptide. For patients who received booster vaccinations, peripheral blood mononuclear cells (PBMCs) were isolated from blood samples collected before vaccination, at week 6 (before the third vaccination), week 18 (three weeks after the sixth vaccination), and every six months thereafter. PBMCs were stored at -160°C and tested in an open-label study. 2 × 10 cells / mL were stimulated overnight with TERT572 or an irrelevant peptide as a negative control or a CEF peptide pool as a positive control or phytohemagglutinin (PHA) as a specific positive control in plates coated with anti-IFNg antibodies (Diaclone) in medium without AIMV serum. 5 PBMC / well (six replicates). The presence of viable PBMCs was verified using PHA. The quality of PBMCs was assessed by measuring responses to the CEF peptide library (a mixture of multi-allelic epitope peptides from common viruses influenza, HPV and CMV). When a sample responds to CEF or all samples from the same patient do not respond to CEF (i.e., CEF reactivity is not present due to the patient's medical history), it is considered a sample. The number of spots was quantified using a counter, and the average of 6 values ​​was calculated for each condition. When a) the difference between the negative control mean and the TERT572 or CEF group mean was greater than 10 spots, and b) there was a statistically significant difference (p < 0.05) between the negative control mean and the TERT572 or CEF group mean, the blood sample was considered to respond to TERT572 or CEF. When a response to TERT572 was detected during the study protocol, patients with no TERT572 response before vaccination were considered immune responders. Patients with pre-vaccination TERT572 reactivity were considered immune responders only if (i) this TERT572 reactivity was expanded (at least twofold) after vaccination or (ii) the patient first lost pre-vaccination TERT572 reactivity and new TERT572 reactivity was subsequently detected during the vaccination regimen.

[0109] Immune responses to other TERT peptides and other antigens were also measured using the IFNg ELISpot assay. The peptides used to detect T cells are described in Table 1 below.

[0110] Table 1: Peptides used to detect specific CTL

[0111]

[0112]

[0113] result

[0114] patient

[0115] A total of 1407 patients were screened and 221 underwent randomization. The main reasons for screening failure were: a) patients were HLA-A*0201 negative, b) no biopsy material suitable for TERT expression assessment, and c) disease progression after first-line chemotherapy.

[0116] Thirty-one patients were excluded from the full analysis set (FAS) analysis because they did not meet the main inclusion criteria (25 patients entered the study with progressive disease, 2 patients with non-NSCLC tumors, and 4 patients with non-metastatic or recurrent disease). Table 2 shows the demographic information of the 190 patients in the FAS.

[0117] Table 2: Demographic information of the evaluated patients. NSQ: non-squamous. SQ: squamous. OR: objective response. SD: stable disease. ECOG: Eastern Cooperative Oncology Group performance status scale.

[0118]

[0119]

[0120] Patients were randomized within 4 weeks after completion of chemotherapy.

[0121] Immune responses in patients with FAS

[0122] Of the 166 evaluable patients, 45 (27.1%) had a detectable TERT572-specific immune response (natural immunity) before vaccination. The percentage of patients with natural immunity was 24.1% in patients who received placebo and 30.4% in patients who received Vx-001.

[0123] Innate immunity is not limited to TERT572 and may also extend to other tumor antigens. Baseline blood samples from 6 patients with innate immunity and 3 patients without innate immunity were tested for 6 additional antigens overexpressed in NSCLC. All patients with innate immunity against TERT572 had T cells reactive against other tumor antigens (e.g., TERT988, TERT540, MAGE248, HER402, survivin 96, NY-ESO96), whereas patients without innate immunity against TERT572 had no response to other tumor antigens, and in the rare cases where a response was detected, the response was very weak ( Figure 2 ).

[0124] These results strongly suggest that patients with natural immunity against TERT572 have immunogenic tumors, whereas patients without natural immunity against TERT572 have non-immunogenic or poorly immunogenic tumors.

[0125] In patients treated with Vx-001, TERT572-specific immune responses were assessed in 79 patients at baseline, 73 patients at W6, 42 patients at W18, and 16 patients who received booster vaccinations. Overall, immune responses were measured in 75 patients at least at W6, W18, or later. TERT572-specific immune responses occurred in 22 patients (29.3%). Surprisingly, this response was significantly more common in non-squamous (NSQ) NSCLC than in squamous (SQ) NSCLC (36% vs 13.3%, p=0.037).

[0126] The frequency of patients with vaccine-induced immune responses did not differ significantly between patients with and without natural immunity to TERT572 (15% vs 36.2%, p=0.14).

[0127] Clinical response

[0128] Analysis of FAS patients showed that there was no significant difference in OS between patients receiving placebo and those receiving Vx-001 (11.3 vs 14.3 months, p = 0.86, HR = 0.97, 95% CI 0.70-1.34). There was no significant difference in TTF (3.5 vs 3.6 months, p = 0.36, HR = 0.88, 95% CI 0.66-1.16) and 12-month survival (49.5% vs 58%, p = 0.24) ( Figure 3 ).

[0129] Subgroup analysis showed no significant differences in OS and 12-month survival in any of the subgroups tested (Table 3), but 12-month survival was significantly higher in males (43% vs 61%, p=0.05).

[0130] Table 3: Subgroup analysis of OS and 12-month survival

[0131]

[0132] We then asked whether there was a correlation between Vx-001-induced immune responses and clinical outcomes, and compared OS and TTF between patients vaccinated with Vx-001 responders and Vx-001 non-responders. There were highly significant differences 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). Figure 4 ).

[0133] Then, we analyzed the effect of natural immunity on OS, TTF and 12-month survival in placebo and Vx-001 treated patients (considered separately). Tables 4 and 5 show that in the placebo treated population, natural immunity is associated with a fairly significant extension 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-month survival is statistically significant (71.4% vs 36.3%, p = 0.006).

[0134] In contrast, 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) in patients treated with Vx-001. There was no significant difference in 12-month survival (55% vs 65%, p=0.46).

[0135] Table 4: Clinical outcomes (OS) of placebo- and Vx-001-treated patients with and without innate immunity

[0136]

[0137] Table 5: Clinical Outcomes for Placebo and Vx-001 Treated Patients with and without Innate Immunity (TTF)

[0138]

[0139] These results suggest that patients with immunogenic tumors have better clinical outcomes than patients with non-immunogenic tumors. Importantly, this difference in clinical outcomes between immunogenic and non-immunogenic tumors disappeared in patients treated with Vx-001. In other words, patients with non-immunogenic tumors treated with Vx-001 had the same clinical outcomes as patients with immunogenic tumors.

[0140] These results led us to focus our attention on patients with non-immunogenic tumors. Table 6 shows the demographic data for the patient population without innate immunity to TERT572. There was no imbalance between placebo and Vx-001 treated patients in terms of histology, response to first-line chemotherapy, first-line chemotherapy, sex, age, and smoking history.

[0141] Table 6: Demographic information of patients without natural immunity

[0142]

[0143] There was no significant difference in OS between the two groups (8.6 vs 13.2 months, p = 0.27, HR = 0.80 95% CI 0.53-1.9), but there was 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-month survival (55% vs 36%, p = 0.045) ( Figure 5 ). These 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% CI0.36-1.0) differences increased when natural immunity was assessed less than 26 days after chemotherapy, while no significant differences were observed in patients whose natural immunity was assessed more than 26 days after chemotherapy (OS 10 vs 13.2 months, p=0.91, HR=0.96 95% CI 0.54-1.78) (nb: 26 days was the median period from the end of chemotherapy to patient randomization (inclusion in group) in our cohort).

[0144] In contrast, there was no difference in OS and 12-month survival between placebo- and Vx-001-treated patients with innate immunity, even when 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% CI 0.82-4.79)) ( Figure 6 ).

[0145] Analysis of different subgroups showed that Vx-001 significantly prolonged the survival of 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 patients with non-immunogenic tumors treated with cisplatin-based (CDDP) chemotherapy (7.1 vs 13.4 months, p = 0.032, HR = 0.51, 95% CI 0.26-0.98). Furthermore, there were significant differences 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).

[0146] The smoking history of patients with non-immunogenic tumors is clearly an important parameter, as Vx-001 significantly prolonged the survival of never-smokers with non-immunogenic tumors (8.6 vs 16.2 months, p=0.0008, HR=0.2, 95% CI 0.02-0.79) ( Figure 7 A) Patients with non-immunogenic tumors who had never smoked or smoked for less than 25 years (7.9 vs 20.7 months, p = 0.0007, HR = 0.29, 95% CI 0.13-0.67) ( Figure 7 B), and patients with non-immunogenic tumors who had never smoked or smoked for less than 20 years (7.9 vs 20.2 months, p = 0.0001, HR = 0.23, 95% CI vs 0.08-0.64) ( Figure 7C) survival. In patients with non-immunogenic tumors who have never smoked or have smoked for less than 25 or 30 years, a significant extension of survival was also observed. In addition, TTF was significantly different in patients with non-immunogenic tumors who have never smoked or smoked for 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 have never smoked or smoked for less than 25 years (3.1 vs 5.6 months, p = 0.006, HR = 0.43, 95% CI 0.20-0.92).

[0147] A significant trend 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 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 were significant differences in 12-month survival in patients with non-immunogenic NSQ tumors (56.7% vs 30.8%, p = 0.036), in patients with non-immunogenic tumors and OR after first-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).

[0148] Table 7: Clinical outcomes (OS) in the subgroup of patients without natural immunity

[0149]

[0150]

[0151] Table 8: Clinical outcomes in the subgroup of patients without natural immunity (TFF)

[0152]

[0153] References

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Claims

1. Use of the peptide of SEQ ID NO: 2 in the preparation of a medicament for treating cancer in HLA-A*0201-positive patients with tumors expressing telomerase reverse transcriptase (TERT), wherein the tumor is non-immunogenic and wherein the peptide of SEQ ID NO: 2 induces a CTL response against the cryptic TERT572 peptide of SEQ ID No:

1.

2. Use of the peptide of SEQ ID NO: 1 in the preparation of a medicament for treating cancer in HLA-A*0201 positive patients having tumors expressing TERT and who have been vaccinated with the peptide of SEQ ID No: 2, wherein the tumor is non-immunogenic.

3. Use of a combination of peptides of SEQ ID No: 1 and SEQ ID No: 2 in the preparation of a medicament for treating cancer in HLA-A*0201 positive patients having tumors expressing TERT, wherein the tumors are non-immunogenic.

4. The use according to any one of claims 1 to 3, wherein the tumor is considered to be non-immunogenic if no CTL response specific to the peptide of SEQ ID NO: 1 and / or no CTL response specific to the peptide of SEQ ID NO: 3 and / or no CTL response specific to the peptide of SEQ ID NO: 5 is detected in a blood sample from the patient before vaccination.

5. The use according to claim 4, wherein the blood sample from the patient is collected less than 2 weeks after the end of first-line platinum-based chemotherapy.

6. The use according to any one of claims 1 to 5, wherein the patient suffers from non-small cell lung cancer (NSCLC).

7. The use according to any one of claims 1 to 6, wherein the patient suffers from non-squamous (NSQ) NSCLC.

8. The use according to any one of claims 6 and 7, wherein the patient is a never smoker or a former smoker who has smoked for less than 30 years, preferably less than 25 years, more preferably less than 20 years, even more preferably less than 10 years.

9. The use according to any one of claims 6 to 8, wherein the patient is a never smoker or a light smoker.

10. Use according to any one of claims 1 to 9, wherein the patient has received and responded to platinum-based chemotherapy, preferably first-line cisplatin-based chemotherapy, prior to vaccination with the peptide of SEQ ID No:

2.

11. The use according to any one of claims 1 to 10, wherein the patient is over 65 years old.

12. The use according to any one of claims 1 to 11, wherein the patient is male.

13. Use of a reagent for measuring CTL responses specific to peptides of SEQ ID NO: 1 and / or SEQ ID NO: 3 and / or SEQ ID NO: 5 in a blood sample in the preparation of a kit for a method for in vitro determining whether an HLA-A*0201-positive patient with a tumor expressing TERT may be a good responder to immunotherapy treatment with a peptide vaccination of SEQ ID NO: 1 and 2, the method comprising measuring CTL responses specific to peptides of SEQ ID NO: 1 and / or SEQ ID NO: 3 and / or SEQ ID NO: 5 in a blood sample, wherein if no CTL responses specific to peptides of SEQ ID NO: 1 and / or SEQ ID NO: 3 and / or SEQ ID NO: 5 are detected in the blood sample of the patient before vaccination, the tumor is non-immunogenic and the patient may be a good responder to the immunotherapy treatment.

14. The use according to claim 13, wherein the CTL response specific to the peptide of SEQ ID NO: 1 and / or the CTL response specific to the peptide of SEQ ID NO: 3 and / or the CTL response specific to the peptide of SEQ ID NO: 5 is measured by ELISpot IFNg assay.

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