NZ737717B2 - Method for inducing early t memory response with short peptides anti-tumor vaccine - Google Patents

Abstract

The present invention relates to a therapeutic peptide T specific immune therapy for use in the treatment of a cancer of an HLA-A2 (Human Leukocyte Antigen A2) positive patient, said treatment comprises a priming period consisting in two to three administrations of said therapeutic peptide T specific immune therapy, thereby inducing a memory T cell response. c immune therapy, thereby inducing a memory T cell response.

Description

(12) Granted patent specificaon (19) NZ (11) 737717 (13) B2 (47) Publicaon date: 2021.12.24 (54) METHOD FOR NG EARLY T MEMORY RESPONSE WITH SHORT PEPTIDES ANTI-TUMOR VACCINE (51) Internaonal Patent Classificaon(s): A61K 39/00 (22) Filing date: (73) Owner(s): 6.29 OSE IMMUNOTHERAPEUTICS (23) Complete specificaon filing date: (74) Contact: 2015.06.29 AJ PARK (86) Internaonal Applicaon No.: (72) Inventor(s): COSTANTINI, Dominique (87) Internaonal Publicaon number: WO/2017/000983 (57) Abstract: The present invenon relates to a euc pepde T specific immune therapy for use in the treatment of a cancer of an HLA-A2 (Human Leukocyte Angen A2) posive paent, said treatment comprises a g period consisng in two to three straons of said therapeuc pepde T specific immune therapy, thereby inducing a memory T cell response.

NZ 737717 B2 Method for inducing early T memory response with short peptides anti- tumor vaccine.

Field ofthe ion The present invention relates to the field of medicine, in ular of oncology, and more particularly the present invention relates to the treatment of cancer.

Background ofthe Invention Memory T cells are a subset of antigen—specific T cells that persist erm after having tered and responded to their cognate antigen. They quickly expand to large numbers of effector T cells upon re—exposure to their cognate antigen, thus providing the immune system with "memory" against past infections as well as cancer cells. At the second ter with the invader or cancer cells, memory T cells can initiate a faster and stronger immune response than the first time. Memory T cells se two subtypes: central memory T cells (TCM cells) and effector memory T cells (TEM cells). Memory cells may be either CD4+ or CD8+.

It has recently been established, for several human cancers, that high densities of or memory CD8+ cytotoxic T cells are associated with a longer overall survival (Fridman W. et al, 2012, Nat Rev Cancer, 12(4), 298—306). Central memory T cells appears however to have an even greater capacity to persist in vivo and are also more efficient in mediating protective immunity because of their increased proliferative capacity (Calarota SA et al, 2013, al and Developmental logy, Article ID 637649).

In the literature, data indicate that an initial proper activation of the CD8+ T cell response by peptide vaccines in mineral oil adjuvant does not ensure long—term effectiveness of these CD8+ T cells (Bijker MS et a1, 2007, J Immunol, 179:5033—5040), although, such a long-term effectiveness would be of huge interest in the field of chronic diseases such as cancer.

Bijker et al bed a specific strategy linked to the use of long peptides to overcome the poor long term effectiveness of cancer vaccines. However, this strategy was shown able by Karkada et a1. (Karkada M et a1, 2014, Biologics: Target and Therapy, 8: 27-38). Indeed, the immune response with such long peptides was not constant at each time point and was decreasing after the first injection.

Thus, there is still a strong need, nowadays, to develop a method of ent with peptide cancer vaccine that would be able to ate memory T cells as early as possible, especially central memory T cells.

The classical administration regimen of short peptide cancer vaccines rely on at least 5-6 administrations of the vaccine. Holmes et al. (defines the optimal scheme of administration of a specific short peptide cancer vaccine as 6 injections of e for each month of treatment s JP et al, 2008, Cancer , 113 , 1666–1675). In a phase two trial, subjects were considering to be effectively part of the ation protocol if they had received at least 6 administrations of the vaccine r S et al, 2012, Nature Medicine, 18 , 1254–126). Seven to ten short peptide vaccine administration were considered by others for an effective treatment (Kantoff PW et al, 2010, Cancer J Clin Oncol, 28 :1099-1105; Schwartzentruber DJ et al, 2011, N Engl J Med, 364 27).

These short peptide vaccine treatments can be painful for the ts and are not devoid of side effects and toxicity. In addition, controlling health care costs and optimizing cancer treatment are key issues in eration of the rise of costs. There is thus a strong need to find new methods allowing to modify the scheme of administration of the vaccine so as to reduce the number of injections while obtaining a memory T response as early as possible.

Summary of the Invention The object of the present invention relates to a new administration regimen of a short peptide cancer vaccine ng an early memory T cells se. In this invention, the inventors singly showed that no more than three administrations of the short peptide ation were sufficient to obtain a T specific Immune response through memory T Cells and this immune response was linked with a longer time to progression (TTP).

The present invention relates to a therapeutic peptide T specific immune therapy for use in the treatment of a cancer of an HLA-A2 (Human Leukocyte Antigen A2) ve patient, wherein the therapeutic peptide T specific immune y comprises the peptide aKXVAAWTLKAAa (SEQ ID No 10, with X and a respectively indicating cyclohexylalanine and d-alanine) and at least 4, 5, 6, 7, 8 or 9 peptides selected from the group consisting of RLLQETELV (SEQ ID No 1), YLQLVFGIEV (SEQ ID No 2), LLTFWNPPV (SEQ ID No 3), KVFGSLAFV (SEQ ID No 4), KLBPVQLWV (SEQ ID No 5, with B indicating α-aminoisobutyric acid), SMPPPGTRV (SEQ ID No 6), IMIGHLVGV (SEQ ID No 7), KVAEIVHFL (SEQ ID No 8), and YLSGADLNL (SEQ ID No 9), and wherein said treatment comprises a priming period consisting in two to three administrations of said therapeutic peptide T specific immune therapy, thereby inducing a central memory T cell response.

In a particular , the present invention provides a use of a therapeutic peptide T specific [FOLLOWED BY PAGE 2a] immune therapy in the manufacture of a medicament for the treatment of a cancer of an HLAA2 (Human Leukocyte Antigen A2) positive patient, wherein the therapeutic peptide T specific immune y comprises the peptide aKXVAAWTLKAAa (SEQ ID No 10, with X and a respectively indicating cyclohexylalanine and d-alanine) and the peptides RLLQETELV (SEQ ID No 1), YLQLVFGIEV (SEQ ID No 2), LLTFWNPPV (SEQ ID No 3), KVFGSLAFV (SEQ ID No 4), KLBPVQLWV (SEQ ID No 5, with B indicating α- aminoisobutyric acid), SMPPPGTRV (SEQ ID No 6), IMIGHLVGV (SEQ ID No 7), KVAEIVHFL (SEQ ID No 8), and LNL (SEQ ID No 9), and wherein said treatment comprises a priming period consisting of three strations of said therapeutic peptide T specific immune therapy, the eutic peptide T specific immune therapy being administered at one dose every three weeks during which a central memory T cell se is induced against one or several peptides of the therapeutic e T specific immunotherapy, and wherein the priming period is followed by a maintenance period during which the administration of said therapeutic e T specific immune therapy occurs every two to eight months.

In a preferred embodiment, the therapeutic peptide T specific immune therapy is the combination of 10 peptides (called herein 01) comprising (or consisting in) the [FOLLOWED BY PAGE 3] following peptides RLLQETELV (SEQ ID No l), YLQLVFGIEV (SEQ ID No 2), LLTFWNPPV (SEQ ID No 3), KVFGSLAFV (SEQ ID No 4), KLBPVQLWV (SEQ ID No 5, with B indicating a—aminoisobutyric acid), SMPPPGTRV (SEQ ID No 6), IMIGHLVGV (SEQ ID No 7), KVAEIVHFL (SEQ ID No 8), YLSGADLNL (SEQ ID No 9), aKXVAAWTLKAAa (SEQ ID No 10, with X and a respectively indicating cyclohexylalanine and d—alanine.

More specifically, said treatment comprises a g period consisting in two to three administrations of the peptide T specific cancer immunotherapy, thereby inducing a central memory T cell response. More preferably, the treatment comprises a priming period consisting in three administrations of the therapeutic peptide T specific immune therapy. In a particular embodiment, in the priming period, the therapeutic peptide T specific immune therapy is administered every one-four weeks, preferably every ree weeks, more preferably every 3 weeks. ally, in the treatment, the priming period is followed by a maintenance period during which the administration of the therapeutic peptide T specific immune therapy occurs every two—eight months, preferably every two-three , more ably every two months through one year and then every three months through one year.

Preferably, the therapeutic peptide T specific immune y is administered parentally, preferably subcutaneously.

Preferably, the peptides are emulsified in incomplete Freund’s nt or the like, preferably Montanide ISA-51.

Preferably, the doses of peptide are ranging from 0.1 to 10 mg of peptide per injection dose.

Optionally, the total peptide dose for each injection is 5.0 mg.

Preferably, the cancer is a cancer selected from the group consisting of lung cancer such as NSCLC (non-small cell lung cancer) and small cell lung cancer, melanoma, mesothelioma, breast cancers, primary brain cancers, ovarian cancer, uterine carcinoma, especially uterine corpus and/or uterine cervix oma, head and neck cancer, colon or ctal cancer, gastro—intestinal cancer, renal cancer, sarcoma, germ cell tumors, ia, lymphoma, testicular cancers and bladder cancers, ably ed from the group consisting of NSCLC, colon cancer, breast cancer, ovarian cancer, and a cancer of the head and/or neck, more ably NSCLC.

The t is patient HLA—A2 positive patient. Optionally, the patient suffers from an advanced or late-stage cancer. Optionally, the patient suffers from metastases, ally brain metastases. Optionally, the patient has a malignant pleural effusion. Optionally, the patient has W0 2017/000983 already received several lines of treatment prior to the treatment with the therapeutic peptide T specific immune therapy.

Optionally, the treatment is used in combination with a treatment with another antitumor drug and/or with radiotherapy, especially with a checkpoint inhibitor, preferably CTLA—4 and/or PD— l/PD-Ll inhibitor such as pembrolizumab, nivolumab, pidilizumab, BMS936559, MEDI4736, AMP-224, AMP—514, MPDL3280A and avelumab, more ularly in order to transform “non immunogenic T memory” patients into “immunogenic T memory” patients.

Detailed description ofthe Invention Previously, only CTL effector cells and HTL effector cells responses were identified following original combination of short peptides epitopes (EPl620456 describing chemical optimization on the binding to the key receptors of T response and original combination of different nature of epitopes) as T cell specific immunotherapy in oncology. Both CTL (Cytotoxic T cytes) and HTL (Helper T cytes) immune responses versus all es were based on effectors T cells after injection of such combination of epitopes from different nature (fixed anchors, heteroclitic and wild type epitope). EPl620456 patent describing the immune response with such original combination was using a standard Elispot measuring effector T cells assay without in vitro expansion in HLA-A2 transgenic models. This rd Elispot assay allows the measurement of T cells capable of immediate secretion of IFN-y upon antigen/ e stimulation. These cells mainly represent effector T cells.

The categorization of memory T cells into different subsets (effector memory (TEM) and central memory (TCM)) was studied in several studies exanimating which type of memory cell is capable of providing optimal protection (Jennifer D t et a1, 2012, Molecular Therapy, 20, 860—869).

In order to standardize these measures, two t assays allow to separate this 2 main populations: the standard ELISPOT assay quantifies or memory T cells whereas the ed ELISPOT assay quantifies expandable memory T cells, representing l memory T cells. Evidence indicates that a different population of T cells, most likely central memory T cells that differentiate into effector T cells during the e period, are measured by the cultured T assay, as compared with the measurement of circulating effector memory T cells that are quantified by the standard ELISPOT (Calarotra S.A. et al . This ed ELISPOT assay is performed by ing lymphocytes with specific antigens for 10 days allowing T cells to expand in response to the antigen. Then, a standard ELISPOT procedure is applied in response to the corresponding ns used for the 10-day stimulation period measuring effector function.

Central memory T cells require antigenic re-stimulation to develop effector function. The cultured ELISPOT assay mainly comprises these central memory T cells because the depletion of central-memory T cell population ablated completely responses in cultured Elispot (S. M.

Todryk, et al., 2009, Immunology, 128, 83—91). Todryk et al explains the predominant role of central memory T cells in the cultured ELISPOT providing CD4+ and CD8+ specific response.

Central memory T cells have a r capacity than effector memory T cells to persist in vivo and are more efficient in mediating protective immunity because of their sed proliferative capacity.

The invention described below l an unmet need in oncology, providing an optimized administration regimen allowing an early memory T cell response in cancer treatment, preferably an early central memory T cell response, and more particularly an early long term memory T cell response.

More particularly, the present ion relates to a new administration regimen of a short peptide cancer therapy based on multi—epitope T specific cancer immunotherapy. Surprisingly, the inventors have demonstrated that the administration of a short peptide multi—epitopes combination leads to an cted strong immune response involving long term memory T cells, in particular central memory T cells. Furthermore, the inventors have also discovered that a very short administration regimen with only three injections is enough to induce this strong and long term response.

Accordingly, the present ion relates to a therapeutic peptide T specific immune therapy as defined herein for use in the ent of a cancer of an HLA-A2 (Human Leukocyte Antigen A2) ve patient, wherein said treatment ses a g period consisting in two to three administrations of said therapeutic peptide T specific immune therapy. It also s to a method of treatment of a cancer of an HLA-A2 (Human Leukocyte Antigen A2) ve patient in need thereof comprising a priming period ting in two to three administrations of said therapeutic peptide T specific immune therapy OSE2101 as defined herein. Finally, the present invention relates to the use of a therapeutic peptide T specific immune therapy 1 as defined herein for the manufacture of a cancer treatment, wherein the therapeutic peptide T specific immune therapy is to be administered two to three times during the g period.

WO 00983 Definitions An "epitope" is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Alternatively, an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell or proteins and/or Major Histocompatibility x (MHC) receptors. Epitopes are present in nature, and can be isolated, purified or otherwise prepared or derived by humans. For example, epitopes can be prepared by isolation from a natural source, or they can be synthesized in accordance with standard protocols in the art. Throughout this disclosure, epitopes may be referred in some cases as peptides or peptide epitopes.

"Human Leukocyte Antigen" or "HLA" is a human class I or class 11 Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et all, IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, CA (1994). HLA molecules are d on the basis of shared peptide—binding icities. For example, HLA—A2 is a particular type of HLA molecules which share similar binding affinity for peptides bearing certain amino acid motifs. The methods for ining the HLA—A2 status in a patient are well—known and easy to obtain (i.e; serological samples) by the one skilled in the art.

A "peptide epitope" is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, peptide epitopes of the invention are capable of binding to an appropriate HLA—A2 molecule and thereafter inducing a cytotoxic T lymphocyte (CTL) response, or a helper T lymphocyte (HTL) response, to the peptide.

A "PanDR peptide" or "PADRE®" peptide is a member of a family of molecules that binds more than one HLA class II molecule. The pattern that defines the PADRE® family of molecules can be referred to as an HLA Class II otif. A PADRE® molecule binds to HLA class II molecules and stimulates in vitro and in viva human HTL responses. PADRE peptides are described in the patent EP735893.

A "CTL and/or an HTL se" is a protective or therapeutic immune response to an n derived from a pathogenic antigen (e‘ g‘ an antigen from an infectious agent or a tumor n), which in some way prevents or at least partially s disease symptoms, side effects or progression. The immune response may also e an antibody response which has been facilitated by the stimulation of helper T cells.

As used herein, the term "memory T cell" is intended to e both the CCR7— (effector memory T cells) and CCR7+ al memory T cells) subpopulations of T cells. This definition also includes both class II-restricted CD4 memory T cells and class I—restricted CD8 memory T cells.

The staging of a cancer describes the severity of a ’s cancer based on the size and/or extent (reach) of the original (primary) tumor and whether or not cancer has spread in the body tasis). NSCLC stages are numbered from 0 to IV. Stages IIIb and IV are the most advanced stages.

"ECOG (Eastern Cooperative Oncology Group) Performance Status" are used by doctors and researchers to assess how a patient's disease is progressing and assess how the e affects the daily living abilities of the patient. ECOG mance Status are numbered from 0 to 5.

A performance status of 0 match to patients who are fully active and able to carry on all pre— disease performance without restriction. A performance status of 1 match to patients who are restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e. g., light house work, office work.

The term "overall survival" (OS) refers to the length of time from the date of the start of treatment that patients are still alive. In a clinical trial, measuring the overall al is one way to see how well a new treatment works.

Therapeutic peptide T specific immunotherapy or peptide vaccine The Multi—epitopes T ic cancer immunotherapy of the invention is able to help the immune system to p immune memory that can have asting, tumor—specific effects.

An effective peptide T specific cancer immunotherapy requires induction of a wide breadth of CTL specificities. This can be best achieved with optimized epitopes targeting multiple Tumor Associated Antigens (TAAs) as a multi—epitopes combination ing at least 5 tumor antigens and based on epitopes combination. Preferably, the at least 5 tumor ns include or are selected among HER2/neu, CEA, MAGEZ, MAGE3 and p53.

The Multi-epitopes T specific cancer immunotherapy comprises a combination of epitopes that can be wild—type epitopes and modified epitopes (heteroclitic and fixed anchors epitopes).

Preferably, the epitopes T specific cancer immunotherapy comprises at least 5 epitopes.

In a preferred embodiment, the combination of epitopes comprises at least 5 epitopes chosen among those disclosed in Table 6 of the US application US2014/01474790 (incorporated herein by reference). More preferably, the Multi—epitopes T ic cancer immunotherapy comprises at least 5 epitopes allowing to target the combination of the following 5 tumor antigens: HER2/neu, CEA, MAGE2, MAGE3 and p53. For instance, Multi—epitopes T specific cancer therapy may comprise at least 5, 6, 7, 8 or 9 epitopes chosen among those disclosed in Table 6 of the US application US2014/01474790.

The original combination used here (OSE-2lOl) is made by wild-type epitopes and modified epitopes (heteroclitic and fixed anchors epitopes). More detailed information on heteroclitic and fixed s epitopes can be found for instance in the patent EPl620456.

OSE—2101 is a multi-epitope T specific cancer immunotherapy composed of 10 synthetic peptides. Nine of the peptides have been designed to induce a CTL response against TAAs.

More particularly, the T specific immune y is ed for administration to patients for the induction of CTL directed against oembryonic antigen (CEA), p53, human epidermal receptor—2/neurological (HER-2/neu) and melanoma antigen 2 and 3 (MAGE—2/3). These TAAs have been chosen based on epidemiology because they are frequently over—expressed in various advanced cancers as colon cancers, ovarian cancers, breast cancers and NSCLC. Each CTL epitope is restricted by HLA—A2 superfamily of major histocompatibility complex class I molecules, thereby providing coverage of approximately 45 % of the general population. The tenth synthetic peptide is the pan-DR epitope (PADRE), a rationally designed helper T— lymphocyte (HTL) epitope included only to increase the magnitude of CTL responses.

OSE-2lOl composition comprises or consists of the following peptides: RLLQETELV SEQ ID No l GIEV SEQ ID No 2 LLTFWNPPV SEQ ID No 3 KVFGSLAFV SEQ ID No 4 KLBPVQLWV SEQ ID No 5, with B indicating u—aminoisobutyric acid SMPPPGTRV SEQ ID No 6 IMIGHLVGV SEQ ID No 7 KVAEIVHFL SEQ ID No 8 YLSGADLNL SEQ ID No 9 WTLKAAa SEQ ID No 10, with X and a tively indicating exylalanine and d-alanine.

Therefore, the therapeutic peptide T specific immune therapy comprises the peptide aKXVAAWTLKAAa (SEQ ID No 10, with X and a respectively indicating cyclohexylalanine and d-alanine) and at least 4, 5, 6, 7, 8 or 9 es selected from the group ting of RLLQETELV (SEQ ID No l), GIEV (SEQ ID No 2), LLTFWNPPV (SEQ ID No 3), KVFGSLAFV (SEQ ID No 4), KLBPVQLWV (SEQ ID No 5, with B indicating 0t- aminoisobutyric acid), SMPPPGTRV (SEQ ID No 6), IMIGHLVGV (SEQ ID No 7), KVAEIVHFL (SEQ ID No 8), and YLSGADLNL (SEQ ID No 9).

The peptides can be sized using standard Boc or Fmoc chemistry for solid phase e synthesis starting with the appropriate resin, and purified by standard methods. Alternatively, the peptide may be produced by c engineering with recombinant cells or by RNA, for instance by in vitro translation system.

The Multi-epitope T specific cancer immunotherapy composition may comprise a pharmaceutically acceptable carrier or excipient. More preferably, the pharmaceutically acceptable carrier is an aqueous carrier, especially a buffer. In particular, it may comprise one or several adjuvants. For instance, adjuvants can be incomplete Freund’s adjuvant, mineral oil adjuvant, aluminum hydroxide, or alum, . Other suitable adjuvants are well-known in the art.

In one embodiment, the Multi-epitope T specific cancer immunotherapy may comprise e pulsed antigen presentating cells, such as dendritic cells.

Preferably, in the Multi-epitope T specific cancer immunotherapy, the peptides are emulsified in incomplete Freund’s adjuvant or the like. In a preferred embodiment, the adjuvant is a mineral oil nt, similar to Incomplete Freund's Adjuvant, manufactured and supplied by Seppic SA, Paris, . In a most preferred embodiment, the nt is Montanide® ISA 5 l .

Each e of the composition can be present at a concentration of 0.1 mg/ml to 1 mg/ml, preferably 0.5 mg/ml. ably, all the es are present in the ition at the same tration.

Preferably, Multi-epitope T specific cancer immunotherapy composition is a sterile, preservative—free emulsion of the 10 peptides at a concentration of 0.5 mg/ml each, formulated in Montanide® ISA 51 adjuvant at a ratio of 1:1 (w:w) and filled into rubber—stoppered glass vials, and refrigerated at 2° to 8°C.

OSE-ZlOl is manufactured under aseptic conditions. Peptides are dissolved in three different solvents, sterile filtered, pooled and then fied in adjuvant via homogenization under controlled conditions. Product release testing included appearance, endotoxin, sterility, viscosity, particle size, peptide concentration of each peptide, , pH and potency. ation of OSE—2101 composition is detailed in W02004/094454, Fig 3A and pages 105— 106, the disclosure of which being incorporated herein by reference.

Optionally, in addition to the 10 peptides of OSE —2101, the peptide composition of the t invention may further comprise additional peptides, in particular peptide epitopes used for inducing of cytotoxic hocyte (CTL) responses and targeting TAAs. For instance, the peptide composition of the present invention may further comprise a peptide as disclosed in W02009/143843 (the disclosure of which being incorporated herein by reference), and more ularly ID05 (SEQ ID No ll).

Administration regimen of the therapeutic peptide T specific immunotherapy: The administration regimen of a vaccine comprises a priming period and optionally a maintenance period.

The term “priming period” refers to the period of the vaccination process during which a central memory T cell response is induced against one or several peptides of the therapeutic peptide T specific immunotherapy.

The term “maintenance period” or “boosting period” refers to the period of the vaccination process following the priming period during which the same therapeutic peptide T specific immunotherapy is administered and the T memory immune response is sustained or enhanced.

According to a red aspect of the present invention, the priming period of a subject under treatment with therapeutic peptide T specific therapy consists in one to three strations of the eutic peptide T ic immunotherapy or vaccine.

In an even more preferred embodiment, the priming period consists in three administrations of the therapeutic peptide T specific immunotherapy or vaccine.

During this priming period, the therapeutic peptide T ic immune therapy is administered every one-four weeks, preferably every two-three weeks, more preferably every 3 weeks.

The priming period is nt for inducing a central memory T cell response against one or l es of the therapeutic peptide T specific immunotherapy, preferably against at least 2, 3, 4 or 5 peptides of the therapeutic peptide T specific immunotherapy.

During the optional maintenance period, which follows the priming period, one or several strations of the therapeutic peptide T specific therapy or vaccine are realized.

During this maintenance , the administration of the vaccine occurs every two-eight 3O months, preferably every two-three months, for instance every two month through one or two years and then every three months through one or two years.

In an alternative ment, the priming period is not followed by a maintenance period.

The priming period and the maintenance period can be separated by a rest period which does not include any the therapeutic peptide T specific immunotherapy administration. This rest period can last four to twelve weeks.

In another alternative embodiment, the g period is followed by a treatment with a checkpoint inhibitor as the T memory cells induced by the therapeutic peptide T specific immunotherapy are rendered immunogenic the tumor environment.

The present invention further relates to a method for treating cancer in an HLA—A2 positive t, comprising administering a therapeutic effective amount of therapeutic peptide T specific immunotherapy as disclosed , wherein said treatment comprise a priming period during which one to three administrations of said peptide vaccine are performed. The method may further comprise a preliminary step of ining the HLA status of the t, selecting the HLA-A2 positive patients and administering a therapeutic ive amount of the therapeutic peptide T specific immunotherapy as disclosed herein to the HLA—A2 positive patients. The method may further comprise a maintenance period as defined above. Preferably, the therapeutic peptide T specific immunotherapy is 1.

Dosage Within the context of the invention, the term “treatment” or “treating” denotes curative, symptomatic, and preventive treatment. Pharmaceutical compositions and preparations of the invention can be used in humans with existing cancer or tumor, preferably at late stages of progression of the cancer. The pharmaceutical compositions and preparations of the invention will not necessarily cure the patient who has the cancer but will delay or slow the progression or prevent further ssion of the disease, ameliorating thereby the patient’s condition. In ular, the pharmaceutical compositions and preparations of the invention reduce the development of tumors, and/or prevent metastasis occurrence or development and cancer e. In treating the cancer, the pharmaceutical composition of the invention is administered in a eutically effective amount.

By "effective amount" it is meant the quantity of the pharmaceutical composition of the invention which prevents, removes or s the deleterious effects of brain metastases. It is understood that the administered dose may be adapted by those skilled in the art ing to the patient, the pathology, the mode of administration, etc. The dosage and regimen depends on the stage and severity of the disease to be treated, the weight and l state of health of the patient and the judgment of the prescribing physician. More ularly, by “therapeutically efficient amount of the therapeutic peptide T specific immunotherapy as disclosed herein” is intended the amount which is sufficient to increase the overall al of a patient. In particular, by “therapeutically efficient amount of the therapeutic peptide T specific immunotherapy as sed herein” is intended the amount which is sufficient to induce a central memory T cell response against one or several peptides of the therapeutic peptide T ic immunotherapy.

Previous cancer trials have tested ting doses of peptide, ranging from 0.1 to 10 mg of peptide per injection dose, emulsified in incomplete Freund’s adjuvant. At all doses tested, the peptide/incomplete ’s adjuvant treatment was deemed to be safe and well tolerated, with no severe dose-related ic toxicities being reported. therapeutic peptide T specific immunotherapy as disclosed herein can be administered by any appropriate route, in ular by eral route such as subcutaneous, intradermal or intramuscular route or by aerosol, transmucosal, intrapleural, or intrathecal routes. In a most preferred embodiment, the es composition is stered subcutaneously. Preferably, the therapeutic peptide T specific immunotherapy as disclosed herein is designed for subcutaneous injection.

Preferably, the doses of peptide are ranging from 0.1 to 10 mg of peptide per injection dose. In a preferred embodiment, the total peptide dose for each ion or stration will be 5.0 mg (1 mL of drug product containing 0.5 mg of each peptide).

Cancer According to a preferred aspect of the present invention, subjects of vaccination with OSE— 2101 composition are patients with a cancer. In a preferred ment, patient’s cancer are due to one of the following cancers: lung cancer such as NSCLC (non—small cell lung cancer) and small cell lung cancer, melanoma, mesothelioma, breast cancers, primary brain cancers, ovarian, uterine carcinoma, especially uterine corpus and/or e cervix carcinoma, head and neck, colon, gastro—intestinal, renal cancers, sarcoma, germ cell tumors, leukemia, lymphoma, testicular cancers and bladder cancers, preferably NSCLC, colon cancer, breast cancer, ovarian cancer, and a cancer of the head and/or neck, more preferably NSCLC.

In another preferred embodiment, patient’s cancer are ed cancer. The term “advanced ” refers to a cancer at an advanced stage of development, i.e. a cancer that has spread in the body (metastasis). Preferably, the patient suffers from brain metastases.

Alternatively, the patient has a malignant pleural effusion, preferably a metastatic pleural effusion, in particular associated with lung cancer, breast , lymphoma or leukemia.

Optionally, the patient has already ed several lines of treatment prior to the vaccination by the peptide composition. In a particular embodiment, the patient has a positive HTL I'CSpOl’lS6.

Combination with another antitumor drug.

Optionally, the treatment is used in combination with a ent with another antitumor drug, in particular a chemotherapy, hormonotherapy and/or immunotherapy.

For instance, the chemotherapy can be selected among cisplatin, carboplatin, cyclophosphamide, etoposide, teniposide, mitomycin, ecan, Vinorelbine, ide, ifosfamide, temozolomide, fluorouracil (5FU), docetaxel, pemetrexed, navelbine, drugs that target tumor blood vessel growth (VEGF) such as bevacizumab, ramucirumab; prednisone; tyrosine kinase inhibitors targeting EGFR such as gefitinib, erlotinib, afatinib ; ALK inhibitors such as crizotinib; ceritinib and any combination thereof.

In a preferred embodiment, the vaccine treatment of the present invention is used in combination with a checkpoint inhibitor, especially a CTLA—4 inhibitor and/or a PD—l or PD— Ll inhibitor; IDO inhibitors. The treatment with the checkpoint inhibitor can be performed before, aneously or after the ent with the therapeutic peptide T specific immunotherapy as disclosed , in particular the priming period of the treatment.

The present invention relates to a kit or product sing (a) the eutic effective amount of eutic peptide T specific immunotherapy as disclosed herein; and (b) a check point inhibitor, preferably a CTLA—4 inhibitor and/or PD-l or PD—Ll inhibitor, as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of cancer.

Preferably, the therapeutic peptide T specific immunotherapy is OSE—2101.

In a preferred embodiment, the treatment with a checkpoint inhibitor is performed after the priming period of the treatment with the eutic effective amount of therapeutic peptide T ic immunotherapy as sed herein.

Several PD—l/PD—Ll inhibitors are already available or under clinical development. For instance, the PD—l/PD—Ll inhibitors can be chosen among the non-exhaustive list including lizumab (Merk), nivolumab (Bristol Myers Squibb), pidilizumab (Cure Tech), BMS936559 (Bristol Myers Squibb), MEDI4736 (Astra Zeneca), AMP-224 (Astra Zeneca), AMP-514 (Astra Zeneca), 80A (Roche), avelumab (also known as 0718C from Merck KgA Serono /Pfizer) . For instance, the PD—l/PD—Ll inhibitors can be chosen among those disclosed in W02013/079174, For instance, the CTLA—4 inhibitors can be chosen among the non—exhaustive list including Tremelimumab (Pfizer Medimmune ) and ipilimumab (BMS).

Further aspects and advantages of this invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.

Description of the Figures Figure 1: Phase 1/2 W9 W18 magnitude of central memory T cell responses induced by OSE2101. figm: Phase 2 Central memory T cells responses induced by OSE2101 for l epitopes through the number of doses.

Figure 3: ation between OSE—2lOl-induced PADRE HTL ses and CTL / central memory T cell responses.

Figure 4: Kaplan—Meier estimate of TTP.

Examples Example 1: early immune response though central memory T cells involvement In the Phase 1 /2 clinical studies, the safety and immunogenicity of a lO-peptide enhanced epitope combination, OSE-2101, rationally designed to induce broad multi-epitope CTL responses in early stage colon and NSCLC patients. In addition to multi-epitope coverage, OSE— 2101 also targeted es from five Tumor antigens which are widely expressed on breast, colon and non—small cell lung, ovarian, tumors (CEA, HER—2/neu, p53, MAGE2 and MAGE3) making this product suitable for therapy against different cancer tions. As a source of T— cell helper for CTL ion, the universal HTL epitope Pan DR e was also included and a mineral oil adjuvant was used for the final ation of this T specific cancer immunotherapy.

Patient eligibility criteria. OSE—2101 was tested in two disease-specific phase I clinical trials enrolling + patients with histologically confirmed stage IA NSCLC or stage III colon cancer. All patients were diagnosed as NED within the past 6 months after undergoing standard therapy. Patients met normal laboratory parameters for blood chemistry and white cell counts and had an Eastern Cooperative Oncology Group performance status of 0 or 1. They were excluded if treated with immunomodulatory agents within one month of study entry or with other cancer immunotherapies. Patients with a history of other cancers, except basal or squamous cell carcinoma of the skin or in situ cervical cancer, were also excluded as were patients with concurrent acute l ions or specified autoimmune diseases.

Peptides. The immunogenicity of all immune monitoring epitopes was confirmed by stimulating CTL induced by treatment of HLA—A2.1/Kb transgenic mice with a pre—clinical lot of OSE-2101 (standard Elispot .

Clinical study design. Both trials were designed as phase 1/2, open label, center, single dose, multiple stration studies to evaluate the safety and immunogenicity of the OSE— 2101 immunotherapy. Patients were treated with 1 ml OSE-2101 every 3 weeks for a total of six doses. Each dose was administered subcutaneously in the same vicinity in the deltoid or upper thigh region, or at a contralateral site if local side—effects were observed. The study duration for each completed patient was 18 weeks. Clinical responses or patient survival were not measured in either phase 1/ 2 s.

Clinical sample processing. Blood or heresis product was ed from patients at the pre-treatment, the Week 9 and Week 18 time points, 3 weeks after the third and sixth dose of OSE—2101 respectively. Peripheral blood mononuclear cells (PBMC) were isolated from s within 24 hours of collection using a Ficoll-Paque density gradient and cryopreserved in containers designed to ensure optimal freezing (Mr. Frosty, e). For use, cells were rapidly thawed at 37°C then transferred to human AB serum-containing medium for work—up.

Measurement of central T memory cells responses and T effector memory cells. The CTL responses in 16 total patients (10 colon and six NSCLC) were monitored for CTL responses t each epitope and t the wild—type epitope of epitopes analogs. For each patient, the pre-treatment, Week 9 and Week 18 time points were batch-tested in the same experiment to allow pre— versus post- treatment response comparison in the same experiment. Central T memory cells responses were measured using an IFN-y enzyme-linked spot (cultured ELISPOT) assay following in vitro stimulation of PBMC for 10 days with each vaccine e.

Briefly, PBMC from each time point were stimulated on day 0 with 10 ug/ml of each epitope individually in replicate in 48—well culture plates (2 x 106 PBMC/well). All cultures were fed with rIL—2 (Endogen, Wobum, MA; 10 U/ml final concentration) on days 1, 4 and 7. Ten days after initiation of e, cells were harvested and tested for activity against specific vaccine peptides and ponding wild—type peptides of vaccine analogs in the T memory ELISPOT assay. Cells were tested in Millipore IP 96—well plates pre—coated with mouse anti—human IFN— 7 antibody (Mabtech USA, Cincinnati, OH). Five x 104 cells and 1.25 x 104 cells from each culture were plated in triplicate wells er with 105 irradiated autologous PBMC and 10 ug/ml peptide. As a control, CTL were also tested against an irrelevant HLA-A2.1—binding HBV peptide. After 20 hours incubation, T memory ELISPOT plates were developed by performing sequential incubations with biotinylated anti—human IFN—y antibody (Mabtech USA), Avidin—Peroxidase Complex (APC, Vector Laboratories, game, CA), and 3— amino—9—ethy1 carbazole (AEC) substrate (Sigma Aldrich, St. Louis, MO). Spot-forming cells (SFC) were enumerated using a computer-assisted image analysis system (Zeiss KS ELISPOT Reader, Carl Zeiss Microlmaging, Thomwood, NJ). Data is reported as the net SFC per 5 x 104 cells after subtracting spots induced with the irrelevant HBV peptide.

The central T memory cultured ELISPOT assay was ied using HLA-A2+ PBMC from patients who demonstrated a recall T effector memory response against an EBV BMLFl CTL epitope (sequence, GLCTLVAML) under cal stimulation conditions used for testing clinical samples. Specific response generated from these donors were titrated at limiting cell doses and a linear response was ed (correlation coefficient = 0.99). The lower detection limit of the assay was ined to be 5 SFC/well and the upper limit was 600 SFC. The inter— experiment reproducibility was evaluated by repeated testing of cryopreserved pre—vaccination PBMC samples from positive donors on separate days, and the coefficient of variation (CV) ranged from 7—12% in these assays. To assess inter—operator reproducibility, two operators tested pre—treatment PBMC from the same patient on different days and the CV was 13—16% at the optimal cell dose. The CV values were considered to be within an acceptable range of variability and strongly supported the use of our assay for analyses of clinical immune responses.

Response criteria. ve response criteria were established prospectively after analyzing the variability of pre-treatment ses t each epitope and the irrelevant HBV control epitope. The mean SFC response in a post-treatment sample was ered a positive epitope— induced T memory cell response if it met all of the following: 1) was greater than 5 SFC above the irrelevant epitope response, 2) was greater than the mean SFC of the irrelevant epitope response, plus 2 SD, and 3) was ld greater than the SFC response in the pre—treatment sample from the same patient, plus 2 SD. The inclusion of “plus two standard deviations” was used to accommodate the assay variability and served to make the criteria more stringent.

RESULTS Patient characterization. Fourteen patients with stage III colon cancer enrolled in the trial and ten patients completed the study after receiving six doses of OSE—2101. In the NSCLC trial ten ts with stage IIB/IIIA disease were enrolled and six patients completed the study.

OSE—2101 was deemed safe and tolerated by patients in the two clinical trials with l side— effects common to peptides prepared in mineral oil adjuvant being observed.

Immunogenicity of OSE-2101 A total of 16 patients (10 colon cancer and 6 NSCLC) receiving the full course of six OSE-2101 doses were evaluated for the frequency, breadth and magnitude of TAA-specific central T memory cell responses d by the specific immunotherapy. To improve detection of central memory T cell responses PBMC were stimulated in vitro for 10 days with each epitope. The effector activity of the in vitro expanded PBMC was measured with an IFN—y ELISPOT assay t the respective epitope and an irrelevant HLA—A*0201— binding HBV epitope. If the epitope was an analog the corresponding wild-type epitope was also tested.

Results from immune monitoring of the 16 ts indicated that OSE-ZlOl was capable of ng a wide breadth of central memory T cell responses in ts. In the colon cancer trial, eight of the 10 ts surprisingly generated CTL responses against four or more vaccine epitopes at the Week 9 and/or Week 18 time points (Figure l). The average magnitude of the ed ELISPOT was in the same range between Week 9 and Week 18. t 607 demonstrated the widest breadth and highest magnitude of central T cell memory responses, displaying responses between 60 — 200 SFC per 5 x 104 cells against seven epitopes at the Week 9 time point. More importantly, five of the seven induced T memory responses in this patient were directed against wild—type epitopes. At the Week 18 time point, patient 607 generated central memory T cell responses, some exceeding 1000 SFC, against six of the same es at Week 9.

Equally noteworthy were the multi-epitope responses observed in colon cancer patients 601, 603, 604 and 606 to five or more epitopes surprisingly at the Week 9 and/or also at Week 18 time .

Multi—epitope central memory T cell responses were also observed in NSCLC patients, surprisingly at the Week 9 and/or also at Week 18 time points. All of the data described utilized a testing protocol where PBMC samples were cultured in vitro for 10 days with individual OSE— 2101 epitopes to expand in viva—primed CTL prior to testing in the ELISPOT assay and the measure central memory T cell response and the in vitro stimulation step as ed Elispot was required to see ses to epitopes.

Immunogenicity of different epitope classes. Analysis of the frequency and magnitude of CTL responses indicated that most of the es and all of the epitope classes ented in the product were immunogenic in patients. Overall, eight of the nine vaccine epitopes induced central memory T cell responses in at least one colon cancer t and six vaccine epitopes were immunogenic in at least one NSCLC patient. Three of the fixed-anchor analogs (CEA.24V9 (SEQ ID No 3), HER2.369V2V9 (SEQ ID No 4) and p53.139L2B3 (SEQ ID No )) and two heteroclitic analogs 91H5 (SEQ ID No 7) and CEA.605D6 (SEQ ID No 9)) were particularly genic as 60-80% of colon cancer patients and 40—80% of the HLA— A*0201—typed NSCLC patients responded to each of the s (Table 1A and 1B week 9 or week 18 responses). The heightened immunogenicity of the analogs was also indicated by average response magnitudes ranging from 70 — 180 SEC at Week 9 and from 90 — 315 SEC at Week 18 in colon cancer patients (Table 1A) and 60 — 138 SEC at Week 18 in NSCLC patients (Table 1B).

Immunological testing of the 16 colon and NSCLC patients who ted treatment indicated that OSE—2101 was successful in inducing a wide breadth of CTL and memory T cell responses in individual patients as soon as after 3 injections, characterized by the aneous induction of CTL specificities directed t several es. The broad CTL/ though central memory T cell responses observed in individual patients in both cohorts indicated the potential long term efficacy for immunotherapy in early and late stage patients. Instead, simultaneous CTL and central Memory T cell responses is addressing a long term clinical benefit of the multi—epitope approach to cancer immunotherapy.

Table 1A $58 Es w. m m. m 0 c a m w m m w a N m w w. a m w .0 form z use: m w w. w a, w o m m m a m N m m m w w m v Enigma m - - ed - i u, - - We - - we. - - Wm - - “I. - - - - u - - -. u - w We a, ad Wm .. .. . _. .. . . . _ . _ _ . , , nnflmfinfl m E . . . , t . . . m . . . , . . . . $ , , : ., , . 05. . . . . - , , . . , 5 2335‘s. , .. . , . i . , . - .

Efiwuaié . . . . . . t - . . . . msflmmwmxu Now NNN he ammo“, Em “3 0.0m fl: so mmm odmm ,. .1. X - NN Hm? Emma. Gum mm New 3 mm» at 0.3m 33:: 5 53m hdm ad Hm: awn Wm. Dom as, 5%. . - flaw . . - mm mwmm fimwfi n3. m. an w . 9mm NE. mama BE. n _ I§.m.fiu . ._, . . , :23 . , . ., . , c . .1 .t . - 3 gnawing 9m mam . . , . . . - a: 9mg Dd Wm out meg mama—ammo.— . , . . ., . .. . , n8. mam _ I . _ :94 new can: mam...“ . . - 3. mam 9% - ., - .. , 5mm 3 mam QB 5mm, we Emma ad cam rm? HHU .

N war Nw ad SE. n.8, Eu 08 Now ad , Wm Maw Emma. 3.” Emma m.m New SE. .3 magmm Enema a m Saga a 3 wow} m we oumwém w me m ma m e, mp m m» m me m mw m 3. m a bum—33m Mom?» “mm? Mom? amp? Eu Mmmfi i3 m3? $33 Sushi mom; x83 uumwimmm mamas M33 Maggi ammo?» owning ougém Mom? ammo?» mum>$m Mme? Mmmg 8978; Mow? x83 a??? cmog Emma Em Sm, mom m8 «8 m8 mam. Em. mom m8 532 mum a) A, CTL were tested t the e analog. b) WT, CTL were tested against the vaccine wild type epitope corresponding to the vaccine analog. c) Value indicates the net pre-vaccination SFC per 50,000 cells tested against the indicated epitope. d) Value indicates the net SFC per 50,000 cells of vaccine positive responses. e) -, tes SFC response did not meet criteria and vaccine response was negative. f) Total numberof vaccine positive se against a vaccine wild type epitope or the wild type epitope corresponding to the vaccine analog. 2015/064746 Table 1B magmomwwz pts o o w. o N w 0 w, o o : w . HER»? o o w a m m a v N m w w NEWE..mE ES m9. I I Nd I I wd I I WE I I 0.0 I I mN I I I I a o mmw \ my M M m 4 I I I I I I I I I I I I I I n K a a.

N m mmflmmmunna .5). ad m I I 3.: I I me I I I I I I I I I, I m md ca a. a. d ad I I 3.: I I m.» Emw mdm 0.0 I NON mumm afim ms 3 0.0 an 9am .mmm add. m w ENwwfiMflfiE ICE mm I a a 0? I a. a. uflfi _ m>me®nNmu DEN 5mm? w w aging ado Wag v v E: EQEU Neg mNfl. N m a I -_ Smoodmu HE. Wm Qmm mam a w nun-Em: mam New me m.m I New NON mgmw w m rm E! a N m m m m m m M. «m _ngm 393E mw we ww ww mw we ww mw m x3? ul ESE-m Mmmg x83 Mom; 3%th Mam?» Moog 39me goo? x00?) Seiko E? «mm; uum>IwE m3; Em; £8? among “mam; mom? .fim SN mom Em mam mmm who :MDE cum .: guacad mflwaocwmm a) A, CTL were tested against the vaccine analog. b) WT, CTL were tested against the vaccine wild type epitope corresponding to the vaccine analog. c) Value indicates the net pre-vaccination SFC per 50,000 cells tested against the indicated epitope. d) Value indicates the net SFC per 50,000 cells of vaccine positive responses. 6) -, indicates SFC se did not meet criteria and vaccine response was negative. f) Total number of vaccine positive response against a vaccine wild type epitope or the wild type epitope corresponding to the vaccine analog.

Example 2: early T memory cell involvement confirmed in phase 2 CTL Immune responses measured though cultured Elispot. The immunogenicity was measured in Phase 2 clinical trial on cryopreserved PBMC from each test sample were thawed in 5% human culture medium (RPMI-1640 medium with 25 mM HEPES, supplemented with % human AB serum, 4 mM L—glutamine, 0.5 mM sodium te, 0.1 mM MEM non— essential amino acids, 100 ug/ml streptomycin, and 100 U/ml penicillin) containing 30 ug/ml DNAase. After centrifugation for 5 minutes at 1200 rpm, the cell pellets were resuspended in % human culture medium and washed 2 times. For the central Memory T cell cultured ELISPOT assay, 2x106/ml PBMC were placed in ate wells in a 48—well plates and stimulated for 10 days with 10 ug/ml of each peptide. As a positive control, PBMC were also stimulated with a pool of recall viral peptides. Human rIL2 (10 U/ml) was added to the peptide— stimulated cultures at day 1, day 4 and day 7. After 10 days the stimulated cells were harvested and plated at a tration of 5X104/we11. Assay wells also ed irradiated autologous PBMC (1x105/well) and 10 ug/ml peptide, either the vaccine peptide, irrelevant peptide or positive control es. Cells stimulated in vitro with each analog epitope were also tested against the corresponding wild—type epitope.

HTL Immune responses measured though standard Elispot. For the HTL ELISPOT assay measuring T helper responses to the PADRE e, 4x106/ml PBMC were placed in a 12 well—plate for overnight culture. Cells were then ted and placed at 2x105/well in flat— bottom 96—well nitrocellulose plates which had been pre—coated with anti—IFN—y monoclonal antibody (mAb) (10 ug/ml; clone 1—D1K; Mabtech). Cells plated in 6—well replicates were stimulated with 10 ug/ml PADRE e or with an irrelevant malaria SSP2 peptide. After 20 hrs incubation at 37°C, the assay plates were washed with PBS/0.05% 20 and 100 ul/well of biotinylated anti—IFN-y mAb (2 ug/ml; clone 7—B6—1; Mabtech) was added to wells.

The plates were incubated for 2 hrs at 37°C then washed 6 times. Finally, spots from IFN—y— secreting cells were developed by sequentially incubating wells with Vectastain ABC and 3— amino—9—ethyl ole (AEC) solutions. Spots were counted by a computer-assisted image analysis reader (Zeiss KS ELISPOT ).

Data analysis and acceptance criteria. Mean and standard deviation (SD) of spots in replicate wells were calculated in all assays by transferring raw ELISPOT data from each experiment to an Excel—based computer program. Positive vaccine-induced T-cell responses were determined according to the criteria described below.

Acceptance Criteria for peptide-Induced specific T—cell response through central memory T cells: The positive ia for CTL responses used in the phase 2 trial was identical to the criteria in the phase l/2 trials which was established prospectively after analyzing the variability of pre—treatment responses in patients against each vaccine epitope and the irrelevant HBV control epitope. A positive peptide-induced CTL/ T memory response to a given epitope met all of the following conditions: 1) was greater than 5 SFC above the irrelevant epitope response, 2) was greater than the mean SFC of the irrelevant epitope response, plus 2 SD, and 3) was two- fold greater than the SFC response in the ccination sample from the same patient, plus 2 SD. The inclusion of “plus two standard deviations” was used to accommodate the assay variability and served to increase the ia stringency.

The criteria for a positive HTL response to the HTL PADRE epitope, ed without prior expansion of PBMC, were as follows: 1) a PADRE—specific response >5 net SFC per 2x105 cells after subtracting background; 2) for each sample tested in 6-well ates, a t-test p value <0.05 when comparing SFC from wells stimulated with the irrelevant HLA—DR binding malaria peptide versus wells stimulated with the PADRE peptide; and 3) a t—test p value <0.05 when comparing SFC d by stimulation with the PADRE e in the pre— versus post— vaccination samples. All three criteria had to be fulfilled before a HTL response was considered to be induced by OSE-2101 ation.

RESULTS Patient enrollment and immune monitoring criteria. A total of 64 HLA—A2+ patients with stage IIIB, IV or recurrent NSCLC in the phase 2 trial were treated with at least one dose of OSE—2lOl. Thirty three patients completed the initial phase of the study ting of six doses of OSE-2101 administered at 3—week intervals and were monitored for epitopes-induced T—cell/ T memory cells responses. Results of tests from this t cohort are described below with a particular attention on early response at W9 (after 3 injections) and W18 (after 6 injections).

The breadth of CTL/central memory T cell responses d in each of the 11 first patients and the immunogenicity profile of individual e epitopes was very similar to the phase 1/2 trials in early-stage NSCLC and colon cancer patient with less burden disease.

Most of the CTL effector/central memory T cells responses in treated patients were d during the initial 3 to 6—dose/18—week treatment phase and surprisingly as early as after 3 weeks (3 doses). Preferably, the CTL/ central memory T cell responses induced during this period, including those directed to Wild Type epitopes, have to be maintained by continued boosting with OSE—2101 sub-cutaneous injections at 2—3 month intervals.

For the remaining 22 patients able to have a leukapheresis and an genicity testing, the overall data indicated that immune monitoring of s against five of the more immunogenically relevant epitopes in the product (CEA.24V9 (SEQ ID No 3), CEA.605D6 (SEQ ID No9), 69V2V9 (SEQ ID No 4), MAGE3.11215 (SEQ ID No 8) and MAGE2. 157 (SEQ ID No 2)) was sufficient for determining T cell and central memory T cell immunogenicity. The clinical timepoints selected for batch testing were the pre—treatment, Week 9, Week 18 and Week 30 timepoints since most CTL/central memory T cells responses observed among the first 11 patients who were tested indicated were already induced by Week 9 and Week 18 and some were maintained at Week 30.

The immunogenicity of OSE 2101 was similar in phase 2 advanced patients (NSCLC HLA A2 positive patients in advanced stage invasive or atic and after at least first line therapy failure) to that observed previously in the two phase 1/ 2 trials in terms of the overall breadth of CTL+/ central memory T cells responses induced in patients and the level of immunogenicity of dual epitopes, thus ming the overall potency of the product in different patient populations. Multi—epitope CTL+/ central memory T cells responses, defined by responses to at least 3 of 5 representative immunogenic epitopes in the OSE—2101 ent, were observed in 22 of the 33 patients (67%) who were monitored in the phase 2 trial and were achieved as early as Week 9.

Table 2 eek 9 week 18 week 30 month 9 month 12 Antigen I °/o °/o o/° °/o °/o CEA CEA24-ANA 13/331 39% 10/321 31% 9/231 28% 4/71 57% 2/41 50% CEA24-WT 12/331 36% 7/321 22% 5/231 22% 2/71 29% 1/41 0% CEA605-ANA 9/331 27% 10/321 31% 8/231 35% 1/71 14% 1/41 25% CEA605-WT 10/331 30% 5/321 16% 3/231 35% 1/71 14% 0/41 0% CEA691-ANA 7/111 64% 10/111 91% 2/61 33% 3/71 43% 2/41 50% CEA691-WT 0/111 0% 0/111 0% 0/61 0% 0/71 0% 0/41 0% HER2 HER2.369-ANA 14/331 42% 14/321 44% 13/231 57% 2/71 29% 2/41 50% HER2.369-WT 11/331 33% 9/321 28% 10/231 43% 2/71 29% 2/41 50% 89-WT 4/11136% 3/11127% 0/61 0% 0/71 0% 0/41 0% MAGE MAGE2.157-WT 9/331 27% 9/321 28% 10/231 43% 4/71 57% 2/41 50% MAGE3.112-ANA 10/331 30% 8/321 25% 10/231 43% 0/71 0% 0/41 0% MAGE3.112-WT 10/331 30% 3/321 9% 51231 22% 0/71 0% 0/41 0% p53 p53.139-ANA 6/111 55% 4/111 36% 3/61 50% 2/71 29% 2/41 50% .53.139-WT 2/111 18% 1/111 9% 0/61 0% 0/71 0% 0/41 0% p53.149-ANA 1/111 9% 0/111 0% 0/61 0% 0/71 0% 0/41 0% o53.149-WT 0/111 0% 1/111 9% 0/61 0% 0/71 0% 0/4: 0% ANA: Analog e, WT=Wi|d-type peptide The first 11 ts were tested with all 9 peptide epitopes and the remaining 22 patients were tested with 5 shaded peptide epitopes (CEA24 (SEQ ID No 3), CEA605 (SEQ ID No 9), HER2.369 (SEQ ID No 4), MAGE2.157 (SEQ ID No 2) and MAGE3.112 (SEQ ID N08)).

Results are also shown in Figure 2.

The early W9 response was at the same level as the W18 response and the long term response was still t at one year for patients able to receive a leukapheresis. The long term response was ined though additive injections.

HTL responses were measured from PBMCs without an in vitro expansion step by standard t. PBMCs were thawed, rested overnight in medium, and 2*105 PBMCs/well were ated with 10 ug/mL Pan DR epitope (HTL) or irrelevant malaria peptide in the eron gamma T assay.

IFN—y producing helper T-cells against PADRE were ed in 18 of 33 patients tested (55%), without short-term in vitro expansion of PBMCs with peptide and was in the same range at Week 9 or Week 18 demonstrating an early HTL response to the epitopes combination.

Then, a correlation between OSE-2lOl-induced PADRE HTL responses and CTL / central memory T cell ses exists (Figure 3). The correlation coefficient for this analysis is 0.405.

Example 3: impact on Time-to-Progression (TTP) though early T memory cells involvement TTP results of the short e combination in advanced NSCLC after at least first line failure (OSE 2101 phase 2 data al report). Clinically in oncology, the presence of high levels of infiltrating memory T cells, evaluated immunohistochemically, correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, and sed survival in 959 specimens of resected colorectal cancer ( Pages, F et al, 2005, N Engl J Med; 353:2654-2666). The TTP is interesting as a coherent surrogate item correlated with or memory T cells for long term and disease free survival.

Sixty-three (63) patients who were HLA—A2 and received OSE-2101 were included in the Time-to—Progression (TTP) analysis. Twenty-eight (28) patients had documented disease progression. Using the Kaplan—Meier estimator, the median time to progression was determined to be 285 days.

The median of the Time to progression (TTP median) after short peptides combination OSE— 2101 was 285 days or 9.4 months (SD 86 days). This important al item is related to the effector memory CD8+cytotoxic T cells early stimulation (TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death).

In the same type of advanced NSCLC population (invasive or metastatic) and after at least first line failure (second line treatment), the Progression-Free Survival (PFS) was described at 2.2 months with a Tyrosine kinase tor as Erlotinib (Shepherd FA, et al,, 2005, N Engl J Med.;353, 123-132.).

Two chemotherapeutic agents, xel and pemetrexed, and nib are currently approved for the second line ent of NSCLC patients. The observed TTP in the two clinical phase 3 for docetaxel was between 10.6 and 26 weeks. From pemetrexed phase 3 results, the median PFS was 2.9 months (M A Bareschino; J Thorac Dis 2011;3z122-l33). The median PFS was 3.5 months in the nivolumab phase 3 study in squamous NSCLC (Julie Brahmer, et al, 2015, New England Journal ofMedicine May 31).

The unexpected early results of CTL ion through central memory T cell induction by short epitope sequences from tumor antigens of 01 vaccine, establish the clinical utility of epitopes combination as T specific immunotherapy against cancer and is of particular interest for short term T memory immune responses providing long term efficacy in poor prognosis cancer patients.

Example 4. Results on advanced cancer patients with malignant pleural effusion (MPE).

Patients present an MPE as a complication of far-advanced cancer or as the l station of an underlying malignancy. Common cancer types causing MPEs include lymphomas, mesotheliomas, and carcinomas of the breast, lung, gastrointestinal tract, and ovaries. The annual incidence of malignant pleural effusions in the United States is estimated to be greater than 150,000 cases. al curves for more than 8,000 patients with non—small-cell lung cancer (NSCLC) with pleural effusion (i.e, stage IIIB) from the SEER database showed that long—terrn al is uncommon in this group. The median survival time is imately 3 months. Pleural effusion restricts ventilation and causes progressive shortness of breath by compression of lung tissue as well as paradoxical movement of the inverted diaphragm. Pleural deposits of tumor cause pleuritic pain. l effusions occur more commonly in patients with advanced—stage tumors, who frequently have ases to the brain, bone, and other organs; logic deficits; malnutrition; debilitation; and other comorbidities. Because of these numerous clinical and pathologic variables, it is difficult to perform prospective trials in patients with pleural effusions. For the same reason, it is often difficult to predict a potential ent outcome or anticipated duration of survival for the specific patient with multiple interrelated clinical problems. Pleural effusion was the first symptom of cancer in 41% of 209 patients with malignant pleural effusion; lung cancer in men (42%) and ovarian cancer in women (27%) were most .

The discovery of malignant cells in pleural fluid and/or parietal pleura signifies inated or advanced disease and a reduced life expectancy in patients with cancer.

In order to understand the role of central memory T cells discovered in phase 2 Cancer patients, the inventors have analyzed retrospectively a sub group of patients presenting Malignant Pleural Effusion. The study was designed to te the safety, efficacy (response and survival), and immunogenicity of OSE—2lOl in ts with advanced NSCLC (stage IIIb and IV) who were HLA-A2 positive. The multi-epitope combination was administered subcutaneously at a dose of 5 mg every 3 weeks for the first 15 weeks, then every 2 months through year 1, then rly through year 2.

A subgroup of 5 patients was presenting pleural on, 2 patients were NSCLC stage IIIB and 3 patients were NSCLC stage IV metastatic.

According to the literature, patients with ant Pleural effusions (MPEs) present a severe prognosis with intrathoracic and extrathoracic malignancies. Median survival after diagnosis of an MPE is between 3— 4 months. These patients were supposed to have the worse survival time and were thus supposed to be the first to die, y in the study.

These 5 patients with MPE were achieving, after receiving the OSE-2101 T specific immunotherapy, a long time without ssion and also a very long term survival.

T_able4A: MPE on NSCLC Patients description 166 (site115) 104 (site121) 172 (site116) 144 (site217) te217) Number NSCLC NSCLC NSCLC NSCLC NSCLC mm- female female “ 58Y 73Y merican NSCLC pleural effusion . . Pleural plus perIcardIal . Pleural efiUSIon Pleural n Pleural effusIon efoSIon effusion Chest RXth Thoracentesis pericardiocentesis Thoracentesis (5040 CY) and 3 lines of Chest Chest RXth Chest and 4 lines Carboplatin + Radiotherapy (70.2 CGY) Radiotherapy of paclitaxel Previous (10300 cGY) and 1 line of (5940 GY) chemotherapy +gemzar treatments and 1 line of chemotherapy and 1 line of latine TRM 1 chemotherapy Carboplatin + chemotherapy +pac|itaxe| investigational carboplatin + paclitaxel cisplatin + VP16 Tarceva drug; paclitaxel Topotecan Tarceva Alimta Table 4B: Time to progression — Survival OS - number of injections Patient 166 121 172 144 170 Number NSCLC NSCLC NSCLC NSCLC NSCLC “20 months 19 months 18 months 6 months 26 months Stable Disease Time to . lost of follow up 19 months 6 months 5 months 3 months progresslon at 20 months CTL + CTL+/ central CTL+/ central central CTL+/ central Memory T memory T memory T memory T versus Not done Not done Versus 4 versus 4 cell 4 epitopes es epitopes responses In s The time to progression achieved in such poor prognosis MPE population is sive with a median over than 6 months (3 to 20 months). The median survival achieved is 18 months (6 to 26 months). These long term clinical results are related to an initial priming of 3 injections though the early involvement of l memory T Cells recruiting effector cells CD8+ T cells.

In addition, 3 patients/5 are presenting strong positive responses versus 4 epitopes.

Claims (23)

1. Use of a therapeutic peptide T specific immune therapy in the manufacture of a medicament for the treatment of a cancer of an HLA-A2 (Human Leukocyte Antigen A2) positive patient, wherein the therapeutic peptide T specific immune therapy comprises the peptide aKXVAAWTLKAAa (SEQ ID No 10, with X and a respectively indicating cyclohexylalanine and d-alanine) and the peptides RLLQETELV (SEQ ID No 1), YLQLVFGIEV (SEQ ID No 2), LLTFWNPPV (SEQ ID No 3), KVFGSLAFV (SEQ ID No 4), KLBPVQLWV (SEQ ID No 5, with B indicating α-aminoisobutyric acid), SMPPPGTRV (SEQ ID No 6), IMIGHLVGV (SEQ ID No 7), KVAEIVHFL (SEQ ID No 8), and YLSGADLNL (SEQ ID No 9), and wherein said ent comprises a priming period consisting of three administrations of said therapeutic peptide T specific immune therapy, the therapeutic peptide T specific immune therapy being administered at one dose every three weeks during which a central memory T cell response is induced against one or several peptides of the therapeutic peptide T specific immunotherapy, and wherein the priming period is followed by a nance period during which the administration of said therapeutic e T ic immune therapy occurs every two to eight months.

2. The use according to of claim 1, wherein the administration of said therapeutic peptide T specific immune therapy in the maintenance period occurs every two to three months.

3. The use according to claim 1 or 2, n the administration of said therapeutic peptide T ic immune y in the maintenance period occurs every two months for one year and then every three months for one year.

4. The use according to any one of claims 1 to 3, wherein said cancer is a cancer selected from the group consisting of lung cancer such as NSCLC (non-small cell lung cancer) and small cell lung , melanoma, elioma, breast cancers, primary brain s, ovarian , uterine carcinoma, uterine corpus and/or uterine cervix carcinoma, head and neck cancer, colon or colorectal cancer, gastro-intestinal cancer, renal cancer, sarcoma, germ cell tumors, leukemia, lymphoma, testicular cancers and bladder cancers.

5. The use according to any one of claims 1 to 3, wherein said cancer is selected from the group consisting of NSCLC, colon cancer, breast cancer, ovarian cancer, and a cancer of the head and/or neck.

6. The use according to any one of claims 1 to 3, wherein said cancer is NSCLC.

7. The use according to any one of claims 1 to 6, wherein said patient suffers from an advanced or late-stage cancer.

8. The use according to any one of claims 1 to 7, wherein said t suffers from metastases.

9. The use according to any one of claims 1 to 8, wherein said t suffers from brain metastases.

10. The use according to any one of claims 1 to 9, wherein said patient has a malignant pleural effusion.

11. The use according to any one of the claims 1 to 10, wherein said patient is HTL (Helper T Cell) positive.

12. The use according to any one of the claims 1 to 11, wherein the therapeutic peptide T specific immune therapy is formulated to be d for a parenteral stration.

13. The use according to any one of the claims 1 to 11, wherein the therapeutic peptide T specific immune therapy is formulated to be adapted for a subcutaneous administration.

14. The use according to any one of the claims 1 to 13, wherein the es are emulsified in incomplete Freund’s adjuvant or the like.

15. The use according to any one of the claims 1 to 13, wherein the peptides are emulsified in Montanide ISA-51.

16. The use according to any one of the claims 1 to 15, n the doses of peptide are ranging from 0.1 to 10 mg of peptide per injection dose.

17. The use according to claim 16, wherein the total e dose for each injection is 5.0 mg.

18. The use according to any one of the claims 1 to 17, wherein said patient has already received several lines of ent prior to the treatment with the therapeutic peptide T specific immune therapy.

19. The use according to any one of the claims 1 to 18, wherein said therapeutic peptide T specific immune therapy is to be used in combination with r antitumor drug and/or with radiotherapy.

20. The use according to any one of the claims 1 to 19, wherein said therapeutic peptide T specific immune therapy is to be used in combination with a checkpoint tor.

21. The use according to claim 20, wherein the checkpoint inhibitor is a CTLA-4 and/or a PD- 1/PD-L1 tor.

22. The use according to claim 21, wherein the CTLA-4 and/or PD-1/PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, BMS936559, MEDI4736, AMP-224, AMP-514 8OA and avelumab.

23. The use according to claim 1, substantially as herein described with reference to any one of the Examples and/or