WO2016189104A1 - New method to produce t cells - Google Patents

Abstract

The present invention relates to an in vitro or ex vivo method to produce high avidity T cells for one or several antigens wherein said T cell obtained from a subject are cultivated with said one or several specific antigens and an anti-PD1 antibody.

Description

NEW METHOD TO PRODUCE T CELLS

FIELD OF THE INVENTION:

The invention relates to an in vitro or ex vivo method to produce high avidity T cells for one or several antigens wherein said T cell obtained from a subject are cultivated with said one or several specific antigens and an anti-PDl antibody. BACKGROUND OF THE INVENTION:

Two powerful immunotherapeutic approaches for the treatment of advanced melanoma rely either on in vivo blockade of check-point inhibitors on immune T cells, such as PD-1 or CTLA-4, or on the transfer of high numbers of ex-vivo expanded specific T lymphocytes (ACT).

The first function assigned to PD-1 was its involvement in immunological peripheral tolerance, maintaining T cell homeostasis by the control of auto-reactive T cells [Nishimura H et al., 1999 and Nishimura H et al., 2001]. PD-1 is expressed on thymocytes and its interaction with its ligand PD-L1, also expressed in the thymus, modulates both positive and negative selection. PD-1 is inducible on many immune cell types, such as T cells, natural killer T cells and B cells. It has two natural ligands: PD-L1, expressed on activated T cells, monocytes and dendritic cells, and PD-L2, whose expression is restricted to dendritic cells and macrophages. Ligation of PD-1 with one of its ligands results in dampening early TCR signaling, through the recruitment of the phosphatases SHP-1 and SHP-2, resulting in direct dephosphorylation of signaling intermediates. Besides its role in maintenance of physiologic self-tolerance, PD-1 is also implicated in the down-regulation of anti-tumor immunity. Indeed, PD-L1 is commonly expressed on a variety of solid tumors including melanomas [Dong H et al., 2002] contributing to immune escape and is often associated with poor prognosis [Zitvogel L et al., 2012]. Furthermore, melanoma infiltrating lymphocytes are often enriched in PD-1 expressing CD8 T cells, which are functionally impaired [Ahmadzadeh M et al., 2009]. Thus, blocking PD-1/PD-L1 pathway appears to be a promising strategy to increase the efficiency of anti-tumor T cell responses. Several clinical trials using blocking anti-PD-1 antibody reported unparalleled effectiveness for cancer immunotherapy, including melanoma, in terms of clinical response rates [Hamid O et al., 2013; Topalian SL et al., 2012; Topalian SL et al., 2014 and Wolchok JD et al., 2013]. However, long-term tumor regression using this treatment cannot be achieved in most patients, and several issues require further improvements such as the therapy to combine with anti-PD-1 treatment and the characterization of biomarkers unequivocally associated with clinical benefit.

Among the growing arsenal of therapeutic strategies, adoptive transfer of tumor specific T cells remains a real option, especially for immunogenic tumors such as melanoma. Adoptive transfer of tumor infiltrating lymphocytes (TIL) has a long clinical history and provided proofs of efficiency in adjuvant setting, with extremely long relapse-free survival for some treated patients [Dreno B et al., 2002; Khammari A et al., 2014 and Khammari A et al., 2007], and also for curative treatment of metastatic melanoma, when associated with prior lymphodepletion [Rosenberg SA et al, 2011]. Now, this approach is evolving towards an increased specificity of infused T cells that can be reached either with the use of genetically modified T cells, such as TCR or CAR-transduced T cells [Kalos M. et al, 2011; Morgan RA et al., 2006 and Robbins et al., 2011] or with enriched or cloned T cells specific for a given HLA-peptide complex [Hunder NN et al., 2008; Khammari A et al., 2009; Mackensen A et al, 2006; Meidenbauer N et al, 2003; Vignard V et al, 2005 and Yee C et al, 2002]. All these approaches could be further improved by the use of specific T cells with optimized functions, such as the avidity of infused T cells and the modulation of inhibitory receptors' expression.

SUMMARY OF THE INVENTION:

In this study, the inventors documented the epigenetic regulation of the inhibitory receptor PD-1 in melanoma specific T cell clones and the impact of PD-1 signaling on both diversity and function of a specific T cell repertoire. These results provide new insights about the role of PD-1 in tumor immunity and have strong implications in the field of cancer immunotherapy, especially in adoptive cell transfer. They notably show that with this new method, antigen specific T cells expanded more efficiently and have a higher avidity for the cognate antigen. Moreover, specific T cells obtained in the examples presented in this application, present a very strong reactive against melanoma cells. Finally, specific T cells amplified with ant-PD-1 antibody exhibited a dampened expression of PD-1 inhibitory receptor, giving them an additional advantage in terms of anti-tumor efficiency in vivo.

Thus, the invention relates to an in vitro or ex vivo method to produce high avidity T cells for one or several antigens wherein said T cell obtained from a subject are cultivated with said one or several specific antigens and an anti-PDl antibody. DETAILED DESCRIPTION OF THE INVENTION: The invention relates to an in vitro or ex vivo method to produce high avidity T cells for one or several antigens wherein said T cell obtained from a subject are cultivated with said one or several specific antigens and an anti-PDl antibody.

As used herein, the term "high avidity T cells for one or several antigens" denotes T cells which recognize a given specific antigen with an EC50 below 0.2 nM. T cells with high avidity are particularly sought and useful for immunotherapeutic strategies, because of their related strong reactivity against tumor cells expressing the target antigens.

Thus, in other words, the invention relates to an in vitro or ex vivo method to produce T cells which recognize one or several antigens wherein said T cells obtained from a subject are cultivated with said one or several antigens and an anti-PDl antibody.

The invention also relates to an in vitro or ex vivo method to produce high avidity and high lytic T cells for one or several antigens wherein said T cell obtained from a subject are cultivate with said one or several specific antigens and an anti-PDl antibody. In one embodiment, the T cells are obtained from Peripheral Blood Mononuclear Cell

(PBMC), derived from HLA-A2 donors or patients.

In one embodiment, the T cells are T CD8⁺ cells.

In another particular embodiment, the T cell obtained from a subject are cultivate with interleukin-2 (IL-2) and/or human serum.

Antibody used according to the invention According to the invention, an anti-PD-1 antibody is used to obtain high avidity T cells for one or several antigens.

Antibodies directed against the PD-1 protein can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against PD-1 protein can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-PD-1 protein single chain antibodies. Compounds useful in practicing the present invention also include anti-PD-1 protein, antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to PD-1 protein.

Humanized anti-PD-1 protein antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). In a particular embodiment, the antibody according to the invention is a blocking anti- PD-1 antibody.

Example of antigens used according to the invention

According to the invention, any type of antigen and particularly peptide antigen can be used to obtain to obtain high avidity T cells for said antigen.

In one embodiment, the antigen used according to the invention may be the immunogenic tumor antigen NY-ESO-1 (see for example Gnjatic S et al, 2006).

In a particular embodiment, antigens are melanoma antigens and especially melanoma antigen peptides. Particularly, the melanoma antigens peptides can be the melanoma antigens Melan-A (SEQ ID NO: 1), MELOE-1 (SEQ ID NO: 2), MELOE-2 (SEQ ID NO: 3) and HLa- A2 restricted peptides derived from these antigens. These antigens allow obtaining T cell specific melanoma.

Sequence of Melan-A (SEQ ID NO:l):

MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGV LLLIGCWYCRRPvNGYRALMDKSLHVGTQCALTRPvCPQEGFD HRDSKVSLQEKNCEPVVPNAPPAYEKLSAEQSPPPYSP

Sequence of MELOE-1 (SEQ ID NO: 2):

MSCVGYPDEATSREQFLPSEGAACPPWHPSERISSTLNDECWPASL

Sequence of MELOE-2 (SEQ ID NO: 3):

MSENAGGAVARTATAFCALVSPTPQPRCPPKPPLAALCQ

In one embodiment, melanoma antigens peptides comprising the amino acids motif derived from Melan-A:

- EX2AGIGILTV (SEQ ID NO: 4)

wherein X2 is leucine, methionine, valine, isoleucine or glutamine

In one embodiment, the melanoma antigens peptides has the sequence SEQ ID NO: 4. In one embodiment, melanoma antigens peptides comprising the amino acids motif derived from MELOE-1 :

- TX2NDECWPX9 (SEQ ID NO: 5)

wherein X2 is leucine, methionine, valine, isoleucine or glutamine and X9 is alanine, valine or leucine,

In one embodiment, melanoma antigens peptides comprising the amino acids motif derived from MELOE-2:

- RX2PPKPPLX9 (SEQ ID NO: 6)

wherein X2 is cysteine, leucine, methionine, valine, isoleucine or glutamine and X9 is alanine, valine or leucine.

As used herein, the term "peptide" refers to an amino acid sequence having less than 50 amino acids. As used herein, the term "peptide" encompasses amino acid sequences having less than 50 amino acids, less than 40 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids or less than 10 amino acids.

In one embodiment of the invention, by "melanoma antigen peptide" is meant a peptide capable of binding to HLA (particularly HLA-A2) molecule and causing a cellular response in a subject against melanoma.

In a first embodiment of the invention, said melanoma antigen peptide may comprise a specific motif such that the polypeptide binds an HLA molecule and induces a CTL response.

In a second embodiment of the invention, said melanoma antigen peptide may comprise a specific motif such that the polypeptide binds an HLA molecule and induces a helper T cell response.

In one embodiment of the invention, said melanoma antigen peptides as described here above are HLA-A2 restricted.

In one embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 50 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above. In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 45 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 40 amino acids long that comprises the amino acid SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 30 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 20 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of less than 15 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is an amino acid sequence of 9, 10 or 11 amino acids long that comprises the amino acid motif SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide is selected in the group consisting of peptides derives from MELOE-1 having the sequence SEQ ID NO: 7 to SEQ ID NO: 21, peptides derives from MELOE-2 having the sequence SEQ ID NO: 22 to SEQ ID NO: 39 and peptide derives from Melan-A having the sequence SEQ ID NO: 40.

MELOE-1 SEQ ID NO 7 X₂ = L X₉ = A TLNDECWPA

MELOE-1 SEQ ID NO 8 X₂ = M X₉ = A TMNDECWPA

MELOE-1 SEQ ID NO 9 X₂ = V X₉ = A TVNDECWPA

MELOE-1 SEQ ID NO 10 X₂ = I X₉ = A TINDECWPA

MELOE-1 SEQ ID NO 11 X₂ = Q X₉ = A TQNDECWPA

MELOE-1 SEQ ID NO 12 X₂ = L X₉ = V TLNDECWPV

MELOE-1 SEQ ID NO 13 X₂ = M X₉ = V TMNDECWPV

MELOE-1 SEQ ID NO 14 X₂ = V X₉ = V TVNDECWPV

MELOE-1 SEQ ID NO 15 X₂ = I X₉ = V TINDECWPV

MELOE-1 SEQ ID NO 16 X₂ = Q X₉ = V TQNDECWPV

MELOE-1 SEQ ID NO 17 X₂ = L X₉ = L TLNDECWPL

MELOE-1 SEQ ID NO 18 X₂ = M X₉ = L TMNDECWPL

MELOE-1 SEQ ID NO 19 X₂ = V X₉ = L TVNDECWPL

MELOE-1 SEQ ID NO 20 X₂ = I X₉ = L TINDECWPL

MELOE-1 SEQ ID NO 21 X₂ = Q X₉ = L TQNDECWPL

MELOE-2 SEQ ID NO 22 X₂ = C X₉ = A RCPPKPPLA

MELOE-2 SEQ ID NO 23 X₂ = L X₉ = A RLPPKPPLA

MELOE-2 SEQ ID NO 24 X₂ = M X₉ = A RMPPKPPLA

MELOE-2 SEQ ID NO 25 X₂ = V X₉ = A RVPPKPPLA

MELOE-2 SEQ ID NO 26 X₂ = I X₉ = A RIPPKPPLA

MELOE-2 SEQ ID NO 27 X₂ = Q X₉ = A RQPPKPPLA

MELOE-2 SEQ ID NO 28 X₂ = C X₉ = V RCPPKPPLV

MELOE-2 SEQ ID NO 29 X₂ = L X₉ = V RLPPKPPLV

MELOE-2 SEQ ID NO 30 X₂ = M X₉ = V RMPPKPPLV

MELOE-2 SEQ ID NO 31 X₂= V X₉ = V RVPPKPPLV

MELOE-2 SEQ ID NO 32 X₂ = I X₉ = V RIPPKPPLV

MELOE-2 SEQ ID NO 33 X₂ = Q X₉ = V RQPPKPPLV

MELOE-2 SEQ ID NO 34 X₂ = C X₉ = L RCPPKPPLL

MELOE-2 SEQ ID NO 35 X₂ = L X₉ = L RLPPKPPLL

MELOE-2 SEQ ID NO 36 X₂ = M X₉ = L RMPPKPPLL

MELOE-2 SEQ ID NO 37 X₂ = V X₉ = L RVPPKPPLL

MELOE-2 SEQ ID NO 38 X₂ = I X₉ = L RIPPKPPLL MELOE-2 SEQ ID NO 39 X₂ = Q X₉ = L RQPPKPPLL

Melan-A SEQ ID NO 40 X2 = L / ELAGIGILTV

In one embodiment, the T cells are cultivated with at least one of the melanoma antigen peptide of SEQ ID NO: 7 and SEQ ID NO: 40. The invention also encompasses peptides that are function-conservative variants of melanoma antigen peptides comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6as described here above.

Typically, the invention encompasses peptides substantially identical to melanoma antigen peptides comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the melanoma antigen peptides comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 as described here above, i.e. being still able to bind to an HLA molecule in substantially the same way as a peptide consisting of the given amino acid sequence.

Examples of conservative substitutions include the substitution of one non-polar

(hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid or another.

The term "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue. "Chemical derivative" refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Examples of such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine. Chemical derivatives also include peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

In one embodiment of the invention, the melanoma antigen peptide consists essentially of an amino acid sequence according to SEQ ID NO: 7 to 40 or a variant thereof.

According to the invention, "consisting essentially of shall mean that a peptide according to the present invention, in addition to the sequence according to any of SEQ ID No. 7 to SEQ ID No. 40 or a variant thereof, contains additional N- and/or C-terminally located stretches of amino acids that are not necessarily forming part of the peptide that functions as core sequence of the peptide comprising the binding motif and as an immunogenic epitope.

According to the invention, the melanoma antigen peptides of the invention can be obtained by synthesizing the peptides according to the method for peptide synthesis known in the art.

Culture Medium, Kit and Method for expending Tree cells

The present invention also relates to a culture medium comprising anti-PDl antibody and one or several antigens.

The culture medium of the present invention is suitable for producing high avidity T cells for said antigens.

The term "culture medium" as used herein refers to a liquid medium suitable for the in vitro culture of T cell, particularly manufactured at clinical grade. Typically, the culture medium of the invention contains:

a source of carbon as energy substrate, such as glucose, galactose or sodium pyruvate;

essential amino-acids;

- vitamins, such as biotin, folic acid, B12...;

at least a purine and a pyrimidine as nucleic acid precursors;

inorganic salts;

The culture medium may also contain pH buffers in order to maintain the pH of the medium at a value suitable for cell growth. The culture medium of the invention may be based on a commercially available medium such as RPMI 1640 supplemented with foetal calf serum.

In a particular embodiment, culture medium of the invention may contain interleukin-2

(IL-2) and/or human serum.

Another aspect of the invention relates to an in vitro method for producing T cells with a high avidity for one or several antigens wherein said method comprises the step of culturing of T cells with the culture medium as described above.

The step of culturing of T cells with the culture medium of the invention shall be carried out for the necessary time required for the production of functional T cells. Typically, the culture of T cells with the medium of the invention shall be carried out for.

Typically, the method according to the invention has three culture steps.

The first culture step is called "stimulation step". PBMC from HLA-A2 donor are seeded in 96 well-plates (0.2106/well) in RPMI medium containing antibody anti-PDl, the stimulating peptide, IL-2 (50U/mL) and human serum.

This stimulation step is a 10 to 20, particularly, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days-culture period. Cultured cells are regularly splitted on the basis of cell concentration (when > 106/mL), with fresh medium containing IL-2 and human serum. Typically 2, 3, 4 or 5 splitting during 14, 15 or 16 days.

The second culture step is a "sorting step" with HLA-peptide coated beads (Labarriere N et al 2013).

The third step is an "amplification step" on irradiated feeder cells, with anti-PDl, PHA and IL-2, of sorted T cells, with anti-PD-1 antibody. This third step is also a 14-16 days- culture period, with regular splitting.

In a particular embodiment, the antibody anti-PD-1 is added to culture medium in the three steps presented above. Another aspect of the invention is a kit comprising: (i) anti-PDl antibody and (ii) one or several antigens according to the inventions.

In a particular embodiment, anti-PD-1 antibody used for the culture of T cells may be added to culture medium several times during the time of culture, to be maintained at a concentration of 10 μ§/ηιΙ., at each cell splitting or medium replacement. Particularly, the anti-PDl antibody may be added 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times during the culture.

In a particular embodiment, the concentration of anti-PD-1 antibody is 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL, 11 μg/mL or 12 μg/mL.

In another particular embodiment, the concentration of anti-PD-1 antibody is selected between 1 μg/mL and 12 μg/mL In a particular embodiment, to amplify the T cell, PHA-L and/or Interleukin 2 may be added to the culture medium.

T cell, therapeutic uses and pharmaceutical composition

In another object, the invention relates to T cell obtainable by the method as above described.

According to the invention, T cells obtained by the method of the invention are useful to treat cancer in a subject in need thereof and specially melanoma when melanoma antigens are used.

In another embodiment, when other kind of cancer antigens is used, T cells obtained by the method of the invention are useful to treat cancer selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non- Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). In a particular embodiment, the cancer is a colorectal cancer.

As used herein, the term "melanoma" includes, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocytes related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, ocular melanoma invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome. Such melanomas in mammals may be caused by, chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, or carcinogenic agents.

As used herein the term "treating" a disorder or a condition refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition. The term "preventing" a disorder or condition refers to preventing one or more symptoms of such disorder or condition.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly a subject according to the invention is a human.

A "therapeutically effective amount" as used herein is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount of the active agent" to a subject is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the subject.

A further aspect of the present invention provides an ex vivo and/or in vivo method for treating or preventing cancer.

Thus, a further aspect of the invention relates to an ex vivo method of treating cancer comprising

(i) removing a blood sample comprising T cells from a subject;

(ii) isolating PBMC from blood sample;

(iii) treating PBMC with (i) an anti-PDl antibody and (ii) one or several antigens specific of the cancer as described above;

(iv) reintroducing the T cells so obtained (with high avidity) into said subject.

A more complete process is described in Labarriere N et al., 2013. This method is particularly useful to obtain T cells useful to treat melanoma.

In one embodiment, the invention relates to T cell obtainable by the method as described above for use in the treatment and prevention of cancer.

Another aspect of the invention relates to an in vivo method for treating or preventing cancer comprising administering to a subject in need thereof a therapeutically effective amount of T cells as described above.

According to another aspect, the present invention provides a pharmaceutical composition comprising T cells as described above and optionally a pharmaceutically acceptable carrier and the use of this pharmaceutical composition in therapy of cancer.

The therapeutic ingredients of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Administration of the T cells:

It will be understood that the total daily usage of the T cells and composition of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

In one embodiment, the cytokine T cells and the composition are administered into the subject simultaneously or sequentially.

The T cells may be administered only as a single dose to the individual. In another aspect, the T cells is administered in multiple doses, the administration of successive doses of the T cells being separated by at least 2, 3 or 4 or more weeks.

The pharmaceutical compositions of the invention may further be combined with other active ingredients, for example chemotherapeutics, anti-metastatic or anti-cancer or antiproliferative agents.

In one specific embodiment, such compound may be combined with cT cells of the invention for cancer therapy, for example, drugs selected from the group consisting of: immunotherapeutic drugs (Imids), therapeutic monoclonal antibodies, and biological therapeutics.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Amplification rates and Melan-A specific T cell diversity in presence of anti-PD-1 blocking antibody. (A) Absolute number of Melan-A specific T cells after the step of PBMC-peptide stimulation. 107 PBMC from two healthy donors and three HLA-A2 melanoma patients were stimulated in 96-well plates (2 x 105 cells/well) during 14 days with 1 μΜ of Melan-AA27Lpeptide, in presence of 10 μg/mL of anti-PD-1 Ab (hatched bars) or with 10μg/mL of control IgG (white bars). At the end of the stimulation period, the absolute number of Melan-A specific T cells was calculated from the total number of expanded T lymphocytes and the percentage of tetramer positive cells. (B, C, D, E, F) Analysis of the VB repertoire of sorted and amplified Melan-A specific T cells from melanoma patient's PBMC. A panel of 24 anti-VB antibodies was used. Empty histograms represent Melan-A specific repertoire expanded in the control condition, and hatched histograms represent the repertoire amplified upon culture in the presence of anti-PD-1 Ab. Arrows indicate VB subfamilies specifically amplified in one or the other of these two culture conditions, used for further analyses.

Figure 2: Avidities of the different VB subfamilies specifically expanded in the two culture conditions. Avidities of specific VB subfamilies amplified in the control condition (dotted lines) or in the presence of anti-PD-1 antibody (solid lines) were evaluated by measuring IFN-γ (A, D), TNF-a production (B, E) and CD 107a membrane expression (C, F) in response to T2 cells loaded with a range of Melan-AA27L peptide, at an E:T ratio of 1 :2. Cytokine production and CD 107a membrane expression were evaluated by double staining with specific anti-VB antibodies and intracellular or membrane labeling.

Table I: Proportion and EC50 of Melan-A specific VB subfamilies amplified in the two different culture conditions

EC50 were determined after activation of Melan-A specific T cell lines by T2 cells loaded with a range of Melan-AA27L peptide (5 hours). The fraction of IFN-γ, TNF-a and CD 107a positive cells among a specific VB subfamily was evaluated by flow cytometry, by double labeling. The % indicates the proportion of each VB subfamily among all Melan-A specific T cells.

EXAMPLE:

Examples of production of Melan-A- and MELOE-l-specific T cells: Material & Methods

PBMC and Cell lines

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation from healthy HLA-A2 donors (Etablissement Francais du Sang (EFS), Nantes, France) or from metastatic melanoma patients (Unit of Skin Cancer, Nantes hospital) after written informed consent (Nantes ethic committee, approval number: DC-2011-1399).

The melanoma cell line Ml 13 was established from metastatic tumor fragments in the Unit of Cell therapy of Nantes and are registered in the BiocoUection PC-U892-NL (CHU Nantes).

The human TAP deficient cell line T2 (174 x CEM.T2) used as a presenting cell was purchased from the ATCC (CRL-1992). Stable cell lines expressing human PD-Ll were established from Ml 13 and T2 cell lines. Briefly, Ml 13 and T2 cells were transfected using lipofectamine, according to the manufacturer's recommendation (Life technologies, France) with an eukaryotic expression vector (pCDNA3) bearing human PD-Ll gene (NM 14143.2), purchased from Sino Biological (Beijing, China). Stable transfectants expressing PD-Ll were selected and cultured in medium containing O^g/mL of G418 antibiotic.

Peptide stimulation of PBMC

PBMC were seeded in 96 well/plates at 2 x 105 cells/well in RPMI 1640 medium supplemented with 8% human serum (HS), 50 IU/mL of IL-2 (Proleukin, Novartis, France) and 10 μg/mL of either anti-PD-1 antibody (Clone EH12.2H7, Biolegend, France) or 10μg/mL of IgGl control istotype (Biolegend, France). PBMC were stimulated by adding 1 μΜ of Melan-AA27L peptide (ELAGIGILTV, SEQ ID NO: 40) or 10 μΜ of MELOE-l₃₆-44 peptide (TLNDECWPA, SEQ ID NO: 7). Peptides were purchased from Proteogenix (Schiltigheim, France). Following stimulation, each microculture was evaluated for the percentage of specific CD8 T lymphocytes by double staining with the relevant HLA-peptide tetramer (from the SFR Sante recombinant protein facility) and anti-CD8 mAb (clone BW135/80, Miltenyi Biotec, France) using a FACS Canto HTS. Microcultures that contained at least 1% of Melan-AA27L or MELOE-136-44 specific T cells were selected, pooled and sorted with the relevant multimer-coated beads (35).

Sorting, amplification and cloning of specific T cells

Sorting of Melan-A and MELOE-1 specific T cells was performed as previously described (35, 40). Sorted specific T cells were seeded at 1000 T cells/well in 96 well plates for polyclonal amplification on feeder cells with 1 μg/mL of PHA-L (Sigma, France) and 150 IU/mL of IL-2 (Novartis) as previously described (35). To isolate and expand Melan-A and MELOE-1 specific T cell clones from specific sorted T cells, we used a limiting dilution cloning method previously described (49). Melan-A and MELOE-1 specific T cell clones, from microcultures showing greater than 95% probability of monoclonality according to the single-hit Poisson law, were selected on the basis on specific tetramer labeling. T cell clones were further expanded into new plates with freshly irradiated feeder cells, IL-2 and PHA-L.

Activation of antigen specific T cells

Phenotypic and functional analyses were performed on resting or activated T cells.

Antigen specific T cells were activated 6 hours in 96 well plates with either coated anti-CD3 antibody (clone OKT3, CRL-8001, ATCC) at ^g/mL, addition of 1 μΜ of Melan-AA27L peptide (ELAGIGILTV, SEQ ID NO: 40) or 10 μΜ of MELOE-136-44 peptide (TLNDECWPA, SEQ ID NO: 7), co-culture with the Ml 13 melanoma cell line presenting specific peptides at two effector/target ratios (1/1 and 1/2) or with addition of ^g/mL of phorbol myristate acetate and calcium ionophore (PMA-Cal) (Sigma Aldrich, USA).

Phenotype and Vfi repertoire of specific T cells

The specificity of stimulated microcultures, sorted T cell lines and T cell clones was assessed by double labeling with MELOE-1 and Melan-A tetramers (10 μg/mL) (Recombinant protein facility, SFR Sante, Nantes, France) and anti-CD8 specific antibody (clone BW135/80, Miltenyi Biotec, France). PD-1 expression was tested on specific T cell clones or sorted T cells at rest and after activation either by double labeling with anti-CD25 (clone M-A251, BD Biosciences, France), as activation marker, and anti-PD-1 antibody (Clone EH12, BD Biosciences), or by a quadruple labeling with specific tetramers, anti-CD8, anti-PD-1 and anti-CD25 antibodies. All the antibodies were used at a concentration of 5μg/mL. Vbeta diversity of sorted Melan-A specific T cell lines was analyzed by labeling with 24 anti-Vb mAbs included in the IOTest Beta Mark TCR V Kit (Beckman-Coulter, Marseille, France). The staining protocol includes a one-step procedure with directly conjugated antibody mixes (45 min at 4°C) and a wash step with PBS, 0.1%BSA. All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).

Real-time PCR

Total RNA was extracted from antigen specific T cells using NucleoSpin RNA II kit

(Macherey-Nagel, France), ^g of total RNA was retrotranscribed using Superscript III reverse transcriptase and oligodT (Life technologies, France). Relative quantification of PD-1 and house keeping genes RPLPO and Cyclophilin-A was performed using brilliant SYBR Green qPCR with an Mx4000 machine (Agilent Technologies France). lOng of each cDNA sample were added to RT2 Sybr Green Master Mix (Agilent Technologies) with 200nM of specific primers. PD-1 specific primers were purchased from Qiagen (catalog number PPH13086G, USA). Thermal cycling was one step at 95°C for 10', followed by 40 cycles at 95°C for 30"and 60°C for 1 '. Duplicate series of 10-fold-diluted cDNA from the Melan-A specific T cell clone HA1 at rest were used to calculate the efficiency of PCR reaction. Mean threshold cycle (CT) values from duplicate PCR reactions were normalized to mean CT values for the two housekeeping genes from the same cDNA preparations. The relative expression ratio of PD-1 gene was calculated based on the PCR efficiency (E) and the CT deviation between a given cell sample (x) and a reference cell sample (calibrator: HA1 resting T cell clone), expressed in comparison with the mean of the housekeeping genes: ratio = (E target) Δ CT target (calibrator - x) / mean ((E housekeeping) Δ CT housekeeping (calibrator - x)).

Methylation status analyses

DNA from specific T cells was extracted using QiaAmp DNA mini kit (Qiagen, France). Methyl-Collector Bisulfite modification kit (Active Motif, Belgium) was used for DNA conversion. DNA converted samples were amplified by two successive PCR with specific primers. Thermal cycles for PCR1 were one step at 95°C for 5', followed by 20 cycles at 95°C for 30", 63°C for 2' and 72°C for 1 '30. Thermal cycles for PCR2 were one step at 95°C for 5', followed by 20 cycles at 95°C for 30", 57°C for 1 ' and 72°C for 1 '30. Amplimers were cloned into pSC-B-Amp/Kan vector (Agilent Technologies France) and a minimum of twelve clones for each sample were sequenced (Eurofms scientific, France).

Specfic T cell avidity and reactivity

IFN-γ secretion of activated T cells was measured by a specific ELISA assay (Human

IFN gamma ELISA Ready-SET-Go, eBioscience, France), according to the supplier recommendation, in supematants, after 6h of activation of specific T cells with either Ml 13 and Ml 13-PD-Llpos melanoma cell line or Tap-deficient T2 cell line and its T2-PD-L1 transfected counterpart, loaded with ΙΟμΜ of MELOE-136-44 peptide. The relative avidity of T cell clones and sorted T cells was measured by intracellular IFN-γ and TNF-a production and CD 107a membrane expression, in response to T2 cells loaded with a range of specific peptides (E/T ratio 1/2). For intracytoplasmic cytokine staining, after a 6h-stimulation period with peptide loaded T2 cells, in presence of brefeldin A at 10 μg/mL (Sigma, St Louis MO, USA), T cells were labeled with PE-conjugated specific anti-VB antibodies (Beckman Coulter). Cells were then fixed for 10 min at room temperature in PBS 4% paraformaldehyde (Sigma, France). Fixed lymphocytes were stained for cytokine production using APC conjugated anti-TNF-a (clone cA2, Miltenyi Biotec) and anti-IFN-γ (clone 45-15, Miltenyi Biotec). Concerning CD 107a mobilization experiment, specific T cells were stimulated at a E/T ratio of 1/2 with peptide loaded T2 cells for 4 h at 37°C in the presence of APC- conjugated mAb specific for CD 107a (clone H4A3, BD Biosciences, France). The T cells were then stained with selected anti-VB antibodies (Beckman coulter) and analyzed by flow cytometry.

Statistics

Statistical analyses were conducted to compare the percentages of global methylation of the 23 CpG dinucleotides of the PD-1 regulatory region, between PD-lpos and PD-lneg T cell clones (at least 33 sequences analyzed in each group), and PD-lpos and PD-lneg Melan- A specific sorted T cells (at least 12 sequences analyzed in each group). These analyses were performed using a non parametric Mann- Whitney t test, with two-tailed p value.

Results

PD-1 is differentially expressed on melanoma specific T cells clones We recently developed a clinical grade procedure leading to the production of high numbers of polyclonal CD8 T lymphocytes specific for the two melanoma antigens Melan-A and MELOE-1, within 30 days of culture using a selection method based on peptide stimulation of PBMC followed by HLA-Peptide specific multimer sorting [Labarriere N et al., 2013]. Produced T lymphocytes were fully specific and reactive against melanoma cell lines expressing these two widely shared melanoma antigens [Godet Y et al., 2008 and Kawakami Y et al, 1994].

In this study, we used this procedure to produce Melan-A and MELOE-1 specific T cells from PBMC from an HLA-A2 healthy donor and a melanoma patient. After the initial peptide stimulation step, lymphocytes enriched in antigen specific T cells were sorted and amplified (data not shown). At the end of the amplification procedure, CD8 T cells were fully specific for the cognate antigen (data not shown). A fraction of specific T cells expressed the PD-1 molecule at rest (attested by the absence of CD25 expression), which could potentially impair their functional reactivity against melanoma tumors in vivo, whereas another fraction was PD-l^neg (data not shown). With the aim to optimize the functions of tumor reactive T cell effectors for adoptive transfer, we sought to investigate molecular mechanisms leading to the absence of PD-1 expression and to thoroughly document the functions of these PD-lneg T cells. We thus derived Melan-A and MELOE-1 specific T cell clones by limiting dilution from polyclonal specific T cells, initially produced according to the procedure mentioned previously. At rest (CD25^neg), the percentage of PD-1 expression was very variable from one clonotype to another but remained very stable for a given clonotype (n=7). Globally, PD-l^pos and PD-l^neg T cell clones exhibited the same phenotype of effector-memory T cells (CD45RO^pos, CD27^neg, CD28^low, CD62-Li_0W) and PD-1 expression was not associated with other exhaustion or inhibition markers (CTLA-4^neg, BTLA^low, Tim-3^low, CD95^low) (data not shown). In order to explore molecular mechanisms explaining the marked difference in PD-1 expression between these specific T cell clones, we selected three pairs of PD-l^pos and PD- l^neg specific T cell clones, from the same healthy donor or melanoma patient. We tested the ability of these T cell clones to express PD-1 when stimulated by various stimuli: specific peptides, anti-CD3 antibody (OKT3), melanoma cell lines expressing Melan-A and MELOE- 1 antigens or PMA-Cal. The fraction of PD-1 expressing T cells increased upon stimulation for PD-l^pos T cell clones, regardless of the stimulation mode, whereas PD-l^neg T cell clones remained unable or poorly able to express this molecule even when bypassing TCR signaling using PMA-Cal stimulation. This suggested either a negative control of PD-1 expression at the transcriptional level or a defect of PD-1 export at the cell surface in these specific T cell clones. We thus further explored the expression of the PD-1 gene in these T cell clones at rest and after stimulation.

PD-1 expression on melanoma specific T cell clones is regulated by epigenetic mechanisms

We analyzed PD-1 expression by RT-qPCR in PD-lpos and PD-lneg T cell clones, at rest and after 6 hours of OKT3 stimulation. Activation status was systematically assessed by CD25 labeling. Results were normalized on PD-1 expression levels detected, at rest, in the Melan-A specific T cell clone HA1 (PD-lpos). At rest, we could not detect PD-1 expression in PD-l^neg T cell clones (2C9, 1D12 and 2A1), whereas PD-1 mRNA was already present in PD-l^pos T cell clones (HA1, 3A12 and 4D1). Upon stimulation, PD-1 relative expression increased in PD-lpos T cell clones, whereas it remained hardly detectable in PD-lneg ones. These results are in accordance with those obtained by flow cytometry, suggesting that the absence of PD-1 expression in PD-l^neg T cell clones was mainly due to a negative transcriptional control. It has been previously described in virus-specific CD8+ T cells that PD-1 expression was controlled by the methylation status of the gene promoter [Youngblood B et al, 2013 and Youngblood B et al, 2011]. We thus investigated this status on genomic DNA derived from both types of T cell clones using bisulfite sequencing methylation analysis of the 23 CpG nucleotides present in the PD-1 conserved regulatory region (data not shown). Globally, at rest and after TCR stimulation, PD-l^neg T cell clones (data not shown) exhibited a significantly higher proportion of methylated CpG dinucleotides within this regulatory region than PD-l^pos T cell clones (data not shown). Results obtained show the methylation status of each CpG position for individual clonotypes, and shows that most CpG nucleotides displayed differences in methylation status between PD-lneg and PD-lpos clonotypes, especially from positions 15 to 21 (data not shown). Upon TCR stimulation, the methylation status of the regulatory region only slightly decreased in one PD-lneg T cell clones (1D12, data not shown), an observation consistent with the low PD-1 expression observed by qPCR in this T cell clone after stimulation (data not shown). These data indicate that there are marked differences in the epigenetic regulation program of the PD-1 gene, among an antigen specific T cell repertoire. We further explored the reactivity and affinity of these T cell clones. pp. ^eg _Sp_ecjfi_c j _ceii clones exhibit lower avidity than PD-l^pos clones

In order to compare the reactivity of PD-lpos and PD-l^neg against a natural target expressing a PD-1 ligand, and since cultured melanoma cell lines lose the expression of endogeneous PD-L1 molecule, we stably transfected a HLA-A2+ melanoma cell line (Ml 13) expressing Melan-A and MELOE-1 antigens (Godet Y et al., 2008 and Bouquie R et al., 2009) with a PD-L1 expression plasmid. The selected cell line stably expressed PD-L1 (data not shown) and similar levels of antigens, together with similar levels of co -stimulation molecules (HLA-A2, ICAM-1, LFA-3) as compared to their non transfected counterparts (data not shown). The reactivity of T cell clones was measured against wild type (data not shown) and transfected cell lines (data not shown) by an IFN-γ specific ELISA test, after a 6hr activation period. As expected, IFN-γ production by Melan-A specific PD-lneg T cell clones (2C9 and ID 12) was not altered by PD-L1 expression on melanoma cells (data not shown) whereas IFN-γ production by PD-lpos T cell clones (specific for Melan-A and MELOE-1) was substantially decreased upon activation with PD-L1 expressing melanoma cell line (solid lines, right panel). Interestingly, we observed that, globally, Melan-A specific PD-lpos T cell clones produced higher levels of IFN-γ compared to PD-lneg clones. Concerning the pair of MELOE-1 specific T cell clones, we did not observed any IFN-γ production by the PD-l^neg T cell clone (2A1) when compared to the PD-l^pos one (4D1). This suggested that the number of specific HLA-peptide complexes naturally presented by the Ml 13 melanoma cell line was too low to activate the 2A1 specific T cell clone, probably due to its low avidity. We thus retested the reactivity of the two MELOE-1 specific T cell clones on TAP-deficient T2 cells stably transfected or not with PD-L1 (data not shown) and loaded with 10 μΜ of MELOE-136-44 specific peptide. Results showed that both clones produced IFN-γ upon stimulation with peptide-pulsed T2 cells and that only the reactivity of 4D1 T cell clone (PD-l^pos) was affected by PD-L1 expression on T2 cells. Furthermore, as observed for Melan-A specific T cell clones, the PD-l^pos T cell clone (4D1) was slightly more reactive than the PD-l^neg one (2A1), in terms of global IFN-γ production on loaded wild type T2 cells. Taken together, these results suggest that PD-l^pos specific T cell clones may be of higher avidity than PD-l^neg ones. We thus formally tested the avidity of these different T cell clones on T2 cells loaded with a range of specific peptides (data not shown). For each pair of specific T cell clones, the PD-l^pos clone was of higher avidity than the PD-l^neg one, as evidenced by EC50 values calculated for IFN-γ and TNF-a production (data not shown). Indeed, EC50 values measured for PD-l^pos specific T cell clones were from 4 to 15 times lower for IFN-γ production, and from 4 to 7 times lower for TNF-a production (data not shown). Therefore, PD-l^neg specific T cell clones would not represent the optimal effectors to select for adoptive transfer. We thus sought to modify the production process of specific T cells in order to allow the expansion of high avidity specific T cells with reduced PD-1 expression. The addition of PD-1 blocking antibody during the peptide stimulation step enhanced the proliferation of antigen specific T cells

As previously mentioned, the procedure used to grow melanoma-specific T cells is a two-step process including a first step of peptide-stimulation of melanoma patient's PBMC, and a second step of sort and amplification of specific T lymphocytes. We first compared the amplification rates of Melan-A-specific T lymphocytes expanded according to the standard peptide-stimulation step or to a modified procedure in which an anti-PD-1 blocking antibody was added together with the peptide. This comparison was performed on PBMC derived from 3 melanoma patients. In each case, the absolute number of Melan-A specific T cells (calculated from the total number of T cells and the fraction of tetramer-positive lymphocytes at the end of the peptide stimulation step) was higher when the PD-1 blocking antibody was added (Figure 1A). This absolute number was from 2 to 9 times greater in this new culture condition, as compared to the control condition. Thus, PD-1 blockade enhances the specific T cell expansion induced by peptide stimulation of patients' PBMC.

The addition of PD-1 blocking antibody modifies the Melan-A specific T cell repertoire expanded in vitro

The increased proliferation of specific T lymphocytes, observed when cultured with anti-PD-1 antibody, prompted us to compare the diversity of the specific T cell repertoire amplified in both conditions. To this aim, specific T cells were sorted as previously described, and amplified on feeder cells with or without anti-PD-1 blocking antibody. After this amplification step, recovered T lymphocytes were fully specific of Melan-A antigen as documented by specific tetramer labeling. Melan-A specific T cell repertoire was significantly modified when T cells were produced in the presence of anti-PD-1 antibody (Figure 1), as compared to the control condition (Figure 1) for healthy donors and patients. Concerning healthy donor HD49 (IE), Melan-A specific T cell repertoire is polyclonal in both conditions, with VB14 lymphocytes as the dominant subfamily in the control condition (28%) and VB7.1 (6.5%) and VB16 (6%) subfamilies specifically amplified in the "anti-PD-1" condition. Specific T cell repertoire was much narrower in HD52 donor (IF), with VB14 subfamily shared in both conditions, and VB23 and VB7.2 subfamilies specifically amplified respectively in the control and "anti-PD-1" conditions. Thus, blocking PD-1 pathway during the amplification process of Melan-A specific T cells appeared to modify specific T cell repertoire. We then conducted the same experiments on PBMC derived from 3 melanoma patients and confirmed this result. For each patient, we observed the specific proliferation of Vbeta subfamilies that were under-represented or absent in the control condition. For the patient P2, Melan-A specific T cells were globally poorly polyclonal, with a dominant (86%) VB13.1 subfamily in the control condition and two distinct dominant Vbeta subtypes in the "anti-PD-1" condition: VB13.2 (15%) and VB14 (75%) (data not shown). Melan-A specific T cells from patient P3 (data not shown) were more polyclonal with common and well- represented Vbeta subtypes in both conditions (VB13.1 and VB14). Nonetheless we also observed for this patient a specific proliferation of peculiar VB subtypes in each given condition. In the control condition, VB3 and VB17 lymphocytes represented respectively 28% and 16% of Melan-A specific T cells whereas there were almost absent in the "anti-PD-1" condition. Conversely, Melan-A specific VB23 T cells were specifically amplified (16%) in this latter culture condition. Finally, specific repertoire differences were even more pronounced for patient P4, with a polyclonal repertoire for the control condition, and a major amplification of VB20 Melan-A specific T cells (85%) for the « anti-PD-1 » condition. However, for this patient, it should be noted that our panel of 24 VB antibodies was insufficient to characterize the full VB repertoire in the control condition since the total of identified VB subtypes represented only 53% of the whole population.

As the use of an anti-PD-1 blocking antibody in our selection and amplification procedure clearly favored the proliferation of peculiar VB subfamilies, we decided to compare the avidity of these subpopulations to that of VB subtypes preferentially expanded in the control condition (Figure IB, C, D, E, F).

The addition of PD-1 blocking antibody favors the amplification of high avidity clonotypes

We tested the affinity of VB subfamilies specifically enriched in the two culture conditions on T2 cells loaded with a range of Melan-AA27L peptide, using double staining with a specific anti-VB antibody together with IFN-γ (left panel), TNF-a (middle panel) or CD 107a labeling. Solid lines on Figure 2 (A-F) represent VB subpopulations specifically expanded with anti-PD-1 antibody and dotted lines VB subpopulations preferentially expanded in the control condition. For healthy donors (HD49 and HD52) VB subfamilies selected in the presence of anti-PD-1 mAb exhibited better functional avidities than those amplified in the control condition with a difference in EC50 ranging from 2 to 15 for each tested function, reaching statistical significance for VB16 (IFNg and CD 107a) and VB7.1 (CD 107a) subfamilies from HD49 and for VB7.2 (for the three tested functions) from HD52. Concerning patient P2, the VB14 subpopulation (amplified in the presence of anti-PD-1 antibody and representing 78% of Melan-A specific T cells) exhibited a slightly better EC50 than the other VB families, both in terms of TNF-a and IFN-γ production (Figure 2 and Table I). However, no difference could be found for CD 107a membrane expression, possibly because this function is less demanding in terms of avidity. Similarly, VB23 specific T lymphocytes expanded from patient P3 in the presence of anti-PD-1 antibody also exhibited a better EC50, for the three tested functions, than the two VB subpopulations (VB17 and VB3) expanded without anti-PD-1. Finally, VB20 lymphocytes, largely represented (85%) in the "anti-PD-1" condition for patient P4, also had a better EC50 than VB14 and VB13.2 T lymphocytes amplified in the control condition (Figure 2 and Table I).

Taken together, these results strongly suggest that PD-1 blockade favors the expansion of PD-lpos specific T lymphocytes with higher avidity, probably because in the absence of PD-1 blocking antibody, PD-1 signaling inhibits the proliferation of these T lymphocytes. The addition of PD-1 blocking antibody dampens PD-1 expression on specific T cells

We further explored the expression of PD-1 on sorted and amplified T cells at rest. As illustrated results, PD-1 expression was clearly down-modulated on Melan-A specific T cells following blockade of PD-1/PD-L1 interaction during the stimulation and expansion steps in vitro. Indeed, Melan-A specific T cells from patients P2 and P3 did not express PD-1 at all, whereas, 76%> and 20%> of specific T cells respectively expressed PD-1 when generated in the control condition. For patient 4, PD-1/PD-L1 blockade had a less pronounced effect on PD-1 expression in terms of percentage of positive cells (56%> with anti-PD-1 vs 67%> without) although we observed a marked difference in the two culture conditions in terms of fluorescence intensity suggesting a decreased density of PD-1 molecules on T cells expanded with the blocking antibody.

In order to exclude that persistent binding of PD-1 antibody to expanded T cells may have prevented subsequent PD-1 staining during the cytometric analysis, we labeled these T cells with a PE-conjugated anti-mouse antibody alone. We did not detect any labeling with this secondary antibody, thus documenting the absence of bound PD-1 antibody on the T cells at the end of the amplification period (data not shown).

We thus investigated the mechanisms responsible for this PD-1 down-modulation. For patient P2, the expression of PD-1 gene, measured by qPCR correlated with membrane expression, with a lower expression of PD-1 gene in T cells produced in the presence of anti- PD-1 antibody (data not shown). For patient P3, the relative expression of PD-1 was low and similar in both culture conditions, and did not reflect the differences seen at the protein level by cytometry (data not shown). For patient P4, PD-1 gene expression was high in both cases. We further analyzed the methylation status of PD-1 promoter in cells generated in the two culture conditions (data not shown). In agreement with the transcriptional analysis, we did not observe significant differences in the PD-1 promoter methylation status between the specific T cells from patients P3 and P4 expanded with or without anti-PDl antibody, although a slight but non significant increased methylation could be noticed for T cells from P3 patient, when amplified with anti-PD-1 antibody. For patient P2, we observed a significant (p<0.5) increase in PD-1 promoter methylation for specific T cells cultured with anti-PD-1 antibody, especially at the CpG positions 20 to 22 (data not shown). This feature probably contributed to the decreased transcription of the PD-1 gene detected in these lymphocytes (data not shown) and suggests that a progressive methylation of PD-1 promoter can occur when PD-1 blockade is maintained throughout the production procedure.

However, in discrepancy with what we previously observed for T cell clones, transcriptional regulation was probably not the only mechanism explaining PD-1 down- modulation. Of note, we observed a progressive re-expression of PD-1 on specific T cells, when activated for longer periods in the absence of PD-1 blocking antibody. This suggested that the presence of the anti-PD-1 blocking antibody in the culture medium was mandatory to maintain the low PD-1 expression on specific T cells. Indeed, we documented the presence of PD-1 antibody in the culture medium all along the amplification period, decreasing proportionally to cell splitting and dilution (data not shown).

Altogether, these results suggested that although transcription of PD-1 may be affected by PD-l/PD-Ll blockade, it is clearly not the sole mechanism responsible for the down modulation of PD-1 expression.

The reactivity of Melan-A specific T cells produced with anti-PD-1 antibody is less or not affected by PD-L1 expression on target cells

Finally, in order to analyze the impact of PD-1 down-modulation on the reactivity of specific T cells towards a PD-L1 expressing target, we assessed the IFN-γ production of the six Melan-A specific T cell lines in response to melanoma cell lines expressing or not the PD- Ll molecule. As shown in the results, the reactivity of Melan-A specific T cells produced without anti-PDl antibody was inhibited in response to Ml 13-PD-Ll^pos melanoma cell line. Inhibition level was proportional to PD-1 expression detected on these T cell lines (data not shown). Consistent with the absence of PD-1 expression on specific T cells amplified with anti-PD-1 antibody from patients P2 and P3, we did not observe any inhibition of IFN-γ production in response to PD-L1 transfected melanoma cell line (data not shown). By contrast, reactivity of specific T cells amplified with anti-PD-1 antibody from patient P4 was inhibited by the presence of PD-L1 on melanoma cells, although to a lesser extent than cells expanded in the control condition. This inhibition was consistent with the residual PD-1 expression observed on these T cells (data not shown). Overall these results suggest that T cells amplified with anti-PD-1 antibody, in addition to their higher avidity, are less sensitive to PD-l/PD-Ll signaling, and therefore could be use in ACT treatments in order to achieve an efficient anti-tumor response.

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Claims

CLAIMS:

An in vitro or ex vivo method to produce high avidity T cells for one or several antigens wherein said T cell obtained from a subject are cultivated with said one or several specific antigens and an anti-PDl antibody.

An in vitro or ex vitro method according to claim 1 wherein the T cell obtained from a subject are cultivated with at least one of the peptides of SEQ ID NO: 7 and SEQ ID NO: 40.

T cell obtainable by the method according to claims 1 to 2.