WO2020048942A1 - Procédés et compositions pharmaceutiques visant à améliorer les réponses immunitaires dépendantes des lymphocytes t cytotoxiques - Google Patents

Procédés et compositions pharmaceutiques visant à améliorer les réponses immunitaires dépendantes des lymphocytes t cytotoxiques Download PDF

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WO2020048942A1
WO2020048942A1 PCT/EP2019/073392 EP2019073392W WO2020048942A1 WO 2020048942 A1 WO2020048942 A1 WO 2020048942A1 EP 2019073392 W EP2019073392 W EP 2019073392W WO 2020048942 A1 WO2020048942 A1 WO 2020048942A1
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cells
malignant
cell
carcinoma
tumor
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Abdel-Majid Khatib
Serge EVRARD
Maria Mercedes TOME MONTESINOS
Fabienne SOULET
Géraldine SIEGFRIED
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université De Bordeaux
Institut Bergonie
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to methods and pharmaceutical compositions for enhancing cytotoxic T lymphocyte-dependent immune responses, in particular, in patients suffering from cancer.
  • T cells can differentiate into multiple types of effector and memory T cells, which help to kill antigen-presenting cells (Kaech and Cui, 2012), directly and indirectly.
  • Immune checkpoints or co-inhibitory receptors such as cytotoxic T lymphocyte antigen (CTLA)-4 and programmed death (PD-l), play important roles in regulating T cell responses. They were proven to be effective targets in treating various cancers; however, prolonged stimulation of T cells due to chronic infections or cancer results in gradual suppression of the cell’s effector function, a process known as“exhaustion” (Wherry, 2011).
  • inhibitory receptors serve as an immune checkpoint which regulate T cell effector function
  • therapies targeting PD-l were clinically effective in various preclinical models and cancer patients, the underlying mechanism involved in total remission of cancers after immunotherapy is presently unclear.
  • majority of patients with solid tumors including colorectal cancers (CRCs, except the microsatellite -instable subset) are refractory to these treatments (Xiao et ah, 2015).
  • CRCs colorectal cancers
  • the failure to respond to anti-PD-l therapy is, in part, due to the presence of irreversibly exhausted T cells.
  • TEE tumor microenvironment
  • NFAT nuclear factor of activated T cells
  • Blimp-l B lymphocyte -induced maturation protein-l
  • Forkhead box protein 01 FoxOl; Box 2
  • Various intracellular signal transduction pathways regulate expression and activation of these factors. They include tyrosine kinase, anti inflammatory signal, and mitochondrial apoptosis pathways.
  • PCs proprotein convertases
  • PC family consists of 7 members, namely, furin, PC1, PC2, PC4, PACE4, PC5 and PC7 that convert their unprocessed substrates into functional molecules by cleaving their basic amino acid motifs (K/R)-(X)n-(K/R)j, where n is 0, 2, 4, or 6 and X is any amino acid (Seidah and Prat, 2012; Scamuffa et ah, 2006; Scamuffa et ah, 2008). These enzymes play an influential role not only in maintaining homeostasis but also in various pathological conditions (Seidah and Prat, 2012; Scamuffa et ah, 2006; Scamuffa et ah, 2008).
  • PCs activate proteins involved in malignant transformation and progression including cell surface-expressed receptors (e.g., integrins), matrix metalloproteinases (MMPs), growth factors and receptors required for tumor angiogenesis including PDGFs, VEGF-C and IGF-l Receptor (Seidah and Prat, 2012; Scamuffa et ah, 2006; Scamuffa et ah, 2008).
  • integrins integrins
  • MMPs matrix metalloproteinases
  • growth factors and receptors required for tumor angiogenesis including PDGFs, VEGF-C and IGF-l Receptor (Seidah and Prat, 2012; Scamuffa et ah, 2006; Scamuffa et ah, 2008).
  • Altered PC levels were reported to be associated with enhanced invasion and proliferation in various tumor cells.
  • the present invention relates to methods and pharmaceutical compositions for enhancing cytotoxic T lymphocyte-dependent immune responses, in particular, in patients suffering from cancer.
  • the present invention is defined by the claims.
  • PC proprotein convertase
  • PD-l immune checkpoint protein
  • the first object of the present invention relates to a method of enhancing the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one proprotein convertase (PC) inhibitor.
  • CTLs cytotoxic T lymphocytes
  • the present invention provides a method of therapy in subjects in need thereof, comprising administering to the subject a therapeutically effective amount at least one proprotein convertase (PC) inhibitor that reduces the expression of an immune checkpoint protein, wherein said administration enhances the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in the subject.
  • PC proprotein convertase
  • Another object of the present invention is a proprotein convertase (PC) inhibitor that reduces the expression of an immune checkpoint protein for use in the treatment of a disease such as a cancer.
  • the PC inhibitor for use according to the invention enhances the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in the subject.
  • CTLs cytotoxic T lymphocytes
  • Another object of the present invention is a pharmaceutically acceptable composition
  • the composition for use according to the invention enhances the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in the subject
  • the present invention provides a method of reducing T cell exhaustion in a subject in need thereof comprising administering to the subject a therapeutically effective amount at least one proprotein convertase (PC) inhibitor.
  • PC proprotein convertase
  • cytotoxic T lymphocyte As used herein, the term“cytotoxic T lymphocyte” or“CTL” has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface.
  • CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. They are MHC class I- restricted, and function as cytotoxic T cells. Cytotoxic T lymphocytes are also called, CD8+ T cells, T-killer cells, cytolytic T cells, or killer T cells.
  • the ability of the proprotein convertase (PC) inhibitor to enhance proliferation, migration, persistence and/or cytotoxic activity of cytotoxic T lymphocytes may be determined by any assay well known in the art.
  • said assay is an in vitro assay wherein cytotoxic T lymphocytes are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by cytotoxic T lymphocytes).
  • target cells e.g. target cells that are recognized and/or lysed by cytotoxic T lymphocytes.
  • the proprotein convertase (PC) inhibitor can be selected for the ability to increase specific lysis by cytotoxic T lymphocytes by more than about 20%, preferably with at least about 30%, at least about 40%, at least about 50%, or more of the specific lysis obtained at the same effector: target cell ratio with cytotoxic T lymphocytes that are contacted by the proprotein convertase (PC) inhibitor of the present invention. Examples of protocols for classical cytotoxicity assays are conventional.
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-l dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et ah, 2011. Nature 480:480- 489).
  • inhibitory checkpoint molecules include B7-H3, B7-H4, BTLA, CTLA-4, CD277, KIR, PD-l, LAG-3, TIM-3, TIGIT and VISTA.
  • B7-H3 also called CD276, was originally understood to be a co stimulatory molecule but is now regarded as co-inhibitory.
  • B7-H4 also called VTCN1
  • B7-H4 is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.
  • B and T Lymphocyte Attenuator (BTLA), also called CD272 is a ligand of HVEM (Herpesvirus Entry Mediator).
  • BTLA T Lymphocyte Attenuator
  • HVEM Herpesvirus Entry Mediator
  • CTLA-4 Cytotoxic T-Lymphocyte- Associated protein 4 and also called CD 152 is overexpressed on Treg cells serves to control T cell proliferation.
  • KIR Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells.
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • TIM-3 short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines.
  • TIM-3 acts as a negative regulator of Thl /Tel function by triggering cell death upon interaction with its ligand, galectin-9.
  • VISTA short for V-domain Ig suppressor of T cell activation, is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors.
  • PD-l has its general meaning in the art and refers to programmed cell death protein 1 (also known as CD279). PD-l acts as an immune checkpoint, which upon binding of one of its ligands, PD-L1 or PD-L2, enables Shp2 to dephosphorylate CD28 and inhibits the activation of T cells.
  • the proprotein convertase (PC) inhibitor is particularly suitable for reducing the expression of PD-l .
  • T cell exhaustion refers to a state of T cell dysfunction.
  • the T cell exhaustion generally arises during many chronic infections and cancer.
  • T cell exhaustion can be defined by poor effector function, sustained expression of inhibitory receptors, and/or a transcriptional state distinct from that of functional effector or memory T cells.
  • T cell exhaustion generally prevents optimal control of infection and tumors. See, e.g., Wherry E J, Nat Immunol. (2011) 12: 492-499, for additional information about T cell exhaustion.
  • T cell exhaustion results from the binding of an immune checkpoint protein to at least one of its ligands (e.g. PD1-1 and one of its ligands PD-L1 or PD-L2).
  • the subject suffers from a cancer, in particular a colorectal cancer, and the method of the present invention is thus suitable for enhancing the proliferation, migration, persistence and/or cytoxic activity of tumor infiltrating cytotoxic T lymphocytes.
  • tumor infiltrating cytotoxic T lymphocyte refers to the pool of cytotoxic T lymphocytes of the patient that have left the blood stream and have migrated into a tumor. Accordingly, the method of the present invention is particularly suitable for the treatment of cancer.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood-bome tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the method of the present invention is suitable for the treatment of a cancer characterized by a high tumor infiltration of cytotoxic T lymphocytes that express an immune checkpoint protein.
  • said tumor-infiltration of cytotoxic T lymphocytes is determined by any conventional method in the art.
  • said determination comprises quantifying the density of cytotoxic T lymphocytes that express at least one immune checkpoint protein (e.g. PD-l) in a tumor sample obtained from the patient.
  • at least one immune checkpoint protein e.g. PD-l
  • tumor tissue sample means any tissue tumor sample derived from the patient. Said tissue sample is obtained for the purpose of the in vitro evaluation.
  • the tumor sample may result from the tumor resected from the patient.
  • the tumor sample may result from a biopsy performed in the primary tumor of the patient or performed in metastatic sample distant from the primary tumor of the patient, for example an endoscopical biopsy performed in the bowel of the patient affected by a colorectal cancer.
  • the tumor tissue sample encompasses (i) a global primary tumor (as a whole), (ii) a tissue sample from the center of the tumor, (iii) a tissue sample from the tissue directly surrounding the tumor which tissue may be more specifically named the“invasive margin” of the tumor, (iv) lymphoid islets in close proximity with the tumor, (v) the lymph nodes located at the closest proximity of the tumor, (vi) a tumor tissue sample collected prior surgery (for follow-up of patients after treatment for example), and (vii) a distant metastasis.
  • the“invasive margin” has its general meaning in the art and refers to the cellular environment surrounding the tumor.
  • the tumor tissue sample irrespective of whether it is derived from the center of the tumor, from the invasive margin of the tumor, or from the closest lymph nodes, encompasses pieces or slices of tissue that have been removed from the tumor center of from the invasive margin surrounding the tumor, including following a surgical tumor resection or following the collection of a tissue sample for biopsy, for further quantification of one or several biological markers, notably through histology or immunohistochemistry methods, through flow cytometry methods and through methods of gene or protein expression analysis, including genomic and proteomic analysis.
  • the tumor tissue sample can, of course, be patiented to a variety of well-known post collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.).
  • the sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).
  • the tumor tissue sample can be used in microarrays, called as tissue microarrays (TMAs).
  • TMA tissue microarrays
  • TMA consists of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. This technology allows rapid visualization of molecular targets in tissue specimens at a time, either at the DNA, RNA or protein level.
  • TMA technology is described in W02004000992, US8068988, Olli et al 2001 Human Molecular Genetics, Tzankov et al 2005, Elsevier; Kononen et al 1198; Nature Medicine.
  • the quantification of density of cytotoxic T lymphocytes that express at least one immune checkpoint protein is determined by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the quantification of the density of cytotoxic T lymphocytes is performed by contacting the tissue tumor tissue sample with a binding partner (e.g. an antibody) specific for a cell surface marker of said cells.
  • the quantification of density of cytotoxic T lymphocytes is performed by contacting the tissue tumor tissue sample with a set of binding partners (e.g. an antibody) specific for CD8 and for the immune checkpoint protein (e.g. PD- 1) ⁇
  • the density of cytotoxic T lymphocytes that express at least one immune checkpoint protein is expressed as the number of these cells that are counted per one unit of surface area of tissue sample, e.g. as the number of cells that are counted per cm 2 or mm 2 of surface area of tumor tissue sample.
  • the density of cells may also be expressed as the number of cells per one volume unit of sample, e.g. as the number of cells per cm 3 of tumor tissue sample.
  • the density of cells may also consist of the percentage of the specific cells per total cells (set at 100%).
  • Immunohistochemistry typically includes the following steps i) fixing the tumor tissue sample with formalin, ii) embedding said tumor tissue sample in paraffin, iii) cutting said tumor tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for the marker, v) rinsing said sections, vi) incubating said section with a secondary antibody typically biotinylated and vii) revealing the antigen-antibody complex typically with avidin-biotin-peroxidase complex. Accordingly, the tumor tissue sample is firstly incubated the binding partners.
  • the labeled antibodies that are bound to a marker of interest are revealed by the appropriate technique, depending of the kind of label being borne by the labeled antibody, e.g. radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously.
  • the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify staining signal) and enzymatic molecules.
  • Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system.
  • Counterstaining may be used, e.g. H&E, DAPI, Hoechst.
  • Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems.
  • one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e the marker).
  • exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof.
  • the label is a quantum dot.
  • Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g.
  • luciferin e.g. luciferin, luciferase
  • haptens e.g. biotin
  • a variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev. Biochem. 41 :843-868.
  • Affinity ligands can also be labeled with enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. 3H, 14C, 32P, 35S or 1251) and particles (e.g. gold).
  • the different types of labels can be conjugated to an affinity ligand using various chemistries, e.g. the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g. aldehydes, carboxylic acids and glutamine.
  • Various enzymatic staining methods are known in the art for detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red.
  • the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody.
  • a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled.
  • the resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining.
  • Methods for image acquisition are well known to one of skill in the art.
  • any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, canning probe microscopes and imaging infrared detectors.
  • the image can be captured digitally.
  • the obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample.
  • Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed).
  • Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS- 200 system (Becton, Dickinson & Co.).
  • detection can be made manually or by image processing techniques involving computer processors and software.
  • the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g., published U.S. Patent Publication No. US20100136549).
  • the image can be quantitatively or semi-quantitatively analyzed and scored based on staining intensity of the sample.
  • Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of the specified biomarker (i.e. the marker).
  • Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically.
  • images can be quantitatively analyzed using a pixel count algorithms (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S.
  • a ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored.
  • the amount of the detected biomarker i.e. the marker
  • the amount is quantified and given as a percentage of positive pixels and/or a score.
  • the amount can be quantified as a percentage of positive pixels.
  • the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels.
  • a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,
  • the method of the present invention comprises the steps consisting in i) providing one or more immunostained slices of tissue section obtained by an automated slide-staining system by using a binding partner capable of selectively interacting with the marker (e.g.
  • step a proceeding to digitalisation of the slides of step a. by high resolution scan capture, iii) detecting the slice of tissue section on the digital picture iv) providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring intensity of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed.
  • quantification of the percentage of cytotoxic T lymphocytes that express at least one immune checkpoint protein is determined by an automatized microscope which allows measurement of morphometric and fluorescence characteristics in the different cell compartments (membrane/ cytoplasm/ nuclei) and quantifying preciously the percent of interest cells. Briefly the quantification of percent of cytotoxic T lymphocytes that expression at least one immune checkpoint protein (e.g.
  • PD-l is performed by following steps: i) providing tissue microarray (TMA) containing RCC samples, ii) TMA samples are stained with anti-CD3, anti-CD8, and anti-PD-l antibodies, iii) the samples are further stained with an epithelial cell marker to assist in automated segmentation of tumour and stroma, iv) TMA slides are then scanned using a multispectral imaging system, v) the scanned images are processed using an automated image analysis software (e.g.
  • Perkin Elmer Technology which allows the detection and segmentation of specific tissues through powerful pattern recognition algorithms, a machine-learning algorithm is trained to segment tumor from stroma and identify cells labelled; vi) the percent of cytotoxic T lymphocytes that expression at least one immune checkpoint protein (e.g. PD-l) within the tumour areas is calculated; vii) a pathologist rates lymphocytes percentage; and vii) manual and automated scoring are compared with survival time of the subject.
  • cytotoxic T lymphocytes that expression at least one immune checkpoint protein (e.g. PD-l) within the tumour areas is calculated.
  • a pathologist rates lymphocytes percentage; and vii) manual and automated scoring are compared with survival time of the subject.
  • the cell density of cytotoxic T lymphocytes is determined in the whole tumor tissue sample, is determined in the invasive margin or centre of the tumor tissue sample or is determined both in the centre and the invasive margin of the tumor tissue sample.
  • a further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising i) quantifying the density of cytotoxic T lymphocytes that express at least one immune checkpoint protein (e.g. PD-l) in a tumor tissue sample obtained from the patient ii) comparing the density quantified at step i) with a predetermined reference value and iii) administering to the patient a therapeutically effective amount of the proprotein convertase (PC) inhibitor when the density quantified at step i) is higher than the predetermined reference value.
  • the term“the predetermined reference value” refers to a threshold value or a cut-off value.
  • a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of cell densities in properly banked historical subject samples may be used in establishing the predetermined reference value.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • ROC curve is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer.
  • ROC curve such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, C RE ATE -ROC. S AS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
  • the subject suffers from a viral infection.
  • viral infections treatable by the present invention include those caused by single or double stranded RNA and DNA viruses, which infect animals, humans and plants, such as retroviruses, poxviruses, immunodeficiency virus (HIV) infection, echovirus infection, parvovirus infection, rubella virus infection, papillomaviruses, congenital rubella infection, Epstein-Barr virus infection, mumps, adenovirus, AIDS, chicken pox, cytomegalovirus, dengue, feline leukemia, fowl plague, hepatitis A, hepatitis B, HSV-l, HSV-2, hog cholera, influenza A, influenza B, Japanese encephalitis, measles, parainfluenza, rabies, respiratory syncytial virus, rotavirus, wart, and yellow fever, adenovirus, a herpesvirus (e.g., HSV-I, HSV-
  • proprotein convertase has its general meaning in the art and refers to a family of Ca+2-dependent endoproteases responsible for the cleavage of precursor proteins by cleavage at a consensus recognition site.
  • the common mammalian PCs described are furin, PC7, PACE4, PC5, PC1 ⁇ 2, PC2 and PC4.
  • furin, PC7, PACE4 and PC5 have a wide tissue distribution and proteolytically process precursors in the constitutive secretory pathway.
  • PC inhibitor refers to any compound natural or not which is capable of inhibiting the activity of proprotein convertases (PCs).
  • PCs proprotein convertases
  • the term encompasses any PC inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the PC.
  • inhibitor of expression Several classes of compound may be used according to the invention as convertase inhibitors. These compounds include: (i) compounds that bind to convertase enzymes and inhibit its activity (e.g.
  • a proprotein convertase inhibitor is any compound or composition that can inhibit the ability of one or more proprotein convertases to cleave one or more of their substrates.
  • Proprotein convertase inhibitors can also be referred to as inhibitors of any or all of the respective proprotein convertase against which the inhibitor is effective.
  • a proprotein convertase inhibitor that can inhibit furin can be referred to as a furin inhibitor. This is the case regardless of whether the inhibitor inhibits only furin or can also inhibit other proprotein convertases.
  • the PC inhibitor of the present invention is a furin inhibitor or a PC7 inhibitor.
  • proprotein convertase inhibitors are known. Examples of such inhibitors include inhibitory prosegments of proprotein convertases, inhibitory variants of anti-trypsin and peptidyl haloalkylketone inhibitors.
  • Representative inhibitory prosegments of proprotein convertases include the inhibitory prosegments of PC5A (also known as PC6A), PC5B (also known as PC6B), PACE4, PCI (also known as PC3), PC2, PC4, PC7 and Furin (Thomas, Nature Reviews Mol. Cell Biol. 3 (2002) 753-766; Zhong et ah, J. Biol. Chem. 274: 33913- 33920, 1999).
  • a representative inhibitory variant of anti-trypsin is a-l antitrypsin Portland, an engineered variant of naturally occurring antitrypsin that inhibits multiple proprotein convertases (Jean et ah, Proc. Natl Acad. Sci. USA 95 (1998) 7293-7298).
  • Representative peptidyl halomethyl ketone inhibitors include decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), decanoyl-Phe-Ala-Lys-Arg-chloromethylketone (Dec-FAKR-CMK), decanoyl-Arg-Glu-Ile-Arg-chloromethylketone (Dec-REIR-CMK), and decanoyl-Arg-Glu- Lys-Arg-chloromethylketone (Dec-REKR-CMK) (Stieneke-Grober, A. et al., EMBO J. 11 (1992) 2407-2414; Jean et al, Proc.
  • Useful proprotein convertases include peptides.
  • the term “peptide” is meant to include both short and long amino acid polymers.
  • the terms “peptide” and “polypeptide” are used interchangeably herein.
  • the disclosed peptide can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more amino acids in length.
  • the disclosed peptide can be less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 amino acids in length.
  • the disclosed peptides are the ability to bind proprotein convertases such as furin.
  • the disclosed peptides can sequester proprotein convertases such as furin and thereby inhibit processing of toxins by said proprotein convertases.
  • the disclosed peptide can bind a proprotein convertase such as furin.
  • the disclosed peptide is not cleaved by a proprotein convertase such as furin.
  • the proprotein convertase inhibitor may be a serine protease inhibitor and is preferably a thiol inhibitor.
  • the thiol inhibitor may be a peptidyl chloroalkylketone having a peptide moiety which mimics at least one convertase enzyme cleavage site. It has been found that peptidyl chloroalkylketones with peptide moieties that mimic the convertase enzyme cleavage site are specific inhibitors of the enzymatic activity.
  • a preferred inhibitor is decanoyl-RVKR-cmk and derivatives thereof.
  • the proprotein convertase inhibitor also can be a small molecule.
  • proprotein convertase inhibitors based on 2,5- dideoxystreptamine are disclosed in Jiao, G., et al. (Proc Natl Acad Sci U S A. 2006 Dec 26;l03(52): 19707-12).
  • alpha 1 -antitrypsin a-l PDX
  • derivatives of alpha 1- antitrypsin such as those comprising the amino acid sequences arg-val-pro-arg, ala-val-arg-arg or arg-val-arg-arg, or nucleic acids encoding the same
  • p-chloromercuribenzoate p-chloromercuribenzoate
  • TPCK tosylamido-phenylethyl chloromethyl ketone
  • D-polyarginines e.g.
  • the PC inhibitor of the present invention is selected from compounds described in DE 102009035593 W02007046781 or in WO2013138666.
  • the PC inhibitor is selected from the group consisting of:
  • the proprotein convertase (PC) inhibitor is an inhibitor of proprotein convertase (PC) expression respectively.
  • An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of Proprotein convertase (PC) mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Proprotein convertase (PC), and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Proprotein convertase (PC) can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g.
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention.
  • Proprotein convertase (PC) gene expression can be reduced by contacting a patient or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Proprotein convertase (PC) gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing Proprotein convertase (PC).
  • PC Proprotein convertase
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • the term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences.
  • the mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomo logous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).
  • NHEJ errorprone nonhomo logous end-joining
  • HDR high-fidelity homology-directed repair
  • the endonuclease is CRISPR- cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the proprotein convertase inhibitor is administered to the patient in a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions 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 subject 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 subject; 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 with the active ingredients; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the proprotein convertase inhibitor of the present invention is administered to the subject in combination with at least one immune checkpoint inhibitor.
  • immune checkpoint inhibitor includes PD-l antagonists, PD-L1 antagonists, PD- L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7- H4 antagonists, and BTLA antagonists.
  • PD-l (Programmed Death- 1) axis antagonists include PD-l antagonist (for example anti-PD-l antibody), PD-L1 (Programmed Death Ligand- 1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody).
  • the anti-PD-l antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-l 106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-01 1 (also known as Pidilizumab, hBAT, and hBAT-l).
  • the PD-l binding antagonist is AMP-224 (also known as B7-DCIg).
  • the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-l 105, and MEDI4736.
  • MDX-l 105 also known as BMS-936559, is an anti-PD-Ll antibody described in W02007/005874.
  • Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010/077634 Al .
  • MEDI4736 is an anti-PD- Ll antibody described in WO2011/066389 and US2013/034559.
  • MDX-l 106 also known as MDX-l 106-04, ONO-4538 or BMS-936558, is an anti-PD-l antibody described in U.S.
  • Merck 3745 also known as MK-3475 or SCH-900475, is an anti-PD-l antibody described in U.S. Pat. No. 8,345,509 and W02009/114335.
  • CT-011 Panizilumab
  • AMP-224 also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
  • Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149.
  • Avelumab is an anti-PD-Ll antibody described in US 20140341917.
  • CA-170 is a PD-l antagonist described in W02015033301 & WO2015033299.
  • Other anti-PD-l antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649.
  • the PD-l inhibitor is an anti-PD-l antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab.
  • PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.
  • CTLA-4 Cytotoxic T-Lymphocyte Antigen-4 antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA- 4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA- 4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA-4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No.
  • CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014.
  • Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98/42752; U.S. Pat.
  • a preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No.
  • CTLA-4 antagonist antibodies
  • Tremelimumab CP- 675,206
  • Ipilimumab Ipilimumab
  • immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211).
  • Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors.
  • the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834).
  • TIM-3 T-cell immunoglobulin domain and mucin domain 3) inhibitors (Lourcade et al., 2010, J. Exp. Med.
  • the term“TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3.
  • the natural ligand of TIM-3 is galectin 9 (Gal9).
  • the term“TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3.
  • the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signalling pathway and/or block the binding of TIM-3 to galectin-9.
  • Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO2011155607, WO2013006490 and WO2010117057.
  • the immune checkpoint inhibitor is an IDO inhibitor.
  • IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1 -methyl-tryptophan (IMT), b- (3-benzofuranyl)-alanine, b-(3- benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 - methyl tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3- diacetate, 9- vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyr
  • the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L- tryptophan, 3-Amino-naphtoic acid and b-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
  • the active ingredient of the present invention e.g. proprotein convertase inhibitor
  • pharmaceutically acceptable excipients e.g. proprotein convertase inhibitor
  • sustained- release matrices such as biodegradable polymers
  • 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 carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • a further object of the present invention relates to an in vitro or ex vivo method of reducing the expression of at least one immune checkpoint protein in a population of immune cells comprising contacting the population of T cells with an amount of at least one proprotein convertase (PC) inhibitor.
  • PC proprotein convertase
  • the method is particularly suitable for reducing the expression of at least one immune checkpoint protein in a population of macrophages, monocytes or dendritic cells.
  • the method is particularly suitable for reducing the expression of at least immune checkpoint protein in a population of immune effector cells.
  • Preferred effector cells include, but are not limited to T cells, natural killer (NK) cells, and natural killer T (NKT) cells.
  • T cells has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity.
  • T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility.
  • TCR T cell receptor for the antigen
  • There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, Gamma delta T cells, and Tregs.
  • the population of T cells is a population of cytotoxic T lymphocytes (as defined above).
  • Naive CD8+ T cells have numerous acknowledged biomarkers known in the art. These include CD45RA+CCR7+HLA-DR-CD8+ and the TCR chain is formed of alpha chain (a) and beta chain (b).
  • Persisting central memory and effector memory
  • non-persisting effector or exhausted subpopulations
  • anergic/tolerant and senescent regulatory CD8+ T cells can be discriminated on their differential expression of surface markers including (but not limited to) CCR7, CD44, CD62L, CD122; CD127; IL15R, KLRG1, CD57, CD137, CD45RO, CD95, PD-l CTLA, Lag3 and transcription factors such as T-bet/Eomes, BCL6, Blimp- 1, STAT3/4/5 ID2/3, NFAT, FoxP3.
  • the population of T cells is a population of CD4+ T cells.
  • CD4+ T cells also called T helper cells or TH cells
  • TH cells refers to T cells which express the CD4 glycoprotein on their surfaces and which assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • CD4+ T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist in the active immune response.
  • APCs antigen-presenting cells
  • TH1, TH2, TH3, TH17, TH9, TFH or Treg which secrete different cytokines to facilitate different types of immune responses.
  • Signaling from the APC directs T cells into particular subtypes.
  • the TH cell surface biomarkers known in the art include CXCR3 (Thl), CCR4, Crth2 (Th2), CCR6 (Thl7), CXCR5 (Tfh) and as well as subtype-specific expression of cytokines and transcription factors including T-bet, GATA3, EOMES, RORyT, BCL6 and FoxP3.
  • the population of T cells is a population of gamma delta T cells.
  • Gamma delta T cells normally account for 1 to 5% of peripheral blood lymphocytes in a healthy individual (human, monkey). They are involved in mounting a protective immune response, and it has been shown that they recognize their antigenic ligands by a direct interaction with antigen, without any presentation by MHC molecules of antigen-presenting cells.
  • Gamma 9 delta 2 T cells (sometimes also called gamma 2 delta 2 T cells) are gamma delta T cells bearing TCR receptors with the variable domains Vy9 and V52. They form the majority of gamma delta T cells in human blood.
  • gamma delta T cells When activated, gamma delta T cells exert potent, non-MHC restricted cytotoxic activity, especially efficient at killing various types of cells, particularly pathogenic cells.
  • These may be cells infected by a virus (Poccia et ah, J. Leukocyte Biology, 1997, 62: 1- 5) or by other intracellular parasites, such as mycobacteria (Constant et al., Infection and Immunity, December 1995, vol. 63, no. 12: 4628-4633) or protozoa (Behr et al., Infection and Immunity, 1996, vol. 64, no. 8: 2892-2896). They may also be cancer cells (Poccia et al., J.
  • the population of T cells is a population of CAR-T cells.
  • CAR-T cell refers to a T lymphocyte that has been genetically engineered to express a CAR.
  • CAR T-cells encompasses all classes and subclasses of T- lymphocytes including CD4+ , CD8+ T cells, gamma delta T cells as well as effector T cells, memory T cells, regulatory T cells, and the like.
  • the T lymphocytes that are genetically modified may be "derived” or “obtained” from the subject who will receive the treatment using the genetically modified T cells or they may "derived” or “obtained” from a different subject.
  • CARs may refer to artificial T-cell receptors T-bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell.
  • CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy.
  • CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain that may vary in length and comprises a tumor associated antigen binding region.
  • CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain.
  • CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD 137, DAP 10, and/or 0X40.
  • molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
  • co-stimulatory molecules including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
  • the population of T cells is specific for an antigen.
  • antigen as used herein refers to protein, peptide, nucleic acid or tissue or cell preparations capable of eliciting a T cell response.
  • the antigen is a tumor- associated antigen (TAA).
  • TAAs include, without limitation, melanoma- associated Ags (Melan-A/MART-l, MAGE-l, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-l (mucin-l) associated with melanoma); CEA (carcino embryonic antigen) which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors, including but not limited to ovarian tumors, testicular tumors and myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and the idiotypic determinants
  • tumor-associated antigens which can be used in the present invention are disclosed in the book“Categories of Tumor Antigens” (Hassane M. et al Holland-Frei Cancer Medicine (2003). 6th edition.) and the review Gregory T. et al (“Novel cancer antigens for personalized immunotherapies: latest evidence and clinical potential” Ther Adv Med Oncol. 2016; 8(1): 4-31) all of which are herein incorporated by reference.
  • the tumor-associated antigen is melanoma-associated Ags.
  • the population of T cells is prepared from a PBMC.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • unfractionated PBMC refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population.
  • Cord blood mononuclear cells are further included in this definition.
  • the PBMC sample according to the invention has not been subjected to a selection step to contain only adherent PBMC (which consist essentially of >90% monocytes) or non-adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors).
  • adherent PBMC which consist essentially of >90% monocytes
  • non-adherent PBMC which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors.
  • a PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells), monocytes, and precursors thereof.
  • lymphocytes B cells, T cells, NK cells, NKT cells
  • monocytes and precursors thereof.
  • these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells.
  • the initial cell preparation consists of PBMCs from fresh or frozen (cytopheresed) blood. Isolated T cell (or APC) can be analysed in flux cytometry.
  • T cells or APC
  • T cells or APC
  • 100 million frozen PBMCs from cytopheresis yield 1 to 5 billion cells with the classical method of preparation.
  • Standard methods for purifying and isolating T cells are well known in the art. For instance, cell sorting is a current protocol that may be used to isolate and purify the obtained CTLs.
  • multimers e.g. tetramers or pentamers
  • the carboxyl terminus of an MHC molecule such as, for example, the HLA A2 heavy chain, is associated with a specific peptide epitope, and treated so as to form a multimer complex having bound hereto a suitable reporter molecule, preferably a fluorochrome such as, for example, fluoroscein isothiocyanate (FITC), phycoerythrin, phycocyanin or allophycocyanin.
  • FITC fluoroscein isothiocyanate
  • phycoerythrin phycocyanin or allophycocyanin.
  • the multimers produced bind to the distinct set of CD8+ T cell receptors (TcRs) on a subset of CD8+ T cells to which the peptide is MHC class I restricted.
  • TcRs CD8+ T cell receptors
  • the number of CD8+ cells binding specifically to the HLA-peptide multimer may be quantified by standard flow cytometry methods, such as, for example, using a FACS Calibur Flow cytometer (Becton Dickinson).
  • the multimers can also be attached to paramagnetic particles or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting. Such particles are readily available from commercial sources (eg. Beckman Coulter, Inc., San Diego, Calif., USA).
  • naive T cells e.g. naive CD8+T cells
  • APCs antigen presenting cells
  • activated T cells preferably are activated in a peptide- specific manner.
  • the ratio of substantially separated naive T cells to APCs may be optimized for the particular individual, e.g., in light of individual characteristics such as the amenability of the individual's lymphocytes to culturing conditions and the nature and severity of the disease or other condition being treated.
  • any culture medium suitable for growth, survival and differentiation of T cells is used for the coculturing step.
  • the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium, all of which are commercially available standard media.
  • the naive T cells are contacted with the APCs of the present invention for a sufficient time to activate a CTL response.
  • one or more selected cytokines that promote activated T cell growth, proliferation, and/or differentiation are added to the culture medium. The selection of appropriate cytokines will depend on the desired phenotype of the activated T cells that will ultimately comprise the therapeutic composition or cell therapy product.
  • cytokines include IL-l , IL-2, IL-7, IL-4, IL-5, IL-6, IL-12, IFN-g, and TNF-a.
  • the culture medium comprises antibodies.
  • Exemplary antibodies include monoclonal anti-CD3 antibodies, such as that marked as ORTHOCLONE OKT®3 (muromonab-CD3).
  • the population of T cells is contacted with the proprotein convertase (PC) inhibitor for a time sufficient for to reduce the expression of checkpoint proteins.
  • the population of T cells and the proprotein convertase (PC) inhibitor are contacted with each other for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 30 hours.
  • the proprotein convertase (PC) inhibitor is added in the culture medium where the population of T cells is cultured.
  • the proprotein convertase (PC) inhibitor is added when the population of T cells is activated (for instance in presence of a population of APC).
  • functionality of the cells may be evaluated according to any standard method which typically include a cytotoxic assay.
  • Cell surface phenotype of the cells with the appropriate binding partners can also be confirmed.
  • Quantifying the secretion of various cytokines may also be performed. Methods for quantifying secretion of a cytokine in a sample are well known in the art. For example, any immunological method such as but not limited to ELISA, multiplex strategies, ELISPOT, immunochromatography techniques, proteomic methods, Western blotting, FACS, or Radioimmunoassays may be applicable to the present invention.
  • the population of T cells obtained by the method of the present invention may find various applications. More particularly, the population of T cells is suitable for the adoptive immunotherapy.
  • the in vitro or ex vivo method of the present invention is particularly suitable for preventing T cell exhaustion when the population of T cells is administered to a patient for adoptive immunotherapy.
  • the term“adoptive immunotherapy” refers the administration of donor or autologous T lymphocytes for the treatment of a disease or disease condition, wherein the disease or disease condition results in an insufficient or inadequate immune response.
  • Adoptive immunotherapy is an appropriate treatment for any disease or disease condition where the elimination of infected or transformed cells has been demonstrated to be achieved by a specific population of T cells.
  • Exemplary diseases, disorders, or conditions that may be treated with the population of T cells as prepared according to the present invention include, for example, include immune disorders, such as immune deficiency disorders, autoimmune disorders, and disorders involving a compromised, insufficient, or ineffective immune system or immune system response; infections, such as viral infections, bacterial infections, mycoplasma infections, fungal infections, and parasitic infections; and cancers.
  • a further object of the present invention relates to method for inducing a CAR-T cells, comprising a step of suppressing the expression of proprotein convertases (PCs) or suppressing the function of proprotein convertases (PCs) in a normal peripheral T cell.
  • the present invention also provides a CAR-T cells inducer, which comprises a substance that suppresses the expression of PCs or the function of PCs.
  • “peripheral normal T cells” are T cells other than regulatory T cells present in the periphery, and include, for example, CD4+ cells and CD8+ cells.
  • cells to be targeted for suppressing PCs expression are particularly preferably CD4+ CD25- cells, such as CD4+CD25-CD45RA+ cells (naive Th cells), CD4+CD25 /low CD45RA- cells (effector Th cells), as well as CD8+ cells are exemplified.
  • RNA molecules such as siRNA, shRNA, miRNA, stRNA and antisense RNA
  • genome editing techniques such as the method of suppressing the expression of PCs using CRISPR/Cas9 are suitably used.
  • Examples of methods for suppressing the function of PCs include, but are not limited to, causing peripheral T cells to act on a neutralizing antibody or a fragment thereof against PCs protein, and acting on a substance that suppresses the activity of PCs, for example, a low molecular substance.
  • the method for inducing a CAR-T cells is described in WO2018117090.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Inhibition of PCs activity represses PD-1 expression in T cells.
  • A PD-l mRNA levels upon PMA/Io stimulation at different time points in 0 and PDX cells by RT- PCR, represented as the fold change to the 3 h time point.
  • B Flow cytometry analysis of PD- 1 expression at different PMA/Io time points in 0 and PDX cells, shown as the percentage of positive cells. Data represented as mean ⁇ SD (C) and mean ⁇ SEM from 3 independent experiments (E, G). *p ⁇ 0.05.
  • A Flow cytometry analysis of PD-l expression in the cells in human lymphocyte population gated from isolated hPBMCs represented as the percentage of positive cells. CMK was added 24 h before anti-CD3 activation for 6h.
  • B Immunofluorescence image quantification of PD-l and CD8 doubled positive cells relative to total CD8 positive cells (3 different images and 3 different areas per image). Data represented as the mean ⁇ SEM from 3 independent experiments (A, C) and 2 independent experiments (E). *p ⁇ 0.05.
  • FIG. 3 Increased T cell survival and proliferation by PC activity blockade. Effect of short (3h) and prolonged PMA/Io stimulation on Jurkat-0 and PDX cell proliferation. Data represented as the mean ⁇ SEM from 3 independent experiments. *p ⁇ 0.05.
  • FIG. 4 PC activity inhibition and T cell cytotoxicity.
  • A Levels of cytolytic protein released by isolated human CD8 T cells measured by a CBA assay. Cells were treated with CMK for 24 h prior to anti-CD3 activation (6 h).
  • B Granzyme B mRNA levels in hPBMCs isolated from three different donors expressed as fold change to control (CD3- and CMK-).
  • C Cytotoxicity flow cytometry-based assay show tumor cell specific lysis (HT-29) after co- culture with hPBMCs previously treated with CMK. Results from three different donors are shown. Data represented as the mean ⁇ SD and the mean ⁇ SEM from 2 independent experiments. *p ⁇ 0.05.
  • FIG. 5 PC inhibition represses tumor growth and increases TILs.
  • A Tumor growth curve of subcutaneously injected CT26-0 and CT26-PDX cells in syngeneic immunocompetent B ALB/c mice.
  • B Flow cytometry analysis of PD-l in CT26-0 and PDX derived tumors. Data represented as the mean ⁇ SD from 6 mice per condition. Flow cytometry data obtained from 2 pools of 3 tumors each per condition (0, PDX), represented as the mean ⁇ SEM. *p ⁇ 0.05.
  • Figure 6 Effect of PC inhibition in others immune checkpoint expression.
  • hPBMCs Human peripheral blood mononuclear cells
  • hPBMCs were cultured in RPMI 1640 (PAN-Biotech) supplemented with 10% FBS (Gibco), 2 mM L-glutamine (Gibco) and penicillin/streptomycin solution (Dominique Dutscher). hPBMCs were directly used for RNA/protein extraction, cultured under indicated experimental conditions, cryopreserved or used for CD8+ T cell isolation. Tumor-infiltrating CD8+ T cells were isolated from colon tumor samples freshly harvested following manufacturer’s instructions (Miltenyi Biotec). Briefly, tumor tissue was cut into small fragments and enzymatically digested. CD8+ T cells were then purified by negative selection with MACS magnetic beads (human CD8+T cell isolation kit from Miltenyi Biotec). CD8+ T cells were tested for purity by flow cytometry and used for RNA extraction or further experiments.
  • the characteristics and the origin of the control (0) and stably al-PDX-expresssing Jurkat cells (Jurkat-PDX) and CT26 cells (CT26-PDX) were described previously (Logeat et al 1998; Scamuffa et al 2008).
  • G418 resistant Jurkat-PDX and CT26-PDX cells were selected and screened for al-PDX expression by western blotting. Further selection was performed by culturing Jurkat-PDX and CT26-PDX cells in the presence of 1 pg/ml Pseudomonas exotoxin A (Khatib et al., 2001). This toxin mediates cell death only after its cleavage by PCs (Inocencio et al., 1994). Cells were grown in the presence of 200 pg/ml G418 to maintain selection.
  • CT26-0 and CT26-PDX cells were transiently transfected with pIRES2- EGFP-V5 empty vector or containing PDGF-A cDNA.
  • PDGF-A is an established PC substrate used to assess the activity of the PCs in tumor cells (Siegfried et al., 2003).
  • Jurkat cell transfections were performed by electroporation (Fogeat et al., 1998) and CT26 cell transfections were carried out using lipofectamine (Invitrogen) as recommended by the manufacturers (Scamuffa et al., 2008).
  • mice Female BAFB/c mice (Charles Rivers) were maintained under pathogen- free conditions until used for experiments. All research animals were housed in our institution (Universite de Bordeaux) in a temperature-controlled environment. All experimental procedures were approved by the Institutional Animal Care and Use Committee, Universite de Bordeaux, and were conducted under the supervision of trained veterinarian. The group sizes used for in vivo studies were those estimated to be the smallest necessary to generate meaningful data. Mice were monitored regularly, and those requiring medical attention were provided with appropriate care and excluded from the studies. B ALB/c mice were inoculated subcutaneously in the right flank with 1 x 10 6 syngeneic colon carcinoma CT26- 0 cells or CT26-PDX cells.
  • Activation of TCR signaling was performed either with phorbol myristate acetate (PMA) (lOOng/ml) and Ionomycin (Io) (lug/ml) or with 5pg/ml plate -bound anti-CD3 (clone OKT3, BioLegend). Activation time ranged between 10 min and 48 h, depending on the experiment.
  • PMA phorbol myristate acetate
  • Io Ionomycin
  • lug/ml 5pg/ml plate -bound anti-CD3
  • Activation time ranged between 10 min and 48 h, depending on the experiment.
  • PC activity was inhibited before TCR activation.
  • hPBMCs were cultured with 100 mM of the general PC inhibitor, Decanoyl-Arg-Val-Lys-Arg- chloromethylketone (CMK) (Bachem), for 24-48 h.
  • CMK-treated cells were activated with anti- CD3.
  • RNA (1 pg) was isolated by the Nucleospin RNA kit (Macherey-Nagel) and used for reverse transcription in a 20 m ⁇ reaction mixture containing 50 mM Tris-HCl (pH 8.3), 30 mM KC1, 8 mM MgCl2, 1 mM dNTPs, and 0.2 U Superscript reverse transcriptase (Invitrogen). Reverse transcription cycle consisted of 25°C 10 min, 2x 37°C 60 min and 85°C 5s, in a Veriti Thermal Cycler (Applied Biosystem).
  • Quantitative real-time PCR of cDNA samples was performed with SYBR or TaqMan primers (Table Sl) using respectively SYBR MesaBlue Master Mix or TaqMan Master Mix (Eurogentec), in a StepOne Plus Real Time PCR system following manufacturer’s instructions (Applied Biosystem). GAPDH was used as housekeeping gene for normalization.
  • Syngeneic mouse tumors from CT26-0 and CT26-PDX cells were collected from mice. Tumors were weighted and cut into smaller pieces depending on tumor size. For immunohistochemistry, tumor samples were fixed in 2% paraformaldehyde for 10 min and cryopreserved in 30% sucrose solution. The samples were embedded in OCT to produce frozen blocks and stored at -80°C.
  • the slides were washed 3 times for 5 min in TBS-tween20 then treated with cold acetone for 5 min, washed and incubated in a blocking solution containing 5% bovine serum albumin (BSA; Euromedex) for 1 h at RT. Sections were then incubated in the primary antibodies diluted 1 :100 in PBS-0.l% BSA, overnight at 4°C.
  • BSA bovine serum albumin
  • PD-l and CD8 detection anti-mouse PD-l (RD Systems), anti-mouse CD8 (BioRad), anti human PD-l (Abeam) and anti-human CD8 (Abeam) were used.
  • Antibodies against PCs namely, anti-PC7 (V-20), anti-PC5 (E-20), anti-furin (H-220, B6) and anti-PACE4 (K-18), were obtained from Santa Cruz Biotech.
  • the sections were incubated with the appropriate fluorophore-conjugated secondary antibody at 1 :500 (Fluoprobes, Interchim) for lh at RT and sections were mounted with ProLong Gold Antifade mounting medium containing DAPI (Invitrogen).
  • Immunocytochemistry was performed in hPBMCs and Jurkat cells in suspension.
  • Cells were washed in PBS with 2% FBS.
  • Surface antigens were detected by incubating with the primary antibody 1 : 100 in TBS with 5% BSA for 1 h at RT (anti-PD-l, anti-CD8), followed by appropriate fluorophore-conjugated secondary antibodies (1 : 100) for 30 min at RT.
  • Cells were then fixed in cold methanol for 7 min on ice.
  • intracellular antigens anti-furin, anti-PC7
  • cells were fixed with methanol before antibody incubations. Finally cells were washed and mounted in Fluoroshield medium containing DAPI (Sigma).
  • PE-anti-PD-l mAh Single cell suspension of hPBMCs and Jurkat cells were stained with fluorophore- conjugated antibodies: PE-anti-PD-l mAh (clone MIH4, eBiosciences), FITC-anti-CD8a mAh (Miltenyi Biotec), PE-anti-CD69 mAh (Beckman Coulter) or APC-anti-CDl07a mAb-(BD Biosciences).
  • the mouse monoclonal antibodies (Miltenyi Biotec) PE- anti-PD-l and APC- or FITC- anti-CD8a were used.
  • PD-l, CD8 and CD69 staining of Jurkat cells and hPBMCs 1-2 x 10 5 cells were collected by centrifugation, washed twice with PBS-5% BSA and incubated with primary antibody at 1 :5 (anti-PD-l), 1 : 10 (anti-CD69) or 1 :50 (anti-CD8) in PBS-5% BSA for 15 min on ice in the dark. Cells were resuspended in PBS- 5% BSA and a viability dye was added 5 min before flow cytometry acquisition, either 7-amino- actinomycin (7AAD) 1 :30 or DAPI 1 : 100.
  • 7AAD 7-amino- actinomycin
  • Jurkat-0 and PDX cells were incubated in the presence and absence of PMA and Ionomycin for 24 h and 48 h. Subsequently, cells were washed with PBS-5% BSA and stained with PE-Annexin V and 7AAD using the Annexin V Apoptosis Detection Kit (BioLegend), according to the manufacturer’s instructions. Cells were analyzed by flow cytometry (BD Accuri C6). The populations Annexin-/7AAD-, Annexin+/7AAD-, Annexin-/7AAD+, and Annexin+/7AAD+ that correspond to live cells, early apoptotic cells, necrotic cells and late apoptotic cells, respectively, were enumerated.
  • Jurkat-0 and PDX cells were plated on 24 wells plate at 1 xl0 5 /well for 24h.
  • PMA and Ionomycin were added and cell number was counted at 0, 3, 24, 48 and 72 h time points.
  • PMA and Ionomycin treatment medium was replaced with fresh medium (no PMA or Ionomycin) for the same time points as for long-term activation.
  • Cells were counted with a Countess II Automated Cell Counter (Invitrogen) and using trypan blue exclusion staining.
  • CMK PC inhibitors
  • pERTKR-MCA fluorogenic peptide
  • cells were incubated with pERTKR-MCA (100 mM) during the indicated time periods in 25 mM Tris (pH 7.4), 25 mM methyl-ethane-sulfonic acid, and 2.5 mM CaCl2, at 37°C, and the fluorometric measurements were performed using a spectrofluorometer (Tecan Infinite® F200 PRO, Tecan Group Ltd. France).
  • JRT3 functional assay The Jurkat T cell line J.RT3-T3.5 (JRT3) stably expressing the human LES -gd TCR (JRT3-LES) was incubated with the colon cancer cell line HT29 overexpressing the endothelial protein C receptor (HT29-EPCR) at 5: 1 (effectordarget) ratio for 4 h at 37°C. Specific recognition and binding of LES -gd TCR to EPCR induces JRT3-LES TCR-mediated activation as previously reported (Willcox et ah, 2012). The activation of JRT3-LES cells was evaluated by the expression of CD69, as assessed by flow cytometry analysis using PE-conjugated anti- CD69 mAh (Beckman Coulter). Data were acquired using a LSR Fortessa and analyses were performed using Diva and FlowJo 9.3.2 softwares (flow cytometry facility of TBM Core).
  • CBA Cytometric Bead Array
  • CD8+ T cells were isolated from hPBMCs from five different donors using a specific CD8+ T cell microbead-cocktail (Miltenyi Biotec). CD8+ T cells were stimulated with plate-bound anti-CD3 for 6 h and supernatants collected for flow cytometry analysis using a LegendPlex human CD8/NK panel CBA (BioLegend) to detect six cytotoxicity-related molecules: Granzyme A, Granzyme B, Perforin- 1, Granulysin, sFas, and sFasL. Flow cytometry data were acquired using a LSR Fortessa. Results were analyzed with the LEGENDplexTM software.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • hPBMCs were then stimulated with plate-bound anti-CD3 antibody (OKT3) 5 Lig/ml for 6 h.
  • CD3 -stimulated hPBMCs were co-cultured at different target: effector (1 : 1, 1 :5) ratios with CFSE-target cells for 24 h.
  • CFSE-target cells were collected for flow cytometry analysis or protein extraction.
  • PCs are involved in many tumors including colon cancer as well as in the inflammation- mediated immune response (Vahatupa et al., 2016).
  • the expression pattern of human secretory pathway PCs namely, furin, PACE4, PC5 and PC7 was analyzed using immunostaining analysis. All of these PCs were expressed in non-cancerous colon tissues with majority of the staining localized to the colon crypts (data not shown).
  • furin a highly diffuse expression throughout the tumor.
  • the immunocheckpoint programmed death 1 (PD-l, also known as CD279) is a co- inhibitory receptor that is inducibly expressed on T cells upon activation (Chen, 2004; Keir et al., 2007). PD-l expression in infiltrated CD8 T cells correlates with their exhausted phenotype and impaired effector function (Ahmadzadeh et al., 2009).
  • PC inhibition we first examined the effects of PC inhibition on PD-l mRNA expression in Jurkat T cells stably expressing the general PC inhibitor, the serpine al-PDX (Seidah and Prat, 2012; Scamuffa et al., 2006; Scamuffa et al., 2008).
  • Phorbol myristate acetate binds to and activates intracellular serine kinases of the PKC family and ionomycin (Io) is a Calcium ionophore that enhances membrane permeability to calcium.
  • PMA/Io ionomycin
  • TCR T cell receptor
  • CMK Decanoyl-Arg-Val-Lys-Arg- chloromethylketone
  • PC activity blockade Increased T cell survival and proliferation by PC activity blockade
  • exhausted T cells expressing PD-l were reported to exhibit proliferation impairment and found to progress to apoptosis because of the defect of differentiating into memory T cells (Yi et ah, 2010).
  • PC inhibition affects T cell functional responses.
  • the cell number was determined at several time points ranging from 3h to 72h, with and without PMA/Io stimulation in Jurkat-0 and PDX cells. First, Jurkat cells were stimulated with PMA/Io for 3h and allowed to proliferate during 72h.
  • Apoptosis of activated T cells requires BIM protein (Hildeman et ah, 2002), a member of BFB-only subset of Bcl-2 family, and their survival was reported to be dependent on the inhibition of the expression of this pro-apoptotic factor (Bouillet et ah, 1999; Sabbagh et ah, 2006).
  • BIM protein Hildeman et ah, 2002
  • BFB-only subset of Bcl-2 family a member of BFB-only subset of Bcl-2 family
  • T cells In addition to high levels of inhibitory receptor expression, T cells impair cytotoxicity in cancer (Jiang et ah, 2015; Wherry, 2011). Cytotoxic CD8 T cells typically utilize two major contact-dependent pathways to kill target cells (Russel and Ley, 2002): the granule exocytosis and Fas-Fas ligand (FasL) pathway. Granulysin and membrane-pore-forming protein, perforin, mediate the delivery of the apoptosis-inducing proteases, granzymes A and B, into the target cells through the granule exocytosis pathway (Lieberman and Fan, 2003; Trapani and Smyth, 2002).
  • Fas-Fas ligand (FasL) pathway activates caspase-mediated apoptosis. Therefore, we analyzed the effect of PC inhibition on these pathways and the ability of T cells to kill cancer cells.
  • the release of cytotoxic proteins was tested by cytometric bead array in conditioned medium from purified human CD8 T cells pretreated with CMK for 24h followed by anti-CD3- mediated activation (Figure 4A).
  • Conditioned medium from non-CMK treated and activated CD8 T cells had higher levels of granzyme A and perforin than non-activated cells.
  • CMK alone was able to increase the amount of granzyme A and granulysin, which increased further upon cell activation (Figure 4A).
  • TCR-deficient Jurkat cells JRT3 expressing a specific TCR (LES) recognizing EPCR protein
  • JRT3-LES TCR-deficient Jurkat cells
  • PC inhibition represses tumor growth and increases T cell infiltration
  • CT26-PDX conditioned media inhibited the cleavage of fluorogenic peptide pERTKR-MCA but not the conditioned media from control cells (data not shown). Similar result was obtained with protein extracts from Jurkat cells cultured with CT26-PDX conditioned media (data not shown).
  • CT26-0 and CT26-PDX cells were analyzed for IGF-IR processing before subcutaneous inoculation of mice to confirm reduced PC activity in CT26- PDX cells (data not shown).
  • TILs tumor infiltrating lymphocytes
  • three weeks after engraftment, tumors from each condition (n 6) were pooled together in groups of 3 for tumor dissociation and analyses. Flow cytometry analyses of CD8 immunofluorescence in dissociated tumors showed two fold increase in CD8- positive T cells in CT26-PDX cells-derived tumors compared to CT26-0 cells-derived tumors (data not shown).
  • PCs proprotein convertases
  • Furin activates Pseudomonas exotoxin A by specific cleavage in vivo and in vitro. J. Biol. Chem. 269, 31831- 31835.
  • Notchl receptor is cleaved constitutively by a furin-like convertase. Proc. Natl. Acad. Sci. U. S. A. 95, 8108-8112.
  • TGFp i -mediated SMAD3 enhances PD-l expression on antigen-specific T cells in cancer. Cancer Discov. 6, 1366-1381.
  • TILs tumour-infiltrating lymphocytes
  • PD-l is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma. Clin. Cancer Res. 13, 1757-1761.
  • T-cell-expressed proprotein convertase FURIN inhibits DMBA/TPA-induced skin cancer development. Oncoimmunology 5.

Abstract

L'activation, l'expansion Et l'infiltration de lymphocytes T cytotoxiques (CTL) dans des tumeurs solides, y compris celles des cancers colorectaux (CRS), sont des événements nécessaires pour une réponse immunitaire efficace. Cependant, les cellules cancéreuses ont la capacité de favoriser un microenvironnement tumoral immunosuppresseur qui protège les cellules malignes de l'action des CTL. Dans la présente invention, les inventeurs ont mis en évidence que l'expression de la proprotéine convertase (PC), principalement La furine et a PC7, est régulée à la hausse dans les lymphocytes T CD8 humains et les CRS. L'inhibition de L'activité PC dans les lymphocytes T réprime l'expression de la protéine de point de contrôle immunitaire (par exemple de la PD -1) qui améliore l'efficacité, augmente l'infiltration tumorale des CTL et la clairance tumorale. Ces découvertes définissent un rôle clé pour les PC dans l'inhibition des réponses de lymphocytes T induites par une tumeur et fournissent une justification pour l'utilisation d'inhibiteurs de PC pour améliorer les thérapies anticancéreuses.
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