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WO2020048942A1 - Methods and pharmaceutical compositions for enhancing cytotoxic t lymphocyte-dependent immune responses - Google Patents

Methods and pharmaceutical compositions for enhancing cytotoxic t lymphocyte-dependent immune responses Download PDF

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Publication number
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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PCT/EP2019/073392
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French (fr)
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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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Publication of WO2020048942A1 publication Critical patent/WO2020048942A1/en

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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.

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Abstract

Activation, expansion and infiltration of cytotoxic T lymphocytes (CTLs) within solid tumors, including colorectal cancers (CRCs), are necessary events for an effective immune response. However, cancer cells have the ability to promote immunosuppressive tumor microenvironment that protects malignant cells from CTLs. Here, the inventors revealed that proprotein convertase (PC) expression, mainly furin and PC7, is upregulated in human CD8 T cells and CRCs. Inhibition of PC activity in T cells represses expression the expression of immune checkpoint protein (e.g. PD-1) which improves efficacy, increases tumor-infiltration of CTLs and tumor clearance. These findings define a key role for PCs in the inhibition of tumor-induced T cell responses and provide a rationale for the use of PC inhibitors to improve anti-cancer therapies.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR ENHANCING CYTOTOXIC T LYMPHOCYTE-DEPENDENT IMMUNE RESPONSES
FIELD OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for enhancing cytotoxic T lymphocyte-dependent immune responses, in particular, in patients suffering from cancer.
BACKGROUND OF THE INVENTION:
To provide protective immunity, 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). The discovery that inhibitory receptors serve as an immune checkpoint which regulate T cell effector function was rapidly exploited for the treatment of wide variety of solid and hematologic cancers (Riley, 2009; Ansell et ah, 2015). Although 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. In addition, majority of patients with solid tumors including colorectal cancers (CRCs, except the microsatellite -instable subset) are refractory to these treatments (Xiao et ah, 2015). The failure to respond to anti-PD-l therapy is, in part, due to the presence of irreversibly exhausted T cells. Understanding the essential mechanism(s) involved in the lack of response to immune checkpoint blockade therapy remains a main challenge. It is well established that solid tumors evade anti-cancer immune control by establishing immune-privileged niches within the tumor microenvironment (TME) that reduce proliferation, viability, or activity of intra-tumoral CTLs (Fearon et ah, 2014). Indeed, the apparent exclusion of CTLs from CRC is associated with a poor prognosis while, conversely, increased accumulation of CTLs within tumors is associated with a favorable outcome (Galon et ah, 2006; Talmadge et ah, 2007; Joyce and Fearon, 2015). Delineating the mechanisms that prevent CTL accumulation within the TME is important to understand its immunosuppressive properties and to increase the efficacy of immunotherapies (Talmadge et al, 2007; Joyce and Fearon, 2015). Upon T cell activation, PD-l expression is induced and is regulated by the transcription factors nuclear factor of activated T cells (NFAT), T-bet, B lymphocyte -induced maturation protein-l (Blimp-l), and Forkhead box protein 01 (FoxOl; Box 2) (Sharpe and Pauken, 2018). Various intracellular signal transduction pathways regulate expression and activation of these factors. They include tyrosine kinase, anti inflammatory signal, and mitochondrial apoptosis pathways. A wide range of proteins involved in these intracellular signal pathways require proteolytic cleavage of their protein precursors by the proprotein convertases (PCs) to be biologically active (Seidah and Prat, 2012; Scamuffa et ah, 2006).
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). Various 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). Altered PC levels were reported to be associated with enhanced invasion and proliferation in various tumor cells. Conversely, inhibition of PC activity by the bioengineered inhibitor, al-PDX (Scamuffa et ah, 2006; Scamuffa et ah, 2008), in various cancer cell lines resulted in reduced processing of various substrates and malignant tumor cells. The implication of PCs as regulators of tumor progression, infection and inflammation has provoked an interest to use them as therapeutic targets (Seidah and Prat, 2012; Scamuffa et ah, 2006; Scamuffa et al., 2008). In a phase I and recent phase II trial (FANG vaccine trial), an autologous tumor-based product targeting furin by shRNAi DNA was found to be beneficial in patients with advanced cancer. The FANG vaccine was safe in patients, which led to prolonged survival (Oh et al, 2016; Senzer et al., 2012).
SUMMARY OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for enhancing cytotoxic T lymphocyte-dependent immune responses, in particular, in patients suffering from cancer. In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION: Activation, expansion and infiltration of cytotoxic T lymphocytes (CTLs) within solid tumors, including colorectal cancers (CRCs), are necessary events for an effective immune response. However, cancer cells have the ability to promote immunosuppressive tumor microenvironment that protects malignant cells from CTLs. Here, the inventors revealed that proprotein convertase (PC) expression, mainly furin and PC7, is upregulated in human CD8+ T cells and CRC cells. Inhibition of PC activity in T cells represses the expression of immune checkpoint protein (e.g. PD-l) which improves efficacy, increases tumor-infiltration of CTLs and tumor clearance. These findings define a key role for PCs in the inhibition of tumor-induced T cell responses and provide a rationale for the use of PC inhibitors to improve anti-cancer therapies.
Accordingly, 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.
More specifically, 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.
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.
Another object of the present invention is a pharmaceutically acceptable composition comprising at least one 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 composition for use according to the invention enhances the proliferation, migration, persistence and/or activity of cytotoxic T lymphocytes (CTLs) in the subject
More particularly, 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.
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. Typically 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). For example, 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.
As used herein the term "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). Examples of 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, 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). Cell surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. 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. As used herein, the term“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.
In some embodiments, the proprotein convertase (PC) inhibitor is particularly suitable for reducing the expression of PD-l .
As used herein, the term“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. Typically, 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).
In some embodiments, 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. As used herein, the term“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.
As used herein, the term "treatment" or "treat" refer 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. By "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. The phrase "maintenance regimen" or "maintenance period" 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.]).
As used herein, the term "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. In addition, 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; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; crythrolcukcmia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In particular, 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. Typically said tumor-infiltration of cytotoxic T lymphocytes is determined by any conventional method in the art. For example, 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.
As used herein, the term“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. In some embodiments, the tumor sample may result from the tumor resected from the patient. In some embodiments, 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. In some embodiments, 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. As used herein the“invasive margin” has its general meaning in the art and refers to the cellular environment surrounding the tumor. In some embodiments, 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 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.
In some embodiments, the quantification of density of cytotoxic T lymphocytes that express at least one immune checkpoint protein is determined by immunohistochemistry (IHC). For example, 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. Typically, 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)·
Typically, the density of cytotoxic T lymphocytes that express at least one immune checkpoint protein (e.g. PD-l) 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 cm2 or mm2 of surface area of tumor tissue sample. In some embodiments, 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 cm3 of tumor tissue sample. In some embodiments, 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. After washing, 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. Alternatively, 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. For example, 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. In some embodiments, 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, 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. In other examples, the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In an indirect IHC assay, 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. For example, once the sample has been stained, 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. In some examples, 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.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, 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. For example, 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. Patent Publication No. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8: 1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). 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) is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels. For example, a sample can have at least or about at least or about 0, 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%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. In some embodiments, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample, and represents the amount of target biomarker (e.g., the marker) present in the sample. Optical density or percentage area values can be given a scaled score, for example on an integer scale. Thus, in some embodiments, 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. an antibody as above described), ii) 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.
In a particular embodiment, quantification of the percentage of cytotoxic T lymphocytes that express at least one immune checkpoint protein (e.g. PD-l) 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.
In some embodiments, 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.
Accordingly 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. As used herein, the term“the predetermined reference value” refers to a threshold value or a cut-off value. Typically, 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). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the cell density in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured densities in samples to be tested, and thus obtain a classification standard having significance for sample classification. 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 (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) 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. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC 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. Existing software or systems in the art may be used for the drawing of the 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.
In some embodiments, the subject suffers from a viral infection. Examples of 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-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picoma virus (e.g., rhino virus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a lentivirus such as HIV).
As used herein, the term“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, PC½, PC2 and PC4. In particular, furin, PC7, PACE4 and PC5 have a wide tissue distribution and proteolytically process precursors in the constitutive secretory pathway. For a review of proprotein convertases, see Thomas, G. (2002) Nat Rev Mol Cell Biol 3:753-766, the entire contents of which are hereby incorporated by reference in their entirety.
As used herein, the term“proprotein convertase inhibitor” or“PC inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of proprotein convertases (PCs). 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. The term also encompasses 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. competitive inhibitors or allosteric inhibitors); (ii) compounds which prevent the transcription, translation or expression of convertase enzymes (e.g. inhibitor of expression); (iii) compounds which increase the rate of degradation of convertase enzymes; (iv) compounds which inhibit the proteolytic activation of the inactive PC (e.g. furin) precursor; and (v) compounds which inhibit a potential intracellular translocation of PC. In particular, 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. Thus, for example, 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. In some embodiments, the PC inhibitor of the present invention is a furin inhibitor or a PC7 inhibitor.
A number of 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. Natl Acad. Sci. USA 95 (1998) 7293-7298; Garten W. et al., Virology 72 (1989) 25-31). For purposes of illustration, the invention will be described hereinafter by reference to exemplary tests using the representative substrate analogue Dec- RVKR-CMK without limiting the invention thereto.
Useful proprotein convertases include peptides. As used herein, the term "peptide" is meant to include both short and long amino acid polymers. Thus, the terms "peptide" and "polypeptide" are used interchangeably herein. Thus, 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. Thus, 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. One advantage of the disclosed peptides is the ability to bind proprotein convertases such as furin. In preferred aspects, the disclosed peptides can sequester proprotein convertases such as furin and thereby inhibit processing of toxins by said proprotein convertases. Thus, the disclosed peptide can bind a proprotein convertase such as furin. In some aspects, 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.
In some embodiments, the proprotein convertase inhibitor also can be a small molecule.
For example, 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).
Further convertase inhibitors suitable for use according to the invention include: (i) alpha 1 -antitrypsin (a-l PDX), or nucleic acids encoding the same; (ii) 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; (iii) p-chloromercuribenzoate; (iv) tosylamido-phenylethyl chloromethyl ketone (TPCK); (v) D-polyarginines (e.g. hexa-arginine and its derivatives); (vi) Acetyl-leu-leu-arg-aldehyde hemisulfate; (vii) S-carboxyphenylethyl- carbamoyl-arg-val-arg-aldehyde; (viii) Threodimercaptobutanediol; and (ix) Tos-Lys- chloromethy lketone .
In some embodiments, the PC inhibitor of the present invention is selected from compounds described in DE 102009035593 W02007046781 or in WO2013138666.
In some embodiments, the PC inhibitor is selected from the group consisting of:
Figure imgf000017_0001
In some embodiments, 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. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, 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. For example, 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. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) 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. In its broadest sense, 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). Typically, 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. In general, 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. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of expression is an endonuclease. 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). In a particular embodiment, the endonuclease is CRISPR- cas. As used herein, the term“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. In some embodiment, 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. In some embodiment, 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).
According to the invention, the proprotein convertase inhibitor is administered to the patient in a therapeutically effective amount. By 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. For example, it is well 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. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, 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.
In some embodiments, the proprotein convertase inhibitor of the present invention is administered to the subject in combination with at least one immune checkpoint inhibitor. Examples of 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.
In some embodiments, 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). In some embodiments, 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). In some embodiments, the PD-l binding antagonist is AMP-224 (also known as B7-DCIg). In some embodiments, 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. Pat. No. 8,008,449 and W02006/121168. 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 (Pidizilumab), also known as hBAT or hBAT-l, is an anti-PD-l antibody described in W02009/101611. 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. In some embodiments, the PD-l inhibitor is an anti-PD-l antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, 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.
In some embodiments, 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. WO 2001/014424, the antibodies disclosed in PCT Publication No. WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional 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. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP- 675206); Mokyr et al, Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00- 9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01/14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP- 675,206) and Ipilimumab.
Other 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. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Lourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, 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). Accordingly, the term“TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, 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.
In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of 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, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a b-carboline derivative or a brassilexin derivative. Preferably 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.
Typically the active ingredient of the present invention (e.g. proprotein convertase inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutical" 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 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. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, 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.
In some embodiments, 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.
In some embodiments, 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.
As used herein, the term” 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. 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.
In some embodiments, 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.
In some embodiments, the population of T cells is a population of CD4+ T cells. The term“CD4+ T cells” (also called T helper cells or 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. These cells can differentiate into one of several subtypes, including 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. In addition to CD4, 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.
In some embodiments, 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. 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. Immunol., 159: 6009-6015; Foumie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147: 338-347). The possibility of modulating the activity of said cells in vitro, ex vivo or in vivo would therefore provide novel, effective therapeutic approaches in the treatment of various pathologies such as infectious diseases (particularly viral or parasitic), cancers, allergies, and even autoimmune and/or inflammatory disorders. In some embodiments, the population of T cells is a population of CAR-T cells. As used herein the term "CAR-T cell" refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of 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. The term“chimeric antigen receptors (CARs),” as used herein, 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. In some embodiments, 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. In particular aspects, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. In some embodiments, CARs comprise domains for additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD 137, DAP 10, and/or 0X40. In some embodiments, 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.
In some embodiments, the population of T cells is specific for an antigen. The term “antigen” (“Ag”) as used herein refers to protein, peptide, nucleic acid or tissue or cell preparations capable of eliciting a T cell response. In some embodiments, the antigen is a tumor- associated antigen (TAA). Examples of 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 of a B- cell lymphoma that can generate tumor-specific immunity (attributed to idiotype-specific humoral immune response), Mesothelin associated with pancreatic, ovarian and lung cancer, P53 associated with ovarian, colorectal, non small cell lung cancer, NY-ESO-l associated with testis, ovarian cancer, EphA2 associated with breast, prostate, lung cancer, EphA3 associated with colorectal carcinoma, Survivin associated with lung, breast, pancreatic, ovarian cancer, HPV E6 and E7 associated with cervical cancer, EGFR associated with NSCL cancer. Moreover, Ags of human T cell leukemia virus type 1 have been shown to induce specific cytotoxic T cell responses and anti-tumor immunity against the virus-induced human adult T- cell leukemia (ATL). Other leukemia Ags can equally be used. 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. In some embodiments, the tumor-associated antigen is melanoma-associated Ags.
Typically, the population of T cells is prepared from a PBMC. The term“PBMC” or “peripheral blood mononuclear cells” or“unfractionated PBMC”, as used herein, 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. Typically, 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). A PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells), monocytes, and precursors thereof. Typically, 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. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known by a skilled person in the art. For example, the initial cell preparation consists of PBMCs from fresh or frozen (cytopheresed) blood. Isolated T cell (or APC) can be analysed in flux cytometry. Several doses of the T cells (or APC) cellular product can be manufactured from one frozen cytopheresis. Typically, 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. Typically, multimers (e.g. tetramers or pentamers) consisting of MHC class 1 molecules loaded with the immunogenic peptide are used. To produce multimers, 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. 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. Following binding, and washing of the T cells to remove unbound or non-specifically bound multimer, 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).
In some embodiments, once the selected naive T cells (e.g. naive CD8+T cells) are purified they are subsequently admixed and incubated the population of antigen presenting cells (APCs) for a time sufficient to activate and enrich for a desired population of activated T cells, such as activated helper T cells, and preferably, CTLs or CD8+ memory T cells. Such 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. Typically, the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium, all of which are commercially available standard media. Typically, the naive T cells are contacted with the APCs of the present invention for a sufficient time to activate a CTL response. In some embodiments, 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. For instance cytokines include IL-l , IL-2, IL-7, IL-4, IL-5, IL-6, IL-12, IFN-g, and TNF-a. In some embodiments, the culture medium comprises antibodies. Exemplary antibodies include monoclonal anti-CD3 antibodies, such as that marked as ORTHOCLONE OKT®3 (muromonab-CD3).
In some embodiments, 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. For instance, 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. Typically, the proprotein convertase (PC) inhibitor is added in the culture medium where the population of T cells is cultured. In some embodiments, the proprotein convertase (PC) inhibitor is added when the population of T cells is activated (for instance in presence of a population of APC).
Once the population of T cells is obtained, 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. As used herein, 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. In the present invention,“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. In the present invention, 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. In order to suppress the expression of PCs, a known technique for controlling the expression of a gene in vivo or in vitro may be used without particular limitation. For example, a method of suppressing expression using RNA molecules such as siRNA, shRNA, miRNA, stRNA and antisense RNA, and a method of suppressing expression using genome editing technology such as CRISPR/Cas9 or TALEN are exemplified. In particular, 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.
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. 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.
Figure 2. Inhibition of PCs activity represses PD-1 expression in primary hPBMCs.
(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.
Figure 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.
Figure 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.
Figure 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. BTLA, TIGIn CTLA4 and LAG3 mRNA levels relative to anti-CD3 or PMA/lonomycinin in Jurkat T cells (A) and PBMCs (B).
EXAMPLE:
Methods
Human PBMCs, T cells and tumor samples
Matched whole blood and freshly resected colon tumor tissues and their non- tumoregenous counterparts were obtained from Bergonie Institute, Bordeaux, France. Fresh whole blood from healthy donors was obtained from Hospital Pellegrin, Bordeaux, France. All specimens were obtained following written informed consent approved by Bergonie Institute. Patient consent forms for all samples were obtained at the time of tissue acquisition. Biopsies were de-identified and processed for RNA and histology studies. Human peripheral blood mononuclear cells (hPBMCs) were isolated from healthy donors and colon cancer patients by density gradient centrifugation with Pancoll (PAN-Biotech; human, density l,077g/ml). 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.
Cell lines
Human colon carcinoma cell lines HT29 and HCT116, B ALB/c syngeneic colon carcinoma CT26 cell line, and human T cell lines Jurkat, J.RT3-T3.5 (JRT3), Myla, SeAx and HUT-78, were cultured in DMEM or RPMI 1640 complete media and grown at 37 °C in a 95% air, 5% C02 humidified incubator. 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). For the generation of Jurkat- PDX and CT26-PDX cells, 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. In some experiments, 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).
Mouse model
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 106 syngeneic colon carcinoma CT26- 0 cells or CT26-PDX cells. Tumors were measured three times per week using calipers, and tumor volume was calculated using the ellipsoid formula: Tumor volume = l/2(length x width2) (Euhus et al., 1986). Animals whose tumors grew larger than 2000 mm3 were considered progressed and were euthanized. After the growth period, tumors were collected from previously euthanized animals, weighted and cut in several pieces depending on tumor size. These pieces were cryopreserved, used for RNA/protein extraction or immunohistochemistry, or freshly used for tumor-infiltrating CD8+ T studies. Tumors were dissociated to obtain single cell suspension with the tumor dissociation kit for mouse from Miltenyi Biotec.
T cell activation
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. In hPBMCs experiments, 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. hPBMCs and collected for flow cytometry analyses, RNA/protein extraction or cytotoxicity assay.
Real-Time PCR
Total RNA (1 pg) was isolated by the Nucleospin RNA kit (Macherey-Nagel) and used for reverse transcription in a 20 mΐ 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.
Immunohistochemistry and immunocytochemistry
Human colon cancer and its corresponding non-cancer colon tissues were cut into smaller pieces and snap frozen in liquid nitrogen until use. Frozen pieces were embedded in OCT (Sakura) prior to sectioning on a microtome-cryostat (Leica CM 1900) at 10 mM thickness. Sections were mounted in gelatine-coated slides and stored at -20°C.
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.
For immunostaining of mouse sections, 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. For 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. For detecting 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). Confocal immunofluorescence images were taken using the inverted microscopes Nikon C2si Eclipse Ti- S with NIS-ElementsAR software (Nikon Instruments Europe B.V.) or Feica DM6 CFS TCS SP8 (Feica Microsystems, Bordeaux Imaging Center (BIC).
Flow cytometry analysis
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). For mouse-derived cells, the mouse monoclonal antibodies (Miltenyi Biotec) PE- anti-PD-l and APC- or FITC- anti-CD8a were used. For PD-l, CD8 and CD69 staining of Jurkat cells and hPBMCs, 1-2 x 105 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. For CDl07a staining, anti-CDl07a antibody (1 :50) and brefeldin A (1 : 100, BD Biosciences) were added to the cells right after stimulation. Flow cytometry data was acquired either with BD Accuri C6 or LSR Fortessa (BD Biosciences). Flow cytometry analyses were performed using BD Accuri C6 software, or Diva (BD Biosciences) and FlowJo 9.3.2 (TreeStar) softwares (flow cytometry facility of TBM Core).
Apoptosis assay
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.
Proliferation assay
Jurkat-0 and PDX cells were plated on 24 wells plate at 1 xl05/well for 24h. For long term activation, PMA and Ionomycin were added and cell number was counted at 0, 3, 24, 48 and 72 h time points. For short-time activation, after 3 h, 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.
Measurement of PC activity
The effect of PC inhibitors (CMK, al-PDX expression in cells) on PC activity in cells and tissues was assessed by the evaluation of the enzymes’ ability to digest the universal PC substrate, the fluorogenic peptide pERTKR-MCA, as previously described (Scamuffa et ah, 2008). Briefly, 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).
Cytometric Bead Array (CBA)
CBA was used to measure the concentration of cytotoxins released by primary human CD8+ T cell populations. 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 LEGENDplex™ software.
Cytotoxicity assay
Susceptibility of cancer target cells (HT29: microsatellite-instable (MSI) cells; and HCT116: microsatellite-stable (MSS) cells) to PBMC-mediated cytotoxicity was determined using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay. CFSE is a dye that irreversibly binds to intracellular proteins and is retained within living cells for a long period. Target cells were stained with 8 mM CFSE (BioLegend) for 20 min at 37°C and plated at 3 x 105 cell/ml. In parallel, hPBMCs (effector cells) were cultured in the presence and absence of CMK for 24 h. 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.
Quantification and statistical analysis
Statistical details can be found in Results, Figure and Figure Legend sections. Data are shown as mean ± S.E.M. or mean ± SD. Analysis of statistical significance was performed using Student's t-test or one-way ANOVA followed by Bonferroni’s comparison as a post hoc test. Statistical significance was estimated when P<0.05.
Results Altered PCs expression in human colon cancer and T cells
PCs are involved in many tumors including colon cancer as well as in the inflammation- mediated immune response (Vahatupa et al., 2016). Firstly, we evaluated the differential expression of PCs in normal and tumoral colon tissues. 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). In the cancerous tissue from the same patients, we observed alterations of the epithelial structure with the loss of crypts, and an altered expression of all four PCs. Furin showed a highly diffuse expression throughout the tumor. Barely detectable levels of PACE4, PC5 and PC7 were observed in the cancerous tissues derived from the same patients (data not shown). Previously, loss of crypts in colon tissue due to malignant changes was associated with enhanced inflammation and lymphocytic infiltration (Galon et al., 2006; Oksanen et al., 2014). Next, we evaluated the potential role of PCs in T cell activity and sought to identify PC family members associated with tumor- infiltrating T cells. We performed real time PCR analyses to investigate the expression of the four PCs in various human T cell lines (Jurkat T, MyLa, SeAx and HUT-78 cells) (data not shown), human peripheral blood mononuclear cells (hPBMCs) (data not shown) and isolated human CD8 T cells (data not shown). We show that furin and PC7 are the main PCs expressed in all these cells. Similarly, immunofluorescence staining of hPBMCs confirmed the expression of furin and PC7 proteins in human CD8 T cells, both in healthy donors and colon cancer patients (data not shown). In addition, colon cancer tissue analysis revealed that although various cells within the tumor expressed mainly furin, a population of tumor infiltrating CD8 T cells expressed furin and weakly PC7 (data not shown). This points to the presence of PCs in tumor-infiltrating lymphocytes (TILs) particularly CD8 T cells, may have a role in their phenotype and response capacity.
Inhibition of PCs activity represses PD-1 expression in T cells
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). To investigate the potential role of PC activity on PD-l expression, 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). In an in vitro enzymatic digestion assay, using the fluorogenic peptide pERTKR-MCA as PCs substrate, we show that the expression of al-PDX in Jurkat cells (Jurkat-PDX) inhibits PC activity compared to cells expressing the empty vector (Jurkat-0) (data not shown). Similarly, in immunoblotting analysis, the expression of al-PDX in Jurkat T cells reduced their ability to convert the PC substrate IGF-I receptor precursor (ProlGF-IR, -200 kDa) (Scamuffa et ah, 2008) into its processed form (-97 kDa) (data not shown). T cells require both calcium and protein kinase C (PKC) signal for activation. Phorbol myristate acetate (PMA) binds to and activates intracellular serine kinases of the PKC family and ionomycin (Io) is a Calcium ionophore that enhances membrane permeability to calcium. Combination of PMA and Io (PMA/Io) mimics T cell receptor (TCR) activation. We found that Jurkat-0 cells stimulated with PMA/Io induced PD-l mRNA expression at various time points (lh, 3h and 24h). In contrast, in Jurkat-PDX cells PD-l mRNA expression was considerably decreased under the same conditions (Figure 1A), suggesting that PC activity is necessary for PD-l transcription. Flow cytometry analysis confirmed the expression of PD-l at the protein level in Jurkat-0 cells after 24h stimulation with a robust increase after 48h, while PD-l was almost undetectable in Jurkat-PDX cells (Figure IB). Immunofluorescence staining and analysis of PD-l expression in Jurkat-0 and PDX cells further confirmed that PC inactivation represses PD-l production in PMA/Io- activated cells (data not shown). These observations indicate that PCs are involved in PD-l expression and that PC inhibitors can impede PD-l expression at the RNA and protein levels. Similarly, al-PDX expression in Jurkat cells led to a significant decrease in PD-l expression at both mRNA (data not shown) and protein levels (data not shown), following anti-CD3- mediated activation. To corroborate these results, isolated hPBMCs from healthy donors were treated with the general convertase pharmacological inhibitor, Decanoyl-Arg-Val-Lys-Arg- chloromethylketone (CMK) (Wang and Pei, 2001), for 24h prior to anti-CD3 -mediated activation. PC inactivation by CMK in hPBMCs led to a significant decrease in PD-l expression, as assessed by flow cytometry (Figure 2A) and immunofluorescence (data not shown) analyses. We confirmed inhibition of PC activity by CMK using the enzymatic digestion assay (data not shown) and the inhibition of pro-IGF-IR processing on protein extracts from hPBMCs (data not shown). Immunofluorescence analysis of hPBMCs derived from colon cancer patients treated with CMK for 48h and activated by anti-CD3 show a 33% reduction of CD8 positive cells, also positive for PD-l (Figure 2B). Total PD-l expression also significantly reduced in CMK-treated hPBMCs compared to non-treated cells after activation (data not shown). No significant difference was observed in the total number of CD8 positive cells between conditions remaining at 25.5%±5.7 (data not shown).
Increased T cell survival and proliferation by PC activity blockade Previously, 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). To determine if PC inhibition affects T cell functional responses, we investigated whether PC activity blockade could rescue activated T cell proliferation and survival. For proliferation measurements, 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. We found that while both Jurkat-0 and PDX cells show similar prolifrative rate in the absence of PMA/Io, the proliferation of Jurkat-0 cells after adding PMA/Io was inhibited. In contrast, the expression of al-PDX in these cells prevented PMA/Io-driven repression of cell proliferation (Figure 3). Furthermore, stimulation of Jurkat-0 cells with PMA/Io for a long period (72h) strongly blocked their proliferation. In contrast, although long-term stimulation also reduced Jurkat-PDX cells proliferation, they still showed a higher proliferation rate than Jurkat-0 cells and were proliferative after 72 h (data not shown). We analyzed the role of PC inhibition in T cell activation-mediated apoptosis using flow cytometry-based detection of Annexin V and 7AAD double staining. In non-stimulated Jurkat-0 and PDX cells, the overall percentage of dead cells (Annexin V+/7AAD+) was very similar (9.l±l .3% and l0.9±0.2, respectively) (data not shown). Upon their activation with PMA/Io for 24h and 48h, we observed a significant increase in the number of Jurkat-0 apoptotic cells (Annexin V+/7AAD-), indicating progression to cell death after PMA/Io stimulation. In Jurkat-0 cells, the percentage of dead cells after 48h of PMA/Io stimulation was 23.9±2.8% while Jurkat-PDX cells showed reduced apoptotic levels with only 7.8±0.7% of dead cells (data not shown). T cell activation either by TCR or PMA/Io stimulation can trigger apoptosis to downregulate T cell activity. The resistance of Jurkat-PDX cells to apoptosis observed by flow cytometry was confirmed by the analyses of the apoptosis effector caspase-3 (Thomberry and Lazebnik, 1998). In Jurkat-0 cells, cleaved caspase-3 was detected after 24h of PMA/Io stimulation and further increased following 48h of treatment data not shown). In Jurkat-PDX cells, cleaved caspase-3 was not detected at 24h or 48h, confirming the protective effect of PC inhibitor in T cells after activation. We also analyzed BCL11B, a survival protein regulated by ERK activation, essential for T cell commitment and specification of mature T cell lineages including cytotoxic CD8 cells (Wakabayashi et ah, 2003a, b). In Jurkat-0 cells, incubation with PMA/Io reduced BCL11 protein expression of in a time-dependent manner; while in Jurkat-PDX cells, high expression of the protein was maintained at all timepoints analyzed (data not shown). 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). However, healthy T cells contain appreciable levels of BIM that remain unaltered even in activated cells undergoing BIM-dependent death. We observed high levels of BIM expression in non-activated Jurkat-0 and PDX cells (data not shown), with lower levels (particularly BIMEL and BIML) in the latter. Stimulation with PMA/Io for 24-48h significantly decreased BIM levels in Jurkat-0 cells and even more in Jurkat-PDX cells. Interestingly, this pronounced reduction correlates to the enhanced ERK phosphorylation (data not shown) confirming the inhibitory effect of upregulated ERK signaling on BIM expression and cell survival (Korfi et ah, 2016).
PCs activity inhibition in T cell cytotoxicity
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). The 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). Similarly, compared to control cells, anti-CD3 -mediated activation of CD8 T cells in the absence or presence of CMK causes the release of sFasL (Figure 4A). The expression levels of the cluster of differentiation CD 107a protein, a cytotoxic T cell degranulation marker, was measured in hPBCMs treated with CMK or not, for 24h and activated with anti-CD3. Flow cytometry analyses showed a similar increase in CD 107a positive cells after activation, in both CMK-treated and non-treated cells (data not shown). Further analysis on anti-CD3 -activated hPBMCs (derived from different donors) in the absence and presence of CMK, revealed high levels of granzyme B mRNA in these cells compared to non-activated cells (Figure 4B). Taken together, these data indicate the preservation of the cytotoxicity pathway in the presence of CMK. To further support the effector function of T cells after PC inhibition, a cell-based cytotoxicity assay was performed using hPBMCs and the human colon cancer cells HT29 (MSS) and HCT116 (MSI) (Duldulao et al., 2012). Before target cell/effector cell contact, hPBMCs were treated with CMK or not, for 24h, followed by anti-CD3 activation for 6h. As expected, the lysis of HT-29 cells was more efficient in activated hPBMCs than in control hPBMCs either in the presence or absence of CMK (Figure 4C). Moreover, in two of the three donors, the percentage of HT-29 cell lysis was higher in CMK- treated hPBMCs. Similarly, the co-culture of human colon cancer cells HCT116 with anti-CD3- activated hPBMCs, previously treated or not with CMK, increased caspase-3 cleavage in both conditions compared to non-activated control cells with or without CMK (data not shown). These findings indicated the ability of CMK-treated hPBMCs to kill MSI and MSS colon cancer cells.
The recognition and binding of antigen-specific TCR to antigen-expressing target cells is essential to activate T cell effector functions. We analyzed the expression of CD69, a T cell activation marker, by flow cytometry to test whether the inhibition of PCs in T cells could affect antigen-TCR binding and therefore, downstream T cell activation. To this end, TCR-deficient Jurkat cells (JRT3) expressing a specific TCR (LES) recognizing EPCR protein, were co culture with EPCR-expressing HT29 cancer cells. As expected, CD69 expression was upregulated when JRT3-LES were co-cultured with EPCR-expressing HT29 cells due to antigen-TCR binding. Similarly, JRT3-LES previously treated with CMK for 24h strongly upregulated CD69 expression in the presence of the cancer cells, while JRT3-LES alone did not induced CD69 expression (data not shown). Western blot analysis of IGF-IR receptor proteolytic cleavage confirmed PC activity inhibition by CMK in JRT3-LES cells as demonstrated by the accumulation of ProlGF-IR (data not shown). The survival factor BCL11B was upregulated in JRT3-LES cells after CMK treatment (data not shown), confirming our previous results in Jurkat-PDX cells (data not shown) and highlighting the survival role of CMK during JRT3-LES and cancer cell interaction. Altogether, these findings show that PC blockade repressed PD-l expression in T cells without affecting their ability to recognize cancer cells and mediate cytotoxicity.
PC inhibition represses tumor growth and increases T cell infiltration
Next, we analyzed whether the effect of PC inhibition that we observed on T cells would translate into a better cancer immunoresponse in vivo, with a particular interest in cytotoxic T cell infiltration and PD-l expression levels within the tumors. We used immunocompetent mice and the syngeneic murine colon cancer cell line CT26 and the same cells stably expressing al- PDX to develop tumors. Since al-PDX is expressed by the tumor cells, we first analyzed the ability of these cells to release al-PDX in cultured media and the ability of this conditioned media to inhibit PC activity in other cells. Thus, we analyzed the processing of two PC substrates, the secreted growth factor PDGF-A and IGF-IR. First, since cells secrete the precursor and processed forms of PDGF-A, we analyzed the processing of this protein by the endogenous activity of the PCs in their conditioned media (Siegfried et ah, 2003). The conditioned media from CT26 control cells and the same cells co- transfected with cDNAs coding for V5-al-PDX and V5-proPDGF-A were analyzed by immunob lotting (data not shown). Using an anti-V5 antibody, we detected secreted al-PDX (50 kDa) and the accumulation of Pro PDGF-A (~24 kDa) in the conditioned medium from CT26-PDX cells. Furthermore we found an accumulation of intracellular IGF-IR precursor (200 kDa) in Jurkat cells after they were cultured with CT26-PDX conditioned media (data not shown). In an enzymatic digestion assay, 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). Two groups of mice (n=6/group) were inoculated and tumor volume was measured at various intervals (Figure 5A). Animals injected with CT26-PDX cells showed a significant reduction in tumor growth compared to controls (0). To analyze tumor infiltrating lymphocytes (TILs), 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). In contrast, intratumoral PD-l immunofluorescence decreased in CT26- PDX cells-derived tumors compared to CT26-0 tumors (Figure 5B). Accordingly, analysis of tumor tissue sections stained for CD8 and PD-l revealed that, in CT26-0 tumors, PD-l positive cells and infiltrated CD8 T cells were mainly located at the periphery in low numbers. On the contrary, in CT26-PDX tumors, a number of dispersed CD8 T cells failed to express PD-l (data not shown). Altogether, these results strongly suggest that al-PDX secreted by CT26-PDX tumor cells may act in a paracrine manner to inhibit PD-l expression and contribute to CD8 T cell infiltration.
Conclusion:
Reactivation of exhausted antitumor immune responses with various immune checkpoint inhibitors has demonstrated potential for the control of various human cancers. However, most patients present immune-related toxicities with these treatments. The development of new immune-checkpoint therapeutic strategies are necessary to overcome the side effects and improve treatments. Here, we demonstrate that proprotein convertases (PCs) regulate PD-l expression in activated cytotoxic T lymphocytes. These findings expand the scope for the role of PCs in immune response and identify PCs as a potential targets for tumor immunotherapy all the more than reduction of expression for other immune checkpoint proteins (e.g. BTLA, TIGIT, CTLA-4, and LAG3) in Jurkat T cells (Figure 6A) and PBMCs (Figure 6B) have been also observed.
REFERENCES:
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Claims

CLAIMS:
1. 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.
2. 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.
3. The method of claim 2 wherein the immune checkpoint protein is PD-l, LAG3, CTLA- 4, TIGIT and BTLA.
4. 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.
5. The method of claim 1, 2 or 4 wherein the subject suffers from a cancer.
6. The method of claim 5 wherein the cancer is selected from the group consisting of 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; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; crythrolcukcmia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
7. The method of claim 5 wherein the cancer is characterized by a high tumor infiltration of cytotoxic T lymphocytes that express an immune checkpoint protein.
8. The method of claim 1, 2 or 4 wherein the subject suffers from a viral infection.
9. The method of claim 1, 2 or 4 wherein the PC inhibitor is a furin inhibitor or a PC7 inhibitor.
10. The method of claim 1, 2, or 4 wherein the PC inhibitor is a peptide, a small molecule or an inhibitor of gene expression such as a siRNA, an antisense oligonucleotide or an endonuclease such as CRISPR/Cas9.
11. The method of claim 1 , 2, or 4 wherein the PC inhibitor is administered to the patient in combination with at least one immune checkpoint inhibitor. Examples of immune checkpoint inhibitor includes PD-l antagonists, PD-L1 antagonisst, 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.
12. A method of treating cancer in a patient in need thereof comprising i) quantifying the density of cytotoxic T lymphocytes that expression 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.
13. 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.
14. The method of claim 13 wherein the population of immune cells is a population of T CD8+ cells, T CD4+ cells, or gamma delta T cells.
15. The method of claim 13 wherein the population of immune cells is a population of C AR-
T cells.
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