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WO2018146128A1 - Detection of kit polymorphism for predicting the response to checkpoint blockade cancer immunotherapy - Google Patents

Detection of kit polymorphism for predicting the response to checkpoint blockade cancer immunotherapy Download PDF

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Publication number
WO2018146128A1
WO2018146128A1 PCT/EP2018/053035 EP2018053035W WO2018146128A1 WO 2018146128 A1 WO2018146128 A1 WO 2018146128A1 EP 2018053035 W EP2018053035 W EP 2018053035W WO 2018146128 A1 WO2018146128 A1 WO 2018146128A1
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antibodies
kit
cancer immunotherapy
checkpoint blockade
polymorphism
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PCT/EP2018/053035
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French (fr)
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Jérôme GALON
Bernhard Mlecnik
Gabriela BINDEA
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Sorbonne Universite
Université Paris Diderot - Paris 7
Universite Paris Descartes
Assistance Publique Hopitaux De Paris
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Publication of WO2018146128A1 publication Critical patent/WO2018146128A1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a method for predicting the response of a patient to checkpoint blockade cancer immunotherapy by detecting a single nucleotide polymorphism (SNP) in the KIT gene.
  • SNP single nucleotide polymorphism
  • Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self- tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage.
  • tumors co- opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors.
  • checkpoint blockade cancer immunotherapy vary among individuals. It is therefore necessary to define reliable predictive bio markers in an effort to better identify patients who are most likely to benefit from such a treatment.
  • checkpoint blockade cancer immunotherapies aim at releasing a brake on the immune system so as to allow the immune cells to infiltrate and attack the tumor.
  • this "brake release" In order for this "brake release" to be efficient, there has to be a pre-existing immune response directed against the tumor. Accordingly, in patients having low intra-tumor immune adaptive response, turning off the intra-tumor immunosuppressive response by using a checkpoint blockade cancer immunotherapy would be useless.
  • the KIT gene also known as the proto-oncogene receptor tyrosine kinase, is widely known in the art: its sequence can be e.g. found under the ref NG 007456 in the NCBI gene database. It encodes the KIT protein which is a 3 transmembrane receptor for MGF (mast cell growth factor, also known as stem cell factor).
  • the SNP of the kit gene according to the present invention consists in a substitution of an adenosine residue into a cytosine residue at position 1621 of the kit gene (A1621C).
  • This polymorphism is accessible under the reference rs3822214 in the Single Nucleotide Polymorphism Database (dbSNP), which is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI).
  • dbSNP Single Nucleotide Polymorphism Database
  • NCBI National Center for Biotechnology Information
  • NHGRI National Human Genome Research Institute
  • the present invention relates to the detection of this particular JQT M541L polymorphism for predicting the response of a patient to a checkpoint blockade cancer immunotherapy.
  • present invention relates to a checkpoint blockade cancer immunotherapy agent for use in a method for treating solid cancer in an individual, wherein said individual has been selected as not displaying the KIT polymorphism consisting of M541L (KIT M541L ).
  • the detection of the KIT polymorphism according to the present invention is thus particularly suitable for discriminating responders from non-responders.
  • the term "responder” refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or stabilized.
  • a non-responder or refractory patient includes patients for whom the cancer does not show reduction or stabilization after the immune checkpoint therapy.
  • the present invention relates to an in vitro method for predicting the response of a patient suffering from cancer to a checkpoint blockade cancer immunotherapy, said method comprising the step consisting of detecting, in a biological sample obtained from said patient, the presence of the KIT polymorphism consisting of M541L (KIT M541L ), wherein the presence of IT M541L indicates that the patient will not respond to checkpoint blockade cancer immunotherapy.
  • the present invention also relates to a method for treating a patient suffering from cancer, wherein said method comprises the steps of:
  • immunocheckpoint protein is widely known in the art and refers to a molecule that is expressed by T cells and that either turns up a signal (stimulatory checkpoint molecules) or turns down a signal (inhibitory checkpoint molecules).
  • Immune checkpoint constitute immune checkpoint pathways such as the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al, 2011. Nature 480:480- 489).
  • inhibitory checkpoint molecules examples include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 TIGIT and VISTA.
  • the Adenosine A2A receptor (A2AR) is considered as an important checkpoint in cancer therapy: the presence of adenosine in the immune microenvironment, leads to an A2a-receptor activation, and induces a negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine.
  • 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
  • B and T Lymphocyte Attenuator (BTLA) and also called CD272 has HVEM (Herpesvirus Entry Mediator) as its ligand.
  • HVEM Herpesvirus Entry Mediator
  • 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. Expression of CTLA-4 on Treg cells serves to control T cell proliferation.
  • IDOl Indoleamine 2,3-dioxygenase 1
  • TDO tryptophan catabolic enzyme
  • Another important molecule is TDO, tryptophan 2,3-dioxygenase.
  • IDOl is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis.
  • KIR Killer-cell Immunoglobulin- like Receptor
  • LAG3, Lymphocyte Activation Gene-3 works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells.
  • PD-1 Programmed Death 1 (PD-1) receptor
  • PD-L1 and PD-L2 This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014.
  • An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment.
  • 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.
  • VISTA Short for V-domain Ig suppressor of T cell activation, VISTA 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.
  • TIGIT also called T cell immunoreceptor with Ig and ITIM domains
  • NK Natural Killer Cells
  • checkpoint blockade cancer immunotherapy agent or “immune checkpoint inhibitor” has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade.
  • Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future.
  • the immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules.
  • CD8+ T cells has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. They are MHC class I-restricted, and function as cytotoxic T cells. "CD8+ T cells” are also called CD8+ T cells are called cytotoxic T lymphocytes (CTL), T-killer cell, cytolytic T cells, CD8+ T cells or killer T cells.
  • CTL cytotoxic T lymphocytes
  • T-killer cell cytolytic T cells
  • CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.
  • the ability of the immune checkpoint inhibitor to enhance T CD8 cell killing activity may be determined by any assay well known in the art.
  • said assay is an in vitro assay wherein CD8+ T cells are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by CD8+ T cells).
  • the immune checkpoint inhibitor of the present invention can be selected for the ability to increase specific lysis by CD8+ T cells 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 CD8+ T cells or CD8 T cell lines that are contacted by the immune checkpoint inhibitor of the present invention, Examples of protocols for classical cytotoxicity assays are conventional.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • the checkpoint blockade cancer immunotherapy agent is an antibody.
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
  • anti-CTLA-4 antibodies examples include anti-CTLA-4 antibodies.
  • One anti-CDLA-4 antibody is tremelimumab, (ticilimumab, CP-675,206).
  • the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4.
  • PD-1 and PD-L1 antibodies are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.
  • the PD-1 blockers include anti- PD-L1 antibodies.
  • the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1 ; CT-011 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade.
  • nivolumab MDX 1106, BMS 936558, ONO 4538
  • a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2
  • 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).
  • TIM3 T-cell immunoglobulin domain and mucin domain 3 inhibitors
  • 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).
  • TIM-3 inhibitor 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 signaling 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 Indoleamine 2,3- dioxygenase (IDO) inhibitor, preferably an IDOl inhibitor. Examples of IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1-methyl- tryptophan (IMT), ⁇ - (3-benzofuranyl)-alanine, P-(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 thiohyl
  • the IDO inhibitor is selected from 1- methyl-tryptophan, ⁇ -(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-Amino-naphtoic acid and ⁇ -[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.
  • the immune checkpoint inhibitor is an anti-TIGIT (T cell immunoglobin and ITIM domain) antibody.
  • the checkpoint blockade cancer immunotherapy agent is a CTLA4 blocking antibody, such as Ipilimumab, or a PD-1 blocking antibody, such as Nivolumab or Pembrolizumab, or a combination thereof.
  • kit nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance.
  • the nucleotide sequence can be obtained from a genomic DNA sample isolated from the biological sample.
  • genomic DNA e.g. not pure red blood cells
  • genomic DNA can be conveniently obtained from blood, semen, saliva, tears, urine, fecal material, sweat, buccal cells, skin, hair or other tissue containing nucleic acid of the individual.
  • the sample contains peripheral blood monocytes.
  • KIT mutations may be detected in a RNA or DNA sample, preferably after amplification.
  • the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a the mutated site or that enable amplification of a the region containing the mutated site.
  • RT-PCR polymerase chain reaction
  • conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the particular KIT SNP according to the invention.
  • RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which the mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • a cDNA obtained from RNA may be cloned and sequenced to identify the SNP according to the present invention in the KIT sequence.
  • RNA sequencing e.g. direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997).
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific oligonucleotides
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and realtime quantitative PCR.
  • the KIT SNP according to the present invention results in a mutated mature KIT protein, in which the methionine in position 541 is substituted by a leucine (KIT M541L ).
  • the SNP in the KIT gene may be detected at the protein level by detecting the IT M541L form of the KIT protein.
  • Suitable samples for detecting the mutated KIT protein according to the present invention are cell or tissue samples.
  • the sample can be a tumor sample obtained from the patient.
  • the tumor sample of the patient may be obtained by biopsy or resection.
  • the biopsy technique applied will depend on the tissue type to be evaluated, the size and type of the tumor, among other factors.
  • Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy.
  • An "excisional biopsy” refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it.
  • An “incisional biopsy” refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
  • This mutated form of the KIT protein may be detected according to any appropriate method known in the art.
  • a sample such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of the IT M541L form, i.e. antibodies that are capable of distinguishing between the IT M541L form and the wild-type protein (or any other protein), to determine the presence or absence of KIT M541L specified by the antibody.
  • the antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies.
  • polyclonal antibodies may be used, monoclonal antibodies are preferred because they are more reproducible in the long run. Procedures for raising "polyclonal antibodies" are also well known. Polyclonal antibodies can be obtained from serum of an animal immunized against the appropriate antigen, which may be produced by genetic engineering for example according to standard methods well-known by one skilled in the art (see e.g. Harlow et al. (1988)).
  • a “monoclonal antibody” in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular epitope.
  • a monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g. a bispecific monoclonal antibody.
  • Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988).
  • Aptamers which are a class of molecule that represents an alternative to antibodies in term of molecular recognition, can also be used for detecting the IT M541L form in the context of the present invention.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • All the probes, primers, aptamers or antibodies used in the context of the present invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the patient suffering from cancer is a mammalian, preferably a human.
  • the cancer is a solid cancer.
  • the cancer is a solid cancer affecting one of the following organs selected from the group consisting of uterus, endometrium, rectum, colon, cervix, esophagus, bladder, stomach, head and neck, liver, lung, bile duct, pancreas, eye, kidney, ovary, brain, breast and the thyroid gland.
  • Figure 1 Graph representing the density of infiltrating CD8+ cells at the invasive margin of the tumor in KIT mutated and non-mutated patients.
  • Example 1 JQX M541L j s correlated with decreased intra-tumor CD8 + cells infiltration Material, methods for KIT mutation detection
  • Genomic DNA from 214 patients has been extracted from frozen tumors using QIAmp DNA mini kit (Qiagen, Courtaboeuf, France) or, if frozen samples were not available, from two 5 ⁇ thick FFPE slides using QIAmp DNA FFPE kit (Qiagen). Quantity of double strand DNA have been evaluated using qubit 2.0 fluorometer (Invitrogen, life Technologies, Saint Aubin, France) and lOng (or 20ng if FFPE) of extracted DNA were amplified using Ion AmpliSeq Cancer HotSpot Panel V2 (Ion Torrent, Life Technologies) according to manufacturer's protocol.
  • hotspot regions of 50 oncogenes or tumor suppressor genes, including MET were amplified using a panel of 207 primer pairs in a 17 cycles PCR reaction (20 cycles for FFPE samples). Amplicon were then digested with FuPa Reagent and samples were separately barcoded with Ion Xpress Barcodes. IonAmpliSeq Adapters were then added to each sample. DNA banks were then purified using Agencourt AMPure XP Reagent (Beckman Coulter, Villepinte, France) and purified library obtained were amplified using Platinum PCR supermix High fidelity enzyme and purified again with Agencourt process, following the manufacturer's instructions (Ion AmpliSeq Library kit 2.0, Ion Torrent, Life Technologies).
  • TMA Tissue microarray
  • IM invasive margin
  • CT computed tomography
  • IM invasive margin
  • Assessment of the invasive margin area was performed on standard paraffin sections and was based on the histomorphological variance of the tissue.
  • the invasive margin was defined as a region centered on the border separating the host tissue from malignant glands, with the extend of 1 mm.
  • TMA sections were incubated (60 min. at room temperature) with monoclonal antibodies against CD8 (4B11, DAKO).
  • Envision+ system enzyme-conjugated polymer backbone coupled to secondary antibodies
  • DAB- chromogen were applied (Dako, Glostrup, Denmark).
  • Double stainings were revealed with phosphate-conjugated secondary antibodies and FastBlue-chromogen.
  • tissue sections were counterstained with Harris hematoxylin (Sigma Aldrich Saint Louis, MO). Isotype-matched mouse monoclonal antibodies were used as negative controls. Slides were analyzed using an image analysis workstation (Spot Browser, Excilone, Elancourt, France). Polychromatic high-resolution spot-images (740x540 pixel, 1.181 ⁇ /pixel resolution) were obtained (x200 fold magnification). The density was recorded as the number of positive cells per unit tissue surface area.
  • the t-test and the Wilcoxon-Mann- Whitney test were the parametric and non-parametric tests used to identify markers with a significantly different cell density among patient groups. P-value smaller than 0.05 was considered as significant.
  • patients displaying the IT M541L mutation have a decreased number of infiltrating CD8+ cells within the invasive margin of the tumor as compared to non-mutated patients.

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Abstract

Blockade of immune checkpoints is one of the most promising approaches for activating therapeutic antitumor immunity. However, the overall benefits of checkpoint blockade cancer immunotherapy vary among individuals. The present inventors demonstrated that a specific single nucleotide polymorphism in the KIT gene is associated with a decrease in intra-tumor CD8 infiltrates. Patients presenting this polymorphism present a low intra-tumor immune adaptive response and treating these patients with a checkpoint blockade cancer immunotherapy would thus be useless. Accordingly the present invention relates to a checkpoint blockade cancer immunotherapy agent for use in a method for treating cancer in an individual who does not display the KIT polymorphism consisting of M541L (KITL541). The present invention further relates to an method for predicting the response of a patient suffering from cancer to a checkpoint blockade cancer immunotherapy by detecting the presence of the M541L KIT polymorphism in a biological sample (such as a tumor sample).

Description

DETECTION OF KIT POLYMORPHISM FOR PREDICTING THE RESPONSE TO CHECKPOINT BLOCKADE CANCER IMMUNOTHERAPY
Field of the invention The present invention relates to a method for predicting the response of a patient to checkpoint blockade cancer immunotherapy by detecting a single nucleotide polymorphism (SNP) in the KIT gene.
Background of the invention
It is well established now that the immune microenvironment of a tumor is critical for determining the likeliness of a patient to be treated with immunotherapies (see e.g. Church et al, Immunity. 2015 Oct 20;43(4):631-3). As explained by Pardoll (Nat Rev Cancer. 2012 Mar 22;12(4):252-64), among the most promising approaches for activating therapeutic antitumor immunity is the blockade of immune checkpoints. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self- tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumors co- opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors.
However, the overall benefits of checkpoint blockade cancer immunotherapy vary among individuals. It is therefore necessary to define reliable predictive bio markers in an effort to better identify patients who are most likely to benefit from such a treatment.
Detailed description
The present inventors have surprisingly discovered that a specific single nucleotide polymorphism in the KIT gene is associated with a decrease in intra-tumor CD8 infiltrates. Patients presenting this polymorphism thus present a low intra-tumor immune adaptive response. As disclosed above, checkpoint blockade cancer immunotherapies aim at releasing a brake on the immune system so as to allow the immune cells to infiltrate and attack the tumor. In order for this "brake release" to be efficient, there has to be a pre-existing immune response directed against the tumor. Accordingly, in patients having low intra-tumor immune adaptive response, turning off the intra-tumor immunosuppressive response by using a checkpoint blockade cancer immunotherapy would be useless.
Accordingly, the inventors have demonstrated that patients displaying this specific single nucleotide polymorphism in the KIT gene cannot advantageously respond to checkpoint blockade cancer immunotherapies.
The KIT gene, also known as the proto-oncogene receptor tyrosine kinase, is widely known in the art: its sequence can be e.g. found under the ref NG 007456 in the NCBI gene database. It encodes the KIT protein which is a 3 transmembrane receptor for MGF (mast cell growth factor, also known as stem cell factor). The SNP of the kit gene according to the present invention consists in a substitution of an adenosine residue into a cytosine residue at position 1621 of the kit gene (A1621C). This polymorphism is accessible under the reference rs3822214 in the Single Nucleotide Polymorphism Database (dbSNP), which is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI). This A1621C polymorphism of the KIT gene encodes for the substitution of a methionine in position 541 by a leucine in the mature KIT protein (also referred to as KITL541 or KITM541L)
In a first aspect, the present invention relates to the detection of this particular JQTM541L polymorphism for predicting the response of a patient to a checkpoint blockade cancer immunotherapy.
Particularly, present invention relates to a checkpoint blockade cancer immunotherapy agent for use in a method for treating solid cancer in an individual, wherein said individual has been selected as not displaying the KIT polymorphism consisting of M541L (KITM541L). The detection of the KIT polymorphism according to the present invention is thus particularly suitable for discriminating responders from non-responders. As used herein, the term "responder" refers to a patient that will achieve a response, i.e. a patient where the cancer is eradicated, reduced or stabilized. A non-responder or refractory patient includes patients for whom the cancer does not show reduction or stabilization after the immune checkpoint therapy. Thus, in a further embodiment, the present invention relates to an in vitro method for predicting the response of a patient suffering from cancer to a checkpoint blockade cancer immunotherapy, said method comprising the step consisting of detecting, in a biological sample obtained from said patient, the presence of the KIT polymorphism consisting of M541L (KITM541L), wherein the presence of ITM541L indicates that the patient will not respond to checkpoint blockade cancer immunotherapy. The present invention also relates to a method for treating a patient suffering from cancer, wherein said method comprises the steps of:
-determining if the patient displays the KIT polymorphism consisting of M541L; and
-administering to said patient a checkpoint blockade cancer immunotherapy agent if said patient does not display the ITM541L polymorphism. The expression "immune checkpoint protein" is widely known in the art and refers to a molecule that is expressed by T cells and that either turns up a signal (stimulatory checkpoint molecules) or turns down a signal (inhibitory checkpoint molecules). Immune checkpoint constitute immune checkpoint pathways such as the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al, 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 TIGIT and VISTA. The Adenosine A2A receptor (A2AR) is considered as an important checkpoint in cancer therapy: the presence of adenosine in the immune microenvironment, leads to an A2a-receptor activation, and induces a negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. 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 tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. 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. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDOl, Indoleamine 2,3-dioxygenase 1, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDOl is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. 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. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. 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, VISTA 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. TIGIT (also called T cell immunoreceptor with Ig and ITIM domains) is an immune receptor on some percentage of T cells and Natural Killer Cells(NK). TIGIT inhibits T cell activation in vivo.
As used herein, the expression "checkpoint blockade cancer immunotherapy agent" or "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. In particular, the immune checkpoint inhibitor of the present invention is administered for enhancing the proliferation, migration, persistence and/or cytoxic activity of CD8+ T cells in the subject and in particular the tumor-infiltrating of CD8+ T cells of the subject. As used herein "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. They are MHC class I-restricted, and function as cytotoxic T cells. "CD8+ T cells" are also called CD8+ T cells are called cytotoxic T lymphocytes (CTL), T-killer cell, cytolytic T cells, CD8+ T cells or killer T cells. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. The ability of the immune checkpoint inhibitor to enhance T CD8 cell killing activity may be determined by any assay well known in the art. Typically said assay is an in vitro assay wherein CD8+ T cells are brought into contact with target cells (e.g. target cells that are recognized and/or lysed by CD8+ T cells). For example, the immune checkpoint inhibitor of the present invention can be selected for the ability to increase specific lysis by CD8+ T cells 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 CD8+ T cells or CD8 T cell lines that are contacted by the immune checkpoint inhibitor of the present invention, Examples of protocols for classical cytotoxicity assays are conventional.
Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin-like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1). Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.
In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.
Examples of anti-CTLA-4 antibodies are described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. One anti-CDLA-4 antibody is tremelimumab, (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4.
Examples of PD-1 and PD-L1 antibodies are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. In some embodiments, the PD-1 blockers include anti- PD-L1 antibodies. In certain other embodiments the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1 ; CT-011 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade.
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 TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade 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 signaling 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 Indoleamine 2,3- dioxygenase (IDO) inhibitor, preferably an IDOl inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl- tryptophan (IMT), β- (3-benzofuranyl)-alanine, P-(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 β- carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1- methyl-tryptophan, β-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-Amino-naphtoic acid and β-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT (T cell immunoglobin and ITIM domain) antibody.
In a preferred embodiment, the checkpoint blockade cancer immunotherapy agent is a CTLA4 blocking antibody, such as Ipilimumab, or a PD-1 blocking antibody, such as Nivolumab or Pembrolizumab, or a combination thereof.
The presence of the Single Nucleotide Polymorphism (SNP) according to the present invention may be detected by analyzing a KIT nucleic acid molecule. In the context of the invention, kit nucleic acid molecules include mRNA, genomic DNA and cDNA derived from mRNA. DNA or RNA can be single stranded or double stranded. These may be utilized for detection by amplification and/or hybridization with a probe, for instance.
The nucleotide sequence can be obtained from a genomic DNA sample isolated from the biological sample. In this case, any biological sample containing genomic DNA (e.g. not pure red blood cells) can be used. For example, and without limitation to, genomic DNA can be conveniently obtained from blood, semen, saliva, tears, urine, fecal material, sweat, buccal cells, skin, hair or other tissue containing nucleic acid of the individual. In a particular embodiment, the sample contains peripheral blood monocytes.
KIT mutations may be detected in a RNA or DNA sample, preferably after amplification. For instance, the isolated RNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for a the mutated site or that enable amplification of a the region containing the mutated site. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the particular KIT SNP according to the invention. Otherwise, RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which the mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. For instance, a cDNA obtained from RNA may be cloned and sequenced to identify the SNP according to the present invention in the KIT sequence.
Several methods known by the skilled person allows detecting a single nucleotide polymorphism in a nucleic acid sequence. Such methods comprise e.g. direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and realtime quantitative PCR. As disclosed above, the KIT SNP according to the present invention results in a mutated mature KIT protein, in which the methionine in position 541 is substituted by a leucine (KITM541L). Thus, according to a further embodiment, the SNP in the KIT gene may be detected at the protein level by detecting the ITM541L form of the KIT protein. Suitable samples for detecting the mutated KIT protein according to the present invention are cell or tissue samples. For instance, the sample can be a tumor sample obtained from the patient. Typically the tumor sample of the patient may be obtained by biopsy or resection. The biopsy technique applied will depend on the tissue type to be evaluated, the size and type of the tumor, among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor.
This mutated form of the KIT protein may be detected according to any appropriate method known in the art. In particular a sample, such as a tissue biopsy, obtained from a subject may be contacted with antibodies specific of the ITM541L form, i.e. antibodies that are capable of distinguishing between the ITM541L form and the wild-type protein (or any other protein), to determine the presence or absence of KITM541L specified by the antibody.
The antibodies may be monoclonal or polyclonal antibodies, single chain or double chain, chimeric antibodies, humanized antibodies, or portions of an immunoglobulin molecule, including those portions known in the art as antigen binding fragments Fab, Fab', F(ab')2 and F(v). They can also be immunoconjugated, e.g. with a toxin, or labelled antibodies.
Whereas polyclonal antibodies may be used, monoclonal antibodies are preferred because they are more reproducible in the long run. Procedures for raising "polyclonal antibodies" are also well known. Polyclonal antibodies can be obtained from serum of an animal immunized against the appropriate antigen, which may be produced by genetic engineering for example according to standard methods well-known by one skilled in the art (see e.g. Harlow et al. (1988)).
A "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g. a bispecific monoclonal antibody. Laboratory methods for preparing monoclonal antibodies are well known in the art (see, for example, Harlow et al., 1988).
Aptamers, which are a class of molecule that represents an alternative to antibodies in term of molecular recognition, can also be used for detecting the ITM541L form in the context of the present invention. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
All the probes, primers, aptamers or antibodies used in the context of the present invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. Typically, the patient suffering from cancer is a mammalian, preferably a human.
The cancer is a solid cancer.
In a preferred embodiment, the cancer is a solid cancer affecting one of the following organs selected from the group consisting of uterus, endometrium, rectum, colon, cervix, esophagus, bladder, stomach, head and neck, liver, lung, bile duct, pancreas, eye, kidney, ovary, brain, breast and the thyroid gland.
Brief Description of the figures
Figure 1: Graph representing the density of infiltrating CD8+ cells at the invasive margin of the tumor in KIT mutated and non-mutated patients.
EXAMPLES
We demonstrate that the polymorphism JQTM541L is correlated with decreased intra-tumor CD8+ cells infiltration. This weak intra-tumor adaptive immune response prevents the patients from responding to checkpoint blockade cancer immunotherapies. Thus, these findings identify the KITM541L mutation as a biomarker for the response of a patient to checkpoint blockade cancer immunotherapy.
Example 1: JQXM541L js correlated with decreased intra-tumor CD8+ cells infiltration Material, methods for KIT mutation detection
Genomic DNA from 214 patients has been extracted from frozen tumors using QIAmp DNA mini kit (Qiagen, Courtaboeuf, France) or, if frozen samples were not available, from two 5μιη thick FFPE slides using QIAmp DNA FFPE kit (Qiagen). Quantity of double strand DNA have been evaluated using qubit 2.0 fluorometer (Invitrogen, life Technologies, Saint Aubin, France) and lOng (or 20ng if FFPE) of extracted DNA were amplified using Ion AmpliSeq Cancer HotSpot Panel V2 (Ion Torrent, Life Technologies) according to manufacturer's protocol. Briefly, "hotspot" regions of 50 oncogenes or tumor suppressor genes, including MET were amplified using a panel of 207 primer pairs in a 17 cycles PCR reaction (20 cycles for FFPE samples). Amplicon were then digested with FuPa Reagent and samples were separately barcoded with Ion Xpress Barcodes. IonAmpliSeq Adapters were then added to each sample. DNA banks were then purified using Agencourt AMPure XP Reagent (Beckman Coulter, Villepinte, France) and purified library obtained were amplified using Platinum PCR supermix High fidelity enzyme and purified again with Agencourt process, following the manufacturer's instructions (Ion AmpliSeq Library kit 2.0, Ion Torrent, Life Technologies). Quality and Quantity of each libraries have been evaluated thanks to High Sensitivity DNA chip (Agilent technologies, Courtaboeuf, France). Patients were then mixed and libraries obtained were amplified and enriched using the ion OneTouch 2 system (Ion PGM Template OT2 200, life technologies). Sequencing was performed with the Ion Torrent PGM system using Ion316 or Ion318 chip and the Ion PGM sequencing 200 kit V2 in a 520 cycles run. Runs were aligned using Variant Caller (V4.2.1.0) plugin compared to Hgl9 database, and results were analysed using Alamut 2.4.1 software (interactive biosoftware, Rouen, France).
Material, methods for immune densities: Tissue Microarray immunohistochemistry.
Tissue microarray (TMA) from the center (CT) and invasive margin (IM) of colorectal tumors (n=415) were constructed. Assessment of the invasive margin area was performed on standard paraffin sections and was based on the histomorphological variance of the tissue. The invasive margin was defined as a region centered on the border separating the host tissue from malignant glands, with the extend of 1 mm. TMA sections were incubated (60 min. at room temperature) with monoclonal antibodies against CD8 (4B11, DAKO). Envision+ system (enzyme-conjugated polymer backbone coupled to secondary antibodies) (Dako, Glostrup, Denmark) and DAB- chromogen were applied (Dako, Glostrup, Denmark). Double stainings were revealed with phosphate-conjugated secondary antibodies and FastBlue-chromogen. For single stainings, tissue sections were counterstained with Harris hematoxylin (Sigma Aldrich Saint Louis, MO). Isotype-matched mouse monoclonal antibodies were used as negative controls. Slides were analyzed using an image analysis workstation (Spot Browser, Excilone, Elancourt, France). Polychromatic high-resolution spot-images (740x540 pixel, 1.181 μιη/pixel resolution) were obtained (x200 fold magnification). The density was recorded as the number of positive cells per unit tissue surface area.
The t-test and the Wilcoxon-Mann- Whitney test were the parametric and non-parametric tests used to identify markers with a significantly different cell density among patient groups. P-value smaller than 0.05 was considered as significant.
As shown in Figure 1, patients displaying the ITM541L mutation have a decreased number of infiltrating CD8+ cells within the invasive margin of the tumor as compared to non-mutated patients.

Claims

1. A checkpoint blockade cancer immunotherapy agent for use in a method for treating solid cancer in an individual, wherein said individual has been selected as not displaying the KIT polymorphism consisting of M541L (KITM451L).
2. A in vitro method for predicting the response of a patient suffering from cancer to a checkpoint blockade cancer immunotherapy, said method comprising the step consisting of detecting, in a biological sample obtained from said patient, the presence of the KIT polymorphism consisting of M541L (KITM541L), wherein the presence of ITM541L indicates that the patient will not respond to checkpoint blockade cancer immunotherapy.
3. The checkpoint blockade cancer immunotherapy agent for use according to claim 1 or the method according to claim 2, wherein said checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti- IDOl antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
4. The checkpoint blockade cancer immunotherapy agent for use according to claim 1 or the method according to claim 2, wherein said checkpoint blockade cancer immunotherapy agent is an antibody selected from anti-CTLA4 antibodies and anti-PDl antibodies.
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