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WO2019207030A1 - Procédés de prédiction d'une réponse à un inhibiteur de point de contrôle immunitaire chez un patient souffrant d'un cancer du poumon - Google Patents

Procédés de prédiction d'une réponse à un inhibiteur de point de contrôle immunitaire chez un patient souffrant d'un cancer du poumon Download PDF

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WO2019207030A1
WO2019207030A1 PCT/EP2019/060591 EP2019060591W WO2019207030A1 WO 2019207030 A1 WO2019207030 A1 WO 2019207030A1 EP 2019060591 W EP2019060591 W EP 2019060591W WO 2019207030 A1 WO2019207030 A1 WO 2019207030A1
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egfr
mutations
stk11
tumor
immune checkpoint
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Inventor
Diane DAMOTTE
Jérôme BITON
Ronald Herbst
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Diderot Paris 7
Universite Sorbonne Paris Nord
Sorbonne Universite
MedImmune LLC
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
Universite Paris Diderot Paris 7
Universite Sorbonne Paris Nord
Sorbonne Universite
MedImmune LLC
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Publication of WO2019207030A1 publication Critical patent/WO2019207030A1/fr
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    • 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
    • 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 method of predicting whether a patient suffering from a lung cancer will achieve a response with an immune checkpoint inhibitor.
  • TMB tumor mutational burden
  • WES heavy whole exome sequencing
  • the inventors performed from lung adenocarcinoma samples an integrative analysis that incorporated immunochemistry (IHC), gene expression, mutational and flow cytometry data. They identified three main tumor immune profiles (TIPs), and found that co- occuring genetic alterations, especially TP53, EGFR and STK11 mutations, are major determinants of the tumor immune composition and of PD-L 1 expression by malignant cells. Moreover, they found that distinct combinations of TP 53, EGFR and STK11 mutations were able to identify best responders to PD-l blockers. Particularly, the inventors showed a prolonged progression-free survival in patients treated with anti-PD-l and harboring TP53- mat/STKll-EGFR-WT tumors.
  • the present invention relates to a method of predicting whether a patient suffering from a lung cancer will achieve a response with an immune checkpoint inhibitor comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient and ii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP 53 and no mutations in STK11 and EGFR.
  • the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:
  • a first object of the present invention relates to a method of predicting whether a patient suffering from a lung cancer will achieve a response with an immune checkpoint inhibitor comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient and ii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP 53 and no mutations in STK11 and EGFR.
  • a supplemental step of measuring the PD-L1 expression in the tumor tissue sample of the patients can be added.
  • the invention relates to a method of predicting whether a patient suffering from a lung cancer will achieve a response with an immune checkpoint inhibitor comprising i) determining the mutations profile of the genes TP 53, STK11 and EGFR in a tumor tissue sample obtained from the patient ii) measuring the PD-L1 expression in said tumor tissue sample and iii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP 53, no mutations in STK11 and EGFR and has a positive expression of PD-L1 on its tumor cells.
  • the lung cancer can be a non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • non-small cell lung cancer As used herein, the term“non-small cell lung cancer” or “NSCLC” has its general meaning in the art and includes a disease in which malignant cancer cells form in the tissues of the lung. Examples of non-small cell lung cancers include, but are not limited to, squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.
  • the term“mutations profile of the genes TP53, STK11 and EGFR” denotes the identification of mutations (at least one) in these genes by well know techniques like PCR or Next Generation Sequencing. According to the invention, the mutations profile of the genes TP 53, STK11 and EGFR is done in a tumor tissue sample obtained from the patient.
  • mutation mean any detectable change in genetic material, e.g. DNA, RNA, cDNA, or any process, mechanism, or result of such a change.
  • a mutation is identified in a subject by comparing the sequence of a nucleic acid or polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population.
  • a mutation in the genetic material may also be“silent”, i.e. the mutation does not result in an alteration of the amino acid sequence of the expression product.
  • TP53 denotes a gene located on the short arm of chromosome 17 (17r13.1). The gene spans 20 kb, with a non-coding exon 1 and a very long first intron of 10 kb.
  • the coding sequence contains five regions showing a high degree of conservation in vertebrates, predominantly in exons 2, 5, 6, 7 and 8, but the sequences found in invertebrates show only distant resemblance to mammalian TP 53.
  • the TP 53 gene is the most frequently mutated gene (>50%) in human cancer, indicating that the TP 53 gene plays a crucial role in preventing cancer formation. Its accession number in the Entrez data base is 7157.
  • STRIP denotes a gene which codes for the serine/threonine kinase 11 which regulates cell polarity and functions as a tumour suppressor. Its accession number in the Entrez data base is 6794.
  • EGFR denotes a gene which codes for the epidermal growth factor receptor which is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family). Over-expression of this receptor is associated with the development of a wide variety of tumors. In lung cancer mutations of EGFR is associated with targeted therapies. Its accession number in the Entrez data base is 1956.
  • a positive expression of PD-L1 on tumor cells denotes that more than 1 percent of all tumor cells express PD-L1 at their surface. In a particular embodiment, the percentage is between 1% and 10%, 1 and 20%, 1 and 30%, 1 and 40% or 1 and 50%. In a particular embodiment, the percentage is more than 50%.
  • the expression "high probability to achieve a response with an immune checkpoint inhibitor” is understood to mean the situation where the patient shows at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% of chance to achieve a response.
  • 50% of chance to achieve a response means that the subject has 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100% of chance to achieve a response.
  • the method is thus particularly suitable for discriminating responder from non responder.
  • the term“responder” in the context of the present disclosure refers to a patient that will achieve a response, i.e. a patient where the lung cancer is eradicated, reduced or improved.
  • the responders have an objective response and therefore the term does not encompass patients having a stabilized cancer such that the disease is not progressing after the treatment with the immune checkpoint inhibitor.
  • a non- responder or refractory patient includes patients for whom the lung cancer does not show reduction or improvement after the treatment with the immune checkpoint inhibitor.
  • the term“non-responder” also includes patients having a stabilized cancer.
  • the characterization of the patient as a responder or non-responder can be performed by reference to a standard or a training set.
  • the standard may be the profile of a patient who is known to be a responder or non-responder or alternatively may be a numerical value.
  • Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media.
  • 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.
  • 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.
  • 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 A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA.
  • the immune checkpoint inhibitor is a PD-l inhibitor or a TIM-3 inhibitor.
  • 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, inhibits the activation of T cells. Accordingly, the term“PD-l inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of PD-l . Lor example, the inhibitor can inhibit the expression or activity of PD-l, modulate or block the PD-l signaling pathway and/or block the binding of PD-l to PD-L1 or PD-L2.
  • the present invention relates to a method of predicting whether a patient suffering from a lung cancer will achieve a response with a PD-l inhibitor comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient and ii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP 53 and no mutations in STK11 and EGFR.
  • a supplemental step of measuring the PD-L1 expression in the tumor tissue sample of the patients can be added.
  • the invention relates to a method of predicting whether a patient suffering from a lung cancer will achieve a response with a PD-l inhibitor comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient ii) measuring the PD-L1 expression in said tumor tissue sample and iii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP53, no mutations in STK11 and EGFR and has a positive expression of PD-L 1 on its tumor cells.
  • a supplemental step of detecting a mutation in the gene KRAS can be added to the method of the invention.
  • the patient with high probability to achieve a response with an immune checkpoint inhibitor has at least one mutations on KRAS.
  • the term“patient” denotes an human suffering from a lung cancer and particularly a non-small cell lung cancer.
  • tumor tissue sample has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection.
  • the tumor tissue sample can be subjected to a variety of well-known post collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.) prior to determining the cell densities.
  • the tumor tissue sample is fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut.
  • Thin slices of material can be then prepared using a microtome, placed on a glass slide and submitted e.g.
  • 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.
  • TP53, STK11 and EGFR in the tumor tissue sample are determined by any well-known method in the art. Accordingly, the mutations may be detected by analysing a nucleic acid molecule.
  • TP53, STK11 and EGFR 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.
  • Mutations in the gene of the invention can be detected in a RNA or DNA sample, preferably after amplification obtained from a tumor tissue sample.
  • the isolated RNA may be subjected to couple 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 mutated site or that enable amplification of a 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 a particular mutation.
  • RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a 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 a mutation in the genes of the invention.
  • nucleic acid molecule may be tested for the presence or absence of a restriction site.
  • a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct enzymatic test for the mutation.
  • RNA sequencing includes, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFFP) 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), high-resolution-melting (HRM) analysis, primer extension (Snapshot), and denaturing high performance liquid chromatography (DHPLC) (Kuklin et ah, 1997).
  • DGGE denaturing gradient gel electrophoresis
  • TGGE temperature denaturing gradient gel electrophoresis
  • SSCP single-stranded conformational polymorphism
  • HRM high-resolution-mel
  • 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 real-time quantitative PCR.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • InvaderTMassay the InvaderTMassay
  • OLA oligonucleotide ligation assay
  • two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the mutation.
  • DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized (Nickerson et ah, 1990).
  • NGS Next Generation Sequencing
  • a lung panel like Ion AmpliSeq Colon and Lung Cancer Research Panel v2 from ThermoFisher (see examples).
  • the expression of PD-L1 in the tumor tissue sample is determined by any well-known method in the art.
  • the expression of PD-L1 in the tumor tissue sample is determined by immunohistochemistry. For example, the determination is performed by contacting the tumor tissue sample with a binding partner (e.g. an antibody) specific PD-L1.
  • a binding partner e.g. an antibody
  • 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 immune checkpoint protein of interest, 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 with the binding partners having for the immune checkpoint protein of interest.
  • the labeled antibodies that are bound to the immune checkpoint protein of interest are revealed by the appropriate technique, depending of the kind of label is 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. Hematoxylin & Eosin, 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 immune checkpoint protein).
  • exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof.
  • 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. 3 H, 14 C, 32 P, 35 S or 125 I) 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 label is a quantum dot.
  • Qdots Quantum dots
  • Qdots are becoming increasingly useful in a growing list of applications including immunohistochemistry, flow cytometry, and plate-based assays, and may therefore be used in conjunction with this invention.
  • Qdot nanocrystals have unique optical properties including an extremely bright signal for sensitivity and quantitation; high photostability for imaging and analysis. A single excitation source is needed, and a growing range of conjugates makes them useful in a wide range of cell-based applications.
  • Qdot Bioconjugates are characterized by quantum yields comparable to the brightest traditional dyes available. Additionally, these quantum dot-based fluorophores absorb 10-1000 times more light than traditional dyes.
  • the emission from the underlying Qdot quantum dots is narrow and symmetric which means overlap with other colors is minimized, resulting in minimal bleed through into adjacent detection channels and attenuated crosstalk, in spite of the fact that many more colors can be used simultaneously.
  • 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 immune checkpoint protein in the sample, or the absolute number of cells positive for the maker of interest, or the surface of cells positive for the maker of interest.
  • Various automated sample processing, scanning and analysis systems suitable for use with IHC 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.
  • 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 quantify the presence of the specified biomarker (i.e. immune checkpoint protein).
  • 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 and tissue recognition pattern (e.g.
  • 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 immune checkpoint protein
  • 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 amount can be quantified as an absolute number of cells positive for the maker of interest.
  • 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 immune checkpoint protein) present in the sample.
  • Optical density or percentage area values can be given a scaled score, for example on an integer scale.
  • 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 PDL-l , ii) proceeding to digitalisation of the slides of step i).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 or the absolute number of stained cells in each unit.
  • the expression level of PDL-l is determined by determining the quantity of mRNA encoding for PD-L1.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the subject
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In some embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • A“detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20l320l6, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos.
  • semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Fife Technologies (Carlshad, Calif.).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence.
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme.
  • the substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podo
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are“specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.
  • the level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • the nCounter® Analysis system is used to detect intrinsic gene expression.
  • the basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3): 317- 325; the contents of which are each incorporated herein by reference in their entireties).
  • the code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed.
  • a pair of probes is designed for each DNA or RNA target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode.
  • the reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over-lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence.
  • each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively.
  • the capture probe can comprise a second target-specific sequence; and a first affinity tag.
  • the capture probe can also comprise one or more label attachment regions.
  • the first target- specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected. Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library".
  • the relative abundance of each target is measured in a single multiplexed hybridization reaction.
  • the method comprises contacting the tumor tissue sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex.
  • the complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution.
  • the tripartite hybridized complexes are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes. This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample.
  • Prep Station NanoString Technologies
  • Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe, electrophoresed to elongate the reporter probes, and immobilized.
  • the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies).
  • the level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface.
  • FOV fields-of-view
  • Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance. Data is output in simple spreadsheet format listing the number of counts per target, per sample.
  • This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No. WO 07/076129 and W007/076132, and US Patent Publication No. 2010/0015607 and 2010/0261026, the contents of which are incorporated herein in their entireties. Further, the term nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No.2010/0047924, incorporated herein by reference in its entirety.
  • Expression level of a gene may be expressed as absolute level or normalized level.
  • levels are normalized by correcting the absolute level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the subject, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. This normalization allows the comparison of the level in one sample, e.g., a subject sample, to another sample, or between samples from different sources.
  • a further object of the present invention relates to a method for determining the survival time of a patient suffering from a lung cancer and treated with an immune checkpoint inhibitor comprising:
  • a further object of the present invention relates to a method for determining the survival time of a patient suffering a lung cancer and treated with an immune checkpoint inhibitor comprising:
  • the fact that the patient will have a short survival time means that the patient is non-responder to the immune checkpoint inhibitor used.
  • the immune checkpoint inhibitor is a PD-l inhibitor.
  • OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or if they've become cancer-free (achieved remission). DSF gives more specific information and is the number of people with a particular cancer who achieve remission.
  • progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) includes people who may have had some success with treatment, but the cancer has not disappeared completely.
  • the expression“short survival time” indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a short survival time, it is meant that the patient will have a“poor prognosis”.
  • the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a long survival time, it is meant that the patient will have a“good prognosis”.
  • Another object of the present invention relates to a method of monitoring a treatment with an immune checkpoint inhibitor in a patient suffering from a lung cancer comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient and ii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP53 and no mutations in STK11 and EGFR.
  • a supplemental step of measuring the PD-L1 expression in the tumor tissue sample of the patients can be added.
  • the invention relates to a method of monitoring a treatment with an immune checkpoint inhibitor in a patient suffering from a lung cancer comprising i) determining the mutations profile of the genes TP53, STK11 and EGFR in a tumor tissue sample obtained from the patient ii) measuring the PD-L1 expression in said tumor tissue sample and iii) concluding that the patient has a high probability to achieve a response with an immune checkpoint inhibitor when the patients has at least one mutation in TP53, no mutations in STK11 and EGFR and has a strong expression of PD-L1 on its tumor cells.
  • a second object of the invention relates to a method of treating a patient suffering from a lung cancer comprising administering to said patient in need thereof a therapeutically effective amount of an immune checkpoint inhibitor and wherein said patient has a high probability to not achieve a response with an immune checkpoint inhibitor as determined by the method described above.
  • the patient will be treated by a different immune checkpoint inhibitor than the first he received.
  • 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 subject 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 subject 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., disease manifestation, etc.]).
  • the term "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the immune checkpoint inhibitor of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the immune checkpoint inhibitor of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the immune checkpoint inhibitor of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician could start doses of the immune checkpoint inhibitor of the present invention employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved.
  • a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled inhibitor of the present invention, fragment or mini-antibody derived from the inhibitor of the present invention.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the human monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of a inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 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,
  • the immune checkpoint inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1 ,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or diglycerides.
  • Faty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include lactose and com starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include, e.g., lactose.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the compositions of this invention may be administered in the form of suppositories for rectal administration.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.
  • the compositions of this invention may also be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.
  • the pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m 2 and 500 mg/m 2 .
  • these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • a pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of the inhibitor of the invention.
  • the immune checkpoint inhibitor (I;e; the different immune checkpoint inhibitor than the first the patient received) is an antibody selected from the group consisting of anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-Galectin 9 antibodies and anti-TIM-3 antibodies.
  • the immune checkpoint inhibitor is an antibody selected from the group consisting of nivolumab (anti-PD-l), pembrolizumab (anti-PD-l) and durvalumab (anti-PD-Ll).
  • antibody is thus used to refer to any antibody- like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" sc
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab’ fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Fe Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.
  • the antibody of the present invention is a single chain antibody.
  • single domain antibody has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also“nanobody®”.
  • single domain antibody are also“nanobody®”.
  • (single) domain antibodies reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends BiotechnoL, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.
  • the term“specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen (e.g. TIM-3, PD-l, galectin-9, PD-F1 or PD-F2), while having relatively little detectable reactivity with other proeins or structures (such as other proteins presented on CD8 T cells, or on other cell types). Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein.
  • an antigen e.g. TIM-3, PD-l, galectin-9, PD-F1 or PD-F2
  • affinity means the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • the affinity constant Ka is defined by l/Kd.
  • Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference.
  • One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.
  • each heavy chain is linked to a light chain by a disulfide bond.
  • Each chain contains distinct sequence domains.
  • the light chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH).
  • variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen.
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs).
  • Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
  • F(ab')2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
  • Fab'“ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
  • a single chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.“dsFv” is a VH::VL heterodimer stabilised by a disulfide bond.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • the antibody is a humanized antibody.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761 , 5,693,762 and 5,859,205, which are hereby incorporated by reference.
  • the antibody is a fully human antibody.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies.
  • the animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.
  • the antibody comprises human heavy chain constant regions sequences but will induce antibody dependent cellular cytotoxicity (ADCC).
  • the antibody of the present invention does not comprise an Fc domain capable of substantially binding to a FcgRIIIA (CD 16) polypeptide.
  • the antibody of the present invention lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of IgG2 or IgG4 isotype.
  • the antibody of the present invention consists of or comprises a Fab, Fab', Fab'-SH, F (ah') 2, Fv, a diabody, single-chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments.
  • the antibody of the present invention is not linked to a toxic moiety.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by ldusogie et al.
  • the antibody of the present invention is a single chain antibody.
  • single domain antibody has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also “nanobody®”.
  • single domain antibody are also “nanobody®”.
  • (single) domain antibodies reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al, Trends BiotechnoL, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.
  • the amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs” which are referred to in the art and herein as “Framework region 1" or “FR1 as “Framework region 2" or “FR2”; as “Framework region 3 " or “FR3”; and as “Framework region 4" or“FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs”, which are referred to in the art as "Complementarity Determining Region for "CDR1”; as “Complementarity Determining Region 2" or “CDR2” and as “Complementarity Determining Region 3" or “CDR3”, respectively.
  • the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.
  • Antibodies having specificity for TIM-3 are well known in the art and typically include those described in WO2011155607, W02013006490 and WO2010117057.
  • Antibodies having specificity for PD-l or PDL-l are well known in the art and typically include those 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: W003042402, WO2008156712, W02010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.
  • the PD-l inhibitors include anti-PD-Ll antibodies.
  • the PD-l inhibitors include anti-PD-l 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-l by its ligands PD-L1 and PD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-l ; CT-011 a humanized antibody that binds PD-l ; 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-l by its ligands PD-L1 and
  • the immune checkpoint inhibitor is a multispecific antibody comprising at least one binding site that specifically binds to a PD-l molecule, and at least one binding site that specifically binds to a TIM-3 molecule.
  • Multispecific antibodies are typically described in WO2011159877. According to the invention the multispecific antibody of the present invention binds to PD-l and TIM-3 and inhibits the ability of PD-l to, for example, bind PD-L1, and inhibits the ability of TIM-3 to, for example, bind galectin-9.
  • Exemplary formats for the multispecific antibody molecules of the present invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to TIM-3 and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically-linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of
  • bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization.
  • Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), FUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
  • the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology.
  • bispecific antibodies In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in W02008119353 and WO 2011131746 (both by Genmab A/S).
  • a bispecific antibody is formed by "Fab-arm" or "half- molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is a antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is a antibody of the present invention and the second antibody has a different binding specificity, or vice versa.
  • the reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2- mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine.
  • Step d) may further comprise restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
  • the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety.
  • the immune checkpoint inhibitor is a polypeptide comprising a functional equivalent of TIM-3 or PD-l .
  • a“functional equivalent of TIM-3 or PD-l” is a polypeptide which is capable of binding to a TIM-3 or PD-l ligand, thereby preventing its interaction with TIM-3 or PD-l .
  • the term "functional equivalent” includes fragments, mutants, and muteins of TIM-3 or PD-l .
  • the term "functionally equivalent” thus includes any equivalent of TIM-3 or PD-l obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to a ligand of TIM-3 or PD-l .
  • Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.
  • Functional equivalents include molecules that bind a ligand of TIM-3 or PD-l and comprise all or a portion of the extracellular domains of TIM-3 or PD-l so as to form a soluble receptor that is capable to trap the ligand of TIM-3 or PD-l .
  • the functional equivalents include soluble forms of the TIM-3 or PD-l .
  • a suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods.
  • the functional equivalent is at least 80% homologous to the corresponding protein.
  • the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm.
  • the term "a functionally equivalent fragment” as used herein also may mean any fragment or assembly of fragments of TIM-3 or PD-l that binds to a ligand of TIM-3 or PD-l .
  • the present invention provides a polypeptide capable of inhibiting binding of TIM-3 or PD-l to a ligand of TIM-3 or PD-l, which polypeptide comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of an extracellular domain of TIM-3 or PD-l, which portion binds to a ligand of TIM-3 or PD-l .
  • the polypeptide corresponds to an extracellular domain of TIM-3 or PD-l .
  • the polypeptide comprises a functional equivalent of TIM-3 or PD-l which is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin.
  • Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use.
  • the Fc region is a native sequence Fc region. In some embodiments, the Fc region is a variant Fc region.
  • the Fc region is a functional Fc region.
  • the term "Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the adhesion portion and the immunoglobulin sequence portion of the immunoadhesin may be linked by a minimal linker.
  • the immunoglobulin sequence typically, but not necessarily, is an immunoglobulin constant domain.
  • the immunoglobulin moiety in the chimeras of the present invention may be obtained from IgGl, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but typically IgGl or IgG3.
  • the functional equivalent of the TIM-3 or PD-l and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker.
  • the term“linker” refers to a sequence of at least one amino acid that links the polypeptide of the invention and the immunoglobulin sequence portion. Such a linker may be useful to prevent steric hindrances.
  • the linker has 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 amino acid residues.
  • the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides.
  • the linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is typically non-immunogenic in the subject to which the immunoadhesin is administered.
  • linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678. Other examples are poly-alanine linker sequences.
  • the immune checkpoint inhibitor is an inhibitor of TIM-3 or PD- 1 expression.
  • 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 TIM-3 or PD-l mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of TIM-3 or PD-l, 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 TIM-3 or PD-l 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.
  • RNAs small inhibitory RNAs
  • TIM-3 or PD-l gene expression can be reduced by contacting a subject 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 TIM-3 or PD-l gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • 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 TIM-3 or PD-l .
  • 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 papilloma viruses
  • herpes virus vaccinia virus
  • polio virus poli
  • the immune checkpoint inhibitor is administered in combination with anti-cancer therapy to treat patient according to the method of the invention.
  • the invention also relates to a method of treating a patient suffering from a lung cancer comprising administering to said patient in need thereof a therapeutically effective amount of an immune checkpoint inhibitor in combination with anti-cancer therapy and wherein said patient has a high probability to not achieve a response with an immune checkpoint inhibitor as determined by the method described above.
  • anti-cancer treatment or“anti-cancer therapy” has its general meaning in the art and refers to anti-cancer agents used in anti-cancer therapy.
  • Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
  • Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
  • radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy such as Ra223 or Pb2l2.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • the term“radiotherapy” for“radiation therapy” has its general meaning in the art and refers the treatment of cancer with ionizing radiation.
  • Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow.
  • One type of radiation therapy commonly used involves photons, e.g. X-rays.
  • the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy.
  • Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay.
  • the radiation therapy is external radiation therapy.
  • external radiation therapy examples include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction
  • the immune checkpoint inhibitor is administered simultaneously, sequentially or concomitantly with one or more therapeutic active agent such as anti-cancer agents.
  • therapeutic active agent such as anti-cancer agents.
  • the term“administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different
  • FIGURES are a diagrammatic representation of FIGURES.
  • PFS progression-free survival
  • C-D data were expressed as mean ⁇ SEM and a nonparametric test (Kruskal-Wallis test followed by a post-hoc Dunn's test) was applied based on Shapiro normality test. *p ⁇ 0.05.
  • A-C Kaplan-Meier curves of overall survival (OS) in patients with TP 53 -Mut/ STK11- EGFR- WT tumors (A), in patients with TP53-STK11-EGFR-WT tumors (B), and in patients with STK11 -Mut/TP53-EGFR-WT tumors (C) (CERTIM-cohort + Rizvi-cohort). Tick marks indicate censoring events. P values ⁇ 0.05 were considered statistically significant.
  • Figure 4 Impact of PD-L1 expression on the PFS of patients treated by anti-PD-1 according to distinct combinations of TP53, EGFR and STK11 mutations.
  • the third cohort was composed of 32 advanced-stage lung adenocarcinoma patients enrolled by the Cochin Immunomodulatory Therapies Multidisciplinary Study group (CERTIM) from February 2015 through August 2016, and treated with nivolumab (anti-PD-l, Bristol Myers Squibb) at a dose of 3 mg per kilogram of body weight every two weeks.
  • RECIST 1.1 criteria were used to monitor response to nivolumab.
  • Written informed consent was obtained from all patients.
  • the protocol was approved by the local ethics committee (CPP Ile de France II, n°2008-l33 and 2012 06-12) in agreement with article L.1121-1 of French law. Additional details are provided in the supplementary methods.
  • FFPE paraffin-embedded
  • Calopix software (Tribvn) was used to count CD66b+ and CD68+ cells in the whole tumor section; CD8 T cells were counted separately in the tumor nests and in tumor stroma.
  • DC-Lamp+ cells were counted manually in the whole tumor section. Areas of the whole tumor section, tumor nests and tumor stroma were determined by using Calopix software.
  • results are expressed as absolute number of positive cells/mm2.
  • the proportion of MHC-I+ and PD-L1+ cells among tumor cells was determined manually by at least two independent observers (JB, AL or DD). The positivity threshold was fixed at > 1%. Additional details are provided in the supplementary methods.
  • the start of follow-up for overall survival was the time of surgery (221 lung adenocarcinoma cohort) or the time of first anti-PD-l injection (CERTIM- cohort).
  • the start date of follow-up for progression-free survival (PFS) was the time of first anti-PD-l injection (CERTIM-cohort).
  • PFS progression-free survival
  • a cohort of patients with advanced-stage lung adenocarcinomas receiving nivolumab was used to assess the effectiveness of this treatment according to patients’ mutational status. Patients entered in the analysis were not part of a clinical trial. All had a metastatic disease. Treatment received was determined during the multidisciplinary thoracic oncology weekly meeting according to the international consensus guidelines. First-line treatment included a platinum derivative given in a doublet of chemotherapy. In case of adenocarcinoma, pemetrexed was combined to platinum. From July 2015, the second- line therapy became nivolumab given as single agent every two weeks because it became available in our hospital for standard patient’s treatment.
  • Treatment could continue beyond initial disease progression if the investigator assessed that patient was having clinical benefit and did have acceptable side effects.
  • First response was assessed after 4 cycles of treatment. Patients with stable disease or response according to RECIST 1.1 criteria could continue treatment. Patients with progressive disease but clinical benefit could also continue treatment and have another evaluation after two more cycles. After 6 cycles of treatment, patients with progressive disease without clinical benefit discontinued nivolumab and were considered as non-responders.
  • Nivolumab response was assessed every month during nivolumab treatment and when nivolumab has been stopped the follow up was continued. Patients were followed for survival continuously while they were receiving treatment and after discontinuation of nivolumab.
  • FFPE lung tumor sample For each FFPE lung tumor sample, two observers, including at least one expert pathologist (AL, DD), selected the tumor section containing the highest density of immune cells on hematoxylin and eosin-safran stained slides. Serial 3 pm tissue sections were deparaffinized, rehydrated and pretreated in appropriate buffer for antigen retrieval, incubated with 5% human serum (ref. S4190, Biowest) for 30 min at room temperature.
  • AL, DD expert pathologist
  • Tissue sections were then incubated for one hour at room temperature with the following primary antibodies, polyclonal anti-CD3 (Dako), anti-MHC-I (EMR8-5, abeam), anti-CD8 (SP16, Spring-bioscience), anti- DC-Lamp (1010.01, Dendritics), anti-CD66b (G10F5, BD bioscience) or anti-CD68 (PG-M1, Dako), followed by an incubation with the appropriate biotinylated secondary antibodies for 30 minutes at room temperature, and with peroxidase-conjugated streptavidin (Dako) for 30 min at room temperature.
  • primary antibodies polyclonal anti-CD3 (Dako), anti-MHC-I (EMR8-5, abeam), anti-CD8 (SP16, Spring-bioscience), anti- DC-Lamp (1010.01, Dendritics), anti-CD66b (G10F5, BD bioscience) or anti-CD68 (PG-M1, Dako)
  • PD-L1 staining For PD-L1 staining, anti-PD-Ll (E1L3N, Cell signaling) antibody was incubated for 1 hour using Leica Bond automat. For single stainings, sections were counterstained with hematoxylin. Slides were scanned using a Nanozoomer scanner (Hamamatsu) and operated with NDPview software. Calopix software (Tribvn) was used to count CD66b + and CD68+ cells in the whole tumor section; CD8 T cells were counted separately in the tumor nests and in tumor stroma. DC-Lamp + cells were counted manually in the whole tumor section. Areas of the whole tumor section, tumor nests and tumor stroma were determined by using Calopix software.
  • results are expressed as absolute number of positive cells/mm2.
  • the percentage of MHC-I + cells and of PD-L1+ cells among tumor cells was determined manually by at least two independent observers (JB, AL or DD).
  • the positivity threshold for PD-L1 expression was fixed at > 1%.
  • cytokine staining For intracellular cytokine staining, cells were stimulated for 4h with or without (unstimulated cells) phorbol l2-myristate 13 -acetate (PM A) and ionomycin (Sigma- Aldrich) in the presence of brefeldin A and monensin (Stimulated and unstimulated cells) (BD Pharmingen). Cell surface staining involved appropriate dilutions of monoclonal antibodies for 30 minutes at 4°C. Cells were then permeabilized by using the Fixation/Permeabilization Solution (BD Biosciences) and stained with appropriate dilutions of various monoclonal antibodies for 30 min at 4oC (data not shown).
  • PM A phorbol l2-myristate 13 -acetate
  • monensin Stimulated and unstimulated cells
  • Unstimulated cells were used as control and stained with the same antibody mix (anti-CD45, anti-CD3, anti-CD8 and anti-IFN-g) than stimulated cells, except for GranzymeB staining for which an isotype control was used.
  • Flow cytometry acquisition was performed on a 15-colors Fortessa cytometer (Becton Dickinson). In most experiments, dead cells were excluded using fixable viability dyes and based on forward- and side-scatter characteristics. Results were analyzed by using DIVA (Becton Dickinson) and/or FlowJo software (TreeStar, Inc).
  • TP53 mutations using the International Agency for Research on Cancer (IARC) database, were classified as missense mutations, nonsense mutations, deletions resulting in frameshift and mutations in splicing sites. Subtypes of TP 53 endpoint mutations were also investigated (IARC). TP 53 missense mutations were classified according to their impact on p53 transcriptional activity as nonfunctional, partially functional and functional (24) (IARC database version Rl 8). The two most frequent EGFR mutations in lung cancer, deletion in exon 19 (Del 19) and L858R mutation in exon 21 which represented approximately 90% of all EGFR gene alterations were studied (25).
  • STK11 mutation subtypes meaning STKllexon i-2 mutations resulting in a potential gain of oncogenic function (GOF) via the synthesis of truncated AN-S1 11 isoforms, and the disruptive STKllexon 3-9 mutations associated with tumor-suppressive function (TSF) were studied (19, 26).
  • GAF oncogenic function
  • TSF tumor-suppressive function
  • TIPs tumor immune profiles
  • the first one (TIP-l) was characterized by the highest density of CD8 T cells, indicating a strong adaptive immune response (data not shown).
  • the main feature of TIP-2 was a strong infiltration of macrophages (data not shown).
  • TIP-3 most tumors could be classified as immuno logically ignored, although some of them exhibited a high density of neutrophils (data not shown).
  • the clinical parameters did not differ among the TIPs except an increased proportion of male in the TIP-l (data not shown).
  • TIP-l was also composed of two subgroups, TIP- la and TIP-lb, which mainly differed regarding CD8Tu cell density, with the highest density of CD8Tu cells observed in TIP-lb (data not shown).
  • TIPs are strongly impacted by TP53, STK11 and EGFR mutations.
  • the differential level of PD-L1 expression by tumor cells in the three identified TIPs might suggest that malignant cells differed at the molecular level in each TIP.
  • TP53 , KRAS, STK11 and EGFR the distribution of 7 gene alterations in each TIP, including that of the four most common mutations in lung adenocarcinoma ( TP53 , KRAS, STK11 and EGFR ) (23).
  • TP53 and STK11 mutations were differentially distributed in the three identified TIPs (data not shown).
  • TP 53 mutations were enriched in TIP-l (data not shown) and to an even greater extent in TIP-lb (data not shown), while STK11 mutations were enriched in TIP-3 (data not shown).
  • 7Y J 53-mutatcd tumors were characterized by higher CD8s densities and PD-L1 expression (data not shown).
  • STK11 -mutated tumors were characterized by higher neutrophil density, lower CD8s and DC-Lamp + cell density, and lower PD-L1 expression (data not shown).
  • EGFR mutations were associated with a lower amount of neutrophils, macrophages, CD8T U cells and PD-L1 expression, together with a higher mature DC density (data not shown), while KRAS mutations did not impact the composition of the tumor immune microenvironment.
  • TP53, STK11 and EGFR mutation subtypes differed in the three TIPs.
  • Most TP53 alterations were missense mutations (data not shown), and among the three TIPs, no significant differences were observed regarding the distribution of TP53 missense mutations, nonsense mutations, deletions resulting in frameshift and mutations in splicing sites (data not shown).
  • the distribution of the different types of TP 53 point mutations was similar in the three TIPs (data not shown).
  • TIPs are strongly influenced by distinct combinations of TP 53, STK11 and EGFR mutations.
  • NSCLC tumors have a high mutational burden with frequent co-occurring mutations, including co-occuring TP53 and STK11 mutations or TP53 and EGFR mutations (27).
  • TP53 and STK11 mutations differentially impacted the immune composition of the tumor microenvironment.
  • the highest densities of CD8T U and CD8s cells, together with the highest expression of PD-L1 by malignant cells were observed in 7Y J 53 -mutated tumors unaffected by additional STK11 nor EGFR mutations (TP53-Mut/STKl 1 -EGFR-WT) (data not shown).
  • TP53-Mut/STKll-EGFR-WT tumors are characterized by an up-regulation of gene signatures associated with T cell chemotaxis, cytotoxicity and antigen presentation by MHC-I.
  • T cell chemotaxis CCL5, CCL9, CCL10, CCL11 and CCL13
  • immune cell cytotoxicity GNLY, GZMA, GZMB and PRF1
  • TAP1, PSMB8, PSMB9, HLA-A and -B a pathway related to antigen processing and peptide presentation by MHC-I
  • TAP1, PSMB8, PSMB9, HLA-A and -B a pathway related to antigen processing and peptide presentation by MHC-I
  • TEP1QA and C1QB was found in TP53-Mn ⁇ JSTKll- EGFR-WT group, signaling an activation of the classical complement pathway.
  • Clinical benefit to anti-PD-1 is strongly influenced by distinct combinations of TP53, STK11 and EGFR mutations.
  • KRAS mutations did not impact PFS from patients having a STK1 l-Mut/TP53-EGFR-WT tumor, or a TP53-STK11-EGFR- WT tumor, while in the TP53-Mut/STKl 1 -EGFR-WT group a non significant trend toward a longer PFS was observed in patients with an additional KRAS alteration (Fig. 3).

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Abstract

L'invention concerne la prédiction d'avoir un répondeur ou un non-répondeur vis-à-vis d'un inhibiteur de point de contrôle immunitaire chez un patient souffrant d'un cancer du poumon. Pour étudier l'inter-jeu entre des cellules malignes et leur micro-environnement immunitaire, les inventeurs ont effectué, à partir d'échantillons d'adénocarcinome pulmonaire, une analyse intégrative qui incorpore l'immunochimie (IHC), l'expression génique et les données de mutation et de cytométrie en flux. Ils ont identifié trois profils immunitaires principaux de tumeur (TIP) et découvert que des modifications génétiques se produisant conjointement, en particulier des mutations TP53, EGFR et STK11, sont des déterminants majeurs de la composition immunitaire tumorale et de l'expression de PD-L1 par des cellules malignes. De plus, il a été découvert que des combinaisons distinctes de mutations TP53, EGFR et STK11 ont pu identifier les meilleurs répondeurs aux bloqueurs PD-l. Ainsi, la présente invention concerne un procédé de prédiction du fait qu'un patient souffrant d'un cancer du poumon va obtenir une réponse à un inhibiteur de point de contrôle immunitaire comprenant la détermination, notamment, du profil de mutations des gènes TP53, STK11 et EGFR et la conclusion que le patient a une probabilité élevée d'obtenir une réponse à un inhibiteur de point de contrôle immunitaire en fonction de son profil de mutations.
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WO2023209035A1 (fr) * 2022-04-26 2023-11-02 F. Hoffmann-La Roche Ag Procédés et compositions pour prédire une réponse à une thérapie anticancéreuse

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KR20220112121A (ko) * 2021-02-03 2022-08-10 연세대학교 산학협력단 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법
WO2022169249A1 (fr) * 2021-02-03 2022-08-11 연세대학교 산학협력단 Méthode de prédiction d'une réponse de traitement à une immunothérapie anticancéreuse dans le cancer du poumon
KR102553128B1 (ko) 2021-02-03 2023-07-06 연세대학교 산학협력단 폐암에서 면역 항암 요법에 대한 치료 반응 예측 방법
WO2023209035A1 (fr) * 2022-04-26 2023-11-02 F. Hoffmann-La Roche Ag Procédés et compositions pour prédire une réponse à une thérapie anticancéreuse

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