Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention relates to methods for predicting the survival time of subjects suffering from melanoma metastases.
• T lymphocytes play a central role in anti-tumor responses.
• Their T cell antigen receptor (TCR) specifically recognizes antigen on tumor cells and elicits tumor destruction.
• T cell responses are tuned by signals that result from the engagement of several other surface receptors that convey positive (co-stimulatory) or negative (co -inhibitory) signals.
• Co- inhibitory molecules such as PD-1 and CTLA-4 dampen TCR activity and their blockade by monoclonal antibodies reinvigorates the activity of the anti-tumor effectors T cells present in tumor infiltrates.
• metastatic melanoma was ones of the poorest prognosis tumors at metastatic stage until the arrival of immunotherapies based on anti-CTLA-4 and anti-PD-1 antibodies that drastically improved progression free survival and overall survival of patients (1-3).
• T cells co-expressing PD-1 together with other co-inhibitory molecules may be more profoundly hyporesponsive than those expressing PD-1 alone, accounting for those patients that do not respond to anti-PD-1 immunotherapies. Therefore, it is likely that combinatorial immunotherapy targeting several appropriate co-inhibitory pathways will be required for maximal therapeutic benefit. It is thus important to characterize novel co- inhibitory pathways and to validate them as target for novel therapy against human tumors.
• HVEM Herpes virus entry mediator
• TNF tumor necrosis factor
• B cells T cells
• natural killer cells TNF
• dendritic cells TNF receptor superfamily
• myeloid cells as well as on the parenchyma of tissues.
• HVEM functions as either a ligand or receptor in diverse physiological and pathological processes.
• HVEM is a ligand for the TNF superfamily members LIGHT and Lymphotoxin a. Ligation of HVEM, expressed by antigen presenting cells, by LIGHT promotes T-cell proliferation and cytokine production via the transcription factor nuclear factor-KB.
• HVEM has a dual functional activity for T-cell activation depending on the receptors engaged. Additionally, HVEM could function also as a receptor by it activating ligation to BTLA and CD 160 (4,5) via the transcription factor nuclear factor-KB.
• HVEM on melanoma cells inhibited IFNy production and the proliferation of tumor-specific CD8 + T cells via engagement of BTLA-expressing T cell, suggesting that inhibitory interactions of HVEM- BTLA play a role in evading host anti-tumor immunity.
• HVEM expression levels on human esophageal squamous cell carcinoma were inversely correlated with the presence of tumor-infiltrating CD4 + and CD8 + T cells and CD45RO memory T cells (6). Similar results have been obtained in colorectal cancer (7) and hepatocellular carcinoma (8).
• the present invention relates to methods for predicting the survival time of subjects suffering from melanoma metastases.
• the present invention is defined by the claims.
• HVEM Herpes Virus Entry Mediator
• HVEM metastases status is an independent prognostic marker in melanoma.
• a high expression of HVEM by melanoma metastases is associated with a significantly poorer survival from the date of excision than a low HVEM expression. Therefore, high levels of HVEM expression on melanoma may dampen anti-tumor immune responses, suggesting that together with its ligands, HVEM constitutes promising targets for antibody-mediated 'checkpoint blockade' therapy.
• a first object of the present invention relates to a method for predicting the survival time of a subject suffering from melanoma metastases comprising i) determining the expression level of HVEM in a metastasis tissue sample ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject will have a short survival time when the expression level determined at step i) is higher than the predetermined reference value or concluding that the subject will have a long survival time when the expression level determined at step i) is lower than the predetermined reference value.
• the method is particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of the cancer subject.
• 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.
• short survival time indicates that the subject will have a survival time that will be lower than the median (or mean) observed in the general population of subjects suffering from said cancer.
• long survival time indicates that the subject will have a survival time that will be higher than the median (or mean) observed in the general population of subjects suffering from said cancer.
• the subject will have a long survival time, it is meant that the subject will have a "good prognosis”.
• the term "metastasis tissue sample” means any tissue metastasis tissue sample derived from a melanoma metastasis. Said tissue sample is obtained for the purpose of the in vitro evaluation.
• the metastasis tissue sample may result from a tumor exicision.
• the metastasis tissue sample may result from a biopsy performed in metastatic sample distant from the primary tumor of the patient.
• the metastasis tissue sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.).
• the sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).
• HVEM is intended to encompass all synonyms including, but not limited to, "Herpes Virus Entry Mediator”, “HVEA”, “Herpes Virus Entry Mediator A”, “TNFRSF14”, “Tumor Necrosis Factor Receptor Superfamily Member 14", “TNR14”, “LIGHTR”, “LIGHT receptor”, “TR2”, “TNF Receptor-like”, “ATAR”, “Another TRAF- Associated Receptor”.
• TNFRSF14 is the HUGO (Human Genome Organization) Gene Nomenclature Committee (HGNC) approved symbol.
• the UniProtKB/Swiss-Prot "Primary Accession Number” for HVEM is Q92956.
• the "Secondary Accession Numbers” are Q8WXR1, Q96J31 and Q9UM65.
• the level of HVEM is determined at the protein level by any well known method in the art.
• such methods comprise contacting the tumor tissue sample with at least one selective binding agent capable of selectively interacting with HVEM.
• the selective binding agent may be polyclonal antibody or monoclonal antibody, an antibody fragment, synthetic antibodies, or other protein-specific agents such as nucleic acid or peptide aptamers.
• the antibodies may be tagged directly with detectable labels such as enzymes, chromogens or fluorescent probes or indirectly detected with a secondary antibody conjugated with detectable labels.
• the expression level of HVEM is determined by immunohistochemistry (IHC). Accordingly, the metastasis tissue sample is firstly incubated the binding partners. After washing, the labeled antibodies that are bound to marker 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, En Vision system. Counterstaining may be used, e.g. H&E, DAPI, Hoechst.
• one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e HVEM).
• exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof.
• the label is a quantum dot.
• Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g.
• chemiluminescent compounds e.g. luminal, imidazole
• bio luminescent proteins e.g. luciferin, luciferase
• haptens e.g. biotin
• horseradish peroxidase alkaline phosphatase, beta-lactamase
• radioisotopes e.g. 3H, 14C, 32P, 35S or 1251
• 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.
• amine reaction or the thiol reaction.
• 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.
• enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red.
• the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody.
• a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled.
• Immunohistochemistry typically includes the following steps i) fixing said metastasis tissue sample with formalin, ii) embedding said metastasis tissue sample in paraffin, iii) cutting said metastasis tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for HVEM, v) rinsing said sections, vi) incubating said section with a biotinylated secondary antibody and vii) revealing the antigen-antibody complex with avidin-biotin-peroxidase complex.
• 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 HVEM in the sample.
• the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g., published U.S. Patent Publication No. US20100136549).
• the image can be quantitatively or semi-quantitatively analyzed and scored based on staining intensity of the sample.
• Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of the specified biomarker (i.e. HVEM).
• Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically.
• images can be quantitatively analyzed using a pixel count algorithms (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S.
• a ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored.
• the amount of the detected biomarker i.e. HVEM
• HVEM 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%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%), 80%), 85%o, 90%), 95% or more positive pixels as compared to the total staining area.
• 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., HVEM) present in the sample.
• target biomarker e.g., HVEM
• 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 HVEM (e.g. an antibody as above descried), ii) proceeding to digitalisation of the slides of step a.
• the level of HVEM is determined at nucleic acid level.
• the level of a gene may be determined by determining the quantity of mRNA.
• 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 fiuorochromes).
• fluorescent molecules or fiuorochromes.
• Numerous fiuorochromes 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).
• Examples of particular fiuorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule 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, naphtho fluorescein, 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.
• a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence.
• Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
• semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics.
• 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 Life 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.
• radioisotopes such as 3 H
• metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
• liposomes 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.
• 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.
• SISH silver in situ hybridization
• 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
• in situ hybridization procedures see, e.g., U.S. Pat. No. 4,888,278.
• Numerous procedures for FISH, CISH, and SISH are known in the art.
• procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al, Proc. Natl.
• CISH is described in, e.g., Tanner et al, Am. .1. Pathol. 157: 1467- 1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
• 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.
• 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 semiquantitative 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.
• All post hybridization steps are handled robotically on a custom liquid-handling robot (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 WO07/076132, 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.
• Expression level of a gene may be expressed as absolute level or normalized level. Typically, 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.
• the predetermined reference value is a threshold value or a cutoff value.
• a threshold value can be determined experimentally, empirically, or theoretically.
• a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of level of HVEM in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
• the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
• ROC Receiver Operating Characteristic
• a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
• AUC area under the curve
• the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
• the AUC value of the ROC curve is between 1.0 and 0.5. When AUO0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
• the predetermined reference value is determined by carrying out a method comprising the steps of a) providing a collection of samples; b) providing, for each ample provided at step a), information relating to the actual clinical outcome for the corresponding subject (i.e.
• the expression level of HVEM has been assessed for 100 samples of 100 subjects.
• the 100 samples are ranked according to the expression level of HVEM.
• Sample 1 has the highest level and sample 100 has the lowest level.
• a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
• the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
• Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
• the predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest.
• the expression level of HVEM corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value.
• the predetermined reference value is not necessarily the median value of levels of HVEM.
• the predetermined reference value thus allows discrimination between a poor and a good prognosis for a subject. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value.
• a range of values is provided instead of using a definite predetermined reference value. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided.
• This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the outcome can be determined by comparing the expression level of HVEM with the range of values which are identified.
• a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a subject may be assessed by comparing values obtained by measuring the expression level of HVEM, where values greater than 5 reveal a poor prognosis and values less than 5 reveal a good prognosis.
• a subject may be assessed by comparing values obtained by measuring the expression level of HVEM and comparing the values on a scale, where values above the range of 4-6 indicate a poor prognosis and values below the range of 4-6 indicate a good prognosis, with values falling within the range of 4-6 indicating an intermediate occurrence (or prognosis).
• the method of the present invention is also suitable for determining whether a patient is eligible to a treatment.
• treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects 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 subject 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 subject during treatment of an illness, e.g., to keep the subject 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 treatment consists in administering to the subject having a short survival time with a therapeutically effective amount of an antagonist of the BTLA/HVEM interaction.
• BTLA has its general meaning in the art and refers to the B- and T-lymphocyte attenuator protein. The term includes any human isoform of BTLA that is capable of binding to HVEM.
• the antagonist is a selected in the group consisting of HVEM ligand. Typically, said antagonist is a soluble HVEM ligand.
• said ligand is selected in the group consisting of BTLA, LIGHT, LTa, glycoprotein D and CD 160.
• said soluble HVEM ligand is recombinant.
• the antagonist is selected from the group consisting of a soluble recombinant BTLA, a soluble recombinant LIGHT, a soluble recombinant LTa, a soluble recombinant glycoprotein D, a soluble recombinant CD 160 or a fragment thereof which blocks the interaction between BTLA and HVEM. More preferably, the antagonist is a recombinant BTLA.
• the antagonist is chosen from antibodies directed against HVEM and fragments thereof which block the interaction between BTLA and HVEM.
• said antagonist is an antibody.
• antibody is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
• Functional fragments include antigen-binding fragments that block the interaction between HVEM and BTLA.
• the antibody is chosen among polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody, or an antibody fragments.
• the term "human antibody” refers to an antibody in which a substantial portion of the antibody molecule resembles, in amino acid sequence or structure, that of an antibody derived from human origin.
• the term “humanized antibody” refers to an antibody which has been modified by genetic engineering or by other means to be similar in structure or amino acid sequence to naturally occurring human antibodies.
• a "human antibody” or a “humanized antibody” may be considered more suitable in instances where it is desirable to reduce the immunogenicity of the antibody for administration to humans for therapeutic, prophylactic or diagnostic purposes.
• Antibodies specifically directed against HVEM may be derived from a number of species including, but not limited to, rodent (mouse, rat, rabbit, guinea pig, hamster, and the like), porcine, bovine, equine or primate and the like.
• Antibodies from primate (monkey, baboon, chimpanzee, etc.) origin have the highest degree of similarity to human sequences and are therefore expected to be less immunogenic.
• Antibodies derived from various species can be "humanized” by modifying the amino acid sequences of the antibodies while retaining their ability to bind the desired antigen.
• Antibodies may also be derived from transgenic animals, including mice, which have been genetically modified with the human immunoglobulin locus to express human antibodies.
• polyclonal antibodies can be obtained from serum of an animal immunized against HVEM, which may be produced by genetic engineering for example according to standard methods well-known by one skilled in the art. Typically, such antibodies can be raised by administering HVEM protein subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
• the antigens can be injected at a total volume of 100 ⁇ per site at six different sites. Each injected material may contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
• the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times at six weeks' interval.
• a sample of serum is then collected 10 days after each boost.
• Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed by (Harlow et al, 1988), which is hereby incorporated in the references.
• monoclonal antibodies were produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of antibody molecules can also be prepared by the methods of the present invention. Laboratory methods for preparing monoclonal antibodies are well known in the art.
• a “monoclonal antibody” or “mAb” in its various names refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular epitope.
• a monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts.
• Monoclonal antibody may also define an antibody molecule which has a plurality of antibody combining sites, each immunospecific for a different epitope. For example, a bispecific antibody would have two antigen binding sites, each recognizing a different interacting molecule, or a different epitope.
• an immunoglobulin molecule such as to permit specific interaction between said molecule and an antigen (e.g. HVEM).
• the portion of an immunoglobulin molecule may include, but is not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of a ligand or counter-receptor (e.g.
• Monoclonal antibodies may be prepared by immunizing a mammal such as mouse, rat, primate and the like, with purified HVEM protein.
• the antibody-producing cells from the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridoma).
• the hybridoma cells producing the monoclonal antibodies are utilized as a source of the desired monoclonal antibody. This standard method of hybridoma culture is described in (Kohler and Milstein, 1975).
• the immunoglobulin genes may be isolated and used to prepare a library for screening for reactive specifically reactive antibodies.
• Many such techniques including recombinant phage and other expression libraries are known to one skilled in the art.
• mAbs can be produced by hybridoma culture the invention is not to be so limited. Also contemplated is the use of mAbs produced by cloning and transferring the nucleic acid cloned from a hybridoma of this invention. That is, the nucleic acid expressing the molecules secreted by a hybridoma of this invention can be transferred into another cell line to produce a transformant.
• the transformant is geno typically distinct from the original hybridoma but is also capable of producing antibody molecules of this invention, including immunologically active fragments of whole antibody molecules, corresponding to those secreted by the hybridoma. See, for example, U.S. Pat. No. 4,642,334 to Reading; PCT Publication No.; European Patent Publications No. 0239400 to Winter et al. and No. 0125023 to Cabilly et al. Antibody generation techniques not involving immunisation are also contemplated such as for example using phage display technology to examine naive libraries (from non-immunised animals); see (Barbas et al, 1992, and Waterhouse et al. (1993).
• Antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, affinity, ion exchange and/or size exclusion chromatography, and the like.
• the antibody of the invention may be a human chimeric antibody.
• Said human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the expression vector by introducing it into an animal cell.
• the CH domain of a human chimeric antibody may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used.
• the CL of a human chimeric antibody may be any region which belongs to Ig, and those of kappa class or lambda class can be used.
• said antibody may be a humanized antibody.
• Said humanized antibody may be produced by obtaining nucleic acid sequences encoding for CDRs domain by inserting them into an expression vector for animal cell having genes encoding a heavy chain constant region identical to that of a human antibody; and a light chain constant region identical to that of a human antibody, and expressing the expression vector by introducing it into an animal cell.
• the humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exist on separate vectors or of a type in which both genes exist on the same vector (tandem type).
• tandem type of the humanized antibody expression vector In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, a tandem type of the humanized antibody expression vector is more preferable (Shitara K et al. 1994).
• Examples of the tandem type humanized antibody expression vector include pKANTEX93 (WO 97/10354), pEE18 and the like.
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5,565,332).
• the general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).
• the fragment of the antibody which blocks the interaction between BTLA and HVEM interaction is chosen among Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments.
• Fab e.g., by papain digestion
• Fab' e.g., by pepsin digestion and partial reduction
• F(ab')2 e.g., by pepsin digestion
• facb e.g., by plasmin digestion
• pFc' e.g., by pepsin or plasmin digestion
• Such fragments may be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein.
• Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
• the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
• Said Fab fragment of the present invention can be obtained by treating an antibody which specifically reacts with human HVEM with a protease, papaine.
• the Fab may be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote to express the Fab.
• Said F(ab')2 of the present invention may be obtained by treating an antibody which specifically reacts with HVEM with a protease, pepsin.
• the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
• Said Fab' may be obtained by treating F(ab')2 which specifically reacts with HVEM with a reducing agent, dithiothreitol.
• the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote to effect its expression.
• Said scFv fragment may be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the scFv.
• CDR grafting involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).
• monoclonal antibodies of the invention are monovalent, bivalent, multivalent, monospecific, bispecific, or multispecific.
• the anti-HVEM antibody is a monoclonal antibody obtainable from the hybridoma deposited at the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France), in accordance with the terms of Budapest Treaty, on May 16, 2013, under the number CNCM I- 4752 such as described in WO2014/184360.
• HVEM 18.10 refers to an isolated HVEM antibody which is obtainable from the hybridoma accessible under CNCM deposit number 1-4752.
• the anti-HVEM antibody is a monoclonal antibody which comprises a variable light chain (VL) comprising all the CDRs of the VL chain of the antibody obtainable from hybridoma deposited as CNCM 1-4752; and a variable heavy chain (VH) comprising all the CDRs of the VH chain of the antibody obtainable from hybridoma deposited as CNCM 1-4752.
• VL variable light chain
• VH variable heavy chain
• the anti-HVEM antibody is a monoclonal antibody obtainable from the hybridoma deposited at the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France), in accordance with the terms of Budapest Treaty, on April 26, 2007, under the numbers CNCM 1-3752, CNCM 1-3753 and CNCM 1-3754 such as described in WO2008/146101.
• CNCM Collection Nationale de Cultures de Microorganismes
• the anti-HVEM antibody is a monoclonal antibody which comprises a variable light chain (VL) comprising all the CDRs of the VL chain of the antibody obtainable from hybridoma deposited as CNCM 1-3752, CNCM 1-3753 or CNCM 1-3754; and a variable heavy chain (VH) comprising all the CDRs of the VH chain of the antibody obtainable from hybridoma deposited as CNCM 1-3752, CNCM 1-3753 or CNCM 1-3754.
• VL variable light chain
• VH variable heavy chain
• said antagonist is chosen from antibodies directed against BTLA and fragments thereof which block the interaction between BTLA and HVEM.
• the previously disclosed technical features apply to said antibodies directed against
• the antagonist is an antibody obtainable from a hybridoma deposited at the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France), in accordance with the terms of Budapest Treaty, on February 4, 2009, under the number CNCM 1-4123 such as described in WO2010/106051 and WO2014/184360.
• BTLA8.2 refers to an isolated BTLA antibody which is obtainable from the hybridoma accessible under CNCM deposit number 1-4123.
• the BTLA antibody which comprises a variable light chain (VL) comprising all the CDRs of the VL chain of the antibody obtainable from hybridoma deposited as CNCM 1-4123; and a variable heavy chain (VH) comprising all the CDRs of the VH chain of the antibody obtainable from hybridoma deposited as CNCM 1-4123.
• VL variable light chain
• VH variable heavy chain
• the treatment consists in administering to the subject having a short survival time with a therapeutically effective amount of an anti-HVEM antibody capable of antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.
• ADCC antigen-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• an anti-HVEM antibody with respect to effector functions, e.g. so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.
• ADCC antigen-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
• cysteine residue(s) may be introduced in the Fc region, thereby allowing inter-chain disulfide bond formation in this region.
• the homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992).
• the treatment consists in administering to the subject having a short survival time with a therapeutically effective amount of an anti-HVEM antibody conjugates to cytotoxic moiety.
• the cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocortico
• the anti-HVEM antibody is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof.
• Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961- 2965.
• auristatin E can be reacted with para-acetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
• auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E).
• Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs, are described in, e.g., U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.
• nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N-hydroxysuccinimide ester or maleimide functionality respectively.
• TDCs cysteine-based site-specific conjugation
• ADCs cysteine-based site-specific conjugation
• Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al, 2012).
• Fc- containing polypeptide engineered with an acyl donor glutamine-containing tag e.g., Gin- containing peptide tags or Q- tags
• endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide).
• a transglutaminase can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine- containing tag or the accessible/exposed/reactive endogenous glutamine (WO 2012059882).
• the subject is administered with a further immune checkpoint inhibitor.
• the term "immune checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function.
• Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.
• Immune checkpoint inhibitors include antibodies or are derived from antibodies.
• the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies (e.g.
• anti-PDl antibodies anti-PDLl antibodies
• anti-TIMP3 antibodies anti-LAG3 antibodies
• anti-B7H3 antibodies anti-B7H4 antibodies
• anti-B7H6 antibodies anti-B7H6 antibodies.
• anti-CTLA-4 antibodies are described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238.
• One anti-CDLA-4 antibody is tremelimumab, (ticilimumab, CP- 675,206).
• the anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonal IgG antibody that binds to CTLA-4.
• Another immune checkpoint protein is programmed cell death 1 (PD-1). Examples of PD-1 and PD-11 blockers are described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: WO03042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.
• the PD-1 blockers include anti-PD-Ll antibodies.
• the PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD- L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4 antibody against PD-1 ; CT-011 a humanized antibody that binds PD-1 ; AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559 (MDX- 1105-01) for PD-L1 (B7-H1) blockade.
• nivolumab MDX 1106, BMS 936558, ONO 4538
• MK-3475 or SCH 900475 lambrolizumab
• CT-011
• immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG- 3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al, 2007, J. Immunol. 179:4202-4211).
• Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors.
• the anti-B7-H3 antibody MGA271 (Loo et al, 2012, Clin. Cancer Res. July 15 (18) 3834).
• TIM3 T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al, 2010, J. Exp. Med. 207:2187-94).
• FIGURES are a diagrammatic representation of FIGURES.
• HVEM expression is heterogeneous in melanoma metastasis.
• A Repartition of HVLE global score in the entire cohort: 61/126 were HVEM low (score 0-5) and 59/126 HVEM high score (6-12).
• B Distribution of high and low HVE expression among metastatic sites.
• C Representative case of low and high HVEM expression in melanoma metastasis.
• Figure 2 Survival from the date of excision of 126 melanoma metastasis in relation to their tumor HVEM status. Patients with high HVEM expression had a significantly poorer prognosis than those with low expression.
• Figure 3 Representation of the intake of HVEM expression to predict prognosis from biopsy: predictive comparison curves.
• HVEM is widely expressed on melanoma cells whereas its ligands are not: flow cytometry.
• Immunohistochemistry (IHC) for HVEM was performed using anti-HVEM antibody (94804, R&D) used at a 1 : 150 dilution. Additional immuno chemistries were performed using Melan-A (A103, Cell-marque) and PS100 (RP035, Cliniscience) to stain melanoma cells and CD68 (KP1, Dako), CD 163 (10D6, Leica) to stain histiocytes/macrophages. Four micrometers thick sections were cut and mounted on SuperFrost Plus adhesive slides (Thermoshandon, Pittsburgh, PA).
• IHC was performed on an automated immunostainer Benchmark XT (Ventana Medical Systems Inc, Roche, Arlington, AZ, USA), using indirect biotin-free system based on polymer (Ultraview universal RED kit, Ventana medical Systems Inc.).
• HVEM signal intensity of each spot was scored from 0 to 3, relatively to normal lymphoid tissue, which was scored 2+. Thus, spot's intensity ranged from 0 (completely negative), 1 (weak: very faint cytoplasmic signal only visible at high magnification and inferior to the signal of the standard), 2 (intermediate signal, equals to the signals of standard lymphocytes) or 3 (the most intense signal noted on the TMA matrix, superior to the signal of the standard) (Fig l.C).
• HVEM HVEM signal heterogeneity
• the 4 spots were analyzed within a given tumor sample using a global score. This global score was calculated secondly to the initial evaluation and corresponded to the sum of the intensity of each of the 4 analyzed TMA spots for each sample and thus ranged from 0 to 12. When less than 4 TMA spots were analyzed, the global score was normalized. In a few cases, whole slides were analyzed and HVEM signal intensity was scored similarly, but using lymphocytes present on the slide as a 2+ standard. To account signal heterogeneity, HVEM was scored on 4 different but representative parts of the slide. For each parts of the slide reviewed, HVEM intensity was interpreted globally and only the highest score was retained when signal heterogeneity was present.
• a global score equal to the sum of the intensity of the 4 part of the slide, was calculated. TMA spots or whole slide cases were excluded of the analysis when no tumor cells were present (confirmed by the absence of staining for Melan-A and/or PS 100), when HVEM + macrophages (identified using CD68 and/or CD 163 expression) were numerous and interfered with interpretation and when melanin pigment would interfere with interpretation.
• HVEM expression was evaluated by immunohistochemistry in 126 melanoma metastases. Cancer cells positive staining for HVEM were observed at the cell membrane, the cytoplasm or both in 115 of 126 samples (91.3 %) and without intra-specimen heterogeneity in 90 % of cases.
• HVEM HVEM expression in melanoma metastases
• 53.2 % of the specimen had a low expression of HVEM ( ⁇ 50 %) whereas 46.8 % of them had a high HVEM expression (>50 %).
• HVEM showed an heterogeneous expression on melanoma metastasis regardless of their site of origin (Fig l.B).
• Immunotherapies strategies based on the blockade of co-inhibitory molecules intend to boost host immune responses against tumors and circumvent tumor evasion strategies.
• blockade of the HVEM-BTLA pathways or enhancement of the HVEM/LIGHT pathway could be of therapeutic value in melanoma. Therefore, it was important to assess whether high levels of HVEM expression by melanoma metastases was associated with a poor clinical outcome or not.
• HVEM was expressed by melanoma metastases in that 91.3 % of the 126 specimens we analyzed, turned out to express HVEM.
• Our data are consistent with a previous study by Derre and colleagues (9) that showed the expression of HVEM on 87.5 % of the 16 analyzed metastases.
• our work genuinely demonstrates the presence of HVEM expression by melanoma cells, that HVEM expression shows heterogeneous levels permitting to define high and low expression groups, regardless of the metastatic localization.
• analyzing 4 representatives tumor sample for each TMA spot and extending in a few cases our analysis to the whole slide revealed that only 10 % of the samples showed an intra-tumor heterogeneity.
• this preliminary study provides new information on the role of HVEM in melanoma, confirming the wide variation existing in HVEM expression among melanoma metastasis and establishing a significant correlation between high expression of HVEM in melanoma metastasis and poor clinical outcomes. Further studies will be needed to decipher the mechanisms regulating the variable levels of HVEM regulation in solid tumors. Pre-clinical studies using mouse models will also permit to establish whether high levels of HVEM expression on melanoma dampen anti-tumor immune responses, making
• HVEM a promising target for antibody-mediated 'checkpoint blockade' therapy.
• Table 1 Clinicopathological characteristics according to HVEM expression in melanoma metastases. Abbreviation: SLNB, Sentinel Lymph Node Biopsy.
• Nb of metastatic sites (>/3 / ⁇ 3) 40/86 2.73 2.25-6.47 ⁇ 0.0001 0.78 0.31-2.00 0.6118
• c- Global tumor volume was assessed on CT scan : Low ( ⁇ 5cm 2 ), Medium (5- 10cm 2 ) and High (> 10cm 2 ).
• HVEM is widely expressed on melanoma cells whereas its ligands are not ( Figure 4).
• the inventors also show that HVEM and BTLA are largely expressed on melanoma TILS while LIGHT is expressed under specific circumstances and CD160 is not ( Figure 5).
• anti-HVEM, anti-BTLA and anti-HVEM+BTLA antibodies induce an increase of TNFa production in CD8+ T cells isolated from Peripheral Blood Mononuclear Cells (PBMC) ( Figure 6).
• HVEM expression contributes to tumor progression and prognosis in human colorectal cancer.

Abstract

Description

Claims

Applications Claiming Priority (4)

Publications (1)

Family

ID=57113342

Family Applications (1)

Country Status (1)

Citations (107)

• 2016
  • 2016-10-07 WO PCT/EP2016/073951 patent/WO2017060397A1/en active Application Filing

Patent Citations (112)

Non-Patent Citations (44)

Similar Documents

Legal Events